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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939058/

Role of magnesium ions in the reaction mechanism at the interface between Tm1631 protein and its DNA ligand

A protein, Tm1631 from the hyperthermophilic organism Thermotoga maritima belongs to a domain of unknown function protein family. It was predicted that Tm1631 binds with the DNA and that the Tm1631–DNA complex is an endonuclease repair system with a DNA repair function (Konc et al. PLoS Comput Biol 9(11): e1003341, 2013 ). We observed that the severely bent, strained DNA binds to the protein for the entire 90 ns of classical molecular dynamics (MD) performed; we could observe no significant changes in the most distorted region of the DNA, where the cleavage of phosphodiester bond occurs. In this article, we modeled the reaction mechanism at the interface between Tm1631 and its proposed ligand, the DNA molecule, focusing on cleavage of the phosphodiester bond. After addition of two Mg 2+ ions to the reaction center and extension of classical MD by 50 ns (totaling 140 ns), the DNA ligand stayed bolted to the protein. Results from density functional theory quantum mechanics/molecular mechanics (QM/MM) calculations suggest that the reaction is analogous to known endonuclease mechanisms: an enzyme reaction mechanism with two Mg 2+ ions in the reaction center and a pentacovalent intermediate. The minimum energy pathway profile shows that the phosphodiester bond cleavage step of the reaction is kinetically controlled and not thermodynamically because of a lack of any energy barrier above the accuracy of the energy profile calculation. The role of ions is shown by comparing the results with the reaction mechanisms in the absence of the Mg 2+ ions where there is a significantly higher reaction barrier than in the presence of the Mg 2+ ions. Graphical abstract A protein, Tm1631 from the hyperthermophilic organism Thermotoga maritima belongs to a domain of unknown function protein family. We modeled the reaction mechanism at the interface between Tm1631 and its proposed ligand, the DNA molecule, focusing on cleavage of the phosphodiester bond Electronic supplementary material The online version of this article (doi:10.1186/s13065-016-0188-6) contains supplementary material, which is available to authorized users. Electronic supplementary material The online version of this article (doi:10.1186/s13065-016-0188-6) contains supplementary material, which is available to authorized users. Background Tm1631 is a member of the domain of unknown function 72 (DUF) family in the Protein family (Pfam) database, a protein domain that has no characterized function [ 1 ]. Protein function can only be unambiguously determined experimentally, but in case of a new protein with no computationally predicted putative function it is difficult to choose the correct experiment. New procedures are necessary to facilitate this research and improve determination of function for all the DUF proteins. A new approach to this problem has been developed by our group and was described in a previous report to predict the function of the Tm1631 protein [ 2 ]. In an earlier paper [ 2 ], we used the binding site comparison capability of the ProBiS algorithm [ 3 , 4 ] to predict the binding site in the Tm1631 protein and to speculate on the nature of the DNA ligand that could bind to the Tm1631 protein. The Tm1631 protein was predicted to be analogous to endonuclease IV despite sharing <10 % sequence identity, and the proposed Tm1631–DNA complex was subjected to 90 ns long classical MD, using the CHARMM simulation package [ 5 ]. This resulted in structures of the Tm1631–DNA complex that are used in the QM/MM study reported in this paper, where we attempted to investigate the catalytic mechanism of the reaction between Tm1631 and DNA. It was of especial interest in this connection to determine how Mg 2+ ions act in this DNA binding site (Fig. 1 ). Endonuclease repair mechanism is an important mechanism that allows organisms to escape DNA damage and plays a major role in the prevention of cancer in higher organisms [ 6 ]. Endonucleases cleave phosphodiester bond of DNA at the damaged site and create a nick in the phosphodiester backbone that is recognized by further repair enzymes in the base excision repair pathway. The endonuclease catalytic mechanism is thought to involve a hydroxide ion derived from water, which forms a bond with phosphorus of the DNA and induces cleavage of the phosphodiester bond. Fig. 1 The proposed reaction mechanism for Tm1631–DNA complex. a Reaction area is encircled . The damaged nucleotide abasic dideoxyribose (3DR), which has no base (abasic site), is on position 7 of the 15 base pair long DNA chain. b Structure-based reaction mechanism of phosphodiester bond cleavage. The abasic site on the DNA is coordinated by the two Mg 2+ ions, of which one also attacks the hydroxyl nucleophile ( left panel ). Pentacovalent transition state [ 20 – 22 ] ( middle panel ) collapses, which leads to the cleavage of the scissile phosphodiester P-O3′ bond, with the transition state and the O3′ leaving group stabilized by the metal ion and inversion of the phosphate configuration ( right panel ) Endonucleases require one, two or three divalent metal ions, such as Mn 2+ or Mg 2+ in the catalytic site [ 7 – 13 ]. It is believed that in thermal environment, such as the one Thermotoga maritima lives in at temperatures around 80 °C, the most suitable metal ion for this kind of system is Mg 2+ [ 14 ]. However, Tm1631 crystal structure (PDB: 1VPQ) does not contain any metal ions in the predicted binding site. On the other hand, there are relatively few crystallographically characterised magnesium binding sites [ 15 ]. For some binding sites, a metal ion is observed in the binding site but which metal ion it is and how many of them are needed for the catalytic activity remains undetermined [ 16 – 19 ]. Here, we report the results of theoretical QM/MM studies of the reaction mechanism for the Tm1631 protein. We postulated a catalytic mechanism with two Mg 2+ ions that resembles the one of the apurinic/apyrimidinic (AP) endonuclease enzyme presented by Mol et al. [ 21 ]. We tested the system with and without the ions, and found that the energetically most favourable pathway of the phosphodiester bond cleavage catalysed by Tm1631 requires presence of Mg 2+ ions. In the proposed catalytic mechanism (Fig. 1 b), Lys73 of the Tm1631 should be in a deprotonated state before water ionizes. One of the Mg 2+ ions attacks the water molecule, and subsequently water ionizes, the proton forms a bond with Lys73 and the OH group forms a bond with the phosphorus atom. The transition state then collapses leading to cleavage of the phosphodiester P-O3′ bond, while the O3′ leaving group is stabilized by the second Mg 2+ ion. Our findings suggest that the catalytic mechanism of Tm1631 requires Mg 2+ ions and is similar to known Steitz's mechanism [ 23 ]. Experimental and computational studies [ 24 , 25 ] point out that the cleavage of the phosphodiester bond occurs via S N 2 nucleophilic substitution in three steps (Fig. 1 b) explained in work by Sgrignani et al. [ 26 ] and Yang et al. [ 27 ]. Methods Calculations were based on the crystal structure of protein Tm1631, (PDB: 1VPQ) and the DNA chains from another crystal structure (PDB: 2NQJ). System setup and MD The structures used in this work were previously equilibrated by classical MD simulation. Since there is no crystal structure of Tm1631 with DNA available in the Protein Data Bank (PDB), we used as the starting structure for this study the predicted Tm1631–DNA complex after 90 ns of classical MD, which was performed in our previous study [ 2 ]. To validate this starting complex structure, we plotted its all-atom, protein Tm1631 and DNA root-mean-square deviations (RMSDs) (compared to the first snapshot at 0 ns of classical MD) dependence against the simulation time, which showed that in the last 20 ns of simulation the RMSDs have reached a plateau, suggesting that the starting structure is well equilibrated (Additional file 1 : Figure S1). In order to obtain good reactant and product structures we probed different Mg 2+ ion and water molecules positions. In this search we required that Mg 2+ initially coordinates with six oxygen atoms (Additional file 1 : Table S1; Distances to Mg 1 2+ and Mg 2 2+ ), and with the distant environment atoms in the same positions for both, the reactant and product conformations. The final energy minimization procedures were performed without any constraints or restraints and we were able to obtain suitable starting positions for the reaction mechanism studies with the above mentioned properties. Model building and QM/MM simulation The starting structure of our simulation was the Tm1631–DNA complex after 90 ns of classical MD simulation. We replaced two water molecules with two Mg 2+ ions and then minimized the system. A minimized structure with Mg 2+ ions was further optimized at quantum mechanics/molecular mechanics (QM/MM) level using the CHARMM software package. The CHARMM force field parameters were used to describe the molecular mechanics (MM) part, while the quantum mechanics (QM) region (42 atoms in total, including both Mg 2+ ions (Fig. 2 a, b) was treated at the density functional theory (DFT) level using the B3LYP functional and the 6–31G* basis set. We used the Replica path (RPATh) method to divide the system into 16 structures equidistantly apart in the RMSD space between the reactants and products and minimized each obtained structure using 3000 steps of adopted basis Newton–Raphson (ABNR) minimization. After minimization we checked the distances between Mg 1 2+ and Mg 2 2+ ions, which were both around 4 à . Both Mg 2+ ions were coordinated with 6 oxygen atoms (reactants, Fig. 2 c; product, Fig. 2 d), and the distances between coordinated atoms and both Mg 2+ ions were approximately 2 à . Fig. 2 Protein Tm1631, Mg 2+ ions, and important amino acids in the QM region. a Protein Tm1631 and b zoom in of the binding pocket used for QM/MM calculation. Protein residues that are considered as QM and 3DR7 residue of the DNA are denoted as stick models, and the two Mg 2+ ions are cyan spheres . One of four link atoms is pink , others are not visible. In c reactant and d product are coordinated with both Mg 2+ ions ( cyan spheres ), each being coordinated with 6 oxygen atoms in total. The distance between O3′ and Mg 2 2+ decreases from reactant (2.302 à ) to product (1.841 à ) QM/MM Ab initio QM/MM [ 28 ] methods were performed with the CHARMM biomolecular program [ 5 ]. In the molecular mechanics part of the calculations, we used CHARMM force field version 36. The ab initio DFT calculations were performed using the general atomic and molecular electronic structure system (GAMESS) [ 29 ] software package, interfaced to the CHARMM program. We used the B3LYP/6–31G* level of theory, which is implemented in the GAMESS program. The ABNR minimization algorithm was used for energy minimizations. Molecular mechanics calculations were performed with a constant dielectric of ε = 1 using a classical force shift method and a cut-off distance of 12 à . Molecular graphic images were produced using the VMD software package [ 30 ]. All ab initio QM/MM and QM calculations were carried out on the CROW clusters at the National Institute of Chemistry in Ljubljana [ 31 ]. RPATh Chain-of-replica methods involve discretising any reaction pathway by defining replicated conformations along the path between the reactants and products [ 32 ]. In order to keep the pathway along the reaction smooth a penalty term is included into the potential function which keeps the RMSD values equidistant between all the neighbouring conformations [ 33 ]. This modified potential function is then used in the geometry minimization procedure to obtain the minimum energy pathway between the reactant and product structures. Since the penalty is in the RMSD space there is no preference in the reaction coordinate to the individual distances among the atoms and is thus an efficient tool to investigate the order of bond breaking and bond making during the reaction process. The RPATh method can be used for both minimizations and the MD simulations. In order to investigate the basic steps of the reaction process, one first explores the potential energy surface by minimum energy pathway from reactants to products by a minimization procedure. In the RPATh setup this means that all the replicas are minimised along with the restraint which forces them to be equidistant in RMSD space. To completely understand the energetics of the reaction process one must calculate the free energy along the reaction pathways. Unfortunately, it is not possible to perform such calculations with the satisfactory accuracy using the currently available computational methods, although much effort is invested to develop practically feasible QM/MM methods to calculate the free energy of a reaction processes [ 34 , 35 ]. Study of concurrent reaction mechanisms The following steps define the procedure for the reaction with two Mg 2+ ions in the binding pocket: For coordination and structural files, we read the last coordinate frame from the file produced by the 90 ns classical MD simulation as explained earlier [ 2 ]. To perform QM/MM calculations, the QM region and if necessary also the boundary between the classical potential and the quantum potential involving link atoms must be defined. We assigned the QM region as follows: Lys73 side chain, two water molecules close to reaction center: water number 1863 (2.710 à ; Fig. 2 and Additional file 1 : Table S1) is involved in the reaction and nearby water 6134, phosphate group with adjacent C5′ atom from abasic dideoxyribose on place 7 in the DNA (residue 3DR7), and the 5-member ring from Cyt6 residue on the DNA. We used 4 link atoms, because we divided QM and MM region in the middle of four covalent bonds: one in Lys73, one in 3DR7, and two in Cyt6 residues. The total number of QM atoms in the system including two Mg 2+ ions was 42 consisting of 170 electrons described by 343 basis functions. The total number of atoms in the system was 13,932, including 2860 water molecules. A reactant was constructed for which the QM/MM minimization procedure was initiated by using RESD [ 36 ] restraints for bonded atoms P (3DR7) and O3′ (CYT6) so that they remained separated at 1.6 à and for OH − (water 1863) and P (3DR7) which remained 3.5 à apart. On both bivalent metal ions, the Mg 2+ harmonical restraint (the "cons harm" command in CHARMM) was used to preserve the coordination of ions. At this stage 100 steps of ABNR minimization were performed. Then we removed CONS HARM restraint from the minimization run of 300 steps with only RESD restraints for bonds left. Subsequently we removed all the restraints and ran 1500 steps of minimization. From the structure of the reactant we made a product, breaking the bond between P (3DR7) and O3′(CYT6) and created a bond between OH − (water 1863) and P (3DR7) 1.6 à using restraints. We also restrained the distance between O5′ (3DR7) and P (3DR7) at 1.6 à . After 300 steps with RESD, we removed all the restraints and ran 1500 steps of a geometry minimization procedure. We replicated the whole system 16 times and produced initial replica conformations by linear interpolation of the coordinates between the reactant and the product. The first and last replica represented reactant and product conformations, respectively, and were fixed throughout the pathway minimizations. With this setup a 3000 steps ABNR minimization was performed. Procedure steps for reaction without Mg 2+ ions in the binding pocket: We started with the minimized reactant from the simulation with two Mg 2+ ions, then removed them from the structure and held the distance between OH − (water 1863) and P (3DR7) at 4 à for 300 steps. Afterwards we removed all RESD restraints and ran 3000 steps of ABNR minimization. We took the product from the previous simulation with two Mg 2+ ions and removed them; subsequently running 3000 steps of an ABNR minimization procedure. We performed an RPATh calculation, with 3000 steps of ABNR minimization procedure using identical number of replicas as in the calculations with ions present in the system. To check the influence of the protein environment on the lowering of the energy barrier, we performed pure QM calculations with the GAMESS program as well as the reaction path calculations with the RPATh method. B3LYP/6–31G* level of theory was also used for all calculations in the pure QM case. Procedure steps for reaction without Mg 2+ ions and pure QM calculations: First we made the reactant state from the already QM/MM minimised structure without Mg 2+ ions. Subsequently we removed the whole protein, DNA structure and water molecules that were not in the QM region. Finally we ran 3000 steps of ABNR geometry minimization with restrains, holding all three protons attached to the Lys73. We made a product from the QM/MM calculations without Mg 2+ ions in the binding pocket. Then we ran 3000 steps of ABNR minimization without restrains. RPATh calculation was finally performed with 3000 steps of ABNR minimization, using 16 replicas. System setup and MD The structures used in this work were previously equilibrated by classical MD simulation. Since there is no crystal structure of Tm1631 with DNA available in the Protein Data Bank (PDB), we used as the starting structure for this study the predicted Tm1631–DNA complex after 90 ns of classical MD, which was performed in our previous study [ 2 ]. To validate this starting complex structure, we plotted its all-atom, protein Tm1631 and DNA root-mean-square deviations (RMSDs) (compared to the first snapshot at 0 ns of classical MD) dependence against the simulation time, which showed that in the last 20 ns of simulation the RMSDs have reached a plateau, suggesting that the starting structure is well equilibrated (Additional file 1 : Figure S1). In order to obtain good reactant and product structures we probed different Mg 2+ ion and water molecules positions. In this search we required that Mg 2+ initially coordinates with six oxygen atoms (Additional file 1 : Table S1; Distances to Mg 1 2+ and Mg 2 2+ ), and with the distant environment atoms in the same positions for both, the reactant and product conformations. The final energy minimization procedures were performed without any constraints or restraints and we were able to obtain suitable starting positions for the reaction mechanism studies with the above mentioned properties. Model building and QM/MM simulation The starting structure of our simulation was the Tm1631–DNA complex after 90 ns of classical MD simulation. We replaced two water molecules with two Mg 2+ ions and then minimized the system. A minimized structure with Mg 2+ ions was further optimized at quantum mechanics/molecular mechanics (QM/MM) level using the CHARMM software package. The CHARMM force field parameters were used to describe the molecular mechanics (MM) part, while the quantum mechanics (QM) region (42 atoms in total, including both Mg 2+ ions (Fig. 2 a, b) was treated at the density functional theory (DFT) level using the B3LYP functional and the 6–31G* basis set. We used the Replica path (RPATh) method to divide the system into 16 structures equidistantly apart in the RMSD space between the reactants and products and minimized each obtained structure using 3000 steps of adopted basis Newton–Raphson (ABNR) minimization. After minimization we checked the distances between Mg 1 2+ and Mg 2 2+ ions, which were both around 4 à . Both Mg 2+ ions were coordinated with 6 oxygen atoms (reactants, Fig. 2 c; product, Fig. 2 d), and the distances between coordinated atoms and both Mg 2+ ions were approximately 2 à . Fig. 2 Protein Tm1631, Mg 2+ ions, and important amino acids in the QM region. a Protein Tm1631 and b zoom in of the binding pocket used for QM/MM calculation. Protein residues that are considered as QM and 3DR7 residue of the DNA are denoted as stick models, and the two Mg 2+ ions are cyan spheres . One of four link atoms is pink , others are not visible. In c reactant and d product are coordinated with both Mg 2+ ions ( cyan spheres ), each being coordinated with 6 oxygen atoms in total. The distance between O3′ and Mg 2 2+ decreases from reactant (2.302 à ) to product (1.841 à ) QM/MM Ab initio QM/MM [ 28 ] methods were performed with the CHARMM biomolecular program [ 5 ]. In the molecular mechanics part of the calculations, we used CHARMM force field version 36. The ab initio DFT calculations were performed using the general atomic and molecular electronic structure system (GAMESS) [ 29 ] software package, interfaced to the CHARMM program. We used the B3LYP/6–31G* level of theory, which is implemented in the GAMESS program. The ABNR minimization algorithm was used for energy minimizations. Molecular mechanics calculations were performed with a constant dielectric of ε = 1 using a classical force shift method and a cut-off distance of 12 à . Molecular graphic images were produced using the VMD software package [ 30 ]. All ab initio QM/MM and QM calculations were carried out on the CROW clusters at the National Institute of Chemistry in Ljubljana [ 31 ]. RPATh Chain-of-replica methods involve discretising any reaction pathway by defining replicated conformations along the path between the reactants and products [ 32 ]. In order to keep the pathway along the reaction smooth a penalty term is included into the potential function which keeps the RMSD values equidistant between all the neighbouring conformations [ 33 ]. This modified potential function is then used in the geometry minimization procedure to obtain the minimum energy pathway between the reactant and product structures. Since the penalty is in the RMSD space there is no preference in the reaction coordinate to the individual distances among the atoms and is thus an efficient tool to investigate the order of bond breaking and bond making during the reaction process. The RPATh method can be used for both minimizations and the MD simulations. In order to investigate the basic steps of the reaction process, one first explores the potential energy surface by minimum energy pathway from reactants to products by a minimization procedure. In the RPATh setup this means that all the replicas are minimised along with the restraint which forces them to be equidistant in RMSD space. To completely understand the energetics of the reaction process one must calculate the free energy along the reaction pathways. Unfortunately, it is not possible to perform such calculations with the satisfactory accuracy using the currently available computational methods, although much effort is invested to develop practically feasible QM/MM methods to calculate the free energy of a reaction processes [ 34 , 35 ]. Study of concurrent reaction mechanisms The following steps define the procedure for the reaction with two Mg 2+ ions in the binding pocket: For coordination and structural files, we read the last coordinate frame from the file produced by the 90 ns classical MD simulation as explained earlier [ 2 ]. To perform QM/MM calculations, the QM region and if necessary also the boundary between the classical potential and the quantum potential involving link atoms must be defined. We assigned the QM region as follows: Lys73 side chain, two water molecules close to reaction center: water number 1863 (2.710 à ; Fig. 2 and Additional file 1 : Table S1) is involved in the reaction and nearby water 6134, phosphate group with adjacent C5′ atom from abasic dideoxyribose on place 7 in the DNA (residue 3DR7), and the 5-member ring from Cyt6 residue on the DNA. We used 4 link atoms, because we divided QM and MM region in the middle of four covalent bonds: one in Lys73, one in 3DR7, and two in Cyt6 residues. The total number of QM atoms in the system including two Mg 2+ ions was 42 consisting of 170 electrons described by 343 basis functions. The total number of atoms in the system was 13,932, including 2860 water molecules. A reactant was constructed for which the QM/MM minimization procedure was initiated by using RESD [ 36 ] restraints for bonded atoms P (3DR7) and O3′ (CYT6) so that they remained separated at 1.6 à and for OH − (water 1863) and P (3DR7) which remained 3.5 à apart. On both bivalent metal ions, the Mg 2+ harmonical restraint (the "cons harm" command in CHARMM) was used to preserve the coordination of ions. At this stage 100 steps of ABNR minimization were performed. Then we removed CONS HARM restraint from the minimization run of 300 steps with only RESD restraints for bonds left. Subsequently we removed all the restraints and ran 1500 steps of minimization. From the structure of the reactant we made a product, breaking the bond between P (3DR7) and O3′(CYT6) and created a bond between OH − (water 1863) and P (3DR7) 1.6 à using restraints. We also restrained the distance between O5′ (3DR7) and P (3DR7) at 1.6 à . After 300 steps with RESD, we removed all the restraints and ran 1500 steps of a geometry minimization procedure. We replicated the whole system 16 times and produced initial replica conformations by linear interpolation of the coordinates between the reactant and the product. The first and last replica represented reactant and product conformations, respectively, and were fixed throughout the pathway minimizations. With this setup a 3000 steps ABNR minimization was performed. Procedure steps for reaction without Mg 2+ ions in the binding pocket: We started with the minimized reactant from the simulation with two Mg 2+ ions, then removed them from the structure and held the distance between OH − (water 1863) and P (3DR7) at 4 à for 300 steps. Afterwards we removed all RESD restraints and ran 3000 steps of ABNR minimization. We took the product from the previous simulation with two Mg 2+ ions and removed them; subsequently running 3000 steps of an ABNR minimization procedure. We performed an RPATh calculation, with 3000 steps of ABNR minimization procedure using identical number of replicas as in the calculations with ions present in the system. To check the influence of the protein environment on the lowering of the energy barrier, we performed pure QM calculations with the GAMESS program as well as the reaction path calculations with the RPATh method. B3LYP/6–31G* level of theory was also used for all calculations in the pure QM case. Procedure steps for reaction without Mg 2+ ions and pure QM calculations: First we made the reactant state from the already QM/MM minimised structure without Mg 2+ ions. Subsequently we removed the whole protein, DNA structure and water molecules that were not in the QM region. Finally we ran 3000 steps of ABNR geometry minimization with restrains, holding all three protons attached to the Lys73. We made a product from the QM/MM calculations without Mg 2+ ions in the binding pocket. Then we ran 3000 steps of ABNR minimization without restrains. RPATh calculation was finally performed with 3000 steps of ABNR minimization, using 16 replicas. Results and discussion The Tm1631 protein with yet unknown function from the organism T. maritima has a similar binding site to that of DNA repair proteins, as established earlier [ 2 ]. By exploring the possible reaction pathways using QM/MM methods we tried to gain insight into the catalytic mechanism of the Tm1631–DNA complex. The similarity of the energetically most favourable pathway of the Tm1631–DNA complex with that of Mol et al. [ 21 ] strongly suggests that the mechanism is the same as in other endonucleases. The mechanism for Mg 2+ ions catalysis that we propose is most likely the so-called Steitz's mechanism [ 23 , 37 ]. A variety of conformations within the active site were energetically evaluated and compared and the following systems were studied: QM/MM calculation for the reaction with 2 × Mg 2+ ions in the binding pocket (Additional file 2 : Video S1). QM/MM calculation for the reaction without Mg 2+ ions in the binding pocket. QM calculation for the reaction without Mg 2+ ions in the binding pocket and environment with no protein or solvent molecules. Quantum mechanics/molecular mechanics and QM calculations were performed using the B3LYP/6–31G* DFT method. As expected, obtained results suggest that the protein has an impact on lowering the reaction barrier and also establish that metal ions are required in the binding pocket. Our interest in this study is focused on the core DNA repair function, leaving the deprotonation of Lys73 for future investigations, however there is evidence that such a state exists [ 8 ]. As explained in the previous section we made sure that the ions are coordinated as expected [ 38 ]. We are aware of the possibility that one, two or three ions may be involved in the DNA repair reaction mechanisms. However we present in this paper the results for the two ion systems and only the core part of the repair mechanism where the hydroxide ion attacks the phosphate and the P-O3′ bond gets cleaved. The hydroxide ion involved in nucleophilic attack on the phosphodiester bond P-O3′ was derived from water, ionization of which is accomplished with the help of an Mg 2+ ion. Then proton from the same water molecule forms a bond with the nitrogen atom of the side chain of the Lys73, which in its deprotonated state can act as proton acceptor in the enzyme's active site [ 39 ], and is part of the nucleic acid repair mechanism [ 8 ]. We calculated the pKa of the Lys73 to be 9.55 using the DEPTH tool [ 40 ], which is the lowest of all lysine residues of the Tm1631 protein. We also calculated the average pKa of the Lys73′s surrounding residues (10.5), which suggested that Lys73 is most likely in a deprotonated state before reaction occurs and can accept a proton from water molecule (Additional file 1 : Figure S2). Literature reports that Mg 2+ plays a functional role in the catalytic mechanism and the stability of protein-DNA complex. Metal ions also lower the local pKa, and this, considering the harsh environment that the organism experiences, is in a good agreement with our study [ 7 , 8 , 41 – 43 ]. Magnesium ions coordination is essential for most phosphoryl transfer enzymes [ 44 ]. The common catalytic mechanism was proposed previously [ 23 ]. This mechanism works on the same principle as our proposed mechanism: Mg 1 2+ coordinates the nucleophile and Mg 2 2+ coordinates the leaving oxygen atom (O3 ′ ). Many similar systems with two Mg 2+ ions in the binding pocket have been studied [ 12 , 37 ]. Distance between both metal ions should be ~4 à and in our case it is 3.85 à (reactant) and 4.18 à (product). Our system is also coordinated in the octahedral shape and most of the angles are 90° between O–Mg–O. Also seen from other enzymatic literature enzymatic phosphate hydrolysis proceeds as S N 2-like nucleophilic attack on the scissile phosphate performed by an hydroxide ion, which is formed upon water activation [ 24 – 26 , 45 ]. The important role of Mg 2+ ions is lowering the pK a of its ligands and also for the presence of second metal ion (which coordinates nucleophilic water or hydroxide in the binding pocket). This leads to a mechanism with early proton transfer [ 46 , 47 ], preceding the cleavage of phosphodiester bond in case on RNase catalytic system. Mg 2+ ions have an essential contribution for the specific catalytic reactions by lowering the pK a of the leaving group and can impose specific geometry for the triphosphate chain—pentacovalent transition state and products [ 44 ]. We observe six critical points in the energy profile for two Mg 2+ ions shown in Fig. 3 . The minimum with the lowest energy is the frame 5, which is very close to a symmetric structure, the so-called pentacovalent intermediate, with P atom in the center, three oxygen atoms (O1P, O2P, O5′) in a planar arrangement, and the two reacting oxygen atoms positioned almost equidistant on the opposite sides of the plane. Usually such a structure would suggest a transition state in the reaction pathway but the Mg 2+ ions effectively stabilize the energy of pentacovalent intermediate to make it a stable minimum. In order to check this structure and its energy we performed a separate calculation. We minimized the geometry of the single number five replica structure. After 10,000 steps of ABNR minimization the geometry had no visible changes to the one in the chain of replicated structures and the energy was lowered by 2.5 kcal/mol due to the removal of the restraint which slightly distorts the distances and makes the energy higher. The effect of the distortion could be made smaller by increasing the number of replicas, however this would not change the overall properties of the reaction mechanism. At the current level of accuracy one can estimate that energy noise level in Fig. 3 is <3 kcal/mol. This makes the reaction kinetically controlled exothermic and not thermodynamically controlled because there are no energy barriers between the reactants and the products. Our calculated energy change (−14.0 kcal/mol) agrees with results of another study [ 26 ], in which energy difference of phosphodiester bong cleavage starting from OH − was found to be −18.1 kcal/mol. The distances to important amino acids are reported in Additional file 1 : Table S1. Fig. 3 Energy profile ( red ) for each frame (1–16) of the QM/MM calculation with two Mg 2+ ions; the values for energy are on the y1 axis . Frame numbers (1–16) represent steps on the reaction path, in which 1 is reactant and 16 is product. Values for distances between atoms Cyt6 03′—P (3DR7) ( green ) and water 1863 OH2-P (3DR7) ( blue ) are marked on the y2 axis Next, we present the results of QM/MM calculations without Mg 2+ ions (Fig. 4 ), which supports two observations: Fig. 4 Energy profile ( red ) for the QM/MM calculation without ions with the values for the energy reported on y1 axis . Values for distances between atoms Cyt6 03′—P (3DR7) ( green ) and water 1863 OH2–P (3DR7) ( blue ) are marked on the y2 axis . The distances to important amino acids are collected in Additional file 1 : Table S2 There is a high 17 kcal/mol barrier in the middle of the reaction path between structures five and nine. This structure has features analogous to those of structure six from Fig. 3 , but in this case it is a transition state structure suggesting that the role of the Mg 2+ ions is to transform this configuration into a stable and low energy pentacovalent intermediate. Many chemical reactions have multiple reaction channels which depend mostly on the positions of the species entering the reaction. In the case where the two Mg 2+ ions are not present the conformational space of the entering species is larger than the one with the two ions present. This makes the reaction energetically less favourable because the system may explore more pathways which are of higher energy than the ones with the ions present. This works in addition to the fact that the environment atoms in the enzyme systems usually lower the energy barriers of any transition state structure. At this point we can add that a possible solution to choose the most favourable reaction channel would be the use of QM/MM molecular dynamics. But there is a sampling problem to be emphasized which, to be resolved, would require tens of nanoseconds of simulation time what translates into tens of millions steps of complete QM/MM calculations. In the present studies less than 100 thousands steps of QM/MM calculations were performed and still it took a few months of CPU time using between 32 and 128 processors depending on the task. To show the impact of the protein, DNA, solvent and ion environment on the studied reaction, we also studied the system in vacuum without Mg 2+ ions. The vacuum calculation was set up keeping just the QM region in the reaction path calculations. From the Fig. 5 one can observe similar behaviour as in the QM/MM calculation without ions, but the barrier is extremely high which suggests that the protein environment indeed significantly contributes to the energy stabilisation of the pentacovalent transition state structure. Fig. 5 Energy profile ( red ) for QM/MM calculation in vacuum without Mg 2+ ions with the values for the energy denoted on the y1 axis . Values for distances between atoms Cyt6 03′—P (3DR7) ( green ) and water 1863 OH2-P (3DR7) ( blue ) are marked on the y2 axis In order to verify the stability of the initial QM/MM setup we performed two additional classical MD 50 ns simulations starting with the reactant and product structures that we used for the minimum energy pathway calculations. The ions in these simulations kept the hexacoordinated structures throughout the simulations. In the case of reactant simulations none of the waters were exchanged and all 12 atoms around the two ions were identical. This means that the atom positions of reactant are in the stable and favorable positions to enter the reaction. Conclusion Protein Tm1631 from the organism T. maritima was predicted to be an endonuclease-like DNA binding protein, and consequently we investigated its function focusing specifically on the role of Mg 2+ ions in its binding pocket. We performed a QM/MM study of Tm1631 in a complex with damaged DNA. We found that Mg 2+ ions are required in the binding pocket in order that the reaction occurs. This allows us to conclude that Tm1631 is indeed an endonuclease binding protein with a reaction mechanism similar to that of other endonucleases. Some reconciliation is still needed regarding the number of metal ions, e.g. it is possible that only one ion suffices for the reaction to take place. The present paper is one of the few theoretical insights in the available literature to study a series of the reactions that play a role in the complex of the endonuclease repair process. Future work should be aimed at determination of the precise number and type of ions that are needed for the reaction to occur. Another interesting study would be to explain the formation of the hydroxide ion in connection with the protonation of Lys73 and the role of ions in such mechanisms. It would also be interesting to compare the present results with the results obtained by similar studies in different proteins (Additional file 3 ). Additional files 10.1186/s13065-016-0188-6 Table of distances between important atoms; RMSD graph; pKa graph. 10.1186/s13065-016-0188-6 Movie of reaction mechanism. 10.1186/s13065-016-0188-6 PDB structures for all studied mechanisms. Acknowledgements We sincerely thank Dr. Walter E. Knapp for hosting Mitja Ogrizek at FU Berlin and Dr. Petra Imhof for fruitful discussion. Competing interests The authors declare that they have no competing interests. Funding sources Financial support was provided through Grants P1-0002, J1-6736 and J1-6743 the Slovenian Research Agency. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759531/

IgE and IgG Antibodies as Regulators of Mast Cell and Basophil Functions in Food Allergy

Food allergy is a major health issue, affecting the lives of 8% of U.S. children and their families. There is an urgent need to identify the environmental and endogenous signals that induce and sustain allergic responses to ingested allergens. Acute reactions to foods are triggered by the activation of mast cells and basophils, both of which release inflammatory mediators that lead to a range of clinical manifestations, including gastrointestinal, cutaneous, and respiratory reactions as well as systemic anaphylaxis. Both of these innate effector cell types express the high affinity IgE receptor, FcϵRI, on their surface and are armed for adaptive antigen recognition by very-tightly bound IgE antibodies which, when cross-linked by polyvalent allergen, trigger degranulation. These cells also express inhibitory receptors, including the IgG Fc receptor, FcγRIIb, that suppress their IgE-mediated activation. Recent studies have shown that natural resolution of food allergies is associated with increasing food-specific IgG levels. Furthermore, oral immunotherapy, the sequential administration of incrementally increasing doses of food allergen, is accompanied by the strong induction of allergen-specific IgG antibodies in both human subjects and murine models. These can deliver inhibitory signals via FcγRIIb that block IgE-induced immediate food reactions. In addition to their role in mediating immediate hypersensitivity reactions, mast cells and basophils serve separate but critical functions as adjuvants for type 2 immunity in food allergy. Mast cells and basophils, activated by IgE, are key sources of IL-4 that tilts the immune balance away from tolerance and towards type 2 immunity by promoting the induction of Th2 cells along with the innate effectors of type 2 immunity, ILC2s, while suppressing the development of regulatory T cells and driving their subversion to a pathogenic pro-Th2 phenotype. This adjuvant effect of mast cells and basophils is suppressed when inhibitory signals are delivered by IgG antibodies signaling via FcγRIIb. This review summarizes current understanding of the immunoregulatory effects of mast cells and basophils and how these functions are modulated by IgE and IgG antibodies. Understanding these pathways could provide important insights into innovative strategies for preventing and/or reversing food allergy in patients. Introduction Global surveillance by the World Allergy Organization, shows that the prevalence of food allergies has been rising over the last decade in both developed and developing countries ( 1 ). This increase has been considered as the "second wave" of the allergy epidemic, following the "first wave" that was driven by allergic respiratory illnesses. In the United States alone, food allergies affect approximately 8% of children and 2%–3% of adults ( 2 ). The most common food allergens in the US are peanut, cow's milk, hen's egg, tree nut, soy, fish, wheat, and shellfish ( 3 ). Food allergies are hypersensitivity reactions that can be mediated by a wide range of humoral and cellular mechanisms. IgE-mediated food allergy is the most common and will be the focus of this review. It occurs in individuals who produce food-specific IgE antibodies. These subjects are often referred to as "sensitized". These IgE antibodies are bound to the innate granulocytic effector cells of anaphylaxis, mast cells and basophils. Upon interaction with allergen and cell-bound IgE, the granule contents of these cells are released and, along with prostaglandin and leukotriene mediators rapidly produced by the same cells, act on a range of target tissues to trigger immediate physiologic responses ( 4 ). In the vasculature, these mediators cause dilation of blood vessels and increased plasma leak which manifest locally in tissues as hives and angioedema (including laryngeal edema) and systemically as hypovolemic shock ( 4 ). The mediators additionally cause smooth muscle constriction, leading to bronchospasm, vomiting, and diarrhea, and also bind to neuronal receptors triggering pain and itch ( 4 ). When multiple organ systems are involved, the reaction is designated systemic anaphylaxis ( 3 ). Acute reactions often resolve within the first few hours; however, some patients experience recurrence of symptoms 8–12 h following the first reaction (biphasic reactions) ( 5 ). The mechanisms of immunological priming leading to IgE production in food allergic subjects are unclear. Some patients have prior histories of ingestion, suggesting immunological sensitization via the gut. However, many children experience adverse reactions following their initial ingestion of a food, suggesting alternate routes of immune priming. Emerging evidence suggests that sensitization can occur following cutaneous contact, especially in the setting of a disrupted skin barrier, as occurs in atopic dermatitis ( 6 , 7 ). Our understanding of the pathways of immunological sensitization, effector cell activation and regulation of IgE-mediated food allergy has grown rapidly since just over 50 years ago when reagin, the fraction of serum responsible for transferring skin test responsiveness from an allergic individual to a naïve recipient, was identified as IgE. The factors regulating IgE-mediated food allergy have been of great interest with a particular emphasis in the role of regulatory T cells (Tregs) in constraining both the emergence of food allergen-specific T helper cells and the production of allergen-specific IgE. However, in recent years, the ability of mast cells and basophils to exert adjuvant functions in immune sensitization to allergens and of IgG antibodies to block IgE-mediated food allergy has been recognized and the role of the inhibitory IgG receptor, FcγRIIb, in potently inhibiting food allergies has really come into focus. In the first part of this review, we briefly discussed the mechanisms, pathophysiology and key players in the disease. In the second part, we cover the evidence for a regulatory functions of mast cells, basophils, IgE and IgG and how they may be targeted clinically to counter food allergy. Mechanisms, Pathophysiology, and Treatment of Food Allergies Food Allergy, a Breakdown of Oral Tolerance Our ability to maintain systemic unresponsiveness to orally ingested antigens is an active process occurring in gut-associated lymphoid tissues. Food antigens can cross the epithelial barrier following damage to the epithelium, through specialized intraepithelial passages, or via sampling by antigen presenting cells (APCs) ( 8 ). Oral exposure promotes the development of Foxp3 + Tregs, including RORγt + Tregs that are induced by microbial signals in a Myd88-dependent manner ( 9 – 11 ). These prevent the development of allergen-specific IgE specialized CD103 + dendritic cells in the gut, via a process involving TGF-β and retinoic acid, promote the differentiation of naïve T cells into Tregs ( 12 ). A break in tolerance can occur when the cytokine environment in the intestine favors the emergence of effector T helper 2 (Th2) cells and/or the reprogramming of Tregs to a pathogenic phenotype. Cytokines produced by gut epithelial cells, including IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), may be particularly important drivers of this shift away from tolerance. IL-25 expression has been shown to be high in the small intestine in mouse models of food allergy, and overexpression of IL-25 increases Th2 cytokine production by type 2 innate lymphoid cells (ILC2s) and amplifies allergic responses. Conversely, lack of IL-25 is protective ( 13 , 14 ). TSLP is similarly expressed in gastrointestinal epithelial cells in the setting of food allergy. Khodoun and colleagues demonstrated that monoclonal antibodies against IL-25, IL-33, or TSLP could each individually prevent the development of food allergy induced by oral gavage with egg white and medium chain triglycerides in mice ( 15 ). IL-33 produced by intestinal epithelial cells leads to an increase in OX40L expression on CD103 + dendritic cells, which skews the immune profile towards Th2 ( 16 , 17 ). IL-33 produced by keratinocytes following epicutaneous allergen exposure to damaged skin in mice, as can occur in atopic dermatitis, has been shown to promote activation of ILC2s, Th2 skewing, intestinal mast cell expansion and susceptibility to anaphylaxis ( 18 ). By increasing the production of IL-4 by ILC2s, IL-33 can also suppress the development of Tregs ( 19 ). Researchers at Stanford have shown that etokimab (anti-IL-33) (NCT02920021) can protect against reactions to oral peanut challenge, reduce allergen-specific IgE, and decrease the levels of cytokines associated with food allergies such as IL-9 and IL-13 ( 20 ). Some of the pro-sensitizing effects for food allergens are exerted by these same cytokines in the skin. In a mouse model of atopic dermatitis driven by the cutaneous application of ovalbumin (OVA) along with the Vitamin D analogue MC903, Noti, and colleagues observed that TSLP produced by keratinocytes promotes basophil expansion and the induction of a Th2 response (driven by basophil-derived IL-4) with food allergy ( 21 , 22 ). Muto and colleagues reported a similar TSLP- and basophil-driven food allergy response in mice epicutaneously sensitized in the presence of SDS ( 23 ). Leyva-Castillo and colleagues have shown that exposure to food allergens via disrupted skin barrier has also been shown to promote mast cell expansion at a distance in the small intestine in a manner dependent on the induction of intestinal ILC2s by keratinocyte-derived IL-33 along with intestinal tuft cell-derived IL-25 ( 18 ). Gastrointestinal Mast Cell Expansion in Food Allergy: IL-4, IL-9, and MMC9 In addition to IL-25 and IL-33, the Th2 cytokines IL-4 and IL-9 have been shown to play an important role in food allergy development. IL-9 is produced both by Th2 cells and by a subset of mast cells, IL-9-producing mucosal mast cells (MMC9). IL-9 promotes mast cell growth and lack of IL-9 prevents the induction of food allergy in mouse models ( 24 , 25 ). Transgenic overexpression of IL-9 promotes the food allergy response by increasing mast cell number in the small intestine and enhancing gut permeability ( 24 , 25 ). Th2 cells contribute to the development of MMC9 that provide a positive feedback loop for the expansion of mast cell numbers following oral challenge ( 26 ). IL-4, in addition to its role in promoting Th2 responses and driving IgE isotype switching in B cells, is also needed to support intestinal mast cell expansion following food allergen ingestion ( 27 ). This cytokine induces the antiapoptotic genes, Bcl-2 and Bcl-X(L), in mast cells in a STAT6-dependent manner ( 27 ). Competitive reconstitution studies using wild type mouse mast cells mixed with mast cells lacking the IL-4 receptor α-chain have revealed a strong competitive advantage of IL-4 receptor-bearing cells. Hogan and colleagues have recently demonstrated that IL-4, signaling via IL-4Rα induces MMC9 in a BATF-dependent mechanism, nicely linking the observed roles of IL-9 and IL-4 in mast cell homeostasis ( 28 ). IL-4 signals can subvert Tregs from their normally suppressive phenotype to one in which they express the Th2 transcription factor GATA-3 and Th2 cytokines, including IL-4, and thereby drive the disease phenotype ( 29 ). Investigators at Stanford and elsewhere are conducting trials exploring the potential of dupilumab (anti-IL-4Rα monoclonal antibody that prevents the binding of IL-4 and IL-13 to its receptor) in combination with anti-IgE (NCT03679676) during peanut oral immunotherapy (OIT). Treatments for Food Allergy Currently, there are no curative treatments for food allergies. The standard approach is education of individuals and families regarding strategies of allergen avoidance. While this is effective in preventing potentially serious reactions, it can significantly impact the quality of life of the patient and their family members. Avoidance paradoxically deprives these patients of one of the best-known paths to achieving tolerance to an antigen, namely ingestion. Furthermore, broad avoidance diets can result in nutritional deficiencies in multi-sensitized children ( 30 , 31 ). Some children will eventually experience natural resolution of their allergy. Still, while this tends to occur for some foods, like egg and dairy, it is rare for others, like peanuts and tree nuts. In addition to practicing allergen avoidance, patients diagnosed with food allergies, must be presented with effective treatment plans on how to manage their reactions in case of accidental exposure. Mild reactions to foods, such as itching and hives, can be treated with anti-histamines, such as diphenhydramine and cetirizine. Though anti-histamines can alleviate the symptoms associated with allergy, they do not hinder the progression of the disease or reverse the disease-associated immune profile. Systemic anaphylaxis, the severe, life threatening reaction, presents as difficulty breathing or swallowing, and is treated immediately with epinephrine. Immunotherapy, which involves exposure to increasing doses of the allergen over a period of time, is the only disease-modifying treatment. Immunotherapy can be administered via several routes, including sublingual (SLIT), epicutaneous (EPIT) and oral (OIT) ( 32 ). Peanut powder (Palforzia ® ) OIT is the only current FDA-approved treatment for immunotherapy. A major limitation of OIT is that the food unresponsive state that is achieved is transient and maintaining it requires continued regular ingestion of the food ( 33 ). Adjunctive anti-cytokine treatments that might enhance safety as well as durability of immunotherapy are actively being explored ( 20 , 34 ). Food Allergy, a Breakdown of Oral Tolerance Our ability to maintain systemic unresponsiveness to orally ingested antigens is an active process occurring in gut-associated lymphoid tissues. Food antigens can cross the epithelial barrier following damage to the epithelium, through specialized intraepithelial passages, or via sampling by antigen presenting cells (APCs) ( 8 ). Oral exposure promotes the development of Foxp3 + Tregs, including RORγt + Tregs that are induced by microbial signals in a Myd88-dependent manner ( 9 – 11 ). These prevent the development of allergen-specific IgE specialized CD103 + dendritic cells in the gut, via a process involving TGF-β and retinoic acid, promote the differentiation of naïve T cells into Tregs ( 12 ). A break in tolerance can occur when the cytokine environment in the intestine favors the emergence of effector T helper 2 (Th2) cells and/or the reprogramming of Tregs to a pathogenic phenotype. Cytokines produced by gut epithelial cells, including IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), may be particularly important drivers of this shift away from tolerance. IL-25 expression has been shown to be high in the small intestine in mouse models of food allergy, and overexpression of IL-25 increases Th2 cytokine production by type 2 innate lymphoid cells (ILC2s) and amplifies allergic responses. Conversely, lack of IL-25 is protective ( 13 , 14 ). TSLP is similarly expressed in gastrointestinal epithelial cells in the setting of food allergy. Khodoun and colleagues demonstrated that monoclonal antibodies against IL-25, IL-33, or TSLP could each individually prevent the development of food allergy induced by oral gavage with egg white and medium chain triglycerides in mice ( 15 ). IL-33 produced by intestinal epithelial cells leads to an increase in OX40L expression on CD103 + dendritic cells, which skews the immune profile towards Th2 ( 16 , 17 ). IL-33 produced by keratinocytes following epicutaneous allergen exposure to damaged skin in mice, as can occur in atopic dermatitis, has been shown to promote activation of ILC2s, Th2 skewing, intestinal mast cell expansion and susceptibility to anaphylaxis ( 18 ). By increasing the production of IL-4 by ILC2s, IL-33 can also suppress the development of Tregs ( 19 ). Researchers at Stanford have shown that etokimab (anti-IL-33) (NCT02920021) can protect against reactions to oral peanut challenge, reduce allergen-specific IgE, and decrease the levels of cytokines associated with food allergies such as IL-9 and IL-13 ( 20 ). Some of the pro-sensitizing effects for food allergens are exerted by these same cytokines in the skin. In a mouse model of atopic dermatitis driven by the cutaneous application of ovalbumin (OVA) along with the Vitamin D analogue MC903, Noti, and colleagues observed that TSLP produced by keratinocytes promotes basophil expansion and the induction of a Th2 response (driven by basophil-derived IL-4) with food allergy ( 21 , 22 ). Muto and colleagues reported a similar TSLP- and basophil-driven food allergy response in mice epicutaneously sensitized in the presence of SDS ( 23 ). Leyva-Castillo and colleagues have shown that exposure to food allergens via disrupted skin barrier has also been shown to promote mast cell expansion at a distance in the small intestine in a manner dependent on the induction of intestinal ILC2s by keratinocyte-derived IL-33 along with intestinal tuft cell-derived IL-25 ( 18 ). Gastrointestinal Mast Cell Expansion in Food Allergy: IL-4, IL-9, and MMC9 In addition to IL-25 and IL-33, the Th2 cytokines IL-4 and IL-9 have been shown to play an important role in food allergy development. IL-9 is produced both by Th2 cells and by a subset of mast cells, IL-9-producing mucosal mast cells (MMC9). IL-9 promotes mast cell growth and lack of IL-9 prevents the induction of food allergy in mouse models ( 24 , 25 ). Transgenic overexpression of IL-9 promotes the food allergy response by increasing mast cell number in the small intestine and enhancing gut permeability ( 24 , 25 ). Th2 cells contribute to the development of MMC9 that provide a positive feedback loop for the expansion of mast cell numbers following oral challenge ( 26 ). IL-4, in addition to its role in promoting Th2 responses and driving IgE isotype switching in B cells, is also needed to support intestinal mast cell expansion following food allergen ingestion ( 27 ). This cytokine induces the antiapoptotic genes, Bcl-2 and Bcl-X(L), in mast cells in a STAT6-dependent manner ( 27 ). Competitive reconstitution studies using wild type mouse mast cells mixed with mast cells lacking the IL-4 receptor α-chain have revealed a strong competitive advantage of IL-4 receptor-bearing cells. Hogan and colleagues have recently demonstrated that IL-4, signaling via IL-4Rα induces MMC9 in a BATF-dependent mechanism, nicely linking the observed roles of IL-9 and IL-4 in mast cell homeostasis ( 28 ). IL-4 signals can subvert Tregs from their normally suppressive phenotype to one in which they express the Th2 transcription factor GATA-3 and Th2 cytokines, including IL-4, and thereby drive the disease phenotype ( 29 ). Investigators at Stanford and elsewhere are conducting trials exploring the potential of dupilumab (anti-IL-4Rα monoclonal antibody that prevents the binding of IL-4 and IL-13 to its receptor) in combination with anti-IgE (NCT03679676) during peanut oral immunotherapy (OIT). Treatments for Food Allergy Currently, there are no curative treatments for food allergies. The standard approach is education of individuals and families regarding strategies of allergen avoidance. While this is effective in preventing potentially serious reactions, it can significantly impact the quality of life of the patient and their family members. Avoidance paradoxically deprives these patients of one of the best-known paths to achieving tolerance to an antigen, namely ingestion. Furthermore, broad avoidance diets can result in nutritional deficiencies in multi-sensitized children ( 30 , 31 ). Some children will eventually experience natural resolution of their allergy. Still, while this tends to occur for some foods, like egg and dairy, it is rare for others, like peanuts and tree nuts. In addition to practicing allergen avoidance, patients diagnosed with food allergies, must be presented with effective treatment plans on how to manage their reactions in case of accidental exposure. Mild reactions to foods, such as itching and hives, can be treated with anti-histamines, such as diphenhydramine and cetirizine. Though anti-histamines can alleviate the symptoms associated with allergy, they do not hinder the progression of the disease or reverse the disease-associated immune profile. Systemic anaphylaxis, the severe, life threatening reaction, presents as difficulty breathing or swallowing, and is treated immediately with epinephrine. Immunotherapy, which involves exposure to increasing doses of the allergen over a period of time, is the only disease-modifying treatment. Immunotherapy can be administered via several routes, including sublingual (SLIT), epicutaneous (EPIT) and oral (OIT) ( 32 ). Peanut powder (Palforzia ® ) OIT is the only current FDA-approved treatment for immunotherapy. A major limitation of OIT is that the food unresponsive state that is achieved is transient and maintaining it requires continued regular ingestion of the food ( 33 ). Adjunctive anti-cytokine treatments that might enhance safety as well as durability of immunotherapy are actively being explored ( 20 , 34 ). Key Players Involved in Food Allergy: Mast Cells And Basophils, lgE, and lgE Receptors Mast Cells and Basophils: Effectors of Immediate Hypersensitivity Mast cells and basophils arise from CD34 + hematopoietic progenitor cells. Mast cells are long-lived tissue resident cells that differentiate locally from bloodborne progenitors ( 35 ). They are often found in vascularized sites that are exposed to the external environment and microbiome, such as the mucosa of the gastrointestinal and respiratory tracts ( 36 ). Basophils are short-lived cells that mature in the bone marrow, enter the circulation and can either be activated intravascularly or traffic to sites of inflammation to exert their functions ( 37 – 39 ). Because of their anatomic localization in barrier tissues, mast cells are likely to be one of the first cell types to encounter and respond to pathogens, making them important effector cells of the innate immune response. Several groups have identified their protective roles in bacterial infection and they are also critical in the immune response to parasites ( 36 , 40 ). They express pathogen recognition receptors and can release anti-microbial peptides upon activation. Mast cell granule proteases play an important role in detoxifying insect, scorpion, and reptile venoms ( 41 – 43 ). Like mast cells, basophils have been demonstrated to play key roles in host defense not only in the setting of Th2 responses, but also in the inflammatory reactions leading to helminth expulsion or tissue encystment and in resistance to ticks ( 44 – 48 ). In addition to acting as effector cells of innate immunity, mast cells and basophils live at the interface of innate and adaptive immunity. Since they express Fc receptors, for IgE and IgG, they are armed with the adaptive immune capability to recognize specific antigens and participate in recall responses. They also regulate the emergence of adaptive responses. Granule components such as histamine can regulate T cell immunity and the antibody response, and mast cells and basophils are major producers of IL-4 and IL-13 ( 49 – 52 ). These two cytokines promote both Th2 cell differentiation and isotype switching of B cells to produce IgE. The classic trigger for mast cell and basophil activation is through the IgE-mediated crosslinking the IgE receptor, FcϵRI. This activation results in the degranulation of the cells, releasing preformed mediators (such as histamine, neutral proteases, and TNF-α), de novo synthesis of pro-inflammatory lipid mediators, and production of growth factors, cytokines, and chemokines ( 53 , 54 ). FcϵRI and Its Downstream Signaling Pathways IgE is the least abundant antibody in circulation. Its concentration in plasma is roughly 10 5 times less (100 ng/ml vs 10 mg/ml) than that of IgG. In addition to being present at such low concentrations, IgE has a notably short half-life, less than 1 day, while that of IgG is around 3 weeks ( 55 ). However, most of the IgE in the body is found in a cell-bound state, owing to the incredibly high affinity for IgE by FcϵRI (K d 1x10 -9 mol/L). IgE effectively remains permanently attached to FcϵRI until internalized, which makes the tissue half-life of IgE to likely be on the order of weeks to months ( 55 , 56 ). There exist two isoforms of the high affinity IgE receptor, FcϵRI. The tetrameric αβγ 2 form is found on mast cells and basophils. The trimeric αγ 2 form, though less abundant than the tetrameric, is present on eosinophils, platelets, monocytes, and dendritic cells. The alpha chain has the ligand binding site, with two Ig-like domains that bind IgE. The β chain contains four transmembrane spanning regions and it amplifies the signal generated by the γ subunit. The γ chains are dimeric disulfide-linked transmembrane proteins. The β and γ chains contain immunoreceptor tyrosine-based activation motifs (ITAMs). An ITAM is a conserved sequence containing tyrosine separated from a leucine or isoleucine by two amino acids (YxxL/I). The ITAM tyrosine is a substrate for phosphorylation by signaling kinases such as Lyn and Syk. Two ITAMs are often found together separated by 6 to 8 amino acids (YxxL/I x(6-8) YxxL/I). When multivalent antigens bind to FcϵRI-bound IgE, proximal FcϵRI aggregate into lipid rafts that are rich in cholesterol, sphingolipids, protein tyrosine kinases (PTKs), and GPI-anchored proteins. Recognition of one antigen molecule by Fab sites on two different IgE molecules bound to neighboring FcϵRI, results in receptor crosslinking and transphosphorylation of the ITAMs. Antigen recognition mediates a rapid cascade of signaling events that induce the activation of PTKs of the SRC and TEC families ( 57 – 59 ). This activation leads to the release of preformed, rapidly formed, and newly synthesized mediators from the effector cells ( 60 ) (see Figure 1 ). Figure 1 Mechanisms of IgG mediated inhibition. Antigen encounter by neighboring FcϵRI (green)-bound IgE (blue) on the surface of mast cells or basophils induces receptor crosslinking, phosphorylation of cytosolic immunoreceptor tyrosine-based activation motif (ITAM) sequences in the tetraspanning β- and disulfide-linked γ-chain dimers, and activation of various signaling pathways involving protein tyrosine kinases, such as Syk, and inositol intermediates, including PIP 3 (left panel). These positive signals culminate in the degranulation of the cell. Allergen specific IgG antibodies (orange) counter the effects of IgE in two ways, receptor-mediated inhibition (center panel) and steric blockade (right panel). In receptor-mediated inhibition, when polyvalent allergens are simultaneously engaged by FcϵRI-bound IgE and FcγRIIb (red)-bound IgG, crosslinking of the two receptors leads to phosphorylation of FcγRIIb cytosolic immunoreceptor tyrosine-based inhibition motifs (ITIMs). These recruit protein tyrosine phosphatases and inositol phosphatases, such as SHPs and SHIPs, respectively. Phosphatases can neutralize phosphoprotein (such as Syk) and phospholipid (such as PIP 3 ) signaling intermediates induced by FcϵRI activation. In steric blockade, IgG antibodies bind the allergen before it reaches receptor-bound IgE. By masking IgE binding epitopes, these blocking IgG antibodies inhibit interaction with IgE and thereby prevent FcϵRI-mediated mast cell activation. Tyrosine residues on ITAMs on β and γ chains are phosphorylated by receptor-associated Lyn tyrosine kinase found constitutively associated with the β chain. Phosphorylation of tyrosine residues in the ITAMs provides docking sites for Src homology 2 (SH2) domain-containing kinases, such as Syk PTK to the γ chains and recruits additional Lyn PTK. Syk is phosphorylated by Lyn and activated by conformational changes ( 61 , 62 ). Phosphorylated Syk subsequently phosphorylates adapter molecules LAT1, LAT2, SLP-76, Grb2, and VAV at tyrosine sites leading to the formation of supramolecular plasma membrane-localized signaling complexes. These signaling events lead to the activation of other signalling pathways such as PI3K, PLC-γ, RAS/ERK, JNK, p38, and AKT. They also lead to the immediate exocytosis of granules and their pre-formed mediators, rapid synthesis of prostaglandins and leukotrienes and eventual transcription of cytokines genes, such as IL-4, IL-6, IL-10, and TNF-α ( 60 ). In addition to driving the allergic effector functions of mast cells and basophils, there is evidence that some IgE antibodies can provide survival signals to mast cells even in the absence of allergen. These have been designated "cytokinergic" IgEs by Kawakami and colleagues ( 63 ). In the absence of the survival cytokines necessary for mast cells, such as SCF, cytokinergic IgE antibodies can provide a survival-enhancing effect ( 64 , 65 ). Furthermore, they have been shown to induce cytokine production, calcium flux, histamine release, and leukotriene synthesis ( 66 – 68 ). Cytokinergic IgE antibodies may act by self-associating or binding to autoantigens in order to aggregate FcϵRIs. Glycosylation of IgE molecules has been shown to correlate with IgE binding to FcϵRI and for effective signaling upon receptor cross-linking. IgE antibodies are very heavily glycosylated when compared with other immunoglobulin isotypes. Anthony and colleagues demonstrated the functional significance of IgE glycans. They found that one at N394 was obligatory for FcϵRI binding ( 69 ). Subsequent comparison of plasma IgE molecules between allergic and non-allergic individuals by mass spectrometry by the same group showed that the "allergic IgE" has more terminal sialylation of its glycans than "non-allergic IgE". The degree of sialylation of IgE does not affect its binding to FcϵRI but modulates the signaling downstream of the receptor and activation of the cell ( 70 ). CD23: The Low Affinity Receptor for IgE IgE can also bind to and exert its effects through its low affinity receptor CD23 (FcϵRII). CD23 is broadly distributed with expression on B cells, dendritic cells, eosinophils, gastrointestinal and respiratory epithelial cells, and others ( 71 ). It is a type II transmembrane protein (N-terminus intracellular) assembled as a multimer with α-helical coiled-coil stalks terminating in C-type lectin heads that bind to IgE (see Figure 2 ) ( 72 ). CD23 is the only Fc receptor that is not part of the immunoglobulin superfamily. In addition to binding IgE, it also interacts with the B cell surface protein, CD21, which functions as complement receptor 2 (CR2) and is the binding site and entry point for Epstein-Barr virus in B cells ( 73 ). Protease-sensitive sites present in the stalks of CD23 can be cleaved by endogenous proteases such as the metalloproteinase, ADAM10, and by protease allergens including the dust mite protease, Der p 1 . The released oligomeric CD23 heads are called soluble CD23 (sCD23) and retain their ability to bind IgE. Figure 2 Structures and functions of the low-affinity IgE receptor, CD23. CD23 is expressed as a multimer of subunits consisting of coiled-coil stalks with lectin-family domain heads that bind to IgE (upper panels). Membrane-bound CD23 can be converted to a soluble form that retains IgE-binding ability following cleavage at protease sites by endogenous (ADAM) or allergen (e.g. Der p 1 ) proteases. Various functions have been attributed to CD23 (lower panels). It can facilitate antigen uptake for presentation by B cells and antigen presenting cells to T cells (left panel) and mediate the transport of allergens across polarized epithelium in the gut and airway (center panel). CD23 also regulates IgE production; the transmembrane form on B cells suppresses their production of IgE and the soluble form, via interactions with B cell surface CD21 and IgE, enhances IgE production (right panel). Mast Cells and Basophils: Effectors of Immediate Hypersensitivity Mast cells and basophils arise from CD34 + hematopoietic progenitor cells. Mast cells are long-lived tissue resident cells that differentiate locally from bloodborne progenitors ( 35 ). They are often found in vascularized sites that are exposed to the external environment and microbiome, such as the mucosa of the gastrointestinal and respiratory tracts ( 36 ). Basophils are short-lived cells that mature in the bone marrow, enter the circulation and can either be activated intravascularly or traffic to sites of inflammation to exert their functions ( 37 – 39 ). Because of their anatomic localization in barrier tissues, mast cells are likely to be one of the first cell types to encounter and respond to pathogens, making them important effector cells of the innate immune response. Several groups have identified their protective roles in bacterial infection and they are also critical in the immune response to parasites ( 36 , 40 ). They express pathogen recognition receptors and can release anti-microbial peptides upon activation. Mast cell granule proteases play an important role in detoxifying insect, scorpion, and reptile venoms ( 41 – 43 ). Like mast cells, basophils have been demonstrated to play key roles in host defense not only in the setting of Th2 responses, but also in the inflammatory reactions leading to helminth expulsion or tissue encystment and in resistance to ticks ( 44 – 48 ). In addition to acting as effector cells of innate immunity, mast cells and basophils live at the interface of innate and adaptive immunity. Since they express Fc receptors, for IgE and IgG, they are armed with the adaptive immune capability to recognize specific antigens and participate in recall responses. They also regulate the emergence of adaptive responses. Granule components such as histamine can regulate T cell immunity and the antibody response, and mast cells and basophils are major producers of IL-4 and IL-13 ( 49 – 52 ). These two cytokines promote both Th2 cell differentiation and isotype switching of B cells to produce IgE. The classic trigger for mast cell and basophil activation is through the IgE-mediated crosslinking the IgE receptor, FcϵRI. This activation results in the degranulation of the cells, releasing preformed mediators (such as histamine, neutral proteases, and TNF-α), de novo synthesis of pro-inflammatory lipid mediators, and production of growth factors, cytokines, and chemokines ( 53 , 54 ). FcϵRI and Its Downstream Signaling Pathways IgE is the least abundant antibody in circulation. Its concentration in plasma is roughly 10 5 times less (100 ng/ml vs 10 mg/ml) than that of IgG. In addition to being present at such low concentrations, IgE has a notably short half-life, less than 1 day, while that of IgG is around 3 weeks ( 55 ). However, most of the IgE in the body is found in a cell-bound state, owing to the incredibly high affinity for IgE by FcϵRI (K d 1x10 -9 mol/L). IgE effectively remains permanently attached to FcϵRI until internalized, which makes the tissue half-life of IgE to likely be on the order of weeks to months ( 55 , 56 ). There exist two isoforms of the high affinity IgE receptor, FcϵRI. The tetrameric αβγ 2 form is found on mast cells and basophils. The trimeric αγ 2 form, though less abundant than the tetrameric, is present on eosinophils, platelets, monocytes, and dendritic cells. The alpha chain has the ligand binding site, with two Ig-like domains that bind IgE. The β chain contains four transmembrane spanning regions and it amplifies the signal generated by the γ subunit. The γ chains are dimeric disulfide-linked transmembrane proteins. The β and γ chains contain immunoreceptor tyrosine-based activation motifs (ITAMs). An ITAM is a conserved sequence containing tyrosine separated from a leucine or isoleucine by two amino acids (YxxL/I). The ITAM tyrosine is a substrate for phosphorylation by signaling kinases such as Lyn and Syk. Two ITAMs are often found together separated by 6 to 8 amino acids (YxxL/I x(6-8) YxxL/I). When multivalent antigens bind to FcϵRI-bound IgE, proximal FcϵRI aggregate into lipid rafts that are rich in cholesterol, sphingolipids, protein tyrosine kinases (PTKs), and GPI-anchored proteins. Recognition of one antigen molecule by Fab sites on two different IgE molecules bound to neighboring FcϵRI, results in receptor crosslinking and transphosphorylation of the ITAMs. Antigen recognition mediates a rapid cascade of signaling events that induce the activation of PTKs of the SRC and TEC families ( 57 – 59 ). This activation leads to the release of preformed, rapidly formed, and newly synthesized mediators from the effector cells ( 60 ) (see Figure 1 ). Figure 1 Mechanisms of IgG mediated inhibition. Antigen encounter by neighboring FcϵRI (green)-bound IgE (blue) on the surface of mast cells or basophils induces receptor crosslinking, phosphorylation of cytosolic immunoreceptor tyrosine-based activation motif (ITAM) sequences in the tetraspanning β- and disulfide-linked γ-chain dimers, and activation of various signaling pathways involving protein tyrosine kinases, such as Syk, and inositol intermediates, including PIP 3 (left panel). These positive signals culminate in the degranulation of the cell. Allergen specific IgG antibodies (orange) counter the effects of IgE in two ways, receptor-mediated inhibition (center panel) and steric blockade (right panel). In receptor-mediated inhibition, when polyvalent allergens are simultaneously engaged by FcϵRI-bound IgE and FcγRIIb (red)-bound IgG, crosslinking of the two receptors leads to phosphorylation of FcγRIIb cytosolic immunoreceptor tyrosine-based inhibition motifs (ITIMs). These recruit protein tyrosine phosphatases and inositol phosphatases, such as SHPs and SHIPs, respectively. Phosphatases can neutralize phosphoprotein (such as Syk) and phospholipid (such as PIP 3 ) signaling intermediates induced by FcϵRI activation. In steric blockade, IgG antibodies bind the allergen before it reaches receptor-bound IgE. By masking IgE binding epitopes, these blocking IgG antibodies inhibit interaction with IgE and thereby prevent FcϵRI-mediated mast cell activation. Tyrosine residues on ITAMs on β and γ chains are phosphorylated by receptor-associated Lyn tyrosine kinase found constitutively associated with the β chain. Phosphorylation of tyrosine residues in the ITAMs provides docking sites for Src homology 2 (SH2) domain-containing kinases, such as Syk PTK to the γ chains and recruits additional Lyn PTK. Syk is phosphorylated by Lyn and activated by conformational changes ( 61 , 62 ). Phosphorylated Syk subsequently phosphorylates adapter molecules LAT1, LAT2, SLP-76, Grb2, and VAV at tyrosine sites leading to the formation of supramolecular plasma membrane-localized signaling complexes. These signaling events lead to the activation of other signalling pathways such as PI3K, PLC-γ, RAS/ERK, JNK, p38, and AKT. They also lead to the immediate exocytosis of granules and their pre-formed mediators, rapid synthesis of prostaglandins and leukotrienes and eventual transcription of cytokines genes, such as IL-4, IL-6, IL-10, and TNF-α ( 60 ). In addition to driving the allergic effector functions of mast cells and basophils, there is evidence that some IgE antibodies can provide survival signals to mast cells even in the absence of allergen. These have been designated "cytokinergic" IgEs by Kawakami and colleagues ( 63 ). In the absence of the survival cytokines necessary for mast cells, such as SCF, cytokinergic IgE antibodies can provide a survival-enhancing effect ( 64 , 65 ). Furthermore, they have been shown to induce cytokine production, calcium flux, histamine release, and leukotriene synthesis ( 66 – 68 ). Cytokinergic IgE antibodies may act by self-associating or binding to autoantigens in order to aggregate FcϵRIs. Glycosylation of IgE molecules has been shown to correlate with IgE binding to FcϵRI and for effective signaling upon receptor cross-linking. IgE antibodies are very heavily glycosylated when compared with other immunoglobulin isotypes. Anthony and colleagues demonstrated the functional significance of IgE glycans. They found that one at N394 was obligatory for FcϵRI binding ( 69 ). Subsequent comparison of plasma IgE molecules between allergic and non-allergic individuals by mass spectrometry by the same group showed that the "allergic IgE" has more terminal sialylation of its glycans than "non-allergic IgE". The degree of sialylation of IgE does not affect its binding to FcϵRI but modulates the signaling downstream of the receptor and activation of the cell ( 70 ). CD23: The Low Affinity Receptor for IgE IgE can also bind to and exert its effects through its low affinity receptor CD23 (FcϵRII). CD23 is broadly distributed with expression on B cells, dendritic cells, eosinophils, gastrointestinal and respiratory epithelial cells, and others ( 71 ). It is a type II transmembrane protein (N-terminus intracellular) assembled as a multimer with α-helical coiled-coil stalks terminating in C-type lectin heads that bind to IgE (see Figure 2 ) ( 72 ). CD23 is the only Fc receptor that is not part of the immunoglobulin superfamily. In addition to binding IgE, it also interacts with the B cell surface protein, CD21, which functions as complement receptor 2 (CR2) and is the binding site and entry point for Epstein-Barr virus in B cells ( 73 ). Protease-sensitive sites present in the stalks of CD23 can be cleaved by endogenous proteases such as the metalloproteinase, ADAM10, and by protease allergens including the dust mite protease, Der p 1 . The released oligomeric CD23 heads are called soluble CD23 (sCD23) and retain their ability to bind IgE. Figure 2 Structures and functions of the low-affinity IgE receptor, CD23. CD23 is expressed as a multimer of subunits consisting of coiled-coil stalks with lectin-family domain heads that bind to IgE (upper panels). Membrane-bound CD23 can be converted to a soluble form that retains IgE-binding ability following cleavage at protease sites by endogenous (ADAM) or allergen (e.g. Der p 1 ) proteases. Various functions have been attributed to CD23 (lower panels). It can facilitate antigen uptake for presentation by B cells and antigen presenting cells to T cells (left panel) and mediate the transport of allergens across polarized epithelium in the gut and airway (center panel). CD23 also regulates IgE production; the transmembrane form on B cells suppresses their production of IgE and the soluble form, via interactions with B cell surface CD21 and IgE, enhances IgE production (right panel). lgG Receptors In addition to FcϵRI, human and mouse mast cells and basophils also express Fcγ receptors (FcγRs) that bind to IgG antibodies. FcγRs belong to the immunoglobulin superfamily and their patterns of expression vary among leukocytes ( 74 – 77 ). The general consensus is that human mast cells and basophils express FcγRIIa and FcγRIIb, while mouse mast cells and basophils express FcγRIIIa and FcγRIIb ( 78 ). FcγRIIb is the only inhibitory IgG receptor, and all others are activating. IgG and FcγRs are involved in a number of immune defense mechanisms including toxin neutralization, antibody dependent cell-mediated cytotoxicity, phagocytosis, and cytokine production. Activating IgG Receptors: IgG-Mediated Anaphylaxis The classical paradigm for allergic reactions centers on antigen aggregation of IgE, bound to FcϵRI on mast cells and basophils, leading to their activation. However, even before the discovery of IgE, IgG antibodies had been shown to be able to activate mast cells. In the 1950s, Ovary, Benacerraf and others found that the ability of serum to transfer cutaneous hypersensitivity from one animal to another resided in both a heat-stable gamma-globulin fraction of serum (IgG) and in a heat-labile reagin fraction ( 79 , 80 ). The relevance of these observations in cutaneous hypersensitivity was confirmed for systemic anaphylaxis more than four decades later when it was reported that active systemic anaphylaxis can be elicited in i.v .-challenged mice lacking the Cϵ exons encoding the IgE heavy chain ( 76 , 81 – 83 ). Like FcϵRI, crosslinking of activating IgG receptors by immune complexes results in the phosphorylation of ITAMs and a cascade of signaling events ( 84 , 85 ). While FcϵRI-mediated anaphylaxis is histamine dependent, FcγRIIIa-mediated anaphylaxis involves the release of platelet activating factor from macrophages ( 86 ). As demonstrated in mice, IgG-mediated anaphylaxis, unlike IgE-mediated anaphylaxis, occurs predominantly following intravascular antigen exposure and has been shown to require much higher doses of the antigen ( 86 ). The existence of IgG-mediated anaphylaxis in humans has not been directly proven and remains somewhat controversial. However, the elicitation of reactions with the physiologic features of anaphylaxis after intravascular administration of agents to which no IgE antibody response can be detected suggests the existence of an IgG pathway. The integration of input signals from activating and inhibitory receptors determines the outcome of the local and systemic inflammatory responses. FcγRIIb: The Inhibitory IgG Receptor FcγRIIb is the only inhibitory IgG receptor and one of its main function is to turn off signals initiated by activating Fc receptors and the B cell receptor (BCR). The importance of IgG-mediated feedback control of humoral immunity by FcγRIIb has long been appreciated ( 87 ). Co-aggregation of FcγRIIb with the BCR increases the BCR activation threshold and suppresses B cell-mediated antigen presentation. In the absence of FcγRIIb, the amount of antigen needed to activate B cells is increased, and production of antibodies to T cell-dependent antigens is suppressed ( 88 ). FcγRIIb signals can inhibit the maturation of dendritic cells as well as their FcγR-mediated antigen presentation and T cell priming ( 89 , 90 ). In macrophages, FcγRIIb inhibits FcγR-mediated phagocytosis and release of cytokines ( 91 – 93 ). Overall, FcγRIIb is the most widely expressed FcγR and its presence on various cell types is required for maintenance of peripheral tolerance. Unlike activating receptors, the aggregation of FcγRIIb alone is of no consequence and binding of IgG or immune complexes solely to FcγRIIb does not inhibit the functions of a resting cell. The inhibitory receptor is expressed on the surface of cells in conjunction with activating receptors, and arming the inhibitory function of FcγRIIb requires an initial licensing signal by an activating receptor. When immunoglobulins on activating and inhibitory receptors recognize independent epitopes in cis on a common antigen, the receptors co-aggregate in lipid rafts for crosslinking to occur ( 94 , 95 ). FcγRIIb lacks the gamma chain and therefore has no ITAMs. Instead it contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytosolic domain. ITIMs recruit protein tyrosine phosphatases and inositol phosphatases ( 96 ). ITIMs were first identified in FcγRIIb and subsequent sequence alignments revealed their presence in a large number of inhibitory receptors ( 96 , 97 ). Following ligand binding and aggregation with an activating receptor, tyrosine residues in these ITIMs are phosphorylated ( 98 , 99 ). These ITIM phosphotyrosines are docking sites for Src homology 2 (SH2) domain-containing protein tyrosine phosphatases (SHPs) and inositol phosphatases (SHIPs). SHIP-1 dephosphorylates phosphatidylinositol (3, 4, 5)-triphosphate (PIP 3 ) to phosphatidylinositol diphosphate (PIP 2 ) (see Figure 1 ). This reaction depletes membrane PIP 3 docking sites for PH-domain-containing signaling intermediates, such as BTK, and consumes the substrate in the formation of IP 3 , which participates in mast cell activation by inducing calcium release from endoplastic reticulum stores. Mast cells deficient in SHIP-1 degranulate at lower concentrations of IgE and antigen, and they also degranulate much more strongly ( 100 ). Therefore, SHIP-1, at baseline, raises the threshold for FcϵRI mediated mast cell activation. It is commonly understood that the inhibitory function of FcγRIIb is mediated by its coaggregation with an activating receptor by the same multivalent antigen. This is described as FcγRIIb-mediated "cis-inhibition". However, Malbec et al. show that FcγRIIb can also mediate inhibition of activating receptors triggered independently of, and not co-aggregated with, FcγRIIb ( 101 ). The authors describe this phenomenon as "trans-inhibition". Trans-inhibition can decrease the release of β-hexosaminidase, histamine, LTC-4, MIP1-α, and TNF-α from mast cells and basophils, and decrease anaphylaxis. The F(ab) 2 portion of IgG was not capable of mediating this inhibitory effect, suggesting that the phenotype is dependent on receptor-mediated action of IgG. FcγRIIb in Disease Due to its known roles in inhibiting the activation of immune cells, FcγRIIb has been considered as a potential regulator in various immunological disorders, especially in autoimmune diseases. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibody production affecting many organs including the skin, brain, joints, and kidneys. Abnormal B cell responses to immune complexes containing autoantigens is a feature of SLE. Immune complexes exert their effects via activating FcγRs, and impaired FcγRIIb function has been shown to be involved in the disease pathogenesis ( 102 ). In rheumatoid arthritis (RA), autoantibodies mediate the destruction of the synovial membrane. Variants in FcγRIIb have been shown to be associated with RA ( 103 ). In a collagen-induced arthritis mouse model of RA, lack of FcγRIIb increases the disease score, cartilage destruction, and concentration of collagen specific IgG. In patients with idiopathic thrombocytopenic purpura (ITP), the immune recognition of autoantigens on platelets triggers their destruction. The same variant form of FcγRIIb identified in patients with RA has been shown to be associated with ITP ( 104 ). In a mouse model of multiple sclerosis, lack of FcγRIIb is associated with increase in disease score and more activation of myelin-specific T cells. Administration of intravenous immunoglobulin (IVIG) has been successfully used to treat patients with autoimmune diseases during acute flares. IVIG induces the expression of FcγRIIb on blood basophils, monocytes, and eosinophils ( 105 ). In contrast, lack of FcγRIIb, or variants of the protein that decrease its function may confer an advantage for fighting infections ( 106 ). For example, FcγRIIb deficiency is associated with resistance to streptococcal infections in mice ( 93 , 107 ). Variants of FcγRIIb associated with SLE (FCGR2B T232 ) have been shown to be more prevalent in areas where malaria is endemic and may confer protection against the infection ( 108 ). Activating IgG Receptors: IgG-Mediated Anaphylaxis The classical paradigm for allergic reactions centers on antigen aggregation of IgE, bound to FcϵRI on mast cells and basophils, leading to their activation. However, even before the discovery of IgE, IgG antibodies had been shown to be able to activate mast cells. In the 1950s, Ovary, Benacerraf and others found that the ability of serum to transfer cutaneous hypersensitivity from one animal to another resided in both a heat-stable gamma-globulin fraction of serum (IgG) and in a heat-labile reagin fraction ( 79 , 80 ). The relevance of these observations in cutaneous hypersensitivity was confirmed for systemic anaphylaxis more than four decades later when it was reported that active systemic anaphylaxis can be elicited in i.v .-challenged mice lacking the Cϵ exons encoding the IgE heavy chain ( 76 , 81 – 83 ). Like FcϵRI, crosslinking of activating IgG receptors by immune complexes results in the phosphorylation of ITAMs and a cascade of signaling events ( 84 , 85 ). While FcϵRI-mediated anaphylaxis is histamine dependent, FcγRIIIa-mediated anaphylaxis involves the release of platelet activating factor from macrophages ( 86 ). As demonstrated in mice, IgG-mediated anaphylaxis, unlike IgE-mediated anaphylaxis, occurs predominantly following intravascular antigen exposure and has been shown to require much higher doses of the antigen ( 86 ). The existence of IgG-mediated anaphylaxis in humans has not been directly proven and remains somewhat controversial. However, the elicitation of reactions with the physiologic features of anaphylaxis after intravascular administration of agents to which no IgE antibody response can be detected suggests the existence of an IgG pathway. The integration of input signals from activating and inhibitory receptors determines the outcome of the local and systemic inflammatory responses. FcγRIIb: The Inhibitory IgG Receptor FcγRIIb is the only inhibitory IgG receptor and one of its main function is to turn off signals initiated by activating Fc receptors and the B cell receptor (BCR). The importance of IgG-mediated feedback control of humoral immunity by FcγRIIb has long been appreciated ( 87 ). Co-aggregation of FcγRIIb with the BCR increases the BCR activation threshold and suppresses B cell-mediated antigen presentation. In the absence of FcγRIIb, the amount of antigen needed to activate B cells is increased, and production of antibodies to T cell-dependent antigens is suppressed ( 88 ). FcγRIIb signals can inhibit the maturation of dendritic cells as well as their FcγR-mediated antigen presentation and T cell priming ( 89 , 90 ). In macrophages, FcγRIIb inhibits FcγR-mediated phagocytosis and release of cytokines ( 91 – 93 ). Overall, FcγRIIb is the most widely expressed FcγR and its presence on various cell types is required for maintenance of peripheral tolerance. Unlike activating receptors, the aggregation of FcγRIIb alone is of no consequence and binding of IgG or immune complexes solely to FcγRIIb does not inhibit the functions of a resting cell. The inhibitory receptor is expressed on the surface of cells in conjunction with activating receptors, and arming the inhibitory function of FcγRIIb requires an initial licensing signal by an activating receptor. When immunoglobulins on activating and inhibitory receptors recognize independent epitopes in cis on a common antigen, the receptors co-aggregate in lipid rafts for crosslinking to occur ( 94 , 95 ). FcγRIIb lacks the gamma chain and therefore has no ITAMs. Instead it contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytosolic domain. ITIMs recruit protein tyrosine phosphatases and inositol phosphatases ( 96 ). ITIMs were first identified in FcγRIIb and subsequent sequence alignments revealed their presence in a large number of inhibitory receptors ( 96 , 97 ). Following ligand binding and aggregation with an activating receptor, tyrosine residues in these ITIMs are phosphorylated ( 98 , 99 ). These ITIM phosphotyrosines are docking sites for Src homology 2 (SH2) domain-containing protein tyrosine phosphatases (SHPs) and inositol phosphatases (SHIPs). SHIP-1 dephosphorylates phosphatidylinositol (3, 4, 5)-triphosphate (PIP 3 ) to phosphatidylinositol diphosphate (PIP 2 ) (see Figure 1 ). This reaction depletes membrane PIP 3 docking sites for PH-domain-containing signaling intermediates, such as BTK, and consumes the substrate in the formation of IP 3 , which participates in mast cell activation by inducing calcium release from endoplastic reticulum stores. Mast cells deficient in SHIP-1 degranulate at lower concentrations of IgE and antigen, and they also degranulate much more strongly ( 100 ). Therefore, SHIP-1, at baseline, raises the threshold for FcϵRI mediated mast cell activation. It is commonly understood that the inhibitory function of FcγRIIb is mediated by its coaggregation with an activating receptor by the same multivalent antigen. This is described as FcγRIIb-mediated "cis-inhibition". However, Malbec et al. show that FcγRIIb can also mediate inhibition of activating receptors triggered independently of, and not co-aggregated with, FcγRIIb ( 101 ). The authors describe this phenomenon as "trans-inhibition". Trans-inhibition can decrease the release of β-hexosaminidase, histamine, LTC-4, MIP1-α, and TNF-α from mast cells and basophils, and decrease anaphylaxis. The F(ab) 2 portion of IgG was not capable of mediating this inhibitory effect, suggesting that the phenotype is dependent on receptor-mediated action of IgG. FcγRIIb in Disease Due to its known roles in inhibiting the activation of immune cells, FcγRIIb has been considered as a potential regulator in various immunological disorders, especially in autoimmune diseases. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibody production affecting many organs including the skin, brain, joints, and kidneys. Abnormal B cell responses to immune complexes containing autoantigens is a feature of SLE. Immune complexes exert their effects via activating FcγRs, and impaired FcγRIIb function has been shown to be involved in the disease pathogenesis ( 102 ). In rheumatoid arthritis (RA), autoantibodies mediate the destruction of the synovial membrane. Variants in FcγRIIb have been shown to be associated with RA ( 103 ). In a collagen-induced arthritis mouse model of RA, lack of FcγRIIb increases the disease score, cartilage destruction, and concentration of collagen specific IgG. In patients with idiopathic thrombocytopenic purpura (ITP), the immune recognition of autoantigens on platelets triggers their destruction. The same variant form of FcγRIIb identified in patients with RA has been shown to be associated with ITP ( 104 ). In a mouse model of multiple sclerosis, lack of FcγRIIb is associated with increase in disease score and more activation of myelin-specific T cells. Administration of intravenous immunoglobulin (IVIG) has been successfully used to treat patients with autoimmune diseases during acute flares. IVIG induces the expression of FcγRIIb on blood basophils, monocytes, and eosinophils ( 105 ). In contrast, lack of FcγRIIb, or variants of the protein that decrease its function may confer an advantage for fighting infections ( 106 ). For example, FcγRIIb deficiency is associated with resistance to streptococcal infections in mice ( 93 , 107 ). Variants of FcγRIIb associated with SLE (FCGR2B T232 ) have been shown to be more prevalent in areas where malaria is endemic and may confer protection against the infection ( 108 ). Regulation of Immune Responses by lgE And lgG Antibodies Acting on Mast Cells and Basophils The opposing effects of IgE antibodies, triggering activating input via FcϵRI, and IgG antibodies, sending ITIM-mediated inhibitory signals via FcγRIIb, are critical in controlling the acute effector functions of mast cells and basophils. These are the pathways that respectively drive immediate hypersensitivity reactions in food allergy, including anaphylaxis, and suppress acute reactions in subjects who have developed food-specific IgG responses. However, the opposing roles of IgE and IgG in food allergy extend significantly beyond these direct effects on effector cell activation, with immunoregulatory influences ranging from modulation of IgE receptor density and signaling thresholds in effector cells, to the IL-4-driven priming and maintenance of effective Th2 responses to food allergens and corresponding suppression and/or pathogenic reprogramming of Tregs. These immediate and downstream functions of food allergen-specific IgE and IgG antibodies extend the rationale for IgE blockade by omalizumab during OIT and form the mechanistic basis for the development of food allergen-specific IgG monoclonal antibodies as novel therapeutics for food allergy. Here we provide a detailed review of the immunomodulatory effects of IgE and IgG antibodies as they relate both to innate effector cell functions and to downstream regulation of adaptive immune responses in food allergy. IgE and Regulation of IgE Receptor Density IgE and its receptors have symbiotic relationships. It has been known for many years that plasma IgE levels and basophil FcϵRI expression are regulated in tandem in atopic patients ( 109 ), but until recently it had not been possible to distinguish between correlation and causation. Saini and colleagues showed that the relationship between circulating IgE and basophil FcϵRI levels holds up across a spectrum of allergic, infectious, and immunodeficiency disorders associated with dysregulated IgE levels, indicating that atopy, itself, is not the driver and suggesting that IgE antibodies themselves have an effect on FcϵRI levels ( 110 ). Such a direct effect of IgE on FcϵRI expression was supported by studies of a rat basophilic leukemia line in which FcϵRI levels increased when IgE antibodies were added to the culture ( 111 ). Subsequent mouse genetic analyses showed that this interaction is indeed operative in vivo at physiologic IgE levels. IgE -/- mice had greatly reduced FcϵRI expression both on circulating basophils and on tissue mast cells, and both cell types rapidly upregulated FcϵRI following IgE infusion ( 112 , 113 ). This phenomenon could be reproduced in culture using freshly isolated primary mast cells, or cultured bone marrow derived mast cells (BMMCs), and has subsequently been confirmed in human mast cells, basophils, and dendritic cells ( 114 – 117 ). IgE-mediated modulation of FcϵRI levels, in turn, regulates effector cell activation thresholds. In the presence of low ambient IgE, decreased basophil surface FcϵRI is associated with an increased threshold for activation by allergen ( 115 ). The presence of IgE stabilizes FcϵRI, preventing internalization and degradation. As FcϵRI continues to be synthesized within a cell, the presence of IgE favors the capture and accumulation of FcϵRI at the cell surface ( 118 ). A similar relationship exists for IgE and CD23. Occupancy of CD23 by IgE protects its protease-sensitive sites from cleavage, and CD23 levels are low in animals lacking IgE and increase directly in relation to ambient IgE levels ( 56 ). IgE and Mast Cell Homeostasis In addition to enhancing FcϵRI levels on the surface of mast cells, IgE antibodies promote mast cell survival and proliferation in vitro and in vivo . The derivation and propagation of mast cells from bone marrow stem cells require the presence of IL-3 and SCF. Without these cytokines, BMMCs undergo apoptotic cell death. However, the addition of IgE both enhances the proliferation of BMMCs in the presence of IL-3 and SCF, and protects them from apoptosis upon withdrawal of IL-3, suggesting an important role for IgE in mast cell expansion in settings of type 2 inflammation, and for survival in growth factor limiting conditions ( 64 , 65 ). Such a function of promoting mast cell expansion and survival has in fact been observed in mouse models of parasitic worm infections. IgE promotes splenic mast cell expansion and parasite clearance in the course of Trichinella spiralis infection ( 119 ). Similarly, in an asthma model, sensitization of mice by inhalation of the fungus Aspergillus fumigatus drives mast cell expansion in the bronchus, trachea, and spleen and this expansion is dependent on the presence of IgE antibodies ( 120 ). Together, these findings reveal that IgE antibodies not only act to trigger mast cell degranulation and regulate FcϵRI levels, but also promote mast cell survival and expansion. Downregulation of IgE Receptor by Omalizumab Omalizumab is a recombinant humanized IgG antibody that recognizes the Fc portion of IgE. Its affinity for free IgE is greater than that of IgE for its receptor so it can effectively compete for free IgE and prevent its binding to cell surface FcϵRI. However, omalizumab cannot effectively disrupt established interactions between IgE and FcϵRI. Once free IgE is captured by omalizumab, immune complexes are formed that are eventually cleared from circulation. By reducing the ambient free IgE concentration, omalizumab leads to a downregulation of FcϵRI. Allergic patients that have been treated with omalizumab have low density of FcϵRI on the surface of basophils, mast cells, and dendritic cells ( 114 – 117 ). Thus, more than the removal of circulating allergen-specific IgE, this secondary effect of omalizumab on FcϵRI density is critical in its mechanism of action in the treatment of hypersensitivity reactions. Mast Cells and Basophils as Producers of Th2 and Pro-Inflammatory Cytokines The role of IgE and mast cells as effectors of immediate hypersensitivity in food allergy is well characterized. However, in addition to rapidly releasing the vasoactive mediators of anaphylaxis, IgE-activated mast cells serve as an important source of immunomodulatory cytokines. Cytokine production requires the activation of a transcriptional program and is therefore delayed. Thus, several hours after FcϵRI is crosslinked, as the symptoms of immediate hypersensitivity abate, IgE-activated mast cells produce a range of chemokines and cytokines important for orchestrating the influx of innate immune cells (eosinophils and basophils) and T cells important in driving type 2 inflammation ( 121 , 122 ). Indeed, IL-4, the critical inducer of Th2 responses, was first reported to be produced by mast cells, in a study conducted by Marshall Plaut, Bill Paul, and colleagues ( 49 ). Mast cells are also prolific inducers of IL-6 and TNF-α, cytokines critical in the activation of APCs and induction of inflammation. Thus, mast cells, present in abundance in the gastrointestinal tract, are prime candidates to be the innate immune inducers of immunological sensitization and Th2 responses. Similar to mast cells, IgE-activated basophils are potent producers of cytokines, particularly IL-4 and, following adjuvant exposure, TSLP and IL-25 ( 50 – 52 , 123 ). Mast Cells, Basophils, ILC2s, and IgE Antibodies Act as Adjuvants in Mouse Models Upon IgE-mediated activation via FcϵRI, mast cells positioned at mucosal surfaces and in the skin as well as basophils recruited to these sites can prime the allergic immune response by influencing both innate and adaptive immune cells. Effective induction of T cell responses to contact sensitizers applied to the skin occurs only when mast cells and IgE are present, and local cutaneous inflammatory responses to superantigens are mast cell dependent ( 124 , 125 ). Mast cell-derived TNF-α is thought to induce Langerhans migration from the epidermis to draining lymph nodes in contact sensitivity models ( 126 ) and mast cell derived histamine may activate Langerhans activation in mice injected intradermally with IgE followed by antigen challenge ( 127 ). Although basophils have not been implicated in pathways of immune priming in contact sensitivity, they have, as already noted, been shown to contribute to immunological priming in the skin in the setting of allergic inflammation induced by M903 application or barrier disruption by SDS ( 21 , 23 ). When basophils are depleted or when basophils lack the ability to produce IL-4, the outcomes of sensitization and challenge are altered, with reduced allergen-induced responses ( 22 , 23 ). In some models of asthma, mast cells, basophils, and IgE play important roles in orchestrating allergic sensitization and effector responses ( 128 – 130 ). The role of mast cells as endogenous adjuvants is most pronounced in settings where artificial adjuvants are not employed, as shown by Galli and colleagues in an alum-independent mouse model of OVA-driven asthma ( 128 ). The constitutive presence of mast cells in the intestinal mucosa has drawn attention to their potential contribution to immunological priming in the gut in food allergy. An adjuvant role of mast cells in food allergy was demonstrated by Burton and colleagues using a mouse model of peanut allergy ( 131 ). They found that two independent strains of mice lacking mast cells, Kit W-sh and Mcpt5 cre iDTR, exhibited decreased peanut-specific IgE production and impaired peanut-specific Th2 responses, but maintained relatively robust induction of Tregs. They further established that signaling pathways downstream of FcϵRI are needed to drive allergic sensitization. For instance, mast cell lineage-specific deletion of Syk kinase in Mctp5 cre -Syk fl/fl mice, and pharmacologic inhibition of Syk kinase function, both separately recapitulated the phenotype of suppressed peanut allergy. Furthermore, blockade of IgE with anti-IgE antibodies and genetic removal of IgE (IgE -/- mice) both independently led to impaired Th2 responses to peanut ingestion and still permitted the development and expansion of Tregs. The observation that mast cell-deficient mice reconstituted with wild type, but not IL-4-deficient, mast cells had restored Th2 responses to peanut strongly implicated mast cells as a key source of IL-4 in this system. IL-4 also negatively impacts the numbers and functions of Tregs so that, while promoting Th2 immunity, it also suppresses and potentially subverts the mechanisms that keep it in check. Chatila and colleagues showed that IL-4 can subvert Tregs to a pathogenic phenotype, expressing Th2 transcription factor GATA-3 as well as IL-4, a state which contributes to, rather than suppresses, allergic disease ( 29 ). Taken together these findings established the critical roles of gut mast cells as inducers of Th2 immunity, a function that, during recurrent allergen exposures and evolving adaptive immune responses, is amplified by food-specific antibodies, which signal via FcϵRI. In contrast to the analyses of basophil contributions to immune sensitization in the skin, the role of basophils in priming immune sensitization to ingested allergens has been less extensively studied. Using basophil depletion (Ba103 anti-CD200R3 mAb) or basophil-deleted Bas-TRECK mice, Kawakami and colleagues demonstrated attenuation of clinical scores, diarrhea incidence and plasma levels of the mast cell protease mMCP-1 following intragastric allergen instillation in mice primed intraperitoneally and then enterally challenged with OVA ( 132 ). Total and OVA-specific IgE responses were not different between basophil-deficient and -sufficient mice arguing against adjuvant immune priming effect in this model. Along with mast cells, basophils also contribute to systemic anaphylaxis. In a mouse model of peanut induced anaphylaxis, selective, or inducible ablation of basophils, without affecting the mast cell compartment, has been shown to reduce hypothermia ( 133 ). In addition to mast cells and basophils, ILC2s have been identified as critical innate immune sources of IL-4 and inducers of Type 2 immune responses. It turns out there is a critical interplay between these cell types, revealed in murine models of food allergy. Like Th2 cells, ILC2s express the transcription factor GATA-3 and secrete Th2 cytokines, including IL-5 and IL-13. Unlike Th2 cells, they lack T cell receptor (TCR) and cannot recognize antigen. In response to epithelial-derived cytokines, such as IL-25 and IL-33, they produce large amounts of IL-5, IL-9, and IL-13 ( 134 ). Recent findings indicate that in addition to being primed by epithelial cell-derived cytokines, ILC2s can also be activated in a mast cell driven manner, and conversely that effects of ILC2s on mast cells can influence the severity of anaphylaxis in food allergy. In mouse models of food allergy using OVA or peanut, the induction of ILC2s was significantly impaired in mice lacking IgE antibodies and those lacking mast cells ( 135 ). Furthermore, in these same murine models, IL-13 produced by ILC2s can regulate the severity of anaphylaxis by increasing sensitivity of target organs to mediators of hypersensitivity reactions ( 135 ). Recent work by Leyva-Castillo et al. in studies of food allergy-induced by epicutaneous food exposure revealed that the interaction between mast cells and ILC2s might be bidirectional. They found that intestinal mast cell expansion driven by mechanical skin injury and allergen exposure requires IL-4 and IL-13 derived from ILC2s ( 18 ). Regulation of Allergic Responses by IgE Antibodies in Humans and Mice With Humanized IgE Receptor Expression The roles of mast cells and IgE in regulating Th2 responses in allergic disease in humans are not as clearly established but there is some evidence for such a connection. Testing the immunomodulatory effects of IgE blockade in OIT with food-allergic subjects offers an opportunity to test this question. In humans, omalizumab has been reported to facilitate more aggressive up-dosing, while also reducing allergic reactions during the course of the treatment ( 136 – 138 ). Stranks et al. hypothesized that IgE blockade might also alter the immunological changes that are induced by OIT. The authors tested this in the PRROTECT cohort of highly peanut-allergic subjects, randomized to receive either standard OIT or OIT in combination with omalizumab ( 139 ). While their analysis was somewhat limited by the fact that patients had the option to switch to open-label omalizumab part way through the study, which most did, the analysis revealed that those initially assigned to the omalizumab group, who therefore received the initial peanut dose escalation in the setting of IgE blockade, exhibited a more robust induction of anti-peanut IgG antibodies, one of the key markers of successful OIT ( 139 ). This finding suggests that in human food allergy IgE antibodies might have an immunoregulatory effect and that blockade of its receptors or their signaling pathways in mast cells might be an effective strategy for preventing or reversing food allergy. In addition to enhancing tissue-resident mast cell production of IL-4, to prime and consolidate local Th2 and IgE responses, IgE antibodies may further promote adaptive immune responses by enhancing the ability of APCs to prime T cells. Both IgE receptors, FcϵRI and CD23, are expressed by APCs and can mediate internalization of allergen complexed with IgE ( 140 ). In humans, the trimeric form of the high affinity IgE receptor, FcϵRI, is expressed on APCs. Studies of the skin of patients affected by atopic dermatitis have revealed the presence of several FcϵRI + APCs. These include Langerhans cells and inflammatory dendritic cells (which do not contain Birbeck granules) in the epidermis, and dermal dendritic cells. FcϵRI is markedly upregulated on these cell types during allergic flares. However, the lack of FcϵRI expression by murine APCs has made it challenging to investigate whether it, like CD23, might promote T cell responses in vivo . Mice with humanized expression of FcϵRI, using an FcϵRI α−chain transgene driven by the CD11c promoter constitutively active in APCs, have proven useful in answering this question. These animals were used to show that allergen-specific IgE, acting via FcϵRI on APCs, instructed naïve T cells to differentiate into Th2 cells, resulting in augmented allergen-specific Th2 responses in vivo ( 141 ). Immunoregulatory Effects of CD23 The ability of CD23 to participate in the priming of T cell responses in vivo was first reported in mice as facilitated antigen presentation, a process whereby IgE antibodies generated in response to a previous allergen encounter, amplify Th2 responses upon re-exposure to that allergen in a mechanism mediated by CD23 (see Figure 2 ). Recent studies of facilitated antigen presentation by Heyman and colleagues suggest that IgE:allergen complexes, bound to circulating B cells via CD23 enter splenic B cell follicles, where antigen is transferred to resident dendritic cells via B cell exosomes generated in a protease (ADAM10) and CD23-dependent process, activating them for efficient antigen presentation ( 142 ). Consistent with this model, exogenous IgE does not augment T cell responses in CD23 –/– mice but does enhance humoral and cellular immunity following reconstitution with CD23 + B cells. Both the diversity of the IgE repertoire for specific allergens (the range of recognized epitopes) and the avidity of the pooled IgE for antigen affect the efficiency of facilitated antigen presentation ( 143 ). CD23 expressed by B cells appears to play a role in regulating IgE synthesis. Ligation of the receptor by IgE suppresses IgE production and CD23-deficient mice exhibit stronger and longer-lasting IgE responses after immunization ( 144 – 146 ). Conversely CD23 transgenic animals exhibit decreased IgE production ( 147 , 148 ). In humans, treatment with lumiliximab, a CD23-blocking monoclonal antibody, lowers IgE levels ( 149 ). In contrast, soluble fragments of CD23 (sCD23) seem to promote IgE synthesis, perhaps by competing for IgE binding with cell-bound CD23 (see Figure 2 ) ( 149 – 151 ). IgE and Regulation of IgE Receptor Density IgE and its receptors have symbiotic relationships. It has been known for many years that plasma IgE levels and basophil FcϵRI expression are regulated in tandem in atopic patients ( 109 ), but until recently it had not been possible to distinguish between correlation and causation. Saini and colleagues showed that the relationship between circulating IgE and basophil FcϵRI levels holds up across a spectrum of allergic, infectious, and immunodeficiency disorders associated with dysregulated IgE levels, indicating that atopy, itself, is not the driver and suggesting that IgE antibodies themselves have an effect on FcϵRI levels ( 110 ). Such a direct effect of IgE on FcϵRI expression was supported by studies of a rat basophilic leukemia line in which FcϵRI levels increased when IgE antibodies were added to the culture ( 111 ). Subsequent mouse genetic analyses showed that this interaction is indeed operative in vivo at physiologic IgE levels. IgE -/- mice had greatly reduced FcϵRI expression both on circulating basophils and on tissue mast cells, and both cell types rapidly upregulated FcϵRI following IgE infusion ( 112 , 113 ). This phenomenon could be reproduced in culture using freshly isolated primary mast cells, or cultured bone marrow derived mast cells (BMMCs), and has subsequently been confirmed in human mast cells, basophils, and dendritic cells ( 114 – 117 ). IgE-mediated modulation of FcϵRI levels, in turn, regulates effector cell activation thresholds. In the presence of low ambient IgE, decreased basophil surface FcϵRI is associated with an increased threshold for activation by allergen ( 115 ). The presence of IgE stabilizes FcϵRI, preventing internalization and degradation. As FcϵRI continues to be synthesized within a cell, the presence of IgE favors the capture and accumulation of FcϵRI at the cell surface ( 118 ). A similar relationship exists for IgE and CD23. Occupancy of CD23 by IgE protects its protease-sensitive sites from cleavage, and CD23 levels are low in animals lacking IgE and increase directly in relation to ambient IgE levels ( 56 ). IgE and Mast Cell Homeostasis In addition to enhancing FcϵRI levels on the surface of mast cells, IgE antibodies promote mast cell survival and proliferation in vitro and in vivo . The derivation and propagation of mast cells from bone marrow stem cells require the presence of IL-3 and SCF. Without these cytokines, BMMCs undergo apoptotic cell death. However, the addition of IgE both enhances the proliferation of BMMCs in the presence of IL-3 and SCF, and protects them from apoptosis upon withdrawal of IL-3, suggesting an important role for IgE in mast cell expansion in settings of type 2 inflammation, and for survival in growth factor limiting conditions ( 64 , 65 ). Such a function of promoting mast cell expansion and survival has in fact been observed in mouse models of parasitic worm infections. IgE promotes splenic mast cell expansion and parasite clearance in the course of Trichinella spiralis infection ( 119 ). Similarly, in an asthma model, sensitization of mice by inhalation of the fungus Aspergillus fumigatus drives mast cell expansion in the bronchus, trachea, and spleen and this expansion is dependent on the presence of IgE antibodies ( 120 ). Together, these findings reveal that IgE antibodies not only act to trigger mast cell degranulation and regulate FcϵRI levels, but also promote mast cell survival and expansion. Downregulation of IgE Receptor by Omalizumab Omalizumab is a recombinant humanized IgG antibody that recognizes the Fc portion of IgE. Its affinity for free IgE is greater than that of IgE for its receptor so it can effectively compete for free IgE and prevent its binding to cell surface FcϵRI. However, omalizumab cannot effectively disrupt established interactions between IgE and FcϵRI. Once free IgE is captured by omalizumab, immune complexes are formed that are eventually cleared from circulation. By reducing the ambient free IgE concentration, omalizumab leads to a downregulation of FcϵRI. Allergic patients that have been treated with omalizumab have low density of FcϵRI on the surface of basophils, mast cells, and dendritic cells ( 114 – 117 ). Thus, more than the removal of circulating allergen-specific IgE, this secondary effect of omalizumab on FcϵRI density is critical in its mechanism of action in the treatment of hypersensitivity reactions. Mast Cells and Basophils as Producers of Th2 and Pro-Inflammatory Cytokines The role of IgE and mast cells as effectors of immediate hypersensitivity in food allergy is well characterized. However, in addition to rapidly releasing the vasoactive mediators of anaphylaxis, IgE-activated mast cells serve as an important source of immunomodulatory cytokines. Cytokine production requires the activation of a transcriptional program and is therefore delayed. Thus, several hours after FcϵRI is crosslinked, as the symptoms of immediate hypersensitivity abate, IgE-activated mast cells produce a range of chemokines and cytokines important for orchestrating the influx of innate immune cells (eosinophils and basophils) and T cells important in driving type 2 inflammation ( 121 , 122 ). Indeed, IL-4, the critical inducer of Th2 responses, was first reported to be produced by mast cells, in a study conducted by Marshall Plaut, Bill Paul, and colleagues ( 49 ). Mast cells are also prolific inducers of IL-6 and TNF-α, cytokines critical in the activation of APCs and induction of inflammation. Thus, mast cells, present in abundance in the gastrointestinal tract, are prime candidates to be the innate immune inducers of immunological sensitization and Th2 responses. Similar to mast cells, IgE-activated basophils are potent producers of cytokines, particularly IL-4 and, following adjuvant exposure, TSLP and IL-25 ( 50 – 52 , 123 ). Mast Cells, Basophils, ILC2s, and IgE Antibodies Act as Adjuvants in Mouse Models Upon IgE-mediated activation via FcϵRI, mast cells positioned at mucosal surfaces and in the skin as well as basophils recruited to these sites can prime the allergic immune response by influencing both innate and adaptive immune cells. Effective induction of T cell responses to contact sensitizers applied to the skin occurs only when mast cells and IgE are present, and local cutaneous inflammatory responses to superantigens are mast cell dependent ( 124 , 125 ). Mast cell-derived TNF-α is thought to induce Langerhans migration from the epidermis to draining lymph nodes in contact sensitivity models ( 126 ) and mast cell derived histamine may activate Langerhans activation in mice injected intradermally with IgE followed by antigen challenge ( 127 ). Although basophils have not been implicated in pathways of immune priming in contact sensitivity, they have, as already noted, been shown to contribute to immunological priming in the skin in the setting of allergic inflammation induced by M903 application or barrier disruption by SDS ( 21 , 23 ). When basophils are depleted or when basophils lack the ability to produce IL-4, the outcomes of sensitization and challenge are altered, with reduced allergen-induced responses ( 22 , 23 ). In some models of asthma, mast cells, basophils, and IgE play important roles in orchestrating allergic sensitization and effector responses ( 128 – 130 ). The role of mast cells as endogenous adjuvants is most pronounced in settings where artificial adjuvants are not employed, as shown by Galli and colleagues in an alum-independent mouse model of OVA-driven asthma ( 128 ). The constitutive presence of mast cells in the intestinal mucosa has drawn attention to their potential contribution to immunological priming in the gut in food allergy. An adjuvant role of mast cells in food allergy was demonstrated by Burton and colleagues using a mouse model of peanut allergy ( 131 ). They found that two independent strains of mice lacking mast cells, Kit W-sh and Mcpt5 cre iDTR, exhibited decreased peanut-specific IgE production and impaired peanut-specific Th2 responses, but maintained relatively robust induction of Tregs. They further established that signaling pathways downstream of FcϵRI are needed to drive allergic sensitization. For instance, mast cell lineage-specific deletion of Syk kinase in Mctp5 cre -Syk fl/fl mice, and pharmacologic inhibition of Syk kinase function, both separately recapitulated the phenotype of suppressed peanut allergy. Furthermore, blockade of IgE with anti-IgE antibodies and genetic removal of IgE (IgE -/- mice) both independently led to impaired Th2 responses to peanut ingestion and still permitted the development and expansion of Tregs. The observation that mast cell-deficient mice reconstituted with wild type, but not IL-4-deficient, mast cells had restored Th2 responses to peanut strongly implicated mast cells as a key source of IL-4 in this system. IL-4 also negatively impacts the numbers and functions of Tregs so that, while promoting Th2 immunity, it also suppresses and potentially subverts the mechanisms that keep it in check. Chatila and colleagues showed that IL-4 can subvert Tregs to a pathogenic phenotype, expressing Th2 transcription factor GATA-3 as well as IL-4, a state which contributes to, rather than suppresses, allergic disease ( 29 ). Taken together these findings established the critical roles of gut mast cells as inducers of Th2 immunity, a function that, during recurrent allergen exposures and evolving adaptive immune responses, is amplified by food-specific antibodies, which signal via FcϵRI. In contrast to the analyses of basophil contributions to immune sensitization in the skin, the role of basophils in priming immune sensitization to ingested allergens has been less extensively studied. Using basophil depletion (Ba103 anti-CD200R3 mAb) or basophil-deleted Bas-TRECK mice, Kawakami and colleagues demonstrated attenuation of clinical scores, diarrhea incidence and plasma levels of the mast cell protease mMCP-1 following intragastric allergen instillation in mice primed intraperitoneally and then enterally challenged with OVA ( 132 ). Total and OVA-specific IgE responses were not different between basophil-deficient and -sufficient mice arguing against adjuvant immune priming effect in this model. Along with mast cells, basophils also contribute to systemic anaphylaxis. In a mouse model of peanut induced anaphylaxis, selective, or inducible ablation of basophils, without affecting the mast cell compartment, has been shown to reduce hypothermia ( 133 ). In addition to mast cells and basophils, ILC2s have been identified as critical innate immune sources of IL-4 and inducers of Type 2 immune responses. It turns out there is a critical interplay between these cell types, revealed in murine models of food allergy. Like Th2 cells, ILC2s express the transcription factor GATA-3 and secrete Th2 cytokines, including IL-5 and IL-13. Unlike Th2 cells, they lack T cell receptor (TCR) and cannot recognize antigen. In response to epithelial-derived cytokines, such as IL-25 and IL-33, they produce large amounts of IL-5, IL-9, and IL-13 ( 134 ). Recent findings indicate that in addition to being primed by epithelial cell-derived cytokines, ILC2s can also be activated in a mast cell driven manner, and conversely that effects of ILC2s on mast cells can influence the severity of anaphylaxis in food allergy. In mouse models of food allergy using OVA or peanut, the induction of ILC2s was significantly impaired in mice lacking IgE antibodies and those lacking mast cells ( 135 ). Furthermore, in these same murine models, IL-13 produced by ILC2s can regulate the severity of anaphylaxis by increasing sensitivity of target organs to mediators of hypersensitivity reactions ( 135 ). Recent work by Leyva-Castillo et al. in studies of food allergy-induced by epicutaneous food exposure revealed that the interaction between mast cells and ILC2s might be bidirectional. They found that intestinal mast cell expansion driven by mechanical skin injury and allergen exposure requires IL-4 and IL-13 derived from ILC2s ( 18 ). Regulation of Allergic Responses by IgE Antibodies in Humans and Mice With Humanized IgE Receptor Expression The roles of mast cells and IgE in regulating Th2 responses in allergic disease in humans are not as clearly established but there is some evidence for such a connection. Testing the immunomodulatory effects of IgE blockade in OIT with food-allergic subjects offers an opportunity to test this question. In humans, omalizumab has been reported to facilitate more aggressive up-dosing, while also reducing allergic reactions during the course of the treatment ( 136 – 138 ). Stranks et al. hypothesized that IgE blockade might also alter the immunological changes that are induced by OIT. The authors tested this in the PRROTECT cohort of highly peanut-allergic subjects, randomized to receive either standard OIT or OIT in combination with omalizumab ( 139 ). While their analysis was somewhat limited by the fact that patients had the option to switch to open-label omalizumab part way through the study, which most did, the analysis revealed that those initially assigned to the omalizumab group, who therefore received the initial peanut dose escalation in the setting of IgE blockade, exhibited a more robust induction of anti-peanut IgG antibodies, one of the key markers of successful OIT ( 139 ). This finding suggests that in human food allergy IgE antibodies might have an immunoregulatory effect and that blockade of its receptors or their signaling pathways in mast cells might be an effective strategy for preventing or reversing food allergy. In addition to enhancing tissue-resident mast cell production of IL-4, to prime and consolidate local Th2 and IgE responses, IgE antibodies may further promote adaptive immune responses by enhancing the ability of APCs to prime T cells. Both IgE receptors, FcϵRI and CD23, are expressed by APCs and can mediate internalization of allergen complexed with IgE ( 140 ). In humans, the trimeric form of the high affinity IgE receptor, FcϵRI, is expressed on APCs. Studies of the skin of patients affected by atopic dermatitis have revealed the presence of several FcϵRI + APCs. These include Langerhans cells and inflammatory dendritic cells (which do not contain Birbeck granules) in the epidermis, and dermal dendritic cells. FcϵRI is markedly upregulated on these cell types during allergic flares. However, the lack of FcϵRI expression by murine APCs has made it challenging to investigate whether it, like CD23, might promote T cell responses in vivo . Mice with humanized expression of FcϵRI, using an FcϵRI α−chain transgene driven by the CD11c promoter constitutively active in APCs, have proven useful in answering this question. These animals were used to show that allergen-specific IgE, acting via FcϵRI on APCs, instructed naïve T cells to differentiate into Th2 cells, resulting in augmented allergen-specific Th2 responses in vivo ( 141 ). Immunoregulatory Effects of CD23 The ability of CD23 to participate in the priming of T cell responses in vivo was first reported in mice as facilitated antigen presentation, a process whereby IgE antibodies generated in response to a previous allergen encounter, amplify Th2 responses upon re-exposure to that allergen in a mechanism mediated by CD23 (see Figure 2 ). Recent studies of facilitated antigen presentation by Heyman and colleagues suggest that IgE:allergen complexes, bound to circulating B cells via CD23 enter splenic B cell follicles, where antigen is transferred to resident dendritic cells via B cell exosomes generated in a protease (ADAM10) and CD23-dependent process, activating them for efficient antigen presentation ( 142 ). Consistent with this model, exogenous IgE does not augment T cell responses in CD23 –/– mice but does enhance humoral and cellular immunity following reconstitution with CD23 + B cells. Both the diversity of the IgE repertoire for specific allergens (the range of recognized epitopes) and the avidity of the pooled IgE for antigen affect the efficiency of facilitated antigen presentation ( 143 ). CD23 expressed by B cells appears to play a role in regulating IgE synthesis. Ligation of the receptor by IgE suppresses IgE production and CD23-deficient mice exhibit stronger and longer-lasting IgE responses after immunization ( 144 – 146 ). Conversely CD23 transgenic animals exhibit decreased IgE production ( 147 , 148 ). In humans, treatment with lumiliximab, a CD23-blocking monoclonal antibody, lowers IgE levels ( 149 ). In contrast, soluble fragments of CD23 (sCD23) seem to promote IgE synthesis, perhaps by competing for IgE binding with cell-bound CD23 (see Figure 2 ) ( 149 – 151 ). The Regulatory Effects of lgG, Signaling via FcγRIIb IN lgE-Mediated Food Allergy Natural Resolution of Food Allergy Is Associated With IgG Induction Many children with low to moderate levels of food-specific IgE antibodies can ingest the foods to which they are sensitized without exhibiting any reaction. On the one hand, this observation creates a tremendous challenge for allergy clinicians trying to establish or rule out food allergy using IgE testing. Oral food challenges, in a clinical setting, are often required to establish with certainty whether a child is tolerant or allergic. On the other hand, the inconsistent correlation between food-specific IgE and reactivity provides an important clue regarding the regulatory factors that might block immune responses to foods. There is now abundant evidence that IgG antibodies account both for natural protection from allergic reactions to foods in patients harboring food allergen-specific IgE, and that the induction of IgG responses underlies, at least in part, the protective effects of OIT. An analogous situation has been described for respiratory allergy. In large population-based cohort studies in Australia and the UK, Custovic and colleagues have established that aeroallergen-sensitized children, with aeroallergen-specific IgE antibodies, often have no symptoms. This led to the concept of "benign Th2 immunity" and further analysis of these subjects revealed that those with higher allergen-specific IgG/IgE ratios had fewer symptoms and their sera inhibited the activation of basophils sensitized with aeroallergen-specific IgE ( 152 , 153 ). One challenge in diagnosis is that circulating IgE and IgG levels may not reflect the amounts of these antibodies present in the mucosal tissues where allergic reactions are initiated. Several investigations have revealed that natural resolution of milk allergy in children is associated with increasing IgG levels ( 154 , 155 ). Similarly, among subjects who test positive for IgE antibodies to peanut, Santos and colleagues found that higher levels of specific IgG4 correlate with tolerance ( 156 ). For the most part, analyses of any protective effects of IgG in food allergy have focused on the IgG4 isotype. A protective role for IgG4 had previously been established to be a very strong biomarker of efficacy in subcutaneous allergen immunotherapy (SCIT) ( 157 ). The IgG4 findings in SCIT led to a focus on this subclass in food allergy studies and it is the only subclass of IgG for which specific tests have been commercially developed. IgG4 is the least abundant isotype of IgG in human serum, often representing only about 5% of total IgG ( 158 , 159 ). However, with chronic antigen exposure, IgG4 can increase to account for a larger fraction of total IgG ( 160 ). Like IgE, IgG4 is induced during Th2 immune responses under the action of IL-4 and IL-13 on B cells. The concept of a "modified Th2 response," has evolved to describe a scenario where IL-10 is present along with IL-4, and IgG4 class switching and production is promoted over IgE ( 159 , 161 – 163 ). IgG4 is the sole subclass of IgG in that it does not mediate common IgG effector functions such as antibody-dependent cell-mediated cytotoxicity or complement dependent-cytotoxicity. IgG4 antibodies also exhibit a unique ability to undergo Fab arm exchange (FAE). In this process, heavy chains of IgG4 antibodies can separate into half antibodies, each consisting of one heavy chain and one light chain. Half antibodies originating from different parent IgG4s can combine to form bispecific antibodies ( 164 ). This re-assortment of Fab regions potentially allows a single IgG4 to recognize two epitopes on an allergen, both increasing overall binding avidity and facilitating crosslinking. Due to its presence in the serum at higher concentration than IgE, its ability to recognize more epitopes and its limited ability to form immune complexes and mediate effector function, it has been proposed that IgG4 might be uniquely suited to function as a blocking antibody, intercepting allergens before they can be engaged by FcϵRI-bound IgE on the surface of effector cells. Induction of High Levels of Specific IgG Following OIT Perhaps the most compelling evidence for a role of IgG antibodies in regulating food allergen responsiveness has come from OIT studies. In OIT, an allergenic food is administered orally in daily doses that incrementally increase, often up-dosing weekly, over the course of several months. Upon completion of OIT, patients can typically tolerate significant amounts of the allergen without reaction. As this acquired ability to ingest the food is temporary and Tregs which maintain immunological tolerance at the T cell level have not been implicated, this state is referred to as food "unresponsiveness" rather than tolerance ( 165 ). Maintenance of this unresponsive status requires ongoing ingestion of the allergenic food. It is striking that at the completion of OIT patients still have very high levels of food allergen-specific IgE antibodies, despite their ability to ingest the allergen without incident. In fact, the IgE titers typically seen after OIT can be unchanged from pre-OIT levels, and are of a magnitude that would strongly predict a significant reaction if obtained in a patient who had not undergone OIT ( 166 ). These observations are highly suggestive that OIT induces a suppressive factor, one that inhibits IgE-mediated anaphylaxis. A clear clue as to the identity of the suppressive factor has been provided by the highly consistent observation that OIT of foods, including milk, egg, and peanut, induces strong food allergen-specific IgG4 responses ( 167 – 172 ). The success of baked-egg challenge has been correlated with these increases in egg-protein IgG4 following OIT ( 173 ). In contrast to prior observations with SCIT, the IgG response in OIT encompasses all IgG subclasses, not just IgG4. For instance, patients undergoing OIT for peanut allergy in the PRROTECT trial had several log increases in levels of peanut-specific IgG1, IgG2, IgG3, IgG4, and IgA, as well as the ratio of peanut-specific IgG4/IgE with the greatest increases evident in peanut-specific IgG2 and IgG4 which were increased by two logs ( 139 , 174 ). The groups of Galli and Nadeau reported that robust IgG responses and elevated IgG4/IgE ratios correlate with sustained unresponsiveness following peanut OIT ( 175 ). When modeled in mice with established peanut allergy, OIT also induces a strong IgG response, inclusive of all the murine IgG subclasses ( 174 ). FcγRIIb-Mediated Suppression of IgE-Mediated Mast Cell Activation by Food-Specific IgG Induced During OIT Mechanisms of OIT and the inhibitory effects of IgG have been studied both in mouse models and in mechanistic investigations in human clinical trials. Since BMMCs can be easily cultured using IL-3 and SCF, and they can be sensitized with food allergen-specific IgE, they provide an excellent tool with which to interrogate the serum of OIT-treated mice for any potential inhibitory activity of induced IgG. Surface expression of the granule marker LAMP-1 (CD107a), which is extruded upon degranulation, is a sensitive marker of mast cell activation. Within minutes of exposure to allergen, mast cells degranulate and an increase in LAMP-1 on the cell surface can be detected. Burton and colleagues reported that sera from mice that underwent OIT for peanut allergy can inhibit the peanut-induced activation of BMMCs sensitized with specific IgE in an IgG-dependent manner ( 174 ). In addition to inhibiting IgE-induced degranulation, OIT-induced food allergen-specific IgG antibodies also inhibit the production of IL-4 and IL-13 by activated BMMCs ( 176 ). Under these conditions, suppression of IgE activation of IgG is not observed in FcγRIIb -/- BMMCs, indicating a requirement for this receptor in IgG-mediated inhibition. At high doses of IgG, however, inhibition of mast cell activation can be exerted even in cells lacking FcγRIIb, indicating that, in addition to sending negative signals via FcγRIIb, IgG can also act as a blocking antibody, sterically preventing the interaction of allergen with FcϵRI-bound IgE (see Figure 1 ) ( 174 ). Prior to the identification of the key role of FcγRIIb in mediating the suppressive actions of IgG on mast cells and basophils, this steric blocking effect had commonly been presumed to be the dominant inhibitory mechanism of IgG in allergy, especially following SCIT, which is why such IgG antibodies are still commonly referred to as blocking IgG. The physiologic relevance of IgG : FcγRIIb-mediated inhibition of IgE-triggered mast cell activation to allergic reactions in vivo , particularly to anaphylaxis, has been demonstrated using mouse models. FcγRIIb-deficient mice have provided an excellent genetic tool to analyze this biology. Sensitized FcγRIIb-deficient mice exhibit enhanced anaphylaxis upon allergen challenge ( 176 ). Furthermore, inhibition of anaphylaxis by passive transfer of allergen-specific IgG occurs in wild type but not FcγRIIb-deficient mice, indicating that restoration of tolerance via IgG requires IgG : FcγRIIb interactions. Although FcγRIIb -/- mice lack the receptor on all cells that would normally express the receptor, reconstitution of the mice with cultured FcγRIIb +/+ mast cells from wild type donors restores the protective effects exerted by passive administration of exogenous allergen-specific IgG, confirming the central role of mast cell FcγRIIb in regulating IgE-mediated anaphylaxis in vivo (see Figure 1 ) ( 176 ). Inhibition of Basophil Activation by Post-OIT Serum in Human Subjects The same receptor-mediated inhibition of IgE-induced activation by IgG has also now been clearly demonstrated in patients undergoing OIT. Using an indirect basophil activation test (iBAT), Burton and colleagues analyzed suppressive activity in the serum of peanut-allergic patients who underwent OIT ( 174 ). In the iBAT, basophils from a non-allergic donor are used to interrogate the sera of study subjects for both activating and suppressive factors (see Figure 3 ). IgE-containing serum, typically from a peanut allergic patient, is added to these basophils in culture. After the addition of peanut extract, granule extrusion is measured by flow cytometric quantitation of CD63, a granule protein that is similar to the LAMP-1 marker used in the murine system. Using this assay, the group found decreased basophil activation by iBAT following completion of OIT. When both pre- and post-OIT serum from the same individual were incubated with donor basophils, the degranulation was lower than that induced by pre-OIT sera alone, indicating that the suppressive activity was OIT-induced ( 176 ). It was determined that this suppression is IgG-mediated and could be blocked by antibodies to FcγRIIb. Figure 3 The indirect basophil activation test (iBAT) as a probe for inhibitory food allergen specific IgG. In this assay, basophils from a non-allergic donor are sensitized with IgE from an allergic donor, then incubated with serum to be queried (typically pre-OIT serum, post-OIT serum, or a mix of the two) and then exposed to allergen. Conditions assayed include (proceeding clockwise starting from top center): (A) basophil incubation with allergen in the absence of serum (no activation), (B) with serum from an allergic donor (full degranulation), (C) with post-OIT serum (suppressed activation), (D) with a mix of pre- and post-OIT serum (suppressed activation if inhibitory activity is present), (E) post-OIT serum with IgG removed (to query the contribution of IgG to suppression) and (F) post-OIT serum with antibodies to FcγRIIb (to test whether inhibition is receptor-mediated). Similar observations were subsequently reported by Santos et al. in a separate peanut-allergic cohort. Rather than using basophils from non-allergic donors to query the OIT sera, this group used the human mast cell line, LAD2 ( 156 ). They too observed IgG-mediated suppressive activity in LAD2 activation by post-OIT sera, and found that specific depletion of IgG4 reduced the suppression leading them to the conclusion that post-OIT suppression is IgG4-mediated. In reviewing the Santos report, however, it is important to note that while IgG4 removal reduced the degree of suppression exerted by post-OIT sera, the effect was incomplete. IgG4-depleted sera from post-OIT subjects in their cohort exerted >50% suppression of basophil activation compared with 80% in sham-depleted sera, which actually suggests that most of the suppressive activity might in fact be accounted for by non-IgG4 isotypes ( 156 ). At this point the argument could be made that suppression of FcϵRI signaling by FcγRIIb in food allergy is not uniquely accounted for by IgG4, but is also exerted by other IgG subclasses, all of which are known to have measurable affinity for FcγRIIb. Like OIT, serum from patients that had undergone SLIT can also inhibit basophil reactivity in an IgG dependent manner ( 177 ). Future studies with monoclonal IgG antibodies, expressed as IgG isotype swap variants, are needed to clearly delineate the potential contributions of these isotypes to FcγRIIb-mediated basophil suppression. The discovery that food-specific IgG antibodies account, at least in part, for suppression of food reactions in IgE + food-tolerant subjects and those who have completed OIT has seeded interest in potential therapeutic applications of IgG in food allergy. Although inhibitory food-specific IgG antibodies are still in development by pharmaceutical companies, and there are not yet any direct data regarding the efficacy of IgG in preventing food anaphylaxis, there are preclinical studies that support the approach. Burton and colleagues recently developed a humanized mouse model in which NOD-scid IL2Rγ null mice (NSG,NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ), given human CD34 + stem cells, exhibit robust T cell expansion, including Foxp3 + CD25 + Tregs, IFNγ + Th1 cells and IL-4 + Th2 cells, and B cell engraftment ( 178 ). These animals are readily sensitized to ingested peanut, producing specific IgE and exhibiting anaphylaxis with elevated plasma tryptase levels upon challenge ( 178 ). If post-OIT serum is administered 24 h prior to allergen challenge, the mice are protected from peanut-induced anaphylaxis ( 179 ). This IgG-mediated inhibition can be blocked by injecting mice with anti-FcγRIIb ( 179 ). However, blocking FcγRIIb does not fully restore the anaphylaxis phenotype, suggesting that IgG is able to exert some inhibition in a manner independent of FcγRIIb, most likely through steric hinderance ( 179 ). Tissue-Specific Variations in FcγRIIb Expression The expression of FcgRIIb on human mast cells is still an active area of investigation including the factors that regulate the expression of the receptor. Comparison of the expression of FcγRIIb on mast cells residing in various human tissues has revealed that FcγRIIb is absent from dermal mast cells, but is expressed on those in the gastrointestinal tract ( 179 , 180 ). This is recapitulated in humanized mice, in which FcγRIIb is detectable by qPCR and flow cytometry in the small intestine and spleen, but not in the skin ( 179 ). As would be anticipated by the lack of FcγRIIb, in vitro IgG-mediated inhibition of mast cell degranulation does not manifest in human skin mast cells. This low, or completely absent, expression of FcγRIIb on dermal mast cells likely explains why patients who have successfully undergone desensitization therapy are able to consume the allergen without symptoms, but they may still exhibit positive skin prick test responses to allergen ( 181 – 184 ). The relevance of FcγRIIb to physiologic regulation of allergic responses is further suggested by genetic associations. An asthma family cohort study that included 370 atopic, 239 non-atopic, and 169 asthmatic subjects, identified a functional SNP in FCGR2B (187Ile>Thr) that was associated with atopy and IgE production ( 185 ). Functional analysis of this variation, that is located in the transmembrane segment of the receptor, showed that 187Ile>Thr FCGR2B is less effective at mediating inhibitory signals than the common allele ( 186 – 188 ). IgG-Mediated Inhibition of Sensitization to Ingested Antigens As one might predict, based on their ability to prevent IgE-induced production of Th2 cytokines by mast cells in culture, IgG antibodies that are administered prophylactically prior to initial allergen ingestion can hinder the development of Th2 responses and IgE antibodies. This possibility was assessed in a recent investigation using mouse model of food allergy involving repeated administration of OVA in Il4raF709 mice, a strain rendered inherently atopic by knock-in of a variant IL-4R α-chain ( 176 ). Administration of allergen-specific, but not control, IgG during the sensitization phase markedly suppressed production of OVA-specific Th2 cells, and was permissive for the expansion of Tregs which was not observed in controls ( 176 ). Allergen-driven mast cell expansion was also suppressed in the IgG-treated mice. As would be expected in the setting of decreased Th2 immunity, IgE responses were suppressed by more than one log. The combination of low IgE and decreased mast cell numbers rendered the OVA IgG-treated animals completely resistant to anaphylaxis, with no signs of hypothermia, a cardinal physiologic feature of anaphylaxis in mice, or elevated levels of plasma mast cell protease-1 (MMCP-1), a granule protease and marker of mast cell activation analogous to tryptase in humans. Taken together, these findings show that, in addition to blocking phenotypes of IgE-mediated immediate hypersensitivity, like anaphylaxis in the setting of established food allergy, IgG antibodies can block the development of food allergy by blunting the Th2 adjuvant function of mast cells (see Figure 4 ). Analysis of FcγRIIb -/- mice in the same study revealed a key role for the inhibitory receptor in mediating the protective effect of IgG. Figure 4 Effects of mast cells on adaptive immune responses to food allergens and the regulation of these effects by IgE and IgG antibodies. Food antigens pass through damaged epithelium, specialized intraepithelial passages ( 8 ), or are sampled by antigen presenting cells (APCs). Epithelial cells subjected to stress or microbial signals secrete cytokines such as IL-25 and IL-33 that promote the activity of various cellular mediators involved in the breakdown of tolerance. Mucosal APCs present antigen to naïve T cells that mature into Th2 cells in the context of a Th2-conducive environment. Th2 cells are known to both depend on IL-4 for their differentiation and survival, and to produce IL-4 that drives IgE isotype switching by B cells and mast cell expansion, while inhibiting the production of regulatory T cells (Tregs) and subverting their function. Mast cells sensitized with allergen-specific IgE and type 2 innate lymphoid cells (ILC2s) provide a priming source of IL-4, initiating and sustaining the Th2 environment. In contrast, allergen-specific IgG antibodies induced during natural allergen resolution or during OIT can inhibit mast cell activation via signaling through FcγRIIb receptor. Inhibition of mast cell activation by IgG can break the positive feedback loop between mast cells and Th2. In a study of fish allergy, active immune induction of IgG antibodies by vaccinating mice with a hypoallergenic mutant of the fish allergen Cyp c 1 , has also been shown to protect against allergy ( 189 ). These findings suggest that an IgG-based preventive strategy might be beneficial as a prophylactic treatment for children at risk for developing food allergies. The recent findings of Oyoshi and colleagues from studies done in mice, in which IgG:allergen immune complexes passed by food allergen-tolerant mothers to their offspring via breast milk, support the induction of Tregs and suppress IgE responses, implicate IgG in physiologic maternal transfer of tolerance ( 190 ). Furthermore, offspring of mothers who have high levels of allergen-specific IgG in their plasma, cord blood, and breast milk, have lower incidence of allergen sensitization ( 191 ). However, the role of mast cells and FcγRIIb in mediating this tolerogenic mechanism have not yet been explored. Restoration of Tolerance During Adjunctive Therapy With IgG During OIT OIT can also be modeled in OVA-sensitized Il4raF709 mice. Mouse models of food allergy using this line have been applied to test the hypothesis that allergen-specific IgG given during the course of OIT might enhance the effectiveness of OIT, by dampening Th2-inducing signals. As expected, OIT, even without adjunctive IgG, resulted in diminished allergen sensitivity, as assessed by anaphylaxis (hypothermia) and MMCP-1 release. However, the protective effects of IgG were dramatically amplified in mice receiving OVA-specific IgG during their OIT with complete abrogation of anaphylaxis and markedly blunted Th2 and IgE responses ( 176 ). IgG Antibodies in Allergic Diarrhea IgE-mediated gastrointestinal reactions, including diarrhea, are common in IgE-mediated food allergy. The effects of food allergen-specific IgG antibodies in both anaphylaxis and diarrhea were investigated by Kucuk and colleagues in a hybrid model of food allergy involving both active sensitization and passive transfer of IgG ( 192 ). Though anaphylaxis and diarrhea are both IgE- and FcϵRI-mediated, the mast cell mediators driving these phenotypes appear to be different. Histamine is associated with anaphylactic shock, while platelet activating factor and serotonin are associated with diarrhea ( 86 , 193 ). To address whether IgG can confer protection against the diarrhea phenotype, Kucuk et al. tested if anti-TNP IgG1 administration prevents the diarrhea induced by TNP-BSA challenge in sensitized mice. When the investigators tested the phenotypes on the FcγRIIb -/- background, protection against anaphylaxis by IgG1 antibodies was no longer observed while IgG1-mediated inhibition of diarrhea was, surprisingly, retained ( 192 ). These findings suggest that while IgG-mediated inhibition of shock is dependent on signaling via FcγRIIb as has been confirmed by others, that inhibition of diarrhea may be due to steric hinderance of antigen-IgE binding rather than a receptor-mediated mechanism (see Figure 1 ). Allergen-Specific IgG and IgE Repertoire Overlap and the Role of IgG + B Cells as Custodians of IgE Memory For optimal choreography of allergen-specific interactions between IgE and IgG antibodies in activating or suppressing food allergen responses one might predict that overlap of their repertoires would be advantageous. Recent findings regarding the relationship between IgE and IgG memory suggest that such coordination exists. The process of B cell isotype class switching to IgG + from IgM + precursors as well as the evolution of high affinity IgG responses to antigens through affinity maturation, and the creation of memory B cell clones all occur in germinal centers of lymph nodes. It turns out that, in contrast to IgG + B cells, mouse germinal center IgE + B cells are susceptible to apoptosis and tend to rapidly transition to a CD138 + plasmablast phenotype. An analysis of sorted human IgE + B cells from allergic subjects by Croote et al. via single cell sequencing revealed that they, like their murine equivalents, almost all have plasmablast transcriptional signatures ( 194 ). This unique fate of IgE + B cells may be related to their very low surface levels of IgE compared with surface IgG or surface IgM on B cells expressing those isotypes ( 195 – 198 ). Both IgE affinity maturation and memory seem to require an intermediate IgG + stage during which this process occurs followed, sequentially, by a second isotype switch from IgG + to IgE + . The presence of hybrid switch sequences, Sµ-Sγ-Sϵ in many IgE + B cells serves as a footprint of their previous existence as IgG + clones. The requirement for the intermediate IgG stage in affinity maturation is demonstrated by the lack of high-affinity IgE responses in mice lacking the Cγ locus ( 199 ). Deep sequencing of millions of peripheral blood IgH genes in allergic subjects by Boyd and colleagues revealed a phylogenetic lineage progression in which all somatically-mutated IgE sequences were derived from identically-mutated IgG parent clones ( 200 ). These findings are consistent with a mechanism in which IgE + and IgG + B cells have a shared allergen specific repertoire, with memory residing in the IgG + population. Furthermore, T follicular helper (Tfh) cells are required for antibody isotype switching by B cells ( 201 , 202 ). IL4 producing T follicular helper (Tfh) cells are needed for IgE production and IgE production can be limited by regulatory T follicular (Tfr) cells ( 203 ). Eisenbarth and colleagues have recently described a specific subset of IL-4- and IL-13-producing Tfh cells that can drive the production of IgE with high affinity to the antigen ( 204 ). Therapeutic Applications of IgG Harnessing the evolving understanding of the importance of allergen specific IgG in regulating both immediate hypersensitivity and chronic type 2 responses in allergy, researchers have developed recombinant allergen specific IgG antibodies or molecules that bind FcγRIIb and could be used to treat IgE-mediated allergies. In a recent clinical trial in cat-allergic subjects, Orengo et al. reported that administration of a single high dose of a pair of Fel d 1 -specific monoclonal IgG4 antibodies in humans prevented symptoms following cat allergen exposure. The dose of IgG4 results in levels in plasma that are in vast excess of the circulating IgE, comparable to the IgG levels induced during successful immunotherapy. In as little as 8 days following injection of Fel d 1 specific IgG4, subjects challenged with cat allergen had a decrease in total nasal symptom score and an increase in peak inspiratory flow compared to controls. By day 29, their skin prick test reactivity was decreased ( 205 ). As animal studies, discussed above, show that allergen-specific IgG exerts immunomodulatory effects, skewing the T cell compartment away from a pro-inflammatory Th2 profile, it would be interesting to see if the protective effects of allergen specific humanized IgG antibodies extend beyond immediate hypersensitivity and whether these monoclonal antibodies might be valuable adjuncts for SCIT in subjects with respiratory allergy ( 176 ). Given the strong induction of allergen-specific IgG antibodies in food-allergic subjects undergoing OIT and the clear immunomodulatory effects of these antibodies, we anticipate that food-specific monoclonal antibodies currently in development will also prove beneficial. A significant limitation of the IgG antibody approach is that any given allergen-specific IgG therapeutic would only target a single allergen. It is unclear if IgG specific for each component allergen would be required for effective therapy. Since the inhibitory signal delivered to the mast cell by IgG against any protein within a food, it is possible that targeting just one component would be sufficient. A variety of alternative approaches have been explored using bispecific antibodies or fusion proteins that bring bridge FcϵRI and FcγRIIb ( 206 , 207 ). Several groups have reported the development of bifunctional FcϵRI crosslinkers composed of the Fc portion of IgG1 and the Fc portion of IgE or an allergen ( 208 , 209 ). However, molecules containing the Fc portion of IgE might be limited in their activity by the availably of free FcϵRI sites. Tam et al. designed a bispecific antibody consisting of a Fab' fragment that recognizes human IgE and a Fab' fragment that recognizes FcγRIIb. Incubation of IgE-sensitized cord blood derived mast cells and basophils with the bispecific antibody blocked histamine release following antigen challenge ( 210 ). Similarly, Jackman and colleagues described a bispecific antibody in which one arm recognizes FcϵRI at a site not blocked by IgE binding, while the other arm binds to FcγRIIb ( 211 ). While these molecules are interesting because of their potential to broadly inhibit allergic responses, they have several limitations. Chronic administration of these compounds is made challenging by their immunogenicity and their short half-life in vivo . Furthermore, experience has shown that even a very low clinical risk of IgE receptor cross linking and anaphylaxis with molecules bearing an FcϵRI-binding moiety can pose very significant barriers to their clinical advancement. Natural Resolution of Food Allergy Is Associated With IgG Induction Many children with low to moderate levels of food-specific IgE antibodies can ingest the foods to which they are sensitized without exhibiting any reaction. On the one hand, this observation creates a tremendous challenge for allergy clinicians trying to establish or rule out food allergy using IgE testing. Oral food challenges, in a clinical setting, are often required to establish with certainty whether a child is tolerant or allergic. On the other hand, the inconsistent correlation between food-specific IgE and reactivity provides an important clue regarding the regulatory factors that might block immune responses to foods. There is now abundant evidence that IgG antibodies account both for natural protection from allergic reactions to foods in patients harboring food allergen-specific IgE, and that the induction of IgG responses underlies, at least in part, the protective effects of OIT. An analogous situation has been described for respiratory allergy. In large population-based cohort studies in Australia and the UK, Custovic and colleagues have established that aeroallergen-sensitized children, with aeroallergen-specific IgE antibodies, often have no symptoms. This led to the concept of "benign Th2 immunity" and further analysis of these subjects revealed that those with higher allergen-specific IgG/IgE ratios had fewer symptoms and their sera inhibited the activation of basophils sensitized with aeroallergen-specific IgE ( 152 , 153 ). One challenge in diagnosis is that circulating IgE and IgG levels may not reflect the amounts of these antibodies present in the mucosal tissues where allergic reactions are initiated. Several investigations have revealed that natural resolution of milk allergy in children is associated with increasing IgG levels ( 154 , 155 ). Similarly, among subjects who test positive for IgE antibodies to peanut, Santos and colleagues found that higher levels of specific IgG4 correlate with tolerance ( 156 ). For the most part, analyses of any protective effects of IgG in food allergy have focused on the IgG4 isotype. A protective role for IgG4 had previously been established to be a very strong biomarker of efficacy in subcutaneous allergen immunotherapy (SCIT) ( 157 ). The IgG4 findings in SCIT led to a focus on this subclass in food allergy studies and it is the only subclass of IgG for which specific tests have been commercially developed. IgG4 is the least abundant isotype of IgG in human serum, often representing only about 5% of total IgG ( 158 , 159 ). However, with chronic antigen exposure, IgG4 can increase to account for a larger fraction of total IgG ( 160 ). Like IgE, IgG4 is induced during Th2 immune responses under the action of IL-4 and IL-13 on B cells. The concept of a "modified Th2 response," has evolved to describe a scenario where IL-10 is present along with IL-4, and IgG4 class switching and production is promoted over IgE ( 159 , 161 – 163 ). IgG4 is the sole subclass of IgG in that it does not mediate common IgG effector functions such as antibody-dependent cell-mediated cytotoxicity or complement dependent-cytotoxicity. IgG4 antibodies also exhibit a unique ability to undergo Fab arm exchange (FAE). In this process, heavy chains of IgG4 antibodies can separate into half antibodies, each consisting of one heavy chain and one light chain. Half antibodies originating from different parent IgG4s can combine to form bispecific antibodies ( 164 ). This re-assortment of Fab regions potentially allows a single IgG4 to recognize two epitopes on an allergen, both increasing overall binding avidity and facilitating crosslinking. Due to its presence in the serum at higher concentration than IgE, its ability to recognize more epitopes and its limited ability to form immune complexes and mediate effector function, it has been proposed that IgG4 might be uniquely suited to function as a blocking antibody, intercepting allergens before they can be engaged by FcϵRI-bound IgE on the surface of effector cells. Induction of High Levels of Specific IgG Following OIT Perhaps the most compelling evidence for a role of IgG antibodies in regulating food allergen responsiveness has come from OIT studies. In OIT, an allergenic food is administered orally in daily doses that incrementally increase, often up-dosing weekly, over the course of several months. Upon completion of OIT, patients can typically tolerate significant amounts of the allergen without reaction. As this acquired ability to ingest the food is temporary and Tregs which maintain immunological tolerance at the T cell level have not been implicated, this state is referred to as food "unresponsiveness" rather than tolerance ( 165 ). Maintenance of this unresponsive status requires ongoing ingestion of the allergenic food. It is striking that at the completion of OIT patients still have very high levels of food allergen-specific IgE antibodies, despite their ability to ingest the allergen without incident. In fact, the IgE titers typically seen after OIT can be unchanged from pre-OIT levels, and are of a magnitude that would strongly predict a significant reaction if obtained in a patient who had not undergone OIT ( 166 ). These observations are highly suggestive that OIT induces a suppressive factor, one that inhibits IgE-mediated anaphylaxis. A clear clue as to the identity of the suppressive factor has been provided by the highly consistent observation that OIT of foods, including milk, egg, and peanut, induces strong food allergen-specific IgG4 responses ( 167 – 172 ). The success of baked-egg challenge has been correlated with these increases in egg-protein IgG4 following OIT ( 173 ). In contrast to prior observations with SCIT, the IgG response in OIT encompasses all IgG subclasses, not just IgG4. For instance, patients undergoing OIT for peanut allergy in the PRROTECT trial had several log increases in levels of peanut-specific IgG1, IgG2, IgG3, IgG4, and IgA, as well as the ratio of peanut-specific IgG4/IgE with the greatest increases evident in peanut-specific IgG2 and IgG4 which were increased by two logs ( 139 , 174 ). The groups of Galli and Nadeau reported that robust IgG responses and elevated IgG4/IgE ratios correlate with sustained unresponsiveness following peanut OIT ( 175 ). When modeled in mice with established peanut allergy, OIT also induces a strong IgG response, inclusive of all the murine IgG subclasses ( 174 ). FcγRIIb-Mediated Suppression of IgE-Mediated Mast Cell Activation by Food-Specific IgG Induced During OIT Mechanisms of OIT and the inhibitory effects of IgG have been studied both in mouse models and in mechanistic investigations in human clinical trials. Since BMMCs can be easily cultured using IL-3 and SCF, and they can be sensitized with food allergen-specific IgE, they provide an excellent tool with which to interrogate the serum of OIT-treated mice for any potential inhibitory activity of induced IgG. Surface expression of the granule marker LAMP-1 (CD107a), which is extruded upon degranulation, is a sensitive marker of mast cell activation. Within minutes of exposure to allergen, mast cells degranulate and an increase in LAMP-1 on the cell surface can be detected. Burton and colleagues reported that sera from mice that underwent OIT for peanut allergy can inhibit the peanut-induced activation of BMMCs sensitized with specific IgE in an IgG-dependent manner ( 174 ). In addition to inhibiting IgE-induced degranulation, OIT-induced food allergen-specific IgG antibodies also inhibit the production of IL-4 and IL-13 by activated BMMCs ( 176 ). Under these conditions, suppression of IgE activation of IgG is not observed in FcγRIIb -/- BMMCs, indicating a requirement for this receptor in IgG-mediated inhibition. At high doses of IgG, however, inhibition of mast cell activation can be exerted even in cells lacking FcγRIIb, indicating that, in addition to sending negative signals via FcγRIIb, IgG can also act as a blocking antibody, sterically preventing the interaction of allergen with FcϵRI-bound IgE (see Figure 1 ) ( 174 ). Prior to the identification of the key role of FcγRIIb in mediating the suppressive actions of IgG on mast cells and basophils, this steric blocking effect had commonly been presumed to be the dominant inhibitory mechanism of IgG in allergy, especially following SCIT, which is why such IgG antibodies are still commonly referred to as blocking IgG. The physiologic relevance of IgG : FcγRIIb-mediated inhibition of IgE-triggered mast cell activation to allergic reactions in vivo , particularly to anaphylaxis, has been demonstrated using mouse models. FcγRIIb-deficient mice have provided an excellent genetic tool to analyze this biology. Sensitized FcγRIIb-deficient mice exhibit enhanced anaphylaxis upon allergen challenge ( 176 ). Furthermore, inhibition of anaphylaxis by passive transfer of allergen-specific IgG occurs in wild type but not FcγRIIb-deficient mice, indicating that restoration of tolerance via IgG requires IgG : FcγRIIb interactions. Although FcγRIIb -/- mice lack the receptor on all cells that would normally express the receptor, reconstitution of the mice with cultured FcγRIIb +/+ mast cells from wild type donors restores the protective effects exerted by passive administration of exogenous allergen-specific IgG, confirming the central role of mast cell FcγRIIb in regulating IgE-mediated anaphylaxis in vivo (see Figure 1 ) ( 176 ). Inhibition of Basophil Activation by Post-OIT Serum in Human Subjects The same receptor-mediated inhibition of IgE-induced activation by IgG has also now been clearly demonstrated in patients undergoing OIT. Using an indirect basophil activation test (iBAT), Burton and colleagues analyzed suppressive activity in the serum of peanut-allergic patients who underwent OIT ( 174 ). In the iBAT, basophils from a non-allergic donor are used to interrogate the sera of study subjects for both activating and suppressive factors (see Figure 3 ). IgE-containing serum, typically from a peanut allergic patient, is added to these basophils in culture. After the addition of peanut extract, granule extrusion is measured by flow cytometric quantitation of CD63, a granule protein that is similar to the LAMP-1 marker used in the murine system. Using this assay, the group found decreased basophil activation by iBAT following completion of OIT. When both pre- and post-OIT serum from the same individual were incubated with donor basophils, the degranulation was lower than that induced by pre-OIT sera alone, indicating that the suppressive activity was OIT-induced ( 176 ). It was determined that this suppression is IgG-mediated and could be blocked by antibodies to FcγRIIb. Figure 3 The indirect basophil activation test (iBAT) as a probe for inhibitory food allergen specific IgG. In this assay, basophils from a non-allergic donor are sensitized with IgE from an allergic donor, then incubated with serum to be queried (typically pre-OIT serum, post-OIT serum, or a mix of the two) and then exposed to allergen. Conditions assayed include (proceeding clockwise starting from top center): (A) basophil incubation with allergen in the absence of serum (no activation), (B) with serum from an allergic donor (full degranulation), (C) with post-OIT serum (suppressed activation), (D) with a mix of pre- and post-OIT serum (suppressed activation if inhibitory activity is present), (E) post-OIT serum with IgG removed (to query the contribution of IgG to suppression) and (F) post-OIT serum with antibodies to FcγRIIb (to test whether inhibition is receptor-mediated). Similar observations were subsequently reported by Santos et al. in a separate peanut-allergic cohort. Rather than using basophils from non-allergic donors to query the OIT sera, this group used the human mast cell line, LAD2 ( 156 ). They too observed IgG-mediated suppressive activity in LAD2 activation by post-OIT sera, and found that specific depletion of IgG4 reduced the suppression leading them to the conclusion that post-OIT suppression is IgG4-mediated. In reviewing the Santos report, however, it is important to note that while IgG4 removal reduced the degree of suppression exerted by post-OIT sera, the effect was incomplete. IgG4-depleted sera from post-OIT subjects in their cohort exerted >50% suppression of basophil activation compared with 80% in sham-depleted sera, which actually suggests that most of the suppressive activity might in fact be accounted for by non-IgG4 isotypes ( 156 ). At this point the argument could be made that suppression of FcϵRI signaling by FcγRIIb in food allergy is not uniquely accounted for by IgG4, but is also exerted by other IgG subclasses, all of which are known to have measurable affinity for FcγRIIb. Like OIT, serum from patients that had undergone SLIT can also inhibit basophil reactivity in an IgG dependent manner ( 177 ). Future studies with monoclonal IgG antibodies, expressed as IgG isotype swap variants, are needed to clearly delineate the potential contributions of these isotypes to FcγRIIb-mediated basophil suppression. The discovery that food-specific IgG antibodies account, at least in part, for suppression of food reactions in IgE + food-tolerant subjects and those who have completed OIT has seeded interest in potential therapeutic applications of IgG in food allergy. Although inhibitory food-specific IgG antibodies are still in development by pharmaceutical companies, and there are not yet any direct data regarding the efficacy of IgG in preventing food anaphylaxis, there are preclinical studies that support the approach. Burton and colleagues recently developed a humanized mouse model in which NOD-scid IL2Rγ null mice (NSG,NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ), given human CD34 + stem cells, exhibit robust T cell expansion, including Foxp3 + CD25 + Tregs, IFNγ + Th1 cells and IL-4 + Th2 cells, and B cell engraftment ( 178 ). These animals are readily sensitized to ingested peanut, producing specific IgE and exhibiting anaphylaxis with elevated plasma tryptase levels upon challenge ( 178 ). If post-OIT serum is administered 24 h prior to allergen challenge, the mice are protected from peanut-induced anaphylaxis ( 179 ). This IgG-mediated inhibition can be blocked by injecting mice with anti-FcγRIIb ( 179 ). However, blocking FcγRIIb does not fully restore the anaphylaxis phenotype, suggesting that IgG is able to exert some inhibition in a manner independent of FcγRIIb, most likely through steric hinderance ( 179 ). Tissue-Specific Variations in FcγRIIb Expression The expression of FcgRIIb on human mast cells is still an active area of investigation including the factors that regulate the expression of the receptor. Comparison of the expression of FcγRIIb on mast cells residing in various human tissues has revealed that FcγRIIb is absent from dermal mast cells, but is expressed on those in the gastrointestinal tract ( 179 , 180 ). This is recapitulated in humanized mice, in which FcγRIIb is detectable by qPCR and flow cytometry in the small intestine and spleen, but not in the skin ( 179 ). As would be anticipated by the lack of FcγRIIb, in vitro IgG-mediated inhibition of mast cell degranulation does not manifest in human skin mast cells. This low, or completely absent, expression of FcγRIIb on dermal mast cells likely explains why patients who have successfully undergone desensitization therapy are able to consume the allergen without symptoms, but they may still exhibit positive skin prick test responses to allergen ( 181 – 184 ). The relevance of FcγRIIb to physiologic regulation of allergic responses is further suggested by genetic associations. An asthma family cohort study that included 370 atopic, 239 non-atopic, and 169 asthmatic subjects, identified a functional SNP in FCGR2B (187Ile>Thr) that was associated with atopy and IgE production ( 185 ). Functional analysis of this variation, that is located in the transmembrane segment of the receptor, showed that 187Ile>Thr FCGR2B is less effective at mediating inhibitory signals than the common allele ( 186 – 188 ). IgG-Mediated Inhibition of Sensitization to Ingested Antigens As one might predict, based on their ability to prevent IgE-induced production of Th2 cytokines by mast cells in culture, IgG antibodies that are administered prophylactically prior to initial allergen ingestion can hinder the development of Th2 responses and IgE antibodies. This possibility was assessed in a recent investigation using mouse model of food allergy involving repeated administration of OVA in Il4raF709 mice, a strain rendered inherently atopic by knock-in of a variant IL-4R α-chain ( 176 ). Administration of allergen-specific, but not control, IgG during the sensitization phase markedly suppressed production of OVA-specific Th2 cells, and was permissive for the expansion of Tregs which was not observed in controls ( 176 ). Allergen-driven mast cell expansion was also suppressed in the IgG-treated mice. As would be expected in the setting of decreased Th2 immunity, IgE responses were suppressed by more than one log. The combination of low IgE and decreased mast cell numbers rendered the OVA IgG-treated animals completely resistant to anaphylaxis, with no signs of hypothermia, a cardinal physiologic feature of anaphylaxis in mice, or elevated levels of plasma mast cell protease-1 (MMCP-1), a granule protease and marker of mast cell activation analogous to tryptase in humans. Taken together, these findings show that, in addition to blocking phenotypes of IgE-mediated immediate hypersensitivity, like anaphylaxis in the setting of established food allergy, IgG antibodies can block the development of food allergy by blunting the Th2 adjuvant function of mast cells (see Figure 4 ). Analysis of FcγRIIb -/- mice in the same study revealed a key role for the inhibitory receptor in mediating the protective effect of IgG. Figure 4 Effects of mast cells on adaptive immune responses to food allergens and the regulation of these effects by IgE and IgG antibodies. Food antigens pass through damaged epithelium, specialized intraepithelial passages ( 8 ), or are sampled by antigen presenting cells (APCs). Epithelial cells subjected to stress or microbial signals secrete cytokines such as IL-25 and IL-33 that promote the activity of various cellular mediators involved in the breakdown of tolerance. Mucosal APCs present antigen to naïve T cells that mature into Th2 cells in the context of a Th2-conducive environment. Th2 cells are known to both depend on IL-4 for their differentiation and survival, and to produce IL-4 that drives IgE isotype switching by B cells and mast cell expansion, while inhibiting the production of regulatory T cells (Tregs) and subverting their function. Mast cells sensitized with allergen-specific IgE and type 2 innate lymphoid cells (ILC2s) provide a priming source of IL-4, initiating and sustaining the Th2 environment. In contrast, allergen-specific IgG antibodies induced during natural allergen resolution or during OIT can inhibit mast cell activation via signaling through FcγRIIb receptor. Inhibition of mast cell activation by IgG can break the positive feedback loop between mast cells and Th2. In a study of fish allergy, active immune induction of IgG antibodies by vaccinating mice with a hypoallergenic mutant of the fish allergen Cyp c 1 , has also been shown to protect against allergy ( 189 ). These findings suggest that an IgG-based preventive strategy might be beneficial as a prophylactic treatment for children at risk for developing food allergies. The recent findings of Oyoshi and colleagues from studies done in mice, in which IgG:allergen immune complexes passed by food allergen-tolerant mothers to their offspring via breast milk, support the induction of Tregs and suppress IgE responses, implicate IgG in physiologic maternal transfer of tolerance ( 190 ). Furthermore, offspring of mothers who have high levels of allergen-specific IgG in their plasma, cord blood, and breast milk, have lower incidence of allergen sensitization ( 191 ). However, the role of mast cells and FcγRIIb in mediating this tolerogenic mechanism have not yet been explored. Restoration of Tolerance During Adjunctive Therapy With IgG During OIT OIT can also be modeled in OVA-sensitized Il4raF709 mice. Mouse models of food allergy using this line have been applied to test the hypothesis that allergen-specific IgG given during the course of OIT might enhance the effectiveness of OIT, by dampening Th2-inducing signals. As expected, OIT, even without adjunctive IgG, resulted in diminished allergen sensitivity, as assessed by anaphylaxis (hypothermia) and MMCP-1 release. However, the protective effects of IgG were dramatically amplified in mice receiving OVA-specific IgG during their OIT with complete abrogation of anaphylaxis and markedly blunted Th2 and IgE responses ( 176 ). IgG Antibodies in Allergic Diarrhea IgE-mediated gastrointestinal reactions, including diarrhea, are common in IgE-mediated food allergy. The effects of food allergen-specific IgG antibodies in both anaphylaxis and diarrhea were investigated by Kucuk and colleagues in a hybrid model of food allergy involving both active sensitization and passive transfer of IgG ( 192 ). Though anaphylaxis and diarrhea are both IgE- and FcϵRI-mediated, the mast cell mediators driving these phenotypes appear to be different. Histamine is associated with anaphylactic shock, while platelet activating factor and serotonin are associated with diarrhea ( 86 , 193 ). To address whether IgG can confer protection against the diarrhea phenotype, Kucuk et al. tested if anti-TNP IgG1 administration prevents the diarrhea induced by TNP-BSA challenge in sensitized mice. When the investigators tested the phenotypes on the FcγRIIb -/- background, protection against anaphylaxis by IgG1 antibodies was no longer observed while IgG1-mediated inhibition of diarrhea was, surprisingly, retained ( 192 ). These findings suggest that while IgG-mediated inhibition of shock is dependent on signaling via FcγRIIb as has been confirmed by others, that inhibition of diarrhea may be due to steric hinderance of antigen-IgE binding rather than a receptor-mediated mechanism (see Figure 1 ). Allergen-Specific IgG and IgE Repertoire Overlap and the Role of IgG + B Cells as Custodians of IgE Memory For optimal choreography of allergen-specific interactions between IgE and IgG antibodies in activating or suppressing food allergen responses one might predict that overlap of their repertoires would be advantageous. Recent findings regarding the relationship between IgE and IgG memory suggest that such coordination exists. The process of B cell isotype class switching to IgG + from IgM + precursors as well as the evolution of high affinity IgG responses to antigens through affinity maturation, and the creation of memory B cell clones all occur in germinal centers of lymph nodes. It turns out that, in contrast to IgG + B cells, mouse germinal center IgE + B cells are susceptible to apoptosis and tend to rapidly transition to a CD138 + plasmablast phenotype. An analysis of sorted human IgE + B cells from allergic subjects by Croote et al. via single cell sequencing revealed that they, like their murine equivalents, almost all have plasmablast transcriptional signatures ( 194 ). This unique fate of IgE + B cells may be related to their very low surface levels of IgE compared with surface IgG or surface IgM on B cells expressing those isotypes ( 195 – 198 ). Both IgE affinity maturation and memory seem to require an intermediate IgG + stage during which this process occurs followed, sequentially, by a second isotype switch from IgG + to IgE + . The presence of hybrid switch sequences, Sµ-Sγ-Sϵ in many IgE + B cells serves as a footprint of their previous existence as IgG + clones. The requirement for the intermediate IgG stage in affinity maturation is demonstrated by the lack of high-affinity IgE responses in mice lacking the Cγ locus ( 199 ). Deep sequencing of millions of peripheral blood IgH genes in allergic subjects by Boyd and colleagues revealed a phylogenetic lineage progression in which all somatically-mutated IgE sequences were derived from identically-mutated IgG parent clones ( 200 ). These findings are consistent with a mechanism in which IgE + and IgG + B cells have a shared allergen specific repertoire, with memory residing in the IgG + population. Furthermore, T follicular helper (Tfh) cells are required for antibody isotype switching by B cells ( 201 , 202 ). IL4 producing T follicular helper (Tfh) cells are needed for IgE production and IgE production can be limited by regulatory T follicular (Tfr) cells ( 203 ). Eisenbarth and colleagues have recently described a specific subset of IL-4- and IL-13-producing Tfh cells that can drive the production of IgE with high affinity to the antigen ( 204 ). Therapeutic Applications of IgG Harnessing the evolving understanding of the importance of allergen specific IgG in regulating both immediate hypersensitivity and chronic type 2 responses in allergy, researchers have developed recombinant allergen specific IgG antibodies or molecules that bind FcγRIIb and could be used to treat IgE-mediated allergies. In a recent clinical trial in cat-allergic subjects, Orengo et al. reported that administration of a single high dose of a pair of Fel d 1 -specific monoclonal IgG4 antibodies in humans prevented symptoms following cat allergen exposure. The dose of IgG4 results in levels in plasma that are in vast excess of the circulating IgE, comparable to the IgG levels induced during successful immunotherapy. In as little as 8 days following injection of Fel d 1 specific IgG4, subjects challenged with cat allergen had a decrease in total nasal symptom score and an increase in peak inspiratory flow compared to controls. By day 29, their skin prick test reactivity was decreased ( 205 ). As animal studies, discussed above, show that allergen-specific IgG exerts immunomodulatory effects, skewing the T cell compartment away from a pro-inflammatory Th2 profile, it would be interesting to see if the protective effects of allergen specific humanized IgG antibodies extend beyond immediate hypersensitivity and whether these monoclonal antibodies might be valuable adjuncts for SCIT in subjects with respiratory allergy ( 176 ). Given the strong induction of allergen-specific IgG antibodies in food-allergic subjects undergoing OIT and the clear immunomodulatory effects of these antibodies, we anticipate that food-specific monoclonal antibodies currently in development will also prove beneficial. A significant limitation of the IgG antibody approach is that any given allergen-specific IgG therapeutic would only target a single allergen. It is unclear if IgG specific for each component allergen would be required for effective therapy. Since the inhibitory signal delivered to the mast cell by IgG against any protein within a food, it is possible that targeting just one component would be sufficient. A variety of alternative approaches have been explored using bispecific antibodies or fusion proteins that bring bridge FcϵRI and FcγRIIb ( 206 , 207 ). Several groups have reported the development of bifunctional FcϵRI crosslinkers composed of the Fc portion of IgG1 and the Fc portion of IgE or an allergen ( 208 , 209 ). However, molecules containing the Fc portion of IgE might be limited in their activity by the availably of free FcϵRI sites. Tam et al. designed a bispecific antibody consisting of a Fab' fragment that recognizes human IgE and a Fab' fragment that recognizes FcγRIIb. Incubation of IgE-sensitized cord blood derived mast cells and basophils with the bispecific antibody blocked histamine release following antigen challenge ( 210 ). Similarly, Jackman and colleagues described a bispecific antibody in which one arm recognizes FcϵRI at a site not blocked by IgE binding, while the other arm binds to FcγRIIb ( 211 ). While these molecules are interesting because of their potential to broadly inhibit allergic responses, they have several limitations. Chronic administration of these compounds is made challenging by their immunogenicity and their short half-life in vivo . Furthermore, experience has shown that even a very low clinical risk of IgE receptor cross linking and anaphylaxis with molecules bearing an FcϵRI-binding moiety can pose very significant barriers to their clinical advancement. Discussion The quest to understand why some individuals with allergen-specific IgE experience life-threatening reactions, while others have no symptoms at all following ingestion has led to the discovery of allergen-specific IgG as the serum factor that is responsible for conferring protection against allergic reactions. Food allergen-specific IgG levels are higher in individuals that are sensitized but unresponsive to the allergen, in those who have outgrown their food allergies and in subjects who have acquired food unresponsiveness after successful completion of OIT. Furthermore, research in animal models has convincingly demonstrated that the presence of allergen specific IgG during sensitization can inhibit the production of allergen specific IgE, and subsequent promotion of Th2 immunity, while promoting effective induction of Tregs. While not studied in animals, IgE and IgG antibodies may be important to sustain food allergy in a maintenance phase, in addition to induction. Similarly, in these models, an adjunctive therapy with IgG during OIT also impairs Th2 responses and promotes the development of Tregs ( 176 ). The factors driving IgE and IgG responses to ingested antigens are complex. Outside the scope of this review but critically important to shaping immune responses in the gut is the microbiome. Mice with reduced numbers of intestinal microbes or diminished microbial diversity (germ free mice or antibiotic-fed mice) have increased susceptibility to sensitization and food allergy ( 212 , 213 ). Germ free mice exhibit higher baseline IL-33 expression in their small intestine and also greater IgE levels in their serum ( 9 , 129 ). Certain species of bacteria, including some in the genus Clostridia , have been shown to confer protection against development of food allergies by contributing to the development of peripherally expanded protective RORγ + T cells in a MYD88-dependent manner ( 9 , 11 , 213 ). The specific contribution of IgG4 relative to other IgG isotypes in the inhibition of IgE-mediated effector cell activation via FcϵRI and in exerting the immunomodulatory effects of IgG requires further study. We believe that investigations in the field have been skewed by the exclusive availability of IgG4 reagents in the ImmunoCAP platform used by most clinical investigators. This has prevented consideration and analysis of the other isotypes. However, as noted in this review, all food-specific antibodies of all four IgG isotypes are induced during OIT and binding to FcγRIIb is clearly not specific to IgG4, begging the question of whether IgG1-3 contribute. As presented in this review, data correlating allergen-specific IgG with protection against food allergy phenotypes and the role of FcγRIIb in blocking mast cell and basophil activation has been repeatedly shown by many groups. Though the antibody and the receptor can work together to block the activity of effector cells, research has also shown that they are also capable of exerting inhibitory effects independent of one another via a number of ways. These observations have been harnessed to engineer antibodies against specific allergen epitopes, and to design small molecules that can aggregate FcγRIIb to FcϵRI-bound IgE bound to antigen. As our understanding of the roles of IgG antibodies both in preventing IgE-triggered anaphylaxis and in regulating Th2 immune responses continues to evolve and as approaches for engineering both allergen-specific and broad-spectrum therapeutics are further developed, we are optimistic that effective strategies will emerge for prevention and treatment in the worldwide problem of food allergies. Author Contributions Under the supervision of HCO, the manuscript was written by CK with YSEA and OLL. All authors contributed to the article and approved the submitted version. Funding This work was supported by NIH grant 1R01AI119918 (HO). CK is supported by a postdoctoral fellowship from the Fonds de recherche du Québec—Santé (258617 and 285834). All figures were created with BioRender.com . Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Abbreviations Mouse names: IgE knockout, Igh-7 -/- ; Kit W-sh mice, B6.Cg- Kit W-sh /HNihrJaeBsmGlliJ; Mcpt5cre iDTR, B6.Tg(Mcpt5 cre ) Gt(ROSA)26Sor tm1(HBEGF)Awai .

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427793/

Cytoskeletal dynamics: A view from the membrane

Many aspects of cytoskeletal assembly and dynamics can be recapitulated in vitro; yet, how the cytoskeleton integrates signals in vivo across cellular membranes is far less understood. Recent work has demonstrated that the membrane alone, or through membrane-associated proteins, can effect dynamic changes to the cytoskeleton, thereby impacting cell physiology. Having identified mechanistic links between membranes and the actin, microtubule, and septin cytoskeletons, these studies highlight the membrane's central role in coordinating these cytoskeletal systems to carry out essential processes, such as endocytosis, spindle positioning, and cellular compartmentalization. Membrane regulation of actin dynamics Cells simultaneously assemble, maintain, and disassemble different F-actin networks within a common cytoplasm; each are tailored to facilitate a particular fundamental process such as motility, polarization, division, or endocytosis ( Chhabra and Higgs, 2007 ; Blanchoin et al., 2014 ). F-actin networks with specified organization and dynamics are produced through the coordinated action of different overlapping sets of diverse actin-binding proteins with an array of complementary properties that include actin monomer (G-actin) binding, assembly, end capping, bundling, and severing/disassembling ( Blanchoin et al., 2014 ). F-actin network assembly, organization, and dynamics are therefore controlled by the spatial and temporal regulation of the activity of actin-binding proteins. The association of these actin-binding proteins with the membrane is multifaceted. In some cases, actin-binding proteins are modulated by binding directly to phosphoinositide lipids. In other cases, membrane-associated proteins modify the activity of actin-binding proteins. Subsets of actin-binding proteins are even integral membrane proteins. Phosphoinositide lipids associate with diverse types of actin-binding proteins, and either inhibit or stimulate their activity (for review see Saarikangas et al., 2010 ). The actin nucleation promotion factors, WAVE and WASP, facilitate actin polymerization via the Arp2/3 complex upon binding PI(4,5)P 2 . In contrast, actin-capping protein, the F-actin–severing protein ADF/Cofilin, and the G-actin–binding protein profilin are all inhibited by binding PI(4,5)P 2 ( Saarikangas et al., 2010 ). Regulation of actin-binding proteins by association with and/or release from phosphoinositide lipids is an exciting possibility that could help explain the self-organization of diverse F-actin networks. However, the importance of phosphoinositide lipid regulation of most actin binding proteins has not been validated in vivo. Membrane regulation of profilin Cells maintain a reserve of up to hundreds of micromolar of unassembled G-actin monomers, which is available for rapid polymerization upon activation of assembly factors and/or production of free actin filament ends ( Pollard et al., 2000 ). Despite the effective critical concentration for actin assembly being only 0.1 µM, a higher concentration of unassembled actin is maintained in part by G-actin–binding proteins that prevent its de novo assembly. Profilin is the primary evolutionarily conserved small G-actin–binding protein ( Carlsson et al., 1977 ), which prevents actin filament assembly by inhibiting the formation of actin dimer and/or trimer nuclei ( Jockusch et al., 2007 ). Actin monomers bound by profilin can only be added to actin filaments that are assembled by actin assembly factors such as Arp2/3 complex, formin, and Ena/VASP ( Dominguez, 2009 ). Profilin-bound actin was assumed to be equally incorporated into F-actin networks assembled by different nucleation factors. However, by simultaneously binding to G-actin and continuous stretches of proline residues that are found on specific actin assembly factors such as formin and Ena/VASP ( Ferron et al., 2007 ), profilin significantly increases the elongation rate of formin-assembled filaments ( Romero et al., 2004 ; Kovar et al., 2006 ). Conversely, profilin inhibits Arp2/3 complex–nucleated branch formation by competing with the nucleation-promoting factor WASP for G-actin ( Suarez et al., 2015 ). As a result, profilin facilitates formin- and Ena/VASP-mediated actin assembly over assembly by the Arp2/3 complex ( Rotty et al., 2015 ; Suarez et al., 2015 ). It is therefore likely that the spatial and temporal regulation of profilin helps govern the type of F-actin network assembled, as profilin activity determines whether G-actin is incorporated into networks generated by one actin assembly factor over another ( Fig. 1 A ). Figure 1. Regulation of actin assembly by membrane lipids. (A) Membrane phosphoinositides such as PI(4,5)P 2 and PI(3,4,5)P 3 might control the spatial and temporal assembly of diverse actin filament networks by regulating profilin activity. Profilin bound to PI(4,5)P 2 cannot associate with actin, which potentially could establish a pool of free actin monomers that might favor the nucleation of branched actin filaments by the Arp2/3 complex, which is activated by binding to the WASP V-CA domain (left). Alternatively, phosphorylated phospholipase C (PLC) releases profilin by hydrolyzing PI(4,5)P 2 , which could facilitate a pool of actin bound to profilin that might favor the elongation of unbranched actin filaments by formin (right) or Ena/VASP (not depicted). (B) Small activated GTPases of the Rho superfamily insert into the membrane via a covalent lipid modification. These GTPases recruit and activate a nucleation-promoting factor such as WASP/WAVE that further modulates Arp2/3 complex activity. F-BAR proteins interact with WASP and either activate or inhibit actin polymerization activity. These activities lead to diverse functions, as indicated in the text boxes. (C) Small activated GTPases of the Rho superfamily directly bind to and recruit formins to the membrane, where they activate actin polymerization. F-BAR proteins can further modulate actin dynamics by either activating or inhibiting formin activity at the membrane to drive processes such as membrane protrusion and cytokinesis. In eukaryotes, such as plants, that lack formins with obvious Rho-binding domains, many formins bind directly to the membrane via an N-terminal PTEN domain (dark blue) that binds to PI(3,5)P 2 , driving polarized growth, or via an N-terminal transmembrane domain (red). Question marks designate hypothetical membrane-associated proteins that negatively or positively regulate formin-mediated actin polymerization. Diverse profilins also bind to membrane phosphoinositides such as PI(3,4,5)P 3 and PI(4,5)P 2 , which inhibits profilin's interactions with G-actin and proline-rich stretches ( Lassing and Lindberg, 1985 , 1988 ; Lu et al., 1996 ; Lambrechts et al., 2002 ; Moens and Bagatolli, 2007 ). Multiple hydrophobic regions of profilin, including the actin- and proline-rich–binding regions, have been implicated in binding to phosphoinositides ( Jockusch et al., 2007 ). Association of profilin with membrane phosphoinositides has been proposed to regulate the temporal and spatial levels of profilin-actin by two possible mechanisms ( Fig. 1 A ). One possibility is that external signal-mediated phosphorylation of phospholipase C hydrolyzes PI(4,5)P 2 , releasing membrane-bound profilin to presumably facilitate actin assembly by formin and Ena/VASP ( Goldschmidt-Clermont et al., 1991 ). Second, sequestration of profilin to membrane regions with high concentrations of PI(4,5)P 2 could increase the level of free G-actin, unbound to profilin, that might preferentially incorporate into branched actin filament networks generated by the Arp2/3 complex. Despite the proposal of these general hypotheses nearly 25 years ago ( Goldschmidt-Clermont et al., 1991 ), there is unfortunately little in vivo evidence that phosphoinositide regulation of profilin occurs ( Saarikangas et al., 2010 ). However, most higher eukaryotes express multiple profilin isoforms that associate with the particular ligands with significantly different affinities, such as actin- or proline-rich ligands like formin, which could tailor them for different cellular roles ( Jockusch et al., 2007 ). Therefore, regulation by phosphoinositides would theoretically be a convenient way for individual profilin isoforms to facilitate self-organization of diverse actin filament networks by favoring particular actin assembly factors at discrete cellular locations ( Neidt et al., 2009 ; Mouneimne et al., 2012 ; Ding and Roy, 2013 ). Further work is required to explore this exciting possibility. Membrane regulation of actin assembly factors Mechanistic insights for the role of the membrane are emerging in the case of the regulation of actin assembly factors. The most well-documented example of this is modulation of actin polymerization by small GTPases of the Rho superfamily. Most actin assembly factors are inherently inactive, but can be activated at the right time and place by small GTPase signaling cascades ( Chesarone and Goode, 2009 ; Campellone and Welch, 2010 ). When activated, these small GTPases dock on the membrane due to exposure of a covalent lipid modification that intercalates into the membrane. Many actin assembly factors have GTPase-binding domains; binding to the active GTPase induces a conformational change, usually relieving an auto-inhibited state ( Fig. 1, B and C ). In the case of Arp2/3 complex, the SCAR/WAVE complex interacts with active GTPases and in turn activates the Arp2/3 complex, which generates filaments. Recently, new insights have emerged with respect to control of actin assembly at specific membrane sites. The WAVE complex was found to interact with a sequence motif found on a large number of diverse membrane proteins, ranging from channels to cell adhesion molecules. Binding occurs on a conserved face of the WAVE complex, which when mutated in flies leads to defects in the organization of the actin cytoskeleton ( Chen et al., 2014 ). Future work is needed to sort out the signaling networks connected to this diverse set of membrane proteins and the specific physiological signals leading to activation of Arp2/3 complex-mediated actin polymerization. While the details of specific membrane recruitment are still being sorted out, it is clear that small GTPases bind to and activate the SCAR/WAVE complex, which in turn activates the Arp2/3 complex. However, another actin assembly factor, the formins, are not always fully activated by binding small GTPases ( Seth et al., 2006 ; Maiti et al., 2012 ). In fact, many formins have other mechanisms to bind to the membrane (for review see Cvrčková, 2013 ). For instance, in plants, formins do not have obvious GTPase-binding domains, and in fact, class I formins are integral membrane proteins themselves. Thus, regulation of these molecules at the membrane is likely mediated by interactions with proteins or specific lipids at the membrane ( Fig. 1 C ). In support of this, moss class II formins contain a PTEN domain that mediates binding to PI(3,5)P 2 ( van Gisbergen et al., 2012 ). Recruitment to PI(3,5)P 2 -rich membrane domains and the ability to rapidly elongate actin filaments is essential for formin function during polarized growth ( Vidali et al., 2009 ; van Gisbergen et al., 2012 ). However, examination of formin molecules at the cell cortex demonstrated that only a fraction of these molecules generate actin filaments ( van Gisbergen et al., 2012 ). Thus, additional molecules associated with PI(3,5)P 2 at the membrane likely modulate the activity of this formin ( Fig. 1 C ). Whether there is a common family of molecules in eukaryotes that regulates membrane activity of actin assembly factors is unclear. However, a possible candidate class of membrane-associated molecules is the Bin-Amphiphysin-Rvs (BAR) domain–containing proteins ( Aspenström, 2009 ; Suetsugu et al., 2010 ; Cvrčková, 2013 ). The positively charged BAR domains, which are found on many different proteins ( Suetsugu et al., 2010 ), form α-helical coiled-coils that fold up into a crescent shape. These domains do not have high specificity for a particular lipid, but rather through their structure can sense or participate in membrane bending ( Suetsugu et al., 2010 , 2014 ). In yeast and animals, a family of proteins with an extended BAR domain, known as F-BAR proteins, are essential scaffolds upon which cytoskeletal proteins can assemble in order to generate specific subcellular structures and functions ( Roberts-Galbraith and Gould, 2010 ). During endocytosis, nucleation-promoting factors for the Arp2/3 complex are recruited to the membrane by interacting with F-BAR proteins. F-BAR proteins not only recruit nucleation-promoting factors, but also modify their activity ( Kamioka et al., 2004 ; Itoh et al., 2005 ; Tsujita et al., 2006 ; Takano et al., 2008 ; Henne et al., 2010 ; Roberts-Galbraith and Gould, 2010 ; Wu et al., 2010 ). In budding yeast, two F-BAR proteins oppositely regulate Las17, a homologue of the WASP actin nucleation–promoting factor ( Fig. 1 B ). Early in endocytosis, Syp1 recruits WASP but maintains it in an inactive state ( Rodal et al., 2003 ; Sun et al., 2006 ; Boettner et al., 2009 ; Feliciano and Di Pietro, 2012 ). Upon vesicle maturation, Bzz1 activates WASP activity ( Sun et al., 2006 ), thereby inducing a burst of actin polymerization mediated by the ARP2/3 complex that promotes internalization of endocytic vesicles. Further physiological support for this model has come from studies in neurons ( Dharmalingam et al., 2009 ) and animal cells ( Tsujita et al., 2006 ). F-BAR proteins also recruit formins to membranes. In fission yeast, the F-BAR proteins Cdc15 and Imp2 help recruit the essential cytokinesis formin Cdc12 to the division site ( Chang et al., 1997 ; Carnahan and Gould, 2003 ; Ren et al., 2015 ). Similarly, the budding yeast Cdc15 homologue Hof1p acts redundantly with Rvs167 (a BAR domain–containing protein also containing a C-terminal SH3) to promote formation of the contractile actin ring ( Nkosi et al., 2013 ). Although F-BAR proteins have clearly defined roles in recruiting formins, several recent studies have revealed how F-BAR proteins directly modulate formin activity. In mammals, the F-BAR protein srGAP2 binds to and directly inhibits the actin-severing activity of the formin FMNL1, which is mediated by its formin homology (FH) 1 domain ( Mason et al., 2011 ). During Drosophila melanogaster embryogenesis, the F-BAR protein Cip4 binds to the formin Dia's FH1 domain and inhibits the ability of Dia to promote actin assembly. Cip4 is a known activator of the WASP–WAVE–Arp2/3 complex pathway. Thus, while Cip4 activates Arp2/3 complex activity, it can simultaneously inhibit Dia activity ( Yan et al., 2013 ). More recently, it was demonstrated in budding yeast that the SH3 domain of the F-BAR protein Hof1p dampens the actin nucleation activity of the formin Bnr1p without displacing Bnr1p from the actin filament end ( Fig. 1 C ; Graziano et al., 2014 ). These studies suggest that F-BAR proteins may have a conserved role in regulating diverse sets of actin nucleation factors at the membrane. Thus, understanding how BAR domain–containing proteins interact with and regulate specific subsets of actin regulators may help to decipher the distinct F-actin domains at the cell cortex. Additionally, since BAR domain–containing proteins are found widely throughout eukaryotes ( Ren et al., 2006 ), it is possible that these molecules may have been an early link between membranes and actin modulation that, with various elaborations, evolved differently in distinct lineages. Connecting actin and microtubules to the membrane enables cortical force generation The cell cortex in animal cells plays a fundamental role in cell division, migration, and polarization ( Kunda et al., 2008 ; Pollard and Cooper, 2009 ; Stewart et al., 2011 ; Abu Shah and Keren, 2014 ). The cortex integrates external stimuli—from extracellular matrix and neighboring cells—and transmits them into the cell to effect cytoskeletal changes crucial for development. A key component of the cortex is the thin F-actin shell underneath the cell membrane that is crucial for providing cortical stiffness and is a key determinant of cell shape ( Pollard and Cooper, 2009 ; Guo et al., 2013 ). Perturbations in cortical F-actin architecture can alter the physical properties of the cortex, thereby affecting cell stiffness and strength. A recent study demonstrates that the bulk of the actin cortex is nucleated by the formin mDia1 and Arp2/3 complex ( Bovellan et al., 2014 ), which suggests that fine-tuning of F-actin cortical structure and mechanics may be mediated by adjusting the relative contribution of each actin assembly factor. Several studies (for reviews see Basu and Chang, 2007 ; Akhshi et al., 2014 ) show that changes in microtubule stability also positively and negatively regulate cortical F-actin structures, including formation of lamellipodia and stress fibers. Here we focus on the converse: regulation of microtubule function by the actin-rich cortex. An excellent example of this regulation is how these two elements set the orientation of the mitotic spindle, which determines the plane of cell division, thereby impacting cell fate and tissue organization. It has been known for quite some time that, during cell division, an intact cortical F-actin meshwork and an intact astral microtubule array are required for spindle orientation ( O'Connell and Wang, 2000 ; Théry et al., 2005 ; Toyoshima and Nishida, 2007 ; Fink et al., 2011 ; Luxenburg et al., 2011 ; Castanon et al., 2013 ). However, how the F-actin cortex is involved in this process, and how the membrane supports the underlying cytoskeletal organization to bring about spindle alignment toward a specialized cortical domain, remains unclear in many cellular systems. The prevailing notion is that the F-actin network provides a platform for a cortical anchor, or a complex of anchoring proteins, that could either mediate attachment (i.e., tethering) of astral microtubules or recruit force generators such as motor proteins that exert pulling forces on the microtubules emanating from the spindle. In this notion, the plus ends of astral microtubules would engage with these cortical platforms through so-called +TIPs (plus tip tracking proteins), including adenomatous polyposis coli protein (APC), CLASP, CLIP170, LIS1, dynactin, and dynein ( Coquelle et al., 2002 ; Rogers et al., 2002 ; Reilein and Nelson, 2005 ; Siller and Doe, 2008 ; Ruiz-Saenz et al., 2013 ). Data to support this idea has been found in several organisms, including Caenorhabditis elegans zygotes ( Couwenbergs et al., 2007 ; Nguyen-Ngoc et al., 2007 ), Drosophila neuroblasts ( Siller et al., 2006 ), and cultured human cells ( Kiyomitsu and Cheeseman, 2012 ). These studies have identified an evolutionarily conserved ternary complex composed of Gαi, the α subunit of heterotrimeric G-protein; LGN, a leucine-glycine-asparagine repeat protein; and NuMA, a nuclear mitotic apparatus protein; as the cortical anchoring complex that recruits dynein as the force generator for spindle orientation. NuMA interacts with LGN ( Du and Macara, 2004 ; Bowman et al., 2006 ; Siller et al., 2006 ), which in turn binds to the myristoylated Gαi that is directly attached to the membrane. NuMA can also bind the membrane directly through a C-terminal PIP-binding domain in a manner independent of LGN and Gαi ( Zheng et al., 2014 ). Intriguingly, when the F-actin meshwork was disrupted, NuMA and Gαi dissociate from the cell cortex ( Luxenburg et al., 2011 ; Machicoane et al., 2014 ; Zheng et al., 2014 ), signifying that their membrane association is weak. These observations raise interesting questions about the physical nature of the anchoring platform, and suggest that additional mechanisms may be required to attach anchoring proteins to the F-actin meshwork or to stabilize them at the cortex. Recent work has shown that the actin-binding proteins ezrin/radixin/moesin (ERM) are probably the missing puzzle pieces at the cell cortex mediating spindle orientation ( Solinet et al., 2013 ; Machicoane et al., 2014 ). ERMs help organize the F-actin meshwork, bridging it to the cell membrane, and this may be necessary for establishing and maintaining the Gαi-LGN-NuMA cortical platform. ERMs, when activated by Ste20-like (SLK) kinase ( Machicoane et al., 2014 ), adopt an open conformation that binds F-actin and the plasma membrane. An N-terminal FERM domain, which binds PI(4,5)P 2 directly ( Fievet et al., 2004 ; Roch et al., 2010 ; Roubinet et al., 2011 ), mediates interaction with the membrane. Interestingly, the FERM domain also binds to and stabilizes microtubules ( Solinet et al., 2013 ), possibly via interaction with CLASP family of +TIPs ( Ruiz-Saenz et al., 2013 ), which suggests that ERMs may function as microtubule-tethering factors. However, evidence suggests that they do more than just tethering microtubules. Depletion of ERMs or inhibition of ERM activation leads to loss of cortical rigidity, mislocalization of LGN and NuMA, and abnormal spindle rocking behavior ( Carreno et al., 2008 ; Machicoane et al., 2014 ). It is interesting to speculate that ERMs may be required to increase membrane rigidity by pinning the F-actin meshwork to the plasma membrane. As proposed ( Zheng et al., 2014 ), this rigidity may enable the cortical platform to counteract astral microtubule–mediated and dynein-generated pulling forces on the cortical anchors. It is noteworthy that the budding yeast version of the dynein cortical anchor, Num1, interacts with the plasma membrane directly via a BAR-like domain and a PH domain ( Farkasovsky and Küntzel, 1995 ; Tang et al., 2009 , 2012 ; Klecker et al., 2013 ; Lackner et al., 2013 ). In budding yeast, actin is dispensable for maintenance of the Num1 cortical platform ( Heil-Chapdelaine et al., 2000a ) or to support dynein-dependent spindle movements ( Heil-Chapdelaine et al., 2000b ), as membrane rigidity is provided by turgor pressure and the cell wall. During animal development, it is conceivable that stabilization of membrane rigidity, as exemplified by ERMs, may represent a general mechanism for modulating pulling forces on astral microtubules ( Fig. 2 ). It is therefore tempting to speculate whether the recently characterized human cortical actin–associated protein, MISP, which has a role in astral microtubule stability and spindle orientation ( Zhu et al., 2013 ), would orchestrate actin cytoskeleton communication with the cell membrane and the astral microtubules in a similar manner. Deciphering how actin-dependent membrane rigidity is controlled locally at specific regions of the cell cortex will surely constitute a major challenge to unraveling the mechanisms governing spatial and temporal regulation of oriented cell division. Figure 2. Regulation of microtubule tethering by actin-dependent membrane rigidity. ERM increases membrane rigidity to support Gαi-LGN-NuMA–dependent anchoring and pulling of astral microtubules by cytoplasmic dynein. Activated ERMs in an open conformation may link F-actin to the cell membrane. Membrane association of the Gαi-LGN-NuMA complex mediated by the lipid anchor on Gαi and the PIP-binding domain on NuMA are presumably weak. Stiffening of the membrane (indicated by straight phospholipid tails) or yet unidentified interactions with F-actin or ERMs may further stabilize the Gαi-LGN-NuMA platform to prevent anchorage detachment. Septins: links between polymer assembly and membrane function An additional layer of membrane compartmentalization is provided by septins. Septins are a component of the cytoskeleton that directly bind to membranes in order to polymerize and in turn help organize cell membranes. Knowing how membranes specify septin assemblies at a particular place and time is essential to understand the mechanistic role of septins in cytokinesis and beyond. Septins were first observed at the plasma membrane in budding yeast ( Byers and Goetsch, 1976 ; Rodal et al., 2005 ; Ong et al., 2014 ). Early work found that human septins exhibit a preference for PI(4,5)P 2 and proposed that a conserved polybasic sequence in septins links them to phospholipids ( Zhang et al., 1999 ). More recently, recombinant budding yeast septins were assembled on lipid monolayers containing high levels (10–50%) of PI(4,5)P 2 ( Bertin et al., 2010 ). Interestingly, the presence of the lipids could promote filament formation even with septin proteins that were otherwise defective for polymerization, which suggests that membranes can facilitate filament assembly. The first dynamic look at septin assembly with reconstituted septin proteins supported lipid bilayers with low levels of PI(4,5)P 2 , and single-molecule total internal reflection fluorescence (TIRF) imaging found that septin filaments elongate through diffusion in two dimensions and annealing ( Bridges et al., 2014 ). The notion that polymerization occurs at the membrane is supported by the finding that cytosolic pools of septins in diverse fungi and mammals consist of minimal heteromeric rods (or heteroligomers) but not filaments ( Sellin et al., 2011 ; Bridges et al., 2014 ). Thus, membranes are intimately involved in septin filament formation ( Fig. 3 A ). Figure 3. Septins assemble on and organize membranes. (A) Membrane association of septin heteroligomers promotes assembly of septin filaments. (B) Septins may play a role in promoting or sensing membrane curvature. Posttranslational modifications (indicated by black stars) are implicated in this function. (C) By providing a diffusion barrier, septins play a role in compartmentalizing the membrane. Septins are frequently found in areas where membranes are highly curved, such as the mother-bud neck in yeast and the bases of dendritic spines and primary cilia ( Fares et al., 1995 ; Xie et al., 2007 ; Hu et al., 2010 ). This raises the possibility that septins sense and/or generate curvature, which is supported by the finding that septin filaments can tubulate phospholipid liposomes in vitro ( Tanaka-Takiguchi et al., 2009 ; Fig. 3 B ). In vivo, septins are recruited to curved blebs of membrane that are pulled back toward the cell center, which suggests that there may be a capacity for them to recognize specific curvatures ( Tanaka-Takiguchi et al., 2009 ; Gilden and Krummel, 2010 ). Recent work has shown that septins can promote the formation of curved and ordered bundles of F-actin at the highly curved membranes of furrow canals during embryo cellularization, which suggests that septins and actin may collaborate for curvature sensing ( Mavrakis et al., 2014 ). In mycelia of filamentous fungal systems, there are septin regulatory kinases that are only required for straight septin filaments without impacting septins that assemble at the curved surfaces, which suggests that posttranslational modifications could influence curvature preference or sensing ( DeMay et al., 2009 ; Fig. 3 B ). Finally, there has been substantial interest in the role of septins as diffusional barriers, and work from yeast to human cilia has suggested the possibility that septins can functionally compartmentalize membranes ( Takizawa et al., 2000 ; Barral et al., 2000 ; Hu et al., 2010 ; Fig. 3 C ). Despite the first observations of a barrier function over a decade ago, the mechanism by which septin compartmentalizes membranes has proven to be highly elusive. The first clues as to a molecular basis for the ER-based barrier have come from several recent studies. Yeast genetics uncovered a link between sphingolipid domains and septin-based ER barriers, and a second study identified a role for one specific septin, Shs1, in these barriers ( Chao et al., 2014 ; Clay et al., 2014 ). Finally, a critical functional role for septins in membrane compartmentalization came from a screen looking at regulators of calcium influx in cultured mammalian cells ( Sharma et al., 2013 ). This study showed that septins are required for establishing PIP 2 -rich microdomains at sites of ER–plasma membrane contacts. These functional studies, along with the development of reconstitution methods for probing the barrier properties in artificial lipid membranes, should pave the way for understanding how septins influence membrane diffusion. But it is clear that a reciprocal relationship between certain membrane domains and septins underlies their organization and function. Conclusions As more mechanistic connections emerge between the membrane and the cytoskeleton, it is becoming clear that a new generation of tools is needed. In particular, being able to track the dynamics and localization of specific lipid species, as well as physical methods to measure membrane rigidity in living cells, is critical. Additionally, most studies have been performed in individual cells, but not in the context of developing tissues or varied extracellular environments. Thus, how mechanical strains on the membrane translate into cytoskeletal reorganization ultimately effecting cell physiology and development constitutes the next generation of questions in cytoskeletal dynamics.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126691/

Indole and 3-indolylacetonitrile inhibit spore maturation in Paenibacillus alvei

Background Bacteria use diverse signaling molecules to ensure the survival of the species in environmental niches. A variety of both Gram-positive and Gram-negative bacteria produce large quantities of indole that functions as an intercellular signal controlling diverse aspects of bacterial physiology. Results In this study, we sought a novel role of indole in a Gram-positive bacteria Paenibacillus alvei that can produce extracellular indole at a concentration of up to 300 μM in the stationary phase in Luria-Bertani medium. Unlike previous studies, our data show that the production of indole in P. alvei is strictly controlled by catabolite repression since the addition of glucose and glycerol completely turns off the indole production. The addition of exogenous indole markedly inhibits the heat resistance of P. alvei without affecting cell growth. Observation of cell morphology with electron microscopy shows that indole inhibits the development of spore coats and cortex in P. alvei . As a result of the immature spore formation of P. alvei , indole also decreases P. alvei survival when exposed to antibiotics, low pH, and ethanol. Additionally, indole derivatives also influence the heat resistance; for example, a plant auxin, 3-indolylacetonitrile dramatically (2900-fold) decreased the heat resistance of P. alvei , while another auxin 3-indoleacetic acid had a less significant influence on the heat resistance of P. alvei . Conclusions Together, our results demonstrate that indole and plant auxin 3-indolylacetonitrile inhibit spore maturation of P. alvei and that 3-indolylacetonitrile presents an opportunity for the control of heat and antimicrobial resistant spores of Gram-positive bacteria. Background Bacteria use diverse signaling molecules to ensure the survival of the species in environmental niches. A variety of both Gram-positive and Gram-negative bacteria produce large quantities of indole that functions as an intercellular signal controlling diverse aspects of bacterial physiology. Results In this study, we sought a novel role of indole in a Gram-positive bacteria Paenibacillus alvei that can produce extracellular indole at a concentration of up to 300 μM in the stationary phase in Luria-Bertani medium. Unlike previous studies, our data show that the production of indole in P. alvei is strictly controlled by catabolite repression since the addition of glucose and glycerol completely turns off the indole production. The addition of exogenous indole markedly inhibits the heat resistance of P. alvei without affecting cell growth. Observation of cell morphology with electron microscopy shows that indole inhibits the development of spore coats and cortex in P. alvei . As a result of the immature spore formation of P. alvei , indole also decreases P. alvei survival when exposed to antibiotics, low pH, and ethanol. Additionally, indole derivatives also influence the heat resistance; for example, a plant auxin, 3-indolylacetonitrile dramatically (2900-fold) decreased the heat resistance of P. alvei , while another auxin 3-indoleacetic acid had a less significant influence on the heat resistance of P. alvei . Conclusions Together, our results demonstrate that indole and plant auxin 3-indolylacetonitrile inhibit spore maturation of P. alvei and that 3-indolylacetonitrile presents an opportunity for the control of heat and antimicrobial resistant spores of Gram-positive bacteria. Background In nature, a key element of the adaptive responses of bacteria is the ability to sense and respond to the local environment, such as nutritional limitation, their population, the presence of toxic chemicals from other bacteria and host signals. Hence, it is important to coordinate the pattern of gene expression, and bacteria have evolved specific mechanisms to ensure the survival of the species in environmental niches. For example, many bacteria use a variety of intercellular signaling systems including quorum sensing. The intercellular signal molecules include N -acyl-homoserine lactones (AHLs) in Gram-negative bacteria, autoinducer 2 (AI-2) and indole in both Gram-negative and Gram-positive bacteria, signal peptides in Gram-positive bacteria, and others; these have been seen to co-ordinate gene expression for bioluminescence, sporulation, plasmid conjugal transfer, competence, virulence factor production, antibiotic production, and biofilm formation [ 1 ]. Indole is an intercellular signal [ 2 , 3 ] as well as an interspecies signal [ 4 ]. A variety of both Gram-positive and Gram-negative bacteria (more than 85 species) [ 2 ] produce indole using tryptophanase (TnaA; EC 4.1.99.1) that can reversibly convert tryptophan into indole, pyruvate, and ammonia according to reaction below [ 5 ]. Indole plays diverse biological roles in the microbial community; for example, indole controls the virulence [ 6 - 8 ], biofilm formation [ 4 , 9 - 11 ], acid resistance [ 4 ], and drug resistance [ 3 , 8 , 12 , 13 ] in Gram-negative bacteria. In a Gram-positive Stigmatella aurantiaca , indole increases its sporulation via indole binding pyruvate kinase [ 14 , 15 ]. Moreover, recent studies suggest that abundant bacterial indole in human intestines plays beneficial roles in the human immune system [ 16 , 17 ]. Also importantly, indole increases Escherichia coli antibiotic resistance, which eventually leads to population-wide resistance [ 3 ]. P. alvei (formerly known as Bacillus alvei ) belongs to the class Bacillales , which includes Bacillus , Listeria , and Staphylococcus and is an endospore-forming Gram-positive bacterium that swarms on routine culture medium. P. alvei is frequently present in cases of European foulbrood (a disease of the honey bee) [ 18 ] and has, on occasion, been the cause of human infections [ 19 - 21 ]. P. alvei is the only indole-producing bacterium among many Bacillus species [ 22 ], and the biosynthesis of indole has been well-studied in P. alvei [ 22 - 24 ]. It has long been thought that indole producing bacteria including P. alvei utilize tryptophanase to synthesize tryptophan and other amino acids from indole as a carbon source [ 24 , 25 ]. However, the equilibrium of the reaction favors the production of indole from tryptophan [ 26 , 27 ]. Hence, we sought here the real biological role of indole in P. alvei physiology. Spore-forming bacteria can respond to nutritional limitation and harsh environmental conditions by forming endospores that are remarkably resistant to heat, desiccation, and various chemicals [ 28 , 29 ]. Spore formation is an elaborate and energy intensive process that requires several hours to complete [ 29 ]. Therefore, sporulation is a last-resort adaptive process that is tightly regulated by complex cell-cell signaling or so-called quorum sensing [ 29 , 30 ]. Bacillus subtilis produces multiple cell-cell signaling molecules to control the sophisticated sporulation [ 30 ] that is often a temporal, spatial, and dynamic decision-making process [ 28 ]. The outermost protective layers of B. subtilis endospores are the coat and the cortex [ 31 ]. The spore coat is a barrier against bactericidal enzymes and destructive chemicals. Therefore, heat resistant spores are also resistant to treatment by various chemicals, such as acids, bases, oxidizing agents, alkylating agents, aldehydes and organic solvents [ 32 ]. Thus, we investigated the role of indole on heat resistance as well as other environmental stresses. In this study, we identified that indole was a stationary phase extracellular molecule in P. alvei and functioned to inhibit spore maturation and to decrease survival rates under several environmental stresses. Additionally, we studied the effect of indole derivatives originated from plants on spore formation in P. alvei . This study provides another important role of indole and indole derivatives. Results Extracellular indole accumulation in P. alvei To be an environmental signal molecule, indole has to be excreted out of cells. Thus, the cell growth of P. alvei and the extracellular indole concentration were measured in Luria-Bertani (LB) medium. Clearly, the level of extracellular indole from P. alvei was growth-dependent (Figure 1A ). Indole production was begun in the middle of exponential growth phase and reached the maximum amount (300 μM) in the stationary phase. Notably, the level of extracellular indole present was stable over time at 37°C (Figure 1A ), which was one of characteristics of the indole molecule [ 2 ] while other signaling molecules, such as AHLs, AI-2, and signal peptides, are only temporally present and heat-unstable [ 2 ]. The accumulation pattern of extracellular indole was similar to that of other bacteria, such as E. coli [ 33 ] and Vibrio cholera [ 10 ], while these two bacteria accumulated up to 500-600 μM of extracellular indole within 24 h in LB [ 10 , 33 ]. The slower accumulation of indole in P. alvei was probably due to the 200-fold lower activity of P. alvei tryptophanase than that of E. coli tryptophanase [ 22 ]. Figure 1 Production of extracellular indole in P. alvei . Cell growth and extracellular indole accumulation in LB (A) and extracellular indole accumulation in LB supplemented with different carbon sources (B) at 37°C at 250 rpm. Cell growth (closed circle) was determined via the optical density at 600 nm (OD 600 ). Glucose (Glu), glycerol (Gly), and lactose (Lac) in 0.5% (w/v) were added at the beginning of the culture and cells were cultured for 36 h and indole production was measured. Experiments were performed in triplicate and one standard deviation is shown. Catabolite repression of P. alvei tryptophanase Since indole production was suppressed by the presence of glucose in E. coli through catabolite repression [ 34 ], the effect of carbon sources on indole production was investigated in P. alvei . Similar to E. coli , the addition of glucose and glycerol (0.5%) in LB medium completely abolished the production of indole in P. alvei for 36 h, while lactose (0.5%) did not affect indole accumulation (Figure 1B ). This result suggested that the indole accumulation in P. alvei was strictly controlled by catabolic repression although transport mechanisms of glucose and glycerol would be different. In other words, P. alvei did not produce indole in the presence of the preferred carbon sources such as glucose and glycerol. Unlike the current observation, it was previously reported that the tryptophanase in B. alvei (renamed as P. alvei ) appeared to be constitutive, and catabolite repression was not operative [ 22 ]. The report studied the effect of only tryptophan on tryptophanase activity and found that the activity of P. alvei tryptophanase was independent of tryptophan [ 22 ]. Indole inhibits the heat-resistant cell numbers of P. alvei The main hypothesis of this study was that a large quantity of extracellular indole would play a quorum sensing role in cell physiology of P. alvei so we investigated the effect of indole on sporulation and biofilm formation which was influenced by cell population and environmental stresses in other Bacillus strains [ 30 ]. In P. alvei , the addition of exogenous indole (0, 0.2, or 1.0 mM) surprisingly decreases the heat-resistant colony-forming unit (CFU) in a dose dependent manner (Figure 2A ). For example, indole (1 mM) decreased the heat-resistant CFU of P. alvei compared to no addition of indole 51-fold at 16 hr (0.26 ± 0.01% vs.13.2 ± 0.9%) and 10-fold at 30 hr (8 ± 6% vs. 77 ± 10%). To confirm the presence of exogenous indole, the indole level in DSM medium was measured with HPLC. The level of exogenous indole (1 mM) was not changed at all over 24 h (data not shown). Hence, the exogenous indole was not utilized as a carbon source and inhibited the heat-resistant CFU of P. alvei . Figure 2 Effect of indole and 3-indolylacetonitrile on the heat-resistant CFU of P. alvei . The cells (an initial turbidity of 0.05 at 600 nm) were grown in spore forming DSM medium for 16 h and 30 h. Exogenous indole (A) and 3-indolylacetonitrile (B) were added at the beginning of the culture to test the effect of indole (Ind) and 3-indolylacetonitrile (IAN) on the heat-resistant CFU. Lysozyme-resistance assays (C) were performed with 30 h-grown cells with and without indole and 3-indolyacetonitrile, and lysozyme (1 mg/mL) was treated for 20 min. Each experiment was repeated three to four times and one standard deviation is shown. Additionally, the temperature effect of indole on the heat resistance of P. alvei was investigated since the environmental temperature affected indole signaling in E. coli [ 12 ]. Unlike in E. coli , the inhibitory effect of indole (1 mM) on the heat-resistant CFU of P. alvei at 30°C (0.3 ± 0.1% vs. 8 ± 2% for 16 h) was similar to that at 37°C in P. alvei (Figure 2 ). Hence, it appeared that the temperature effect of indole on the heat-resistant CFU of P. alvei was not significant under the tested laboratory conditions. Indole inhibits the development of spore coat and cortex The effect of indole on the morphology of sporulating cells was examined by transmission electron microscopy. Surprisingly, the proportion of sporulating cells in the total number of cells was similar between with and without treatment of indole (upper panel in Figure 3 ). However, exogenous addition of indole influenced the morphology of the spore coat and the cortex. Cells with exogenous indole formed endospores with a thin spore coat and a thin spore cortex, while using no indole treatment resulted in a thick spore coat and cortex (lower panel in Figure 3 ). Because the spore coat and cortex were important for heat resistance and chemical resistance [ 31 ], we concluded that indole caused an immature spore that negatively contributed to the heat resistance of P. alvei . Figure 3 Electron microscopy analysis of P. alvei endospore formation . DMSO (0.1% v/v) was used as a control (None). 1 mM indole and 1 mM 3-indolylacetonitrile (IAN) dissolved in DMSO were added at the beginning of culture, and cells (an initial turbidity of 0.05 at 600 nm) were grown in DSM for 30 h. The scale bar indicates 500 nm in the upper panel and 100 nm in the lower panel. Abbreviations: SC, spore coat; Cx, cortex; SPC, spore core. Effect of indole derivatives on the heat resistance of P. alvei In the natural environment, indole can be easily oxidized into hydroxyindoles by diverse oxygenases, and indole derivatives often show different effects on bacterial physiology [ 2 ]. Thus, P. alvei can often encounter many kinds of indole-like compounds that are synthesized from tryptophan in other bacteria, plants, and even animals. Therefore, seven indole derivatives have been further investigated for the heat resistance of P. alvei . As a negative control, glucose was used since glucose decreased the sporulation of B. subtilis [ 35 ]. Similar to B. subtilis , glucose (0.5%) clearly decreased the heat-resistant CFU by 600-fold in P. alvei (Figure 4A ). However, L-tryptophan as the main substrate of the indole biosynthesis did not have much influence on the heat-resistant CFU, which supported that indole rather than tryptophan specifically influenced the heat resistance of P. alvei (Figure 4A ). Figure 4 Effect of indole derivatives on the heat-resistant CFU of P. alvei . The cells (an initial turbidity of 0.05 at 600 nm) were grown in spore forming DSM medium for 16 h. Exogenous indole derivatives (1 mM) and glucose (0.5% w/v) were added at the beginning of the culture. Tryptophan (Trp) was dissolved in water, and indole (Ind), 3-indolylacetonitrile (IAN), indole-3-carboxyaldehyde (I3C), 3-indoleacetic acid (IAA), indole-3-acetamide (I3A), tryptamine (TM), and 2-oxindole (OI) were dissolved in dimethyl sulfoxide (DMSO). DMSO (0.1% v/v) was used as a control (None). Each experiment was repeated three to four times and one standard deviation is shown. The structures of Trp, Ind, IAA, I3CA, IAN, I3A, TM, and OI are shown. The asterisk indicates statistical significance determined using a Student t test (P < 0.05). Most interestingly, a plant auxin, 3-indolylacetonitrile dramatically (up to 2900-fold) decreased the heat-resistant CFU of P. alvei in a dose dependent manner at 16 and 30 hr (Figure 2B and Figure 4A ), while another auxin 3-indoleacetic acid had a less significant influence, and tryptamine and 2-oxindole had no effect (Figure 4A ). Therefore, these results suggest that the functional groups of indole derivatives may control the development of P. alvei spores. Similar to indole, the proportion of sporulating cells in the total number of cells was similar with and without treatment of 3-indolylacetonitrile (upper panel in Figure 3 ). Also, 3-indolylacetonitrile produced an irregular spore coat, while no treatment produced sturdy coat (Figure 3 ). Therefore, it appeared that indole and 3-indolylacetonitrile inhibited spore maturation rather than sporulation initiation. In order to understand how most spores (upper panel in Figure 3 ) in the presence of indole and 3-indolylacetonitrile could not survive against heat treatment, the lysozyme resistance assay [ 36 ] was performed with 30-hour grown cells since the lysozyme treatment could release all spores. As a result, indole and 3-indolylacetonitrile produced a large portion of lysozyme-resistant cells (47 ± 8% with indole and 50 ± 3% with 3-indolylacetonitrile) which are probably the number of total spores, while indole and 3-indolylacetonitrile produced only 6.7 ± 0.9% and 1.5 ± 0.1% heat-resistant cells (Figure 2C ); hence it appeared that a large number of spores have some spore defect for heat resistance. Therefore, it appeared that the low heat-resistant CFU was caused by some spore defect or the altered spore structure. Furthermore, the effect of indole and 3-indolylacetonitrile was investigated using another spore-forming medium, Brain Heart Infusion (BHI) agar for a longer incubation time (here, 14 days) when sporulation process would be completed. Similar to DSM medium, indole (1 mM) and 3-indolylacetonitrile (1 mM) inhibited the heat-resistant CFU of P. alvei (17 ± 10% and 16 ± 1%), compared to no addition of exogenous indole (77 ± 3%). Therefore, the inhibitory impact of indole and 3-indolylacetonitrile was effective in different media for a long term, while their effect on heat resistance was attenuated with a longer incubation time. Effect of indole and indole derivatives on cell growth To test the toxicity of indole and indole derivatives, cell turbidity at 16 hr and the specific growth rates with indole and 3-indolylacetonitrile were measured. Most indole derivatives at the concentration tested (1 mM) did not have much of an inhibition effect on the cell growth of P. alvei , while indole-3-acetamide and 2-oxindole (P < 0.05) slightly decreased cell growth (Figure 4B ). The growth rate of P. alvei was 1.38 ± 0.08/h in the absence of the indole derivatives in LB medium, whereas the growth rate was 1.30 ± 0.01/h with indole (1 mM) and 1.27 ± 0.01/h with 3-indolylacetonitrile (1 mM). In DSM medium, the growth rate of P. alvei was 0.19 ± 0.01/h in the absence of the indole derivatives, whereas the growth rate was 0.17 ± 0.01/h with indole (1 mM) and 0.15 ± 0.01/h with 3-indolylacetonitrile (1 mM). Therefore, indole and 3-indolylacetonitrile were not toxic to P. alvei and the inhibitory effect of the heat resistance was mostly due to the function of indole and 3-indolylacetonitrile rather than growth inhibition. Indole contributes to low survival against environmental stresses Since endospores are remarkably resistant to heat as well as various chemicals [ 28 , 29 ], we presumed that indole also decreased the resistance to environmental stresses, such as treatment with antibiotics, ethanol and low pH. As expected, indole decreased the survival rates with three antibiotics (tetracycline, erythromycin, and chloramphenicol) and when exposed to low pH and 70% ethanol (Figure 5 ). For example, indole decreased tetracycline resistance 5.4-fold, erythromycin resistance 6.7-fold, and chloramphenicol resistance 4-fold, and the survival rates with ethanol 8.5-fold and pH 4.0 21-fold, respectively. These results are a good match with the sporulation results (Figure 2 ). Figure 5 Effect of indole on stress-resistance of P. alvei . The cells (an initial turbidity of 0.05 at 600 nm) were grown in spore forming DSM medium for 16 h. After the 16 h incubation, cells (1 ml) were placed in contact with antibiotics, 70% ethanol, and pH 4.0 LB for 1 h. Tet, Em, and Cm stand for tetracycline (1 mg/ml), erythromycin (5 mg/ml), and chloramphenicol (1 mg/ml), respectively. EtOH and pH 4.0 stand for 70% ethanol and pH 4.0 LB, respectively. Each experiment was repeated two to four times and one standard deviation is shown. Effect of indole on the survival of B. subtilis spores Since P. alvei belongs to the same Bacillales order including B. subtilis (the most studied spore-forming bacterium), the effect of indole and 3-indolylacetonitrile was investigated in B. subtilis that did not produce indole (data not shown). Unlike P. alvei , indole and 3-indolylacetonitrile had no impact on the heat resistance in B. subtilis , while glucose treatment as a negative control significantly decreased the heat-resistant CFU (Figure 6 ). Hence, it appeared that the action mechanism of indole was different between indole-producing P. alvei and non-indole-producing B. subtilis . Figure 6 Effect of indole and 3-indolylacetonitrile on the heat-resistant CFU of B. subtilis . Glucose (0.5% w/v), indole (1 mM) and 3-indolylacetonitrile (1 mM) were added at the beginning of culture, and cells (an initial turbidity of 0.05 at 600 nm) were grown in spore forming DSM medium at 37°C for 16 h. Glucose (Glu) was dissolved in water, and indole (Ind) and 3-indolylacetonitrile (IAN) were dissolved in DMSO. DMSO (0.1% v/v) was used as a control (None). DMSO (0.1% (v/v)) alone did not affect cell growth and the heat-resistant CFU. Each experiment was repeated three to four times and one standard deviation is shown. Extracellular indole accumulation in P. alvei To be an environmental signal molecule, indole has to be excreted out of cells. Thus, the cell growth of P. alvei and the extracellular indole concentration were measured in Luria-Bertani (LB) medium. Clearly, the level of extracellular indole from P. alvei was growth-dependent (Figure 1A ). Indole production was begun in the middle of exponential growth phase and reached the maximum amount (300 μM) in the stationary phase. Notably, the level of extracellular indole present was stable over time at 37°C (Figure 1A ), which was one of characteristics of the indole molecule [ 2 ] while other signaling molecules, such as AHLs, AI-2, and signal peptides, are only temporally present and heat-unstable [ 2 ]. The accumulation pattern of extracellular indole was similar to that of other bacteria, such as E. coli [ 33 ] and Vibrio cholera [ 10 ], while these two bacteria accumulated up to 500-600 μM of extracellular indole within 24 h in LB [ 10 , 33 ]. The slower accumulation of indole in P. alvei was probably due to the 200-fold lower activity of P. alvei tryptophanase than that of E. coli tryptophanase [ 22 ]. Figure 1 Production of extracellular indole in P. alvei . Cell growth and extracellular indole accumulation in LB (A) and extracellular indole accumulation in LB supplemented with different carbon sources (B) at 37°C at 250 rpm. Cell growth (closed circle) was determined via the optical density at 600 nm (OD 600 ). Glucose (Glu), glycerol (Gly), and lactose (Lac) in 0.5% (w/v) were added at the beginning of the culture and cells were cultured for 36 h and indole production was measured. Experiments were performed in triplicate and one standard deviation is shown. Catabolite repression of P. alvei tryptophanase Since indole production was suppressed by the presence of glucose in E. coli through catabolite repression [ 34 ], the effect of carbon sources on indole production was investigated in P. alvei . Similar to E. coli , the addition of glucose and glycerol (0.5%) in LB medium completely abolished the production of indole in P. alvei for 36 h, while lactose (0.5%) did not affect indole accumulation (Figure 1B ). This result suggested that the indole accumulation in P. alvei was strictly controlled by catabolic repression although transport mechanisms of glucose and glycerol would be different. In other words, P. alvei did not produce indole in the presence of the preferred carbon sources such as glucose and glycerol. Unlike the current observation, it was previously reported that the tryptophanase in B. alvei (renamed as P. alvei ) appeared to be constitutive, and catabolite repression was not operative [ 22 ]. The report studied the effect of only tryptophan on tryptophanase activity and found that the activity of P. alvei tryptophanase was independent of tryptophan [ 22 ]. Indole inhibits the heat-resistant cell numbers of P. alvei The main hypothesis of this study was that a large quantity of extracellular indole would play a quorum sensing role in cell physiology of P. alvei so we investigated the effect of indole on sporulation and biofilm formation which was influenced by cell population and environmental stresses in other Bacillus strains [ 30 ]. In P. alvei , the addition of exogenous indole (0, 0.2, or 1.0 mM) surprisingly decreases the heat-resistant colony-forming unit (CFU) in a dose dependent manner (Figure 2A ). For example, indole (1 mM) decreased the heat-resistant CFU of P. alvei compared to no addition of indole 51-fold at 16 hr (0.26 ± 0.01% vs.13.2 ± 0.9%) and 10-fold at 30 hr (8 ± 6% vs. 77 ± 10%). To confirm the presence of exogenous indole, the indole level in DSM medium was measured with HPLC. The level of exogenous indole (1 mM) was not changed at all over 24 h (data not shown). Hence, the exogenous indole was not utilized as a carbon source and inhibited the heat-resistant CFU of P. alvei . Figure 2 Effect of indole and 3-indolylacetonitrile on the heat-resistant CFU of P. alvei . The cells (an initial turbidity of 0.05 at 600 nm) were grown in spore forming DSM medium for 16 h and 30 h. Exogenous indole (A) and 3-indolylacetonitrile (B) were added at the beginning of the culture to test the effect of indole (Ind) and 3-indolylacetonitrile (IAN) on the heat-resistant CFU. Lysozyme-resistance assays (C) were performed with 30 h-grown cells with and without indole and 3-indolyacetonitrile, and lysozyme (1 mg/mL) was treated for 20 min. Each experiment was repeated three to four times and one standard deviation is shown. Additionally, the temperature effect of indole on the heat resistance of P. alvei was investigated since the environmental temperature affected indole signaling in E. coli [ 12 ]. Unlike in E. coli , the inhibitory effect of indole (1 mM) on the heat-resistant CFU of P. alvei at 30°C (0.3 ± 0.1% vs. 8 ± 2% for 16 h) was similar to that at 37°C in P. alvei (Figure 2 ). Hence, it appeared that the temperature effect of indole on the heat-resistant CFU of P. alvei was not significant under the tested laboratory conditions. Indole inhibits the development of spore coat and cortex The effect of indole on the morphology of sporulating cells was examined by transmission electron microscopy. Surprisingly, the proportion of sporulating cells in the total number of cells was similar between with and without treatment of indole (upper panel in Figure 3 ). However, exogenous addition of indole influenced the morphology of the spore coat and the cortex. Cells with exogenous indole formed endospores with a thin spore coat and a thin spore cortex, while using no indole treatment resulted in a thick spore coat and cortex (lower panel in Figure 3 ). Because the spore coat and cortex were important for heat resistance and chemical resistance [ 31 ], we concluded that indole caused an immature spore that negatively contributed to the heat resistance of P. alvei . Figure 3 Electron microscopy analysis of P. alvei endospore formation . DMSO (0.1% v/v) was used as a control (None). 1 mM indole and 1 mM 3-indolylacetonitrile (IAN) dissolved in DMSO were added at the beginning of culture, and cells (an initial turbidity of 0.05 at 600 nm) were grown in DSM for 30 h. The scale bar indicates 500 nm in the upper panel and 100 nm in the lower panel. Abbreviations: SC, spore coat; Cx, cortex; SPC, spore core. Effect of indole derivatives on the heat resistance of P. alvei In the natural environment, indole can be easily oxidized into hydroxyindoles by diverse oxygenases, and indole derivatives often show different effects on bacterial physiology [ 2 ]. Thus, P. alvei can often encounter many kinds of indole-like compounds that are synthesized from tryptophan in other bacteria, plants, and even animals. Therefore, seven indole derivatives have been further investigated for the heat resistance of P. alvei . As a negative control, glucose was used since glucose decreased the sporulation of B. subtilis [ 35 ]. Similar to B. subtilis , glucose (0.5%) clearly decreased the heat-resistant CFU by 600-fold in P. alvei (Figure 4A ). However, L-tryptophan as the main substrate of the indole biosynthesis did not have much influence on the heat-resistant CFU, which supported that indole rather than tryptophan specifically influenced the heat resistance of P. alvei (Figure 4A ). Figure 4 Effect of indole derivatives on the heat-resistant CFU of P. alvei . The cells (an initial turbidity of 0.05 at 600 nm) were grown in spore forming DSM medium for 16 h. Exogenous indole derivatives (1 mM) and glucose (0.5% w/v) were added at the beginning of the culture. Tryptophan (Trp) was dissolved in water, and indole (Ind), 3-indolylacetonitrile (IAN), indole-3-carboxyaldehyde (I3C), 3-indoleacetic acid (IAA), indole-3-acetamide (I3A), tryptamine (TM), and 2-oxindole (OI) were dissolved in dimethyl sulfoxide (DMSO). DMSO (0.1% v/v) was used as a control (None). Each experiment was repeated three to four times and one standard deviation is shown. The structures of Trp, Ind, IAA, I3CA, IAN, I3A, TM, and OI are shown. The asterisk indicates statistical significance determined using a Student t test (P < 0.05). Most interestingly, a plant auxin, 3-indolylacetonitrile dramatically (up to 2900-fold) decreased the heat-resistant CFU of P. alvei in a dose dependent manner at 16 and 30 hr (Figure 2B and Figure 4A ), while another auxin 3-indoleacetic acid had a less significant influence, and tryptamine and 2-oxindole had no effect (Figure 4A ). Therefore, these results suggest that the functional groups of indole derivatives may control the development of P. alvei spores. Similar to indole, the proportion of sporulating cells in the total number of cells was similar with and without treatment of 3-indolylacetonitrile (upper panel in Figure 3 ). Also, 3-indolylacetonitrile produced an irregular spore coat, while no treatment produced sturdy coat (Figure 3 ). Therefore, it appeared that indole and 3-indolylacetonitrile inhibited spore maturation rather than sporulation initiation. In order to understand how most spores (upper panel in Figure 3 ) in the presence of indole and 3-indolylacetonitrile could not survive against heat treatment, the lysozyme resistance assay [ 36 ] was performed with 30-hour grown cells since the lysozyme treatment could release all spores. As a result, indole and 3-indolylacetonitrile produced a large portion of lysozyme-resistant cells (47 ± 8% with indole and 50 ± 3% with 3-indolylacetonitrile) which are probably the number of total spores, while indole and 3-indolylacetonitrile produced only 6.7 ± 0.9% and 1.5 ± 0.1% heat-resistant cells (Figure 2C ); hence it appeared that a large number of spores have some spore defect for heat resistance. Therefore, it appeared that the low heat-resistant CFU was caused by some spore defect or the altered spore structure. Furthermore, the effect of indole and 3-indolylacetonitrile was investigated using another spore-forming medium, Brain Heart Infusion (BHI) agar for a longer incubation time (here, 14 days) when sporulation process would be completed. Similar to DSM medium, indole (1 mM) and 3-indolylacetonitrile (1 mM) inhibited the heat-resistant CFU of P. alvei (17 ± 10% and 16 ± 1%), compared to no addition of exogenous indole (77 ± 3%). Therefore, the inhibitory impact of indole and 3-indolylacetonitrile was effective in different media for a long term, while their effect on heat resistance was attenuated with a longer incubation time. Effect of indole and indole derivatives on cell growth To test the toxicity of indole and indole derivatives, cell turbidity at 16 hr and the specific growth rates with indole and 3-indolylacetonitrile were measured. Most indole derivatives at the concentration tested (1 mM) did not have much of an inhibition effect on the cell growth of P. alvei , while indole-3-acetamide and 2-oxindole (P < 0.05) slightly decreased cell growth (Figure 4B ). The growth rate of P. alvei was 1.38 ± 0.08/h in the absence of the indole derivatives in LB medium, whereas the growth rate was 1.30 ± 0.01/h with indole (1 mM) and 1.27 ± 0.01/h with 3-indolylacetonitrile (1 mM). In DSM medium, the growth rate of P. alvei was 0.19 ± 0.01/h in the absence of the indole derivatives, whereas the growth rate was 0.17 ± 0.01/h with indole (1 mM) and 0.15 ± 0.01/h with 3-indolylacetonitrile (1 mM). Therefore, indole and 3-indolylacetonitrile were not toxic to P. alvei and the inhibitory effect of the heat resistance was mostly due to the function of indole and 3-indolylacetonitrile rather than growth inhibition. Indole contributes to low survival against environmental stresses Since endospores are remarkably resistant to heat as well as various chemicals [ 28 , 29 ], we presumed that indole also decreased the resistance to environmental stresses, such as treatment with antibiotics, ethanol and low pH. As expected, indole decreased the survival rates with three antibiotics (tetracycline, erythromycin, and chloramphenicol) and when exposed to low pH and 70% ethanol (Figure 5 ). For example, indole decreased tetracycline resistance 5.4-fold, erythromycin resistance 6.7-fold, and chloramphenicol resistance 4-fold, and the survival rates with ethanol 8.5-fold and pH 4.0 21-fold, respectively. These results are a good match with the sporulation results (Figure 2 ). Figure 5 Effect of indole on stress-resistance of P. alvei . The cells (an initial turbidity of 0.05 at 600 nm) were grown in spore forming DSM medium for 16 h. After the 16 h incubation, cells (1 ml) were placed in contact with antibiotics, 70% ethanol, and pH 4.0 LB for 1 h. Tet, Em, and Cm stand for tetracycline (1 mg/ml), erythromycin (5 mg/ml), and chloramphenicol (1 mg/ml), respectively. EtOH and pH 4.0 stand for 70% ethanol and pH 4.0 LB, respectively. Each experiment was repeated two to four times and one standard deviation is shown. Effect of indole on the survival of B. subtilis spores Since P. alvei belongs to the same Bacillales order including B. subtilis (the most studied spore-forming bacterium), the effect of indole and 3-indolylacetonitrile was investigated in B. subtilis that did not produce indole (data not shown). Unlike P. alvei , indole and 3-indolylacetonitrile had no impact on the heat resistance in B. subtilis , while glucose treatment as a negative control significantly decreased the heat-resistant CFU (Figure 6 ). Hence, it appeared that the action mechanism of indole was different between indole-producing P. alvei and non-indole-producing B. subtilis . Figure 6 Effect of indole and 3-indolylacetonitrile on the heat-resistant CFU of B. subtilis . Glucose (0.5% w/v), indole (1 mM) and 3-indolylacetonitrile (1 mM) were added at the beginning of culture, and cells (an initial turbidity of 0.05 at 600 nm) were grown in spore forming DSM medium at 37°C for 16 h. Glucose (Glu) was dissolved in water, and indole (Ind) and 3-indolylacetonitrile (IAN) were dissolved in DMSO. DMSO (0.1% v/v) was used as a control (None). DMSO (0.1% (v/v)) alone did not affect cell growth and the heat-resistant CFU. Each experiment was repeated three to four times and one standard deviation is shown. Discussion Indole is an abundant environmental signal in both Gram-positive and Gram-negative bacteria [ 2 ]. Currently, the diverse roles of indole as an intercellular signal are beginning to be revealed in various indole-producing-bacteria, such as E. coli [ 2 , 3 ], Vibrio cholerae [ 10 ], Stigmatella aurantiaca [ 14 , 15 ], Fusobacterium nuceatum [ 11 ], and Porphyromonas gingivalis [ 37 ], as well as in non-indole-producing bacteria, such as Pseudomonas aeruginosa [ 8 ] and Salmonella enterica [ 13 , 38 ]. The current study shows that the environmental signal indole also has a role in Gram-positive P. alvei . Interestingly, the role of indole seems to be substantially divergent in different microorganisms, reflecting adaptation to different environments and niche-specific challenges. For example, indole differently controls (increases or decreases) biofilm formation in different E. coli strains [ 2 ], Vibrio cholerae [ 10 ], and Fusobacterium nuceatum [ 11 ]. Also, indole and indole derivatives induced sporulation in Stigmatella aurantiaca [ 14 ], while this study shows that indole reduced the integrity of spores in P. alvei (Figure 3 ). Therefore, the results suggest that different bacterial species have developed their unique systems to beneficially utilize indole in their microbial community. Previously, it was reported that indole derivatives, such as 3-indoleacetic acid, 3-indolylacetonitrile, tryptamine, and 2-oxindole, but not indole, decreased the percentages of spore germination and appressorium formation, which inhibited all stages of infection behaviors in a rice pathogen Magnaporthe grisea [ 39 ]. These results and the current study suggest that indole derivatives, such as 3-indolylacetonitrile, can be used as protective compounds against spore-forming P. alvei . Since indole influenced the biofilm formation of several indole-producing bacteria, such as E. coli [ 2 ], Vibrio cholerae [ 10 ], and Fusobacterium nuceatum [ 11 ], and the sporulation transcription factor SpoA was required for biofilm development in B. subtilis [ 40 ], the effect of indole on the biofilm formation of P. alvei was investigated. However, indole did not show an effect on P. alvei biofilm formation in the 96-well plate biofilm assay in LB or DSM media either at 30°C and at 37°C (data not shown). Therefore, the indole-involving mechanism of P. alvei biofilm formation is different from that in other strains. Glucose obviously prevented the development of CFU of P. alvei presumably by preventing sporulation (Figure 4 ) as well as in B. subtilis via catabolite repression [ 35 ]. Because indole is a stationary phase signal (Figure 1A ) and because the production of indole is tightly regulated by carbon sources (Figure 1B ), the role of indole on spore formation could be closely affected by the catabolite repression. Thus, indole serves not only as an indicator of cell population, but also as an indicator of starvation. This dual function of indole may reflect the status of cells in the environment. Because the accumulation of extracellular indole can be dramatically affected by many environmental factors (pH, temperature, and the presence of antibiotics) in addition to carbon sources [ 41 ], the action of indole would be governed by the environment in a sophisticated manner. Nevertheless, the question remains as to why P. alvei produces copious amount of extracellular indole, as it causes immature spore formation (Figure 3 ). One possible explanation can be found in the previous study in that bacteria utilize indole as a defense tool against non-indole producing pathogenic P. aeruginosa to diminish its virulence [ 8 ]. Another possible answer is that indole intentionally lowers integrity of spores in order to make cells easy to resume growth when the environment is favorable again at a later date. Hence, a large quantity of indole is an indicator of a favorable environment in which other unfavorable species are scare and indole may control the timing of germination in natural environments. Although highly speculative, another possibility is that indole signal negatively controls spore maturation, while other quorum sensing molecules positively regulates sporulation of Bacillus , even using multiple signaling molecules [ 30 ]. Also, there is the possibility that indole is affecting spore germination since indole lowered the survival against environmental stresses (Figure 5 ) while the number of spore was not affected by indole (Figure 3 ). However, it is unclear, so far, how the indole signal influences sporulation in P. alvei . It is necessary to identify the operon of P. alvei tryptophanase to understand the genetic regulation of indole biosynthesis. For further transcriptional study, the P. alvei chromosome should be sequenced. Also, one of future work would be to study which stage of the sporulation cascade or what genetic mechanism is being affected by indole. For example, it is interesting to find indole-interacting proteins in P. alvei , as previously identified indole-binding PykA of S. aurantiaca [ 15 ]. Endospore formation is an altruistic behavior of mother cells that provides the maximum chance of survival for the group (daughter cells) over any its neighbor species [ 28 ]. However, the formation of an environmentally resistant spore of pathogenic bacteria, such as Bacillus anthracis and various Clostridium app., are problematic to human health [ 28 ]. Hence it is important to find a tool which controls sporulation as a disinfectant or sporocide. The current study has revealed the natural action of sporulation reduction by indole and the plant auxin 3-indolylacetonitrile. Previously, 3-indolylacetonitrile from cruciferous vegetables ( Brassica ), such as broccoli, cauliflower, and cabbage, was seen to decrease the biofilm formation of two pathogenic bacteria, E. coli O157:H7 and P. aeruginosa by inhibiting polymeric matrix production [ 42 ]. Hence, indole and 3-indolylacetonitrile are possible spore maturation inhibitors against spore-forming P. alvei and biofilm inhibitors against pathogenic biofilm formation. Currently, various indole derivatives from plants and numerous synthetic indole derivatives are commercially available and work is in progress to identify universal and stronger sporocides and to understand their genetic mechanism in action. Conclusions The current study demonstrates that i) indole is an extracellular stationary phase molecule in a Gram-positive bacteria P. alvei , ii) indole clearly inhibits spore maturation and survival rates under several stresses in P. alvei without affecting cell growth, iii) plant auxin 3-indolylacetonitrile dramatically decreased the heat resistance of P. alvei , iv) electron microscopy shows that indole and 3-indolylacetonitrile inhibit the development of spore coats and cortex in P. alvei . This study shows that indole, as a signaling molecule in quorum-sensing manner, plays a role in sporulation of P. alvei and that 3-indolylacetonitrile can be useful to control of heat and antimicrobial resistant spores of Gram-positive bacteria. Methods Bacterial strains, materials and growth rate measurements P. alvei (ATCC 6344) and B. subtilis strain (ATCC6633) were obtained from Korean Culture Center of Microorganisms. The strain was originally isolated from European foulbrood [ 43 ]. Luria-Bertani (LB) [ 44 ] was used as a basic medium for growth unless indicated. DSM medium (Difco sporulation medium [ 45 ]) was used for spore formation and cell survival tests with antibiotics. DSM medium contains 8 g of Bacto nutrient broth (Difco), 10 ml of 10% KCl, 10 ml of 1.2% MgSO 4 ·7H 2 O, 1.5 ml of 1 M NaOH, 1 ml of 1 M Ca(NO 3 ) 2 , 1 ml of 0.01 M MnCl 2 and 1 ml of 1 mM FeSO 4 per liter. BHI agar medium (Difco brain heart infusion agar) was also used for long-term spore formation. Indole, tryptophan, 3-indoleacetic acid, indole-3-carboxyaldehyde, 3-indolylacetonitrile, indole-3-acetamide, tryptamine, 2-oxindole, tetracycline, erythromycin, chloramphenicol, and streptomycin were purchased from Sigma-Aldrich Co. (Missouri, USA). Ethanol and dimethyl sulfoxide (DMSO) were purchased from Duksan Pure Chemical Co. (Ansan, Korea). Bacterial strains were initially streaked from -80°C glycerol stocks on LB plates, and a fresh single colony was inoculated into LB medium (25 ml) in 250 ml flasks and routinely cultured at 250 rpm at 37°C unless otherwise indicated. Overnight cultures were diluted in a 1:100 ratio using LB medium for cell growth and indole production or DSM medium for the test of spore surviving. For cell growth measurements, the optical density was measured at 600 nm (OD 600 ) with a spectrophotometer (UV-160, Shimadzu, Japan). When the value of OD 600 was above 0.7, the culture sample was diluted to fit within a linear range of between 0.2 and 0.7. In order to measure cell viability and cell number, diluted cells were enumerated with LB agar plates. Indole assays To measure the concentration of extracellular indole, P. alvei was grown in LB medium at 250 rpm for 36 h. The extracellular indole concentrations were measured with reverse-phase HPLC [ 4 ] using a 100 × 4.6 mm Chromolith Performance RP-18e column (Merck KGaA, Darmstadt, Germany) and elution with H 2 O-0.1% (v/v) trifluoroacetic acid and acetonitrile (50:50) as the mobile phases at a flow rate of 0.5 ml/min (50:50). Under these conditions, the retention time and the absorbance maximum were 5.1 min/271 nm for indole. Each experiment was performed with three independent cultures. Sporulation assay Sporulation assays were performed in the spore-forming DSM medium and on BHI agar plates. The overnight culture of P. alvei grown in LB was diluted in a 1:100 ratio in DSM and then re-grown to a turbidity of 0.5 at 600 nm. The cells were re-inoculated in a 1:10 ratio in DSM (an initial turbidity of 0.05 at 600 nm) and grown for 16 hr and 30 hr at 30°C and 37°C. To test the effect of indole and indole derivatives on the heat-resistant CFU, the indole or indole derivatives were added at the beginning of the culture in DSM medium. After incubation for 16 hr and 30 hr, aliquots of each culture (1 ml) were incubated in a water bath at 80°C for 10 min [ 46 ], the cells were then immediately diluted with phosphate buffer (pH 7.4) to cool down, and then the cells were enumerated with LB agar plates. To study the long-term effect of indole and indole derivatives, BHI agar was used and the previous assay [ 47 ] was adapted. The percentage of heat-resistant cells was calculated as the number of CFU per ml remaining after heat treatment divided by the initial CFU per ml at time zero. Since glucose decreased sporulation rate in B. subtilis via catabolite repression [ 35 ], glucose was used as a negative control. Stress resistance assays All survival assays were performed in DSM medium as the sporulation assay. In order to test the effect of indole and indole derivatives, indole or 3-indolylacetonitrile (1 mM) were added at the beginning of the culture in DSM, and the cells were grown for 16 h in DSM. After the incubation, four antibiotics (tetracycline at 1 mg/ml, erythromycin at 5 mg/ml, and chloramphenicol at 1 mg/ml) were mixed with the cells (1 ml) and incubated at 37°C for 1 h without shaking, and then cells were enumerated with LB agar plates. To determine the impact of indole on ethanol resistance and acid resistance, 16 h-grown cells were mixed with 70% ethanol and LB (pH 4.0) and incubated at 37°C for 1 h without shaking, and cells were enumerated with LB agar plates. For lysozyme-resistance assays, 30 h-grown cells with and without indole and 3-indolyacetonitrile were treated with lysozyme (1 mg/mL) in buffer (20 mM Tris-HCl [pH 8.0], 300 mM NaCl) and incubated at 37°C for 20 min [ 36 ]. Aliquots of serial dilutions in PBS buffer were then spotted on LB agar plates to determine the number of survivors. Each experiment was performed with three independent cultures. Crystal-violet biofilm assay A static biofilm formation assay was performed in a 96-well polystyrene plate (Fisher Scientific, Pittsburg, USA) as previously reported [ 48 ]. Briefly, cells were inoculated at an initial turbidity at 600 nm of 0.05 and incubated for 24 h without shaking at both 30°C and 37°C. Cell density (turbidity at 620 nm) and total biofilm (absorbance at 540 nm) were measured using crystal violet staining. Transmission electron microscopy (TEM) To examine the spore structure, TEM was used and a previous method [ 49 ] was modified. Briefly, P. alvei cells were grown in DSM as performed in sporulation assays. After culturing P. alvei cells with and without indole or 3-indolylacetonitrile for 30 h, 2.5% glutaraldehyde and 2% formaldehyde were added to pre-fix the cells and incubated overnight at 4°C. Then, cells were collected by centrifugation and post-fixed in 2% osmium tetroxide overnight at 4°C, and washed four times with 0.2 M phosphate buffer (pH 7.2). Then, cells were mixed with warm 2% agarose and polymerized. Cell block was sliced into 0.5 × 0.5 × 0.1 cm, dehydrated with ethanol and embedded in Epon resin (Hatfield, USA). Ultrathin sections were obtained using a MT-X ultramicrotome (Tucson, USA) and stained with 3% uranyl acetate. TEM images were obtained using a Hitachi H-7600 electron microscope (Tokyo, Japan). Bacterial strains, materials and growth rate measurements P. alvei (ATCC 6344) and B. subtilis strain (ATCC6633) were obtained from Korean Culture Center of Microorganisms. The strain was originally isolated from European foulbrood [ 43 ]. Luria-Bertani (LB) [ 44 ] was used as a basic medium for growth unless indicated. DSM medium (Difco sporulation medium [ 45 ]) was used for spore formation and cell survival tests with antibiotics. DSM medium contains 8 g of Bacto nutrient broth (Difco), 10 ml of 10% KCl, 10 ml of 1.2% MgSO 4 ·7H 2 O, 1.5 ml of 1 M NaOH, 1 ml of 1 M Ca(NO 3 ) 2 , 1 ml of 0.01 M MnCl 2 and 1 ml of 1 mM FeSO 4 per liter. BHI agar medium (Difco brain heart infusion agar) was also used for long-term spore formation. Indole, tryptophan, 3-indoleacetic acid, indole-3-carboxyaldehyde, 3-indolylacetonitrile, indole-3-acetamide, tryptamine, 2-oxindole, tetracycline, erythromycin, chloramphenicol, and streptomycin were purchased from Sigma-Aldrich Co. (Missouri, USA). Ethanol and dimethyl sulfoxide (DMSO) were purchased from Duksan Pure Chemical Co. (Ansan, Korea). Bacterial strains were initially streaked from -80°C glycerol stocks on LB plates, and a fresh single colony was inoculated into LB medium (25 ml) in 250 ml flasks and routinely cultured at 250 rpm at 37°C unless otherwise indicated. Overnight cultures were diluted in a 1:100 ratio using LB medium for cell growth and indole production or DSM medium for the test of spore surviving. For cell growth measurements, the optical density was measured at 600 nm (OD 600 ) with a spectrophotometer (UV-160, Shimadzu, Japan). When the value of OD 600 was above 0.7, the culture sample was diluted to fit within a linear range of between 0.2 and 0.7. In order to measure cell viability and cell number, diluted cells were enumerated with LB agar plates. Indole assays To measure the concentration of extracellular indole, P. alvei was grown in LB medium at 250 rpm for 36 h. The extracellular indole concentrations were measured with reverse-phase HPLC [ 4 ] using a 100 × 4.6 mm Chromolith Performance RP-18e column (Merck KGaA, Darmstadt, Germany) and elution with H 2 O-0.1% (v/v) trifluoroacetic acid and acetonitrile (50:50) as the mobile phases at a flow rate of 0.5 ml/min (50:50). Under these conditions, the retention time and the absorbance maximum were 5.1 min/271 nm for indole. Each experiment was performed with three independent cultures. Sporulation assay Sporulation assays were performed in the spore-forming DSM medium and on BHI agar plates. The overnight culture of P. alvei grown in LB was diluted in a 1:100 ratio in DSM and then re-grown to a turbidity of 0.5 at 600 nm. The cells were re-inoculated in a 1:10 ratio in DSM (an initial turbidity of 0.05 at 600 nm) and grown for 16 hr and 30 hr at 30°C and 37°C. To test the effect of indole and indole derivatives on the heat-resistant CFU, the indole or indole derivatives were added at the beginning of the culture in DSM medium. After incubation for 16 hr and 30 hr, aliquots of each culture (1 ml) were incubated in a water bath at 80°C for 10 min [ 46 ], the cells were then immediately diluted with phosphate buffer (pH 7.4) to cool down, and then the cells were enumerated with LB agar plates. To study the long-term effect of indole and indole derivatives, BHI agar was used and the previous assay [ 47 ] was adapted. The percentage of heat-resistant cells was calculated as the number of CFU per ml remaining after heat treatment divided by the initial CFU per ml at time zero. Since glucose decreased sporulation rate in B. subtilis via catabolite repression [ 35 ], glucose was used as a negative control. Stress resistance assays All survival assays were performed in DSM medium as the sporulation assay. In order to test the effect of indole and indole derivatives, indole or 3-indolylacetonitrile (1 mM) were added at the beginning of the culture in DSM, and the cells were grown for 16 h in DSM. After the incubation, four antibiotics (tetracycline at 1 mg/ml, erythromycin at 5 mg/ml, and chloramphenicol at 1 mg/ml) were mixed with the cells (1 ml) and incubated at 37°C for 1 h without shaking, and then cells were enumerated with LB agar plates. To determine the impact of indole on ethanol resistance and acid resistance, 16 h-grown cells were mixed with 70% ethanol and LB (pH 4.0) and incubated at 37°C for 1 h without shaking, and cells were enumerated with LB agar plates. For lysozyme-resistance assays, 30 h-grown cells with and without indole and 3-indolyacetonitrile were treated with lysozyme (1 mg/mL) in buffer (20 mM Tris-HCl [pH 8.0], 300 mM NaCl) and incubated at 37°C for 20 min [ 36 ]. Aliquots of serial dilutions in PBS buffer were then spotted on LB agar plates to determine the number of survivors. Each experiment was performed with three independent cultures. Crystal-violet biofilm assay A static biofilm formation assay was performed in a 96-well polystyrene plate (Fisher Scientific, Pittsburg, USA) as previously reported [ 48 ]. Briefly, cells were inoculated at an initial turbidity at 600 nm of 0.05 and incubated for 24 h without shaking at both 30°C and 37°C. Cell density (turbidity at 620 nm) and total biofilm (absorbance at 540 nm) were measured using crystal violet staining. Transmission electron microscopy (TEM) To examine the spore structure, TEM was used and a previous method [ 49 ] was modified. Briefly, P. alvei cells were grown in DSM as performed in sporulation assays. After culturing P. alvei cells with and without indole or 3-indolylacetonitrile for 30 h, 2.5% glutaraldehyde and 2% formaldehyde were added to pre-fix the cells and incubated overnight at 4°C. Then, cells were collected by centrifugation and post-fixed in 2% osmium tetroxide overnight at 4°C, and washed four times with 0.2 M phosphate buffer (pH 7.2). Then, cells were mixed with warm 2% agarose and polymerized. Cell block was sliced into 0.5 × 0.5 × 0.1 cm, dehydrated with ethanol and embedded in Epon resin (Hatfield, USA). Ultrathin sections were obtained using a MT-X ultramicrotome (Tucson, USA) and stained with 3% uranyl acetate. TEM images were obtained using a Hitachi H-7600 electron microscope (Tokyo, Japan). Authors' contributions YK carried out most of the experiments and helped to draft the manuscript. J-HL participated in the design of study and interpretation of the data. MHC participated in discussion of the study. JL conceived of the study, participated in its design and coordination, and wrote much of the manuscript. All authors read and approved the final manuscript. Acknowledgements This research was supported was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0021871).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152212/

Fever in Returned Travelers

Predominant causes of fever vary by different geographic areas of exposure. Malaria is the most common overall cause of systemic febrile illness in travelers returning from tropical areas; dengue is the most common cause in travelers to some regions. The approach to a febrile patient must consider travel and exposure history, incubation period, mode of exposure, and impact of pretravel vaccination. Initial symptoms of self-limited and life-threatening infections may be similar; focal signs and symptoms can help to limit the differential diagnosis. Routine laboratory results can provide clues to the final diagnosis. Introduction While fever may be the manifestation of a self-limited infection, it can also presage an infection that could be rapidly progressive and lethal. International travel expands the list of infections that must be considered but does not eliminate common, cosmopolitan infections. Initial attention should focus most urgently on infections that are treatable, transmissible, and that may cause serious sequelae or death. 1 The characteristics of the places visited and the recency of travel will affect the urgency and extent of the initial workup. The recent emergence of the Middle East respiratory syndrome coronavirus (MERS-CoV) in the Arabian Peninsula and of Ebola in West Africa and the recent epidemics of chikungunya and Zika virus diseases underline the necessity of being aware of the possible implication of emerging pathogens in imported fever. 2 This chapter will focus on identifying the cause of fever in a returned traveler. The reader should refer to other sources for the specifics of therapy. Epidemiology of Fever in Travelers Prevalence of Fever in Travelers Fever in the absence of other prominent findings has been reported in 2%–3% of European and American travelers to developing countries. Among 784 American travelers who traveled for 3 months or less to developing countries, 3% reported fever unassociated with other illness. 3 These results are similar to those reported by Steffen et al. 4 in which 152 of 7886 (almost 2%) of Swiss travelers with short-term travel to developing countries reported "high fever over several days" on questionnaires completed several months after return. Of those with fever, 39% reported fever only while abroad, 37% had fever while abroad and at home, and 24% had fever at home only. Analysis of the GeoSentinel Surveillance Network database found that 28% of ill returned travelers seeking care at a GeoSentinel clinic had fever as a chief reason for seeking care. 5 Among patients with travel-related hospitalization, febrile illnesses predominated, accounting for 77% of admissions in a study from Israel. 6 Prevalence of Fever in Travelers Fever in the absence of other prominent findings has been reported in 2%–3% of European and American travelers to developing countries. Among 784 American travelers who traveled for 3 months or less to developing countries, 3% reported fever unassociated with other illness. 3 These results are similar to those reported by Steffen et al. 4 in which 152 of 7886 (almost 2%) of Swiss travelers with short-term travel to developing countries reported "high fever over several days" on questionnaires completed several months after return. Of those with fever, 39% reported fever only while abroad, 37% had fever while abroad and at home, and 24% had fever at home only. Analysis of the GeoSentinel Surveillance Network database found that 28% of ill returned travelers seeking care at a GeoSentinel clinic had fever as a chief reason for seeking care. 5 Among patients with travel-related hospitalization, febrile illnesses predominated, accounting for 77% of admissions in a study from Israel. 6 Causes of Fever in Travelers Findings from eight studies, each with at least 100 cases, that examined causes of fever after tropical travel are shown in Table 56.1 . 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 The geographic region of exposure helps to explain the marked differences in the relative likelihood of various diagnoses, as has been shown in a study by Freedman et al. 14 Malaria was the most common diagnosis among those requiring hospitalization for fever in most recently published series. In a GeoSentinel study including 3655 cases of potentially life-threatening tropical diseases, 91% of which had fever as a symptom, 77% were caused by malaria 15 ; in the study by Bottieau and colleagues 9 falciparum malaria was the only tropical disease that was fatal ( n = 5). In a GeoSentinel study 17% of febrile illnesses were caused by infections that are preventable with vaccines or specific chemoprophylaxis (e.g., falciparum malaria). 14 Common cosmopolitan infections were found in 34% of returned febrile travelers in the Bottieau study. Infections, such as respiratory tract infections, hepatitis, diarrheal illness, urinary tract infections, and pharyngitis, with a broad or worldwide distribution, account for more than half of fevers in some series, 8 and the cause of fever remained undefined in about one-quarter of cases. 5 , 9 , 10 While, overall, malaria is the most common specific infection causing systemic febrile illness, dengue fever, mononucleosis, rickettsial infections, and enteric fever are also important infections. Their relative rank varies by geographic location, with top three diagnoses being falciparum malaria, rickettsial infections, and dengue after travel to sub-Saharan Africa; dengue, falciparum, and vivax malaria after travel to Southeast Asia; enteric fever, dengue, and vivax malaria after travel to South Central Asia; and dengue, vivax malaria, and enteric fever after travel to Latin America and Caribbean. 16 Leptospirosis is likely underrecognized because of difficulty in confirming the diagnosis in many laboratories. The major increase in chikungunya virus infections in Indian Ocean Islands, Asia, and now the Americas has been reflected in an increase in cases in travelers to those regions (and even local spread of infection introduced by travelers in Europe). 17 TABLE 56.1 Summary Data From Major Studies of Fever in Returned Travelers TABLE 56.1 Study Patient Population (Location) Most Common Specific Infections Most Frequently Visited Regions Wilson et al. 2007 5 24,920 ill returned travelers, 6957 of whom had fever (Multicenter, Global) Malaria (21%) Acute TD (15%) RTI (14%) Dengue (6%) Dermatologic illness (4%) Enteric fever (2%) Rickettsioses (2%) Acute UTI (2%) Acute hepatitis (1%) Sub-Saharan Africa (37%) Southeast Asia (18%) Latin America/Caribbean (15%) South Central Asia (13%) North Africa (3%) Bottieau et al. 2006 9 1743 outpatients presenting with fever after tropical travel (Belgium) Malaria (27.7%) RTI (10.5%) Bacterial enteritis (6.2%) Mononucleosis-like syndrome (3.9%) Skin/soft tissue infection (3.6%) GU infection/STD (3.4%) Rickettsioses (3.3%) Dengue (3%) Sub-Saharan Africa (68%) Southeast Asia (12%) Latin America (7%) Indian subcontinent (6%) North Africa (4%) Doherty et al. 1995 10 195 inpatients presenting with fever after tropical travel (United Kingdom) Malaria (42%) Nonspecific viral syndrome (25%) Dengue (6%) Bacterial dysentery (5%) RTI (4%) Hepatitis A (3%) UTI (2%) Typhoid (1.5%) Sub-Saharan Africa (60%) Indian subcontinent (13%) Far East (8%) South America (3%) Europe (0.5%) O'Brien et al. 2001 11 232 inpatients admitted for management of fever after overseas travel (Australia) Malaria (27%) RTI (24%) Gastroenteritis (14%) Dengue (8%) Typhoid (3%) Hepatitis A (3%) Rickettsioses (2%) Tropical ulcer (2%) Asia (61%) The Pacific (20%) Africa (15%) Latin America (2%) Antinori et al. 2004 12 147 inpatients admitted for fever after tropical travel (Italy) Malaria (48%) Presumptive viral illness (12%) Viral hepatitis (9%) Gastroenteritis (5%) Schistosomiasis (5%) Typhoid (4%) Dengue (3%) RTI (3%) UTI (1%) Africa (61%) Asia (22%) Central and South America (13%) Oceania (2%) Middle East (2%) Parola et al. 2006 13 613 inpatients admitted for fever after tropical travel (France) Malaria (75%) RTI (4%) Foodborne/waterborne infection (4%) Dengue (2%) Viral hepatitis (1%) Indian Ocean (55%) West Africa (22%) Central Africa (9%) Southeast Asia (4%) Indian subcontinent (3%) North Africa (2%) Central America/Caribbean (0.5%) West and Riordan 2003 8 162 pediatric inpatients admitted with fever following travel to tropics and subtropics (United Kingdom) Viral illness (34%) Diarrheal illness (27%) Malaria (14%) Pneumonia (8.5%) Hepatitis A (5%) UTI (4%) Enteric fever (3%) Indian subcontinent (82%) Middle East (6%) Africa (4%) Southeast Asia (2%) Siikamaki et al. 2011 7 462 febrile adults returned from malaria-endemic area; emergency room of tertiary hospital; 54% hospitalized (Finland) Diarrheal disease (27%) Systemic febrile illness (21%) (sepsis 3%; enteric fever and other bacteria 3.7%; dengue 3%; other viral including EBV and HIV 5%; rickettsiosis 1.3%) RTI (15%) UTI (4%) Other GI (3%) Sub-Saharan Africa (42%) Southeast Asia (28%) Central Asia and Indian subcontinent (20%) South and Central America and Caribbean (6%) Other (6%) Unknown (1%) Steinlauf et al. 2005 6 211 inpatient adults after tropical travel, of whom 163 were febrile (Israel) Malaria (33%) Dengue (17%) RTI (6%) Diarrhea (6%) Enteric fever (3%) Hepatitis (2%) East Asia (48%) Sub-Saharan Africa (34%) Latin America (16%) Jensenius et al. 2013 15 82,825 ill returned travelers, 3655 of whom had acute and potentially life-threatening tropical diseases and 91% had fever (Multicenter, Global) Falciparum malaria (77%) Typhoid fever (12%) Paratyphoid fever (6%) Leptospirosis (2%) Rickettsiosis (2%) Dengue hemmorrhagic fever and dengue shock syndrome (1%) Sub-Saharan Africa (74%) South Central Asia (14%) Southeast Asia (5%) Latin America/Caribbean (4%) North Africa (1%) EBV, Epstein–Barr virus; GI, gastrointestinal; GU, genitourinary; HIV, human immunodeficiency virus; RTI, respiratory tract infection; STD, sexually transmitted disease; TD, travelers' diarrhea; UTI, urinary tract infection. Adapted from Wilson M, Boggild A. Fever and systemic symptoms. In: Guerrant R, Walker D, Weller P, editors: Tropical infectious diseases: principles, pathogens and practice. 3rd ed. Edinburgh: Saunders Elsevier; 2011. Pp. 925–38. Approach to the Patient With Fever The Travel and Exposure History The fever pattern and clinical findings for many infections are similar. A detailed history of where a person has lived and traveled (including intermediate stops and modes of travel), dates of travel and time since return, activities during travel (such as types of accommodation, food habits, exposures [including sexual exposures, needle and blood exposures, animal and arthropod bites, water exposures]), and vaccinations and other preparation before travel and prophylaxis or treatment during or after travel are essential in developing a list of what infections are possible based on potential exposures and usual incubation periods. Relevant exposures can also occur in transit (e.g., on an airplane flight or cruise ship). 18 During the workup the clinician should keep in mind that fever after exotic travel may reflect infection with a common, cosmopolitan pathogen acquired during travel or after return home. At the same time it should be noted that unfamiliar infections can be acquired in industrialized countries (such as plague, Rocky Mountain spotted fever, tularemia, Lyme disease, hantavirus pulmonary syndrome in North America, and visceral leishmaniasis, hemorrhagic fever with renal syndrome and other hantaviral infections, and tickborne encephalitis in Europe). A detailed review of the clinical course, supplemented by the physical examination and laboratory data, will help to determine more likely causes and also to identify any infections that might require urgent interventions, hence expedited diagnostic studies. The process involved in the evaluation can be summarized in the following questions: • What diagnoses are possible based on the geographic areas visited? • What diagnoses are possible based on the time of travel, taking into account incubation periods? • What diagnoses are more likely based on activities, exposures, host factors, and clinical and laboratory findings? • Among the possible diagnoses, what is treatable, transmissible, or both? Incubation Period Incubation time is a valuable tool in evaluating a febrile patient. Knowledge of the incubation periods can allow one to exclude infections that are not biologically plausible. For example, dengue fever typically has an incubation of 3–14 days. Thus fever that begins >2 weeks after return from Thailand is not likely to be related to dengue fever. Remote travel is sometimes relevant, but most severe, acute life-threatening infections result from exposures that have occurred within the past 3 months. Important treatable infections that may occur >3 months after return include malaria, amebic liver abscess, and visceral leishmaniasis. In the study by O'Brien et al. 11 analyzing patients hospitalized with fever after travel, 96% were seen within 6 months of return from travel; in the study by Bottieau et al. 9 of patients referred for fever after tropical travel, fever occurred during travel or within 1 month of return home in 78%. Although the initial focus should be on travel within the past 3–6 months, the history should extend to include exposures a year or more earlier, if the initial investigation is unrevealing. More than a third of malaria-infected travelers in a study from Israel and the United States had illness that developed >2 months after return from endemic areas. 19 Onset of illness >6 months after return occurred in 2.3% of malaria patients reported to the CDC in 2009. 20 Table 56.2 lists many of the infections seen in travelers by time of onset of symptoms relative to the exposure and the initial clinical presentation. In assessing potential incubation period one must take into account the duration of the trip (and points of potential exposure during travel) and time since return. TABLE 56.2 Common Infections, by Incubation Periods TABLE 56.2 Disease Usual Incubation Period (Range) Distribution Incubation 1 month to 1 year) Widespread in tropics/subtropics Influenza 1–3 days Worldwide; can also be acquired en route Middle East respiratory syndrome (MERS) 5 days (2–14 days) Middle East Acute human immunodeficiency virus (HIV) 10–28 days (10 days to 6 weeks) Worldwide Legionellosis 5–6 days (2–10 days) Widespread Encephalitis, arboviral (e.g., Japanese encephalitis, tickborne encephalitis, West Nile virus, other) 3–14 days (1–20 days) Specific agents vary by region Incubation 14 Days to 6 Weeks Malaria, enteric fever, leptospirosis See above incubation periods for relevant diseases See above distribution for relevant diseases Hepatitis A 28–30 days (15–50 days) Most common in developing countries Hepatitis E 26–42 days (2–9 weeks) Widespread Acute schistosomiasis (Katayama syndrome) 4–8 weeks Most common after travel to sub-Saharan Africa Amebic liver abscess Weeks to months Most common in developing countries Incubation >6 Weeks Malaria, amebic liver abscess, hepatitis E, hepatitis B See above incubation periods for relevant diseases See above distribution for relevant diseases Tuberculosis Primary, weeks; reactivation, years Global distribution; rates and levels of resistance vary widely Leishmaniasis, visceral 2–10 months (10 days to years) Asia, Africa, South America Adapted from Centers for Disease Control and Prevention. CDC Health Information for International Travel 2016. New York. By permission of Oxford University Press, USA. Mode of Exposure Infections that can be acquired by a single bite of an infective arthropod, ingestion of contaminated food or beverages, swimming in contaminated water, or from direct contact with an infected person or animal are most often seen in short-term travelers. Casual sexual contact with new partners is common in travelers (5%–50% among short-term travelers) and inquiry about sexual exposures should be included as part of the history of an ill traveler. A Canadian study found that 15% of travelers reported sex with a new partner, or potential exposure to blood and body fluids through injections, dental work, tattoos, or other skin-perforating procedures during international travel. 21 Thus history is important to review even in returned travelers who are not acutely ill. In many instances travelers will be unaware of exposures. For example, patients with mosquito-borne and tickborne infections may not recall any bites. In contrast, patients who have had freshwater exposure (such as swimming, wading, bathing, or rafting) that places them at risk for schistosomiasis will typically recall the exposure with focused questioning, though they may have been unaware that the exposure carried any risk for infection. The provider should also inquire about medical care during travel. Travel for the purpose of seeking medical care (medical tourism) has expanded; travelers may undergo extensive surgery including cardiac surgery and organ transplantation overseas. In the course of medical care, patients may become colonized or infected with bacteria that are extremely resistant to usual antibiotic therapy, as has recently been reported with the New Delhi metallo-β-lactamase resistance mechanism, 22 or they may have other hospital-acquired infections. Impact of Pretravel Vaccination The history should include a review of pretravel vaccines, including dates of vaccination, types of vaccines received, and number of doses for multidose vaccines. Vaccines vary greatly in efficacy, and knowledge of vaccine status can influence the probability that certain infections will be present. For example, hepatitis A and yellow fever vaccines have high efficacy and only rare instances of infection have been reported in vaccinated travelers. In contrast, the typhoid fever vaccines (oral and parenteral) give incomplete protection. 23 The protective efficacy with the available typhoid vaccines was estimated to be 60%–72% in field trials in endemic regions. 24 The Travel and Exposure History The fever pattern and clinical findings for many infections are similar. A detailed history of where a person has lived and traveled (including intermediate stops and modes of travel), dates of travel and time since return, activities during travel (such as types of accommodation, food habits, exposures [including sexual exposures, needle and blood exposures, animal and arthropod bites, water exposures]), and vaccinations and other preparation before travel and prophylaxis or treatment during or after travel are essential in developing a list of what infections are possible based on potential exposures and usual incubation periods. Relevant exposures can also occur in transit (e.g., on an airplane flight or cruise ship). 18 During the workup the clinician should keep in mind that fever after exotic travel may reflect infection with a common, cosmopolitan pathogen acquired during travel or after return home. At the same time it should be noted that unfamiliar infections can be acquired in industrialized countries (such as plague, Rocky Mountain spotted fever, tularemia, Lyme disease, hantavirus pulmonary syndrome in North America, and visceral leishmaniasis, hemorrhagic fever with renal syndrome and other hantaviral infections, and tickborne encephalitis in Europe). A detailed review of the clinical course, supplemented by the physical examination and laboratory data, will help to determine more likely causes and also to identify any infections that might require urgent interventions, hence expedited diagnostic studies. The process involved in the evaluation can be summarized in the following questions: • What diagnoses are possible based on the geographic areas visited? • What diagnoses are possible based on the time of travel, taking into account incubation periods? • What diagnoses are more likely based on activities, exposures, host factors, and clinical and laboratory findings? • Among the possible diagnoses, what is treatable, transmissible, or both? Incubation Period Incubation time is a valuable tool in evaluating a febrile patient. Knowledge of the incubation periods can allow one to exclude infections that are not biologically plausible. For example, dengue fever typically has an incubation of 3–14 days. Thus fever that begins >2 weeks after return from Thailand is not likely to be related to dengue fever. Remote travel is sometimes relevant, but most severe, acute life-threatening infections result from exposures that have occurred within the past 3 months. Important treatable infections that may occur >3 months after return include malaria, amebic liver abscess, and visceral leishmaniasis. In the study by O'Brien et al. 11 analyzing patients hospitalized with fever after travel, 96% were seen within 6 months of return from travel; in the study by Bottieau et al. 9 of patients referred for fever after tropical travel, fever occurred during travel or within 1 month of return home in 78%. Although the initial focus should be on travel within the past 3–6 months, the history should extend to include exposures a year or more earlier, if the initial investigation is unrevealing. More than a third of malaria-infected travelers in a study from Israel and the United States had illness that developed >2 months after return from endemic areas. 19 Onset of illness >6 months after return occurred in 2.3% of malaria patients reported to the CDC in 2009. 20 Table 56.2 lists many of the infections seen in travelers by time of onset of symptoms relative to the exposure and the initial clinical presentation. In assessing potential incubation period one must take into account the duration of the trip (and points of potential exposure during travel) and time since return. TABLE 56.2 Common Infections, by Incubation Periods TABLE 56.2 Disease Usual Incubation Period (Range) Distribution Incubation 1 month to 1 year) Widespread in tropics/subtropics Influenza 1–3 days Worldwide; can also be acquired en route Middle East respiratory syndrome (MERS) 5 days (2–14 days) Middle East Acute human immunodeficiency virus (HIV) 10–28 days (10 days to 6 weeks) Worldwide Legionellosis 5–6 days (2–10 days) Widespread Encephalitis, arboviral (e.g., Japanese encephalitis, tickborne encephalitis, West Nile virus, other) 3–14 days (1–20 days) Specific agents vary by region Incubation 14 Days to 6 Weeks Malaria, enteric fever, leptospirosis See above incubation periods for relevant diseases See above distribution for relevant diseases Hepatitis A 28–30 days (15–50 days) Most common in developing countries Hepatitis E 26–42 days (2–9 weeks) Widespread Acute schistosomiasis (Katayama syndrome) 4–8 weeks Most common after travel to sub-Saharan Africa Amebic liver abscess Weeks to months Most common in developing countries Incubation >6 Weeks Malaria, amebic liver abscess, hepatitis E, hepatitis B See above incubation periods for relevant diseases See above distribution for relevant diseases Tuberculosis Primary, weeks; reactivation, years Global distribution; rates and levels of resistance vary widely Leishmaniasis, visceral 2–10 months (10 days to years) Asia, Africa, South America Adapted from Centers for Disease Control and Prevention. CDC Health Information for International Travel 2016. New York. By permission of Oxford University Press, USA. Mode of Exposure Infections that can be acquired by a single bite of an infective arthropod, ingestion of contaminated food or beverages, swimming in contaminated water, or from direct contact with an infected person or animal are most often seen in short-term travelers. Casual sexual contact with new partners is common in travelers (5%–50% among short-term travelers) and inquiry about sexual exposures should be included as part of the history of an ill traveler. A Canadian study found that 15% of travelers reported sex with a new partner, or potential exposure to blood and body fluids through injections, dental work, tattoos, or other skin-perforating procedures during international travel. 21 Thus history is important to review even in returned travelers who are not acutely ill. In many instances travelers will be unaware of exposures. For example, patients with mosquito-borne and tickborne infections may not recall any bites. In contrast, patients who have had freshwater exposure (such as swimming, wading, bathing, or rafting) that places them at risk for schistosomiasis will typically recall the exposure with focused questioning, though they may have been unaware that the exposure carried any risk for infection. The provider should also inquire about medical care during travel. Travel for the purpose of seeking medical care (medical tourism) has expanded; travelers may undergo extensive surgery including cardiac surgery and organ transplantation overseas. In the course of medical care, patients may become colonized or infected with bacteria that are extremely resistant to usual antibiotic therapy, as has recently been reported with the New Delhi metallo-β-lactamase resistance mechanism, 22 or they may have other hospital-acquired infections. Impact of Pretravel Vaccination The history should include a review of pretravel vaccines, including dates of vaccination, types of vaccines received, and number of doses for multidose vaccines. Vaccines vary greatly in efficacy, and knowledge of vaccine status can influence the probability that certain infections will be present. For example, hepatitis A and yellow fever vaccines have high efficacy and only rare instances of infection have been reported in vaccinated travelers. In contrast, the typhoid fever vaccines (oral and parenteral) give incomplete protection. 23 The protective efficacy with the available typhoid vaccines was estimated to be 60%–72% in field trials in endemic regions. 24 Clinical Presentations Many febrile infections are associated with focal signs or symptoms, which may help to limit the differential diagnosis. Undifferentiated fever can be more challenging. The following sections discuss common clinical presentations, with focus on more common diseases causing each. Other chapters provide more detailed discussions of diarrhea, skin diseases, and respiratory diseases. Undifferentiated Fever Always Look for Malaria. Malaria remains the most important infection to consider in anyone with fever after visiting or living in a malarious area. In nonimmune travelers falciparum malaria can be fatal if not diagnosed and treated urgently. Although most patients with malaria will report fever, as many as 40% or more may not have fever at the time of initial medical evaluation. 25 Risk of malaria varies greatly from one endemic region to another, but in general risk is highest in parts of sub-Saharan Africa; most severe and fatal cases in travelers follow exposure in this region. Tests to look for malaria should be done urgently (same day) and repeated in 8–24 hours if the initial blood smears are negative. In recent years rapid diagnostic tests for malaria have become valuable tools for the diagnosis of malaria in both endemic and nonendemic areas. 26 Prompt evaluation is most critical in persons who have visited areas with falciparum malaria in recent weeks. In the United States in 2009, 81% of reported patients with acute falciparum malaria had onset of symptoms within a month of return to the country; another 15% had onset of illness before arriving in the country. 20 Use of chemoprophylaxis may ameliorate symptoms or delay onset. No chemoprophylactic agent is 100% effective, so malaria tests should be done even in persons who report taking chemoprophylaxis. Many antimicrobials (e.g., TMP-SMX, azithromycin, doxycycline, clindamycin) have some activity against plasmodia. Taking these drugs for reasons unrelated to malaria may delay the onset of symptoms of malaria or modify the clinical course. Although fever and headache are commonly reported in malaria, gastrointestinal (GI) and pulmonary symptoms may be prominent and may misdirect the initial attention toward other infections. Thrombocytopenia and absence of leukocytosis are common laboratory findings. A prospective study of 335 travelers and migrants with suspected malaria found white blood cell (WBC) count 50% of cases). Rashes may be present, but many rickettsial infections (even among the SFG) are spotless. R. australis, R. africae , and rickettsialpox can cause a vesicular rash that may be mistaken for varicella, monkeypox, or even smallpox. High fever, headache, and normal or low WBC cell count and thrombocytopenia are characteristic. Lymphadenopathy may be present. Infections may be confused with dengue fever. Rickettsiae multiply in and damage endothelial cells and cause disseminated vascular lesions. Without treatment, the illness may persist for 2–3 weeks. Response to tetracyclines is generally prompt. Patients with suspected rickettsial infections should be treated empirically while awaiting laboratory confirmation. Other tickborne infections, human monocytic ehrlichiosis, and human granulocytic ehrlichiosis (granulocytotropic anaplasmosis), 42 are most commonly diagnosed in the United States but are also found in Europe, Africa, and probably Asia. Clinical findings include prominent fever and headache. These infections may also be associated with leukopenia and thrombocytopenia, and respond to treatment with tetracyclines. When epidemiologic and clinical aspects of rickettsial diseases were investigated in 280 international travelers reported to the GeoSentinel Surveillance Network during 1996–2008, 231 (82.5%) had spotted fever (SFG) rickettsiosis, 16 (5.7%) scrub typhus, 11 (3.9%) Q fever, 10 (3.6%) typhus group (TG) rickettsiosis, 7 (2.5%) bartonellosis, 4 (1.4%) indeterminable SFG/TG rickettsiosis, and 1 (0.4%) human granulocytic anaplasmosis; 197 (87.6%) of SFG rickettsiosis cases were acquired in sub-Saharan Africa and were associated with higher age, male gender, travel to southern Africa, late summer season travel, and travel for tourism. 43 Enteric Fever. Enteric fever (typhoid and paratyphoid fever) is another infection that causes fever and headache and can be associated with an unremarkable physical examination, though a faint rash (rose spots) may appear at the end of the first week of illness. Laboratory findings include a normal or low WBC count, thrombocytopenia, and elevation (usually modest) of liver enzymes. GI symptoms such as diarrhea, constipation, and vague abdominal discomfort may be present, as well as dry cough. In contrast to the abrupt onset of fevers in dengue and rickettsial infections, the onset of typhoid fever may be insidious. Leukocytosis in a patient with typhoid fever should raise suspicion of intestinal perforation or other complication. Diagnosis should be confirmed by recovery of Salmonella typhi (or S. paratyphi ) from blood or stool. 44 Culture of bone marrow aspirate may have a higher yield than blood or feces but is generally not favored by clinicians and patients. Serologic tests lack sensitivity and specificity. Increasing resistance of S. typhi to many antimicrobials makes it important to isolate the organism and to do sensitivity testing. The emergence of multidrug resistance and decreased ciprofloxacin susceptibility in Salmonella enterica serovar typhi in South Asia have rendered older drugs, including ampicillin, chloramphenicol, trimethoprim sulfamethoxazole, ciprofloxacin, and ofloxacin, ineffective or suboptimal for typhoid fever. 45 Multiple studies have identified the Indian subcontinent as a destination with relatively high risk for enteric fever in travelers, especially those visiting friends and relatives (VFRs). 46 The efficacy of typhoid vaccines in published studies varies widely depending on the type of vaccine, number of doses, and population studied. As noted, the efficacy of commonly used vaccines may be 60%–70%. 24 The important observation for clinicians evaluating returned travelers is that typhoid fever remains a concern (albeit lower) in persons who have received a typhoid vaccine. Infections with S. paratyphi may be relatively more common as a cause of typhoid fever in vaccinated populations because vaccine protects mainly against S. typhi . 44 Notably, the course of S. paratyphi A was not found to be milder than that of S. typhi infection. 46 Leptospirosis. Although leptospirosis has a broad geographic distribution, infections in humans are more common in tropical and subtropical regions. Recreational activities of travelers, including whitewater rafting in Costa Rica and other sports involving water exposures, have been associated with sporadic cases and large outbreaks. 47 Among 158 competitive swimmers in the Eco-Challenge in Malaysia in 2000, 44% met the case definition for acute leptospirosis. 48 Although clinical manifestations may be protean, common findings include fever, myalgia, and headache. Among 353 cases reported from Hawaii, 39% had jaundice and 28% conjunctival suffusion. 49 Other findings such as meningitis, rash, uveitis, pulmonary hemorrhage, oliguric renal failure, and refractory shock may be present. A summary of 72 sporadic leptospirosis cases in travelers from Europe and Israel shows that the majority were reported from Southeast Asia, were male (84%), the disease was associated with water activities in 91%, and 90% were hospitalized with no mortality. 50 Multiple different serovars exist, and clinical presentation and severity vary with infecting serovar. In Israeli travelers 55% had severe leptospirosis, usually associated with ictero-hemorrhagic serogroup. 51 Owing to lack of sensitive and specific diagnostic tests to confirm infection early in the course in most institutions, early empiric therapy is recommended for suspected infection, especially if severe. Agents used include doxycycline (and other tetracyclines), penicillins, and ceftriaxone. Acute Schistosomiasis. Acute schistosomaisis (Katayama syndrome) follows exposure to fresh water infested with cercariae that penetrate intact skin. The disease, seen primarily in nonimmunes, manifests 3–8 weeks after exposure. Clinical manifestations include high fever, myalgia, lethargy, and intermittent urticaria. 52 Dry cough and dyspnea, sometimes with pulmonary infiltrates, are noted in the majority of patients. 53 Eosinophilia, often high grade, is usually present. In one outbreak involving 12 travelers the median duration of fever was 12 days (range 4–46 days) and 10 of 12 had eosinophilia during the first 10 weeks of infection. 52 In most cases the disease is acquired in Africa (not only sub-Saharan); however, in the last decade an important focus was documented in Laos with infection due to S. mekongi. 54 Amebic Liver Abscess. An amebic abscess can cause fever and chills that develop over days to weeks. Although focal findings may not be prominent, 85%–90% of patients will report abdominal discomfort and about 70%–80% will have right upper quadrant tenderness on examination. 55 Extension of infection to the diaphragmatic surface of the liver may lead to cough, pleuritic or shoulder pain, and right basilar abnormalities on chest x-ray, which may initially suggest a pulmonary process. The abscess can be seen on ultrasound and serology for Entamoeba histolytica is usually positive. Hemorrhagic Fevers Several infections in addition to exotic infections such as Ebola and Marburg can cause fever and hemorrhage in travelers and many are treatable. Ebola and Marburg are transmitted mostly through direct contact with patient body fluids and are rarely seen in international travelers. During the recent Ebola epidemic in West Africa, falciparum malaria was the most frequent cause of fever in travelers to the affected area. 56 Leptospirosis, meningococcemia, and other bacterial infections can cause hemorrhage. Rickettsial infections can produce a petechial rash or purpura, and severe malaria may be associated with disseminated intravascular coagulation. Many viral infections, in addition to dengue, can cause hemorrhage. Most are arthropod-borne (especially mosquito or tick) or have rodent reservoir hosts. Among those reported in travelers are dengue fever (DHF), yellow fever, Lassa fever, Crimean Congo hemorrhagic fever, Rift Valley fever, hemorrhagic fever with renal syndrome (and other hantavirus-associated infections), Kyasanur Forest disease, Omsk hemorrhagic fever, and several viruses in South America (Junin, Machupo, Guanarito, Sabia). Lassa fever responds to ribavirin therapy if started early. Several of the viruses can be transmitted during medical care, so it is important to institute barrier isolation in a private room pending a specific diagnosis. Identification of viral agents causing hemorrhage may require the assistance of staff working in special laboratories, such as one available at CDC. (Assistance is available through the Special Pathogens Branch, Division of Viral and Rickettsial Diseases, CDC, Atlanta, GA 404-639-1511 and other specialized laboratories.) Even when specific treatment is not available, good supportive care can save lives. Fever and CNS Changes Neurologic findings in the febrile patient indicate the need for prompt workup. High fever alone or in combination with metabolic alternations precipitated by systemic infections can cause changes in the mental status in the absence of CNS invasion. One must consider common, cosmopolitan bacterial, viral, and fungal infections that cause fever and CNS changes. Additional considerations in travelers include Japanese encephalitis, rabies, West Nile, polio, tickborne encephalitis, and a number of other geographically focal viral infections, such as Nipah virus. Outbreaks of meningococcal infections (meningococcemia and meningitis) have been associated with the annual hajj pilgrimage to Mecca in Saudi Arabia. Beginning in 2000, for the first time ever, infection with Neisseria meningitidis serogroup W-135 caused outbreaks of meningococcal disease in pilgrims and subsequently in their contacts in multiple countries. Pilgrims vaccinated with the quadrivalent meningococcal vaccine (serogroups A, C, W-135, and Y) can still carry N. meningitidis in the nasopharynx. Dengue fever can cause neurologic findings that mimic Japanese encephalitis. In a study in Vietnam, dengue-associated encephalopathy was found in 0.5% of 5400 children admitted with DHF. 57 Meningitis may be present in leptospirosis. The parasite Angiostrongylus cantonensis causes sporadic infection in many countries and was responsible for an outbreak of eosinophilic meningoencephalitis in travelers to Jamaica in 2000. 58 African trypanosomiasis (sleeping sickness), transmitted by an infective tsetse fly, initially causes a nonspecific febrile illness. A chancre marks the site of the bite. If untreated, trypanosomes can infect the CNS and cause lethargy. Several cases have been seen in travelers after exposures, especially in Tanzania and Kenya. Patients with malaria, typhoid fever, and rickettsial infections often have severe headache, but cerebrospinal fluid (CSF) is typically unremarkable in these infections. Cerebral malaria causes altered mental status and can progress to seizures and coma. Mefloquine taken for malaria chemoprophylaxis has rarely been associated with seizures and other neuropsychiatric side effects, but fever typically is absent. Neuroschistosomiasis can be seen in travelers, but fever usually is not present at the time of the focal neurologic changes, caused by tissue reaction to ectopic schistosome egg deposition in the nervous system. Sexually transmitted infections such as HIV and syphilis, whether acquired at home or during travel, can involve the CNS. Lyme and ehrlichiosis are other treatable infections that can cause prominent neurologic findings. Other treatable infections that are unfamiliar to clinicians in many geographic areas include Q fever, relapsing fever, brucellosis, bartonellosis, anthrax, and plague. Persistent and Relapsing Fevers Diagnoses to be considered in patients with persistent or relapsing fevers include nonfalciparum malaria, typhoid fever, tuberculosis, brucellosis, cytomegalovirus (CMV), toxoplasmosis, louseborne relapsing fever (Borrelia recurrentis), melioidosis (Burkholderia pseudomallei), Q fever (Coxiella burnetii), visceral leishmaniasis, histoplasmosis (and other fungal infections), African trypanosomiasis, and infections that may be unrelated to exposures during travel, such as endocarditis. For fever with prominent respiratory symptoms, please refer to 59 , 60 , 61 , 62 , 63 , 64 and Chapter 59. Undifferentiated Fever Always Look for Malaria. Malaria remains the most important infection to consider in anyone with fever after visiting or living in a malarious area. In nonimmune travelers falciparum malaria can be fatal if not diagnosed and treated urgently. Although most patients with malaria will report fever, as many as 40% or more may not have fever at the time of initial medical evaluation. 25 Risk of malaria varies greatly from one endemic region to another, but in general risk is highest in parts of sub-Saharan Africa; most severe and fatal cases in travelers follow exposure in this region. Tests to look for malaria should be done urgently (same day) and repeated in 8–24 hours if the initial blood smears are negative. In recent years rapid diagnostic tests for malaria have become valuable tools for the diagnosis of malaria in both endemic and nonendemic areas. 26 Prompt evaluation is most critical in persons who have visited areas with falciparum malaria in recent weeks. In the United States in 2009, 81% of reported patients with acute falciparum malaria had onset of symptoms within a month of return to the country; another 15% had onset of illness before arriving in the country. 20 Use of chemoprophylaxis may ameliorate symptoms or delay onset. No chemoprophylactic agent is 100% effective, so malaria tests should be done even in persons who report taking chemoprophylaxis. Many antimicrobials (e.g., TMP-SMX, azithromycin, doxycycline, clindamycin) have some activity against plasmodia. Taking these drugs for reasons unrelated to malaria may delay the onset of symptoms of malaria or modify the clinical course. Although fever and headache are commonly reported in malaria, gastrointestinal (GI) and pulmonary symptoms may be prominent and may misdirect the initial attention toward other infections. Thrombocytopenia and absence of leukocytosis are common laboratory findings. A prospective study of 335 travelers and migrants with suspected malaria found white blood cell (WBC) count 50% of cases). Rashes may be present, but many rickettsial infections (even among the SFG) are spotless. R. australis, R. africae , and rickettsialpox can cause a vesicular rash that may be mistaken for varicella, monkeypox, or even smallpox. High fever, headache, and normal or low WBC cell count and thrombocytopenia are characteristic. Lymphadenopathy may be present. Infections may be confused with dengue fever. Rickettsiae multiply in and damage endothelial cells and cause disseminated vascular lesions. Without treatment, the illness may persist for 2–3 weeks. Response to tetracyclines is generally prompt. Patients with suspected rickettsial infections should be treated empirically while awaiting laboratory confirmation. Other tickborne infections, human monocytic ehrlichiosis, and human granulocytic ehrlichiosis (granulocytotropic anaplasmosis), 42 are most commonly diagnosed in the United States but are also found in Europe, Africa, and probably Asia. Clinical findings include prominent fever and headache. These infections may also be associated with leukopenia and thrombocytopenia, and respond to treatment with tetracyclines. When epidemiologic and clinical aspects of rickettsial diseases were investigated in 280 international travelers reported to the GeoSentinel Surveillance Network during 1996–2008, 231 (82.5%) had spotted fever (SFG) rickettsiosis, 16 (5.7%) scrub typhus, 11 (3.9%) Q fever, 10 (3.6%) typhus group (TG) rickettsiosis, 7 (2.5%) bartonellosis, 4 (1.4%) indeterminable SFG/TG rickettsiosis, and 1 (0.4%) human granulocytic anaplasmosis; 197 (87.6%) of SFG rickettsiosis cases were acquired in sub-Saharan Africa and were associated with higher age, male gender, travel to southern Africa, late summer season travel, and travel for tourism. 43 Enteric Fever. Enteric fever (typhoid and paratyphoid fever) is another infection that causes fever and headache and can be associated with an unremarkable physical examination, though a faint rash (rose spots) may appear at the end of the first week of illness. Laboratory findings include a normal or low WBC count, thrombocytopenia, and elevation (usually modest) of liver enzymes. GI symptoms such as diarrhea, constipation, and vague abdominal discomfort may be present, as well as dry cough. In contrast to the abrupt onset of fevers in dengue and rickettsial infections, the onset of typhoid fever may be insidious. Leukocytosis in a patient with typhoid fever should raise suspicion of intestinal perforation or other complication. Diagnosis should be confirmed by recovery of Salmonella typhi (or S. paratyphi ) from blood or stool. 44 Culture of bone marrow aspirate may have a higher yield than blood or feces but is generally not favored by clinicians and patients. Serologic tests lack sensitivity and specificity. Increasing resistance of S. typhi to many antimicrobials makes it important to isolate the organism and to do sensitivity testing. The emergence of multidrug resistance and decreased ciprofloxacin susceptibility in Salmonella enterica serovar typhi in South Asia have rendered older drugs, including ampicillin, chloramphenicol, trimethoprim sulfamethoxazole, ciprofloxacin, and ofloxacin, ineffective or suboptimal for typhoid fever. 45 Multiple studies have identified the Indian subcontinent as a destination with relatively high risk for enteric fever in travelers, especially those visiting friends and relatives (VFRs). 46 The efficacy of typhoid vaccines in published studies varies widely depending on the type of vaccine, number of doses, and population studied. As noted, the efficacy of commonly used vaccines may be 60%–70%. 24 The important observation for clinicians evaluating returned travelers is that typhoid fever remains a concern (albeit lower) in persons who have received a typhoid vaccine. Infections with S. paratyphi may be relatively more common as a cause of typhoid fever in vaccinated populations because vaccine protects mainly against S. typhi . 44 Notably, the course of S. paratyphi A was not found to be milder than that of S. typhi infection. 46 Leptospirosis. Although leptospirosis has a broad geographic distribution, infections in humans are more common in tropical and subtropical regions. Recreational activities of travelers, including whitewater rafting in Costa Rica and other sports involving water exposures, have been associated with sporadic cases and large outbreaks. 47 Among 158 competitive swimmers in the Eco-Challenge in Malaysia in 2000, 44% met the case definition for acute leptospirosis. 48 Although clinical manifestations may be protean, common findings include fever, myalgia, and headache. Among 353 cases reported from Hawaii, 39% had jaundice and 28% conjunctival suffusion. 49 Other findings such as meningitis, rash, uveitis, pulmonary hemorrhage, oliguric renal failure, and refractory shock may be present. A summary of 72 sporadic leptospirosis cases in travelers from Europe and Israel shows that the majority were reported from Southeast Asia, were male (84%), the disease was associated with water activities in 91%, and 90% were hospitalized with no mortality. 50 Multiple different serovars exist, and clinical presentation and severity vary with infecting serovar. In Israeli travelers 55% had severe leptospirosis, usually associated with ictero-hemorrhagic serogroup. 51 Owing to lack of sensitive and specific diagnostic tests to confirm infection early in the course in most institutions, early empiric therapy is recommended for suspected infection, especially if severe. Agents used include doxycycline (and other tetracyclines), penicillins, and ceftriaxone. Acute Schistosomiasis. Acute schistosomaisis (Katayama syndrome) follows exposure to fresh water infested with cercariae that penetrate intact skin. The disease, seen primarily in nonimmunes, manifests 3–8 weeks after exposure. Clinical manifestations include high fever, myalgia, lethargy, and intermittent urticaria. 52 Dry cough and dyspnea, sometimes with pulmonary infiltrates, are noted in the majority of patients. 53 Eosinophilia, often high grade, is usually present. In one outbreak involving 12 travelers the median duration of fever was 12 days (range 4–46 days) and 10 of 12 had eosinophilia during the first 10 weeks of infection. 52 In most cases the disease is acquired in Africa (not only sub-Saharan); however, in the last decade an important focus was documented in Laos with infection due to S. mekongi. 54 Amebic Liver Abscess. An amebic abscess can cause fever and chills that develop over days to weeks. Although focal findings may not be prominent, 85%–90% of patients will report abdominal discomfort and about 70%–80% will have right upper quadrant tenderness on examination. 55 Extension of infection to the diaphragmatic surface of the liver may lead to cough, pleuritic or shoulder pain, and right basilar abnormalities on chest x-ray, which may initially suggest a pulmonary process. The abscess can be seen on ultrasound and serology for Entamoeba histolytica is usually positive. Always Look for Malaria. Malaria remains the most important infection to consider in anyone with fever after visiting or living in a malarious area. In nonimmune travelers falciparum malaria can be fatal if not diagnosed and treated urgently. Although most patients with malaria will report fever, as many as 40% or more may not have fever at the time of initial medical evaluation. 25 Risk of malaria varies greatly from one endemic region to another, but in general risk is highest in parts of sub-Saharan Africa; most severe and fatal cases in travelers follow exposure in this region. Tests to look for malaria should be done urgently (same day) and repeated in 8–24 hours if the initial blood smears are negative. In recent years rapid diagnostic tests for malaria have become valuable tools for the diagnosis of malaria in both endemic and nonendemic areas. 26 Prompt evaluation is most critical in persons who have visited areas with falciparum malaria in recent weeks. In the United States in 2009, 81% of reported patients with acute falciparum malaria had onset of symptoms within a month of return to the country; another 15% had onset of illness before arriving in the country. 20 Use of chemoprophylaxis may ameliorate symptoms or delay onset. No chemoprophylactic agent is 100% effective, so malaria tests should be done even in persons who report taking chemoprophylaxis. Many antimicrobials (e.g., TMP-SMX, azithromycin, doxycycline, clindamycin) have some activity against plasmodia. Taking these drugs for reasons unrelated to malaria may delay the onset of symptoms of malaria or modify the clinical course. Although fever and headache are commonly reported in malaria, gastrointestinal (GI) and pulmonary symptoms may be prominent and may misdirect the initial attention toward other infections. Thrombocytopenia and absence of leukocytosis are common laboratory findings. A prospective study of 335 travelers and migrants with suspected malaria found white blood cell (WBC) count 50% of cases). Rashes may be present, but many rickettsial infections (even among the SFG) are spotless. R. australis, R. africae , and rickettsialpox can cause a vesicular rash that may be mistaken for varicella, monkeypox, or even smallpox. High fever, headache, and normal or low WBC cell count and thrombocytopenia are characteristic. Lymphadenopathy may be present. Infections may be confused with dengue fever. Rickettsiae multiply in and damage endothelial cells and cause disseminated vascular lesions. Without treatment, the illness may persist for 2–3 weeks. Response to tetracyclines is generally prompt. Patients with suspected rickettsial infections should be treated empirically while awaiting laboratory confirmation. Other tickborne infections, human monocytic ehrlichiosis, and human granulocytic ehrlichiosis (granulocytotropic anaplasmosis), 42 are most commonly diagnosed in the United States but are also found in Europe, Africa, and probably Asia. Clinical findings include prominent fever and headache. These infections may also be associated with leukopenia and thrombocytopenia, and respond to treatment with tetracyclines. When epidemiologic and clinical aspects of rickettsial diseases were investigated in 280 international travelers reported to the GeoSentinel Surveillance Network during 1996–2008, 231 (82.5%) had spotted fever (SFG) rickettsiosis, 16 (5.7%) scrub typhus, 11 (3.9%) Q fever, 10 (3.6%) typhus group (TG) rickettsiosis, 7 (2.5%) bartonellosis, 4 (1.4%) indeterminable SFG/TG rickettsiosis, and 1 (0.4%) human granulocytic anaplasmosis; 197 (87.6%) of SFG rickettsiosis cases were acquired in sub-Saharan Africa and were associated with higher age, male gender, travel to southern Africa, late summer season travel, and travel for tourism. 43 Enteric Fever. Enteric fever (typhoid and paratyphoid fever) is another infection that causes fever and headache and can be associated with an unremarkable physical examination, though a faint rash (rose spots) may appear at the end of the first week of illness. Laboratory findings include a normal or low WBC count, thrombocytopenia, and elevation (usually modest) of liver enzymes. GI symptoms such as diarrhea, constipation, and vague abdominal discomfort may be present, as well as dry cough. In contrast to the abrupt onset of fevers in dengue and rickettsial infections, the onset of typhoid fever may be insidious. Leukocytosis in a patient with typhoid fever should raise suspicion of intestinal perforation or other complication. Diagnosis should be confirmed by recovery of Salmonella typhi (or S. paratyphi ) from blood or stool. 44 Culture of bone marrow aspirate may have a higher yield than blood or feces but is generally not favored by clinicians and patients. Serologic tests lack sensitivity and specificity. Increasing resistance of S. typhi to many antimicrobials makes it important to isolate the organism and to do sensitivity testing. The emergence of multidrug resistance and decreased ciprofloxacin susceptibility in Salmonella enterica serovar typhi in South Asia have rendered older drugs, including ampicillin, chloramphenicol, trimethoprim sulfamethoxazole, ciprofloxacin, and ofloxacin, ineffective or suboptimal for typhoid fever. 45 Multiple studies have identified the Indian subcontinent as a destination with relatively high risk for enteric fever in travelers, especially those visiting friends and relatives (VFRs). 46 The efficacy of typhoid vaccines in published studies varies widely depending on the type of vaccine, number of doses, and population studied. As noted, the efficacy of commonly used vaccines may be 60%–70%. 24 The important observation for clinicians evaluating returned travelers is that typhoid fever remains a concern (albeit lower) in persons who have received a typhoid vaccine. Infections with S. paratyphi may be relatively more common as a cause of typhoid fever in vaccinated populations because vaccine protects mainly against S. typhi . 44 Notably, the course of S. paratyphi A was not found to be milder than that of S. typhi infection. 46 Leptospirosis. Although leptospirosis has a broad geographic distribution, infections in humans are more common in tropical and subtropical regions. Recreational activities of travelers, including whitewater rafting in Costa Rica and other sports involving water exposures, have been associated with sporadic cases and large outbreaks. 47 Among 158 competitive swimmers in the Eco-Challenge in Malaysia in 2000, 44% met the case definition for acute leptospirosis. 48 Although clinical manifestations may be protean, common findings include fever, myalgia, and headache. Among 353 cases reported from Hawaii, 39% had jaundice and 28% conjunctival suffusion. 49 Other findings such as meningitis, rash, uveitis, pulmonary hemorrhage, oliguric renal failure, and refractory shock may be present. A summary of 72 sporadic leptospirosis cases in travelers from Europe and Israel shows that the majority were reported from Southeast Asia, were male (84%), the disease was associated with water activities in 91%, and 90% were hospitalized with no mortality. 50 Multiple different serovars exist, and clinical presentation and severity vary with infecting serovar. In Israeli travelers 55% had severe leptospirosis, usually associated with ictero-hemorrhagic serogroup. 51 Owing to lack of sensitive and specific diagnostic tests to confirm infection early in the course in most institutions, early empiric therapy is recommended for suspected infection, especially if severe. Agents used include doxycycline (and other tetracyclines), penicillins, and ceftriaxone. Acute Schistosomiasis. Acute schistosomaisis (Katayama syndrome) follows exposure to fresh water infested with cercariae that penetrate intact skin. The disease, seen primarily in nonimmunes, manifests 3–8 weeks after exposure. Clinical manifestations include high fever, myalgia, lethargy, and intermittent urticaria. 52 Dry cough and dyspnea, sometimes with pulmonary infiltrates, are noted in the majority of patients. 53 Eosinophilia, often high grade, is usually present. In one outbreak involving 12 travelers the median duration of fever was 12 days (range 4–46 days) and 10 of 12 had eosinophilia during the first 10 weeks of infection. 52 In most cases the disease is acquired in Africa (not only sub-Saharan); however, in the last decade an important focus was documented in Laos with infection due to S. mekongi. 54 Amebic Liver Abscess. An amebic abscess can cause fever and chills that develop over days to weeks. Although focal findings may not be prominent, 85%–90% of patients will report abdominal discomfort and about 70%–80% will have right upper quadrant tenderness on examination. 55 Extension of infection to the diaphragmatic surface of the liver may lead to cough, pleuritic or shoulder pain, and right basilar abnormalities on chest x-ray, which may initially suggest a pulmonary process. The abscess can be seen on ultrasound and serology for Entamoeba histolytica is usually positive. Hemorrhagic Fevers Several infections in addition to exotic infections such as Ebola and Marburg can cause fever and hemorrhage in travelers and many are treatable. Ebola and Marburg are transmitted mostly through direct contact with patient body fluids and are rarely seen in international travelers. During the recent Ebola epidemic in West Africa, falciparum malaria was the most frequent cause of fever in travelers to the affected area. 56 Leptospirosis, meningococcemia, and other bacterial infections can cause hemorrhage. Rickettsial infections can produce a petechial rash or purpura, and severe malaria may be associated with disseminated intravascular coagulation. Many viral infections, in addition to dengue, can cause hemorrhage. Most are arthropod-borne (especially mosquito or tick) or have rodent reservoir hosts. Among those reported in travelers are dengue fever (DHF), yellow fever, Lassa fever, Crimean Congo hemorrhagic fever, Rift Valley fever, hemorrhagic fever with renal syndrome (and other hantavirus-associated infections), Kyasanur Forest disease, Omsk hemorrhagic fever, and several viruses in South America (Junin, Machupo, Guanarito, Sabia). Lassa fever responds to ribavirin therapy if started early. Several of the viruses can be transmitted during medical care, so it is important to institute barrier isolation in a private room pending a specific diagnosis. Identification of viral agents causing hemorrhage may require the assistance of staff working in special laboratories, such as one available at CDC. (Assistance is available through the Special Pathogens Branch, Division of Viral and Rickettsial Diseases, CDC, Atlanta, GA 404-639-1511 and other specialized laboratories.) Even when specific treatment is not available, good supportive care can save lives. Fever and CNS Changes Neurologic findings in the febrile patient indicate the need for prompt workup. High fever alone or in combination with metabolic alternations precipitated by systemic infections can cause changes in the mental status in the absence of CNS invasion. One must consider common, cosmopolitan bacterial, viral, and fungal infections that cause fever and CNS changes. Additional considerations in travelers include Japanese encephalitis, rabies, West Nile, polio, tickborne encephalitis, and a number of other geographically focal viral infections, such as Nipah virus. Outbreaks of meningococcal infections (meningococcemia and meningitis) have been associated with the annual hajj pilgrimage to Mecca in Saudi Arabia. Beginning in 2000, for the first time ever, infection with Neisseria meningitidis serogroup W-135 caused outbreaks of meningococcal disease in pilgrims and subsequently in their contacts in multiple countries. Pilgrims vaccinated with the quadrivalent meningococcal vaccine (serogroups A, C, W-135, and Y) can still carry N. meningitidis in the nasopharynx. Dengue fever can cause neurologic findings that mimic Japanese encephalitis. In a study in Vietnam, dengue-associated encephalopathy was found in 0.5% of 5400 children admitted with DHF. 57 Meningitis may be present in leptospirosis. The parasite Angiostrongylus cantonensis causes sporadic infection in many countries and was responsible for an outbreak of eosinophilic meningoencephalitis in travelers to Jamaica in 2000. 58 African trypanosomiasis (sleeping sickness), transmitted by an infective tsetse fly, initially causes a nonspecific febrile illness. A chancre marks the site of the bite. If untreated, trypanosomes can infect the CNS and cause lethargy. Several cases have been seen in travelers after exposures, especially in Tanzania and Kenya. Patients with malaria, typhoid fever, and rickettsial infections often have severe headache, but cerebrospinal fluid (CSF) is typically unremarkable in these infections. Cerebral malaria causes altered mental status and can progress to seizures and coma. Mefloquine taken for malaria chemoprophylaxis has rarely been associated with seizures and other neuropsychiatric side effects, but fever typically is absent. Neuroschistosomiasis can be seen in travelers, but fever usually is not present at the time of the focal neurologic changes, caused by tissue reaction to ectopic schistosome egg deposition in the nervous system. Sexually transmitted infections such as HIV and syphilis, whether acquired at home or during travel, can involve the CNS. Lyme and ehrlichiosis are other treatable infections that can cause prominent neurologic findings. Other treatable infections that are unfamiliar to clinicians in many geographic areas include Q fever, relapsing fever, brucellosis, bartonellosis, anthrax, and plague. Persistent and Relapsing Fevers Diagnoses to be considered in patients with persistent or relapsing fevers include nonfalciparum malaria, typhoid fever, tuberculosis, brucellosis, cytomegalovirus (CMV), toxoplasmosis, louseborne relapsing fever (Borrelia recurrentis), melioidosis (Burkholderia pseudomallei), Q fever (Coxiella burnetii), visceral leishmaniasis, histoplasmosis (and other fungal infections), African trypanosomiasis, and infections that may be unrelated to exposures during travel, such as endocarditis. For fever with prominent respiratory symptoms, please refer to 59 , 60 , 61 , 62 , 63 , 64 and Chapter 59. Laboratory Clues Routine Laboratory Studies Results of routine laboratory findings may provide clues to the diagnosis in the febrile traveler. An elevated WBC count may suggest a bacterial infection, but a number of bacterial infections, such as uncomplicated typhoid fever, brucellosis, and rickettsial infections, are associated with a normal or low WBC count. Elevated Liver Enzymes In the past hepatitis A virus was the most common cause of hepatitis after travel to developing regions. With the wide use of the hepatitis A vaccine, acute hepatitis A now is seen primarily in persons who failed to receive vaccine (or immunoglobulin) before travel. Hepatitis B remains a risk for unvaccinated persons. Hepatitis E, transmitted via fecally contaminated water or food, clinically resembles acute hepatitis A. Cases have been reported in travelers. 65 Mortality may be 20% or higher in women infected during the third trimester of pregnancy. Many common as well as unusual systemic infections cause fever and elevation of liver enzymes. Among those that may be a concern, depending on geographic exposures, are yellow fever, dengue and other hemorrhagic fevers, typhoid fever, leptospirosis, rickettsial infections, toxoplasmosis, Q fever, syphilis, psittacosis, and brucellosis. Transaminases are often elevated in these infections. Parasites that directly invade the liver and bile ducts (e.g., amebic liver abscess and liver flukes) often cause right upper quadrant pain, tender liver, and elevated alkaline phosphatase. Drugs and toxins (sometimes found in herbal drugs or nutritional supplements) can damage the liver, so a careful review of these agents should be part of the history. Fever and Eosinophilia Eosinophilia is sometimes an incidental finding on laboratory testing. When it is found in a person who has visited or lived in tropical, developing countries, it is a clue that should suggest several specific parasitic infections. 66 For further details see Chapter 58. Initial Diagnostic Workup A careful, complete physical examination should be carried out, looking with special care for rashes or skin lesions, lymphadenopathy, retinal or conjunctival changes, enlargement of liver or spleen, genital lesions, and neurologic findings. The initial laboratory evaluation in a febrile patient with a history of tropical exposures should generally include all or most of the following: • Complete blood count with a differential and estimate of platelets • Liver enzymes • Blood cultures • Malaria and dengue rapid diagnostic tests • Urinalysis and urine culture • Chest radiograph If malaria is suspected, it is essential not only to request the appropriate tests for malaria but also to make certain that tests are done expeditiously and by knowledgeable persons. In patients with diarrhea or GI symptoms (or if enteric fever is suspected), stool culture should be requested. In a patient with persisting fever a repeat physical examination will sometimes identify new findings (e.g., new rash, splenomegaly) that can provide useful clues to the diagnosis. Table 56.3 lists tests used to diagnose common infections in febrile returned travelers. TABLE 56.3 Common Clinical Findings and Associated Infections TABLE 56.3 Common Clinical Findings Infections to Consider After Tropical Travel Fever and rash Dengue, chikungunya, Zika, rickettsioses, enteric fever (skin lesions may be sparse or absent), acute HIV infection, measles Fever and abdominal pain Enteric fever, amebic liver abscess Undifferentiated fever and normal or low white blood cell count Dengue, malaria, rickettsial infection, enteric fever, chikungunya, Zika Fever and hemorrhage Viral hemorrhagic fevers (dengue, Ebola, and others), meningococcemia, leptospirosis, rickettsial infections Fever and eosinophilia Acute schistosomiasis; drug hypersensitivity reaction; fascioliasis and other parasitic infections (rare) Fever and pulmonary infiltrates Common bacterial and viral pathogens; legionellosis, acute schistosomiasis, Q fever, melioidosis, MERS, leptospirosis Fever and altered mental status Cerebral malaria, viral or bacterial meningoencephalitis, rabies, African trypanosomiasis Mononucleosis syndrome Epstein–Barr virus, cytomegalovirus, toxoplasmosis, acute HIV Fever persisting >2 weeks Malaria, enteric fever, Epstein–Barr virus, cytomegalovirus, toxoplasmosis, acute HIV, acute schistosomiasis, brucellosis, tuberculosis, Q fever, visceral leishmaniasis (rare) Fever with onset >6 weeks after travel Vivax or ovale malaria, acute hepatitis (B, C, or E), tuberculosis, amebic liver abscess, rabies HIV, Human immunodeficiency virus; MERS, Middle East respiratory syndrome. Adapted from Centers for Disease Control and Prevention. CDC Health Information for International Travel 2016. New York. By permission of Oxford University Press, USA. The process of travel may lead to medical problems. The immobility associated with travel may predispose to deep vein thrombosis; sinusitis may flare up during or after air travel, related to changes in pressure during ascent and descent. Noninfectious disease causes of fever, such as drug fever, and pulmonary emboli, should also be considered if initial studies do not confirm the presence of an infection. In the study by Bottieau et al. 9 noninfectious causes accounted for 2.2% of the fevers. Management Prompt diagnosis and urgent treatment may be necessary to save the patient's life. Fig. 56.1 provides an algorithm for the approach to a febrile patient following travel. Useful algorithms based on expert opinion and review of published literature are also available. 67 , 68 During the evaluation and treatment, the clinician should also keep in mind the public health impact. Familiar infections (e.g., salmonellosis, campylobacteriosis, gonorrhea) may be caused by multidrug-resistant organisms. It is especially important to recognize the potential for multidrug resistance in infections, such as typhoid fever, that can be lethal. Fig. 56.1 Flowchart for the management of a febrile patient. RDT, Rapid diagnostic test. Fig. 56.1 Absence of response to what should be appropriate treatment should lead the clinician to consider drug resistance, the possibility of the wrong diagnosis, or the presence of two infections. Particularly in patients with acute undifferentiated fever, rickettsial diseases must be considered, and patients with severe disease may be treated empirically. Use of empirical doxycycline treatment for patients with fever of unknown origin should be discussed, especially when empirical treatment with β-lactams has failed or, in severe cases, in association with β-lactams. 69 A number of case reports document the simultaneous presence of malaria and typhoid fever, amebic liver abscess and hepatitis A, and other dual infections. 70 , 71 Routine Laboratory Studies Results of routine laboratory findings may provide clues to the diagnosis in the febrile traveler. An elevated WBC count may suggest a bacterial infection, but a number of bacterial infections, such as uncomplicated typhoid fever, brucellosis, and rickettsial infections, are associated with a normal or low WBC count. Elevated Liver Enzymes In the past hepatitis A virus was the most common cause of hepatitis after travel to developing regions. With the wide use of the hepatitis A vaccine, acute hepatitis A now is seen primarily in persons who failed to receive vaccine (or immunoglobulin) before travel. Hepatitis B remains a risk for unvaccinated persons. Hepatitis E, transmitted via fecally contaminated water or food, clinically resembles acute hepatitis A. Cases have been reported in travelers. 65 Mortality may be 20% or higher in women infected during the third trimester of pregnancy. Many common as well as unusual systemic infections cause fever and elevation of liver enzymes. Among those that may be a concern, depending on geographic exposures, are yellow fever, dengue and other hemorrhagic fevers, typhoid fever, leptospirosis, rickettsial infections, toxoplasmosis, Q fever, syphilis, psittacosis, and brucellosis. Transaminases are often elevated in these infections. Parasites that directly invade the liver and bile ducts (e.g., amebic liver abscess and liver flukes) often cause right upper quadrant pain, tender liver, and elevated alkaline phosphatase. Drugs and toxins (sometimes found in herbal drugs or nutritional supplements) can damage the liver, so a careful review of these agents should be part of the history. Fever and Eosinophilia Eosinophilia is sometimes an incidental finding on laboratory testing. When it is found in a person who has visited or lived in tropical, developing countries, it is a clue that should suggest several specific parasitic infections. 66 For further details see Chapter 58. Initial Diagnostic Workup A careful, complete physical examination should be carried out, looking with special care for rashes or skin lesions, lymphadenopathy, retinal or conjunctival changes, enlargement of liver or spleen, genital lesions, and neurologic findings. The initial laboratory evaluation in a febrile patient with a history of tropical exposures should generally include all or most of the following: • Complete blood count with a differential and estimate of platelets • Liver enzymes • Blood cultures • Malaria and dengue rapid diagnostic tests • Urinalysis and urine culture • Chest radiograph If malaria is suspected, it is essential not only to request the appropriate tests for malaria but also to make certain that tests are done expeditiously and by knowledgeable persons. In patients with diarrhea or GI symptoms (or if enteric fever is suspected), stool culture should be requested. In a patient with persisting fever a repeat physical examination will sometimes identify new findings (e.g., new rash, splenomegaly) that can provide useful clues to the diagnosis. Table 56.3 lists tests used to diagnose common infections in febrile returned travelers. TABLE 56.3 Common Clinical Findings and Associated Infections TABLE 56.3 Common Clinical Findings Infections to Consider After Tropical Travel Fever and rash Dengue, chikungunya, Zika, rickettsioses, enteric fever (skin lesions may be sparse or absent), acute HIV infection, measles Fever and abdominal pain Enteric fever, amebic liver abscess Undifferentiated fever and normal or low white blood cell count Dengue, malaria, rickettsial infection, enteric fever, chikungunya, Zika Fever and hemorrhage Viral hemorrhagic fevers (dengue, Ebola, and others), meningococcemia, leptospirosis, rickettsial infections Fever and eosinophilia Acute schistosomiasis; drug hypersensitivity reaction; fascioliasis and other parasitic infections (rare) Fever and pulmonary infiltrates Common bacterial and viral pathogens; legionellosis, acute schistosomiasis, Q fever, melioidosis, MERS, leptospirosis Fever and altered mental status Cerebral malaria, viral or bacterial meningoencephalitis, rabies, African trypanosomiasis Mononucleosis syndrome Epstein–Barr virus, cytomegalovirus, toxoplasmosis, acute HIV Fever persisting >2 weeks Malaria, enteric fever, Epstein–Barr virus, cytomegalovirus, toxoplasmosis, acute HIV, acute schistosomiasis, brucellosis, tuberculosis, Q fever, visceral leishmaniasis (rare) Fever with onset >6 weeks after travel Vivax or ovale malaria, acute hepatitis (B, C, or E), tuberculosis, amebic liver abscess, rabies HIV, Human immunodeficiency virus; MERS, Middle East respiratory syndrome. Adapted from Centers for Disease Control and Prevention. CDC Health Information for International Travel 2016. New York. By permission of Oxford University Press, USA. The process of travel may lead to medical problems. The immobility associated with travel may predispose to deep vein thrombosis; sinusitis may flare up during or after air travel, related to changes in pressure during ascent and descent. Noninfectious disease causes of fever, such as drug fever, and pulmonary emboli, should also be considered if initial studies do not confirm the presence of an infection. In the study by Bottieau et al. 9 noninfectious causes accounted for 2.2% of the fevers. Management Prompt diagnosis and urgent treatment may be necessary to save the patient's life. Fig. 56.1 provides an algorithm for the approach to a febrile patient following travel. Useful algorithms based on expert opinion and review of published literature are also available. 67 , 68 During the evaluation and treatment, the clinician should also keep in mind the public health impact. Familiar infections (e.g., salmonellosis, campylobacteriosis, gonorrhea) may be caused by multidrug-resistant organisms. It is especially important to recognize the potential for multidrug resistance in infections, such as typhoid fever, that can be lethal. Fig. 56.1 Flowchart for the management of a febrile patient. RDT, Rapid diagnostic test. Fig. 56.1 Absence of response to what should be appropriate treatment should lead the clinician to consider drug resistance, the possibility of the wrong diagnosis, or the presence of two infections. Particularly in patients with acute undifferentiated fever, rickettsial diseases must be considered, and patients with severe disease may be treated empirically. Use of empirical doxycycline treatment for patients with fever of unknown origin should be discussed, especially when empirical treatment with β-lactams has failed or, in severe cases, in association with β-lactams. 69 A number of case reports document the simultaneous presence of malaria and typhoid fever, amebic liver abscess and hepatitis A, and other dual infections. 70 , 71 Conclusion It should be reminded that some febrile illnesses in travelers are still associated with high mortality and should be rapidly suspected and treated. Place of exposure and local epidemiology are key elements in the diagnosis process. Knowledge of the epidemiology of infections in a given geographic area is valuable, but detailed, up-to-date information about a specific location may be unavailable. Electronic databases are a useful source of current information about disease outbreaks and alerts about antimicrobial resistance patterns.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655995/

Template-Driven Spatial-Temporal Outbreak Simulation for Outbreak Detection Evaluation

We developed a non-disease specific template-driven spatial-temporal outbreak simulator for evaluating outbreak detection algorithms. With only a few outbreak parameter settings, our simulator can generate different patterns of outbreak cases either temporally or spatial-temporally using three different generation algorithms: deterministic, independent, Poisson process. Our simulator is flexible, easy to implement and provides case event times rather than aggregated counts. We provide examples of outbreak simulations using linear template functions. Our Template-Driven Simulator is a useful tool for evaluating of outbreak detection algorithms.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3559376/

Both OsRecQ1 and OsRDR1 Are Required for the Production of Small RNA in Response to DNA-Damage in Rice

Small RNA-mediated gene silencing pathways play important roles in the regulation of development, genome stability and various stress responses in many eukaryotes. Recently, a new type of small interfering RNAs (qiRNAs) approximately 20–21 nucleotides long in Neurospora crassa have been shown to mediate gene silencing in the DNA damage response (DDR) pathway. However, the mechanism for RNA silencing in the DDR pathway is largely unknown in plants. Here, we report that a class of small RNAs (qiRNAs) derived from rDNA was markedly induced after treatment by DNA-damaging agents [ethyl methanesulphonate (EMS and UV-C)], and that aberrant RNAs (aRNAs) as precursors were also highly induced after the DNA damage treatment in rice. However, these RNAs were completely abolished in OsRecQ1 (RecQ DNA helicase homologue) and OsRDR1 (RNA-dependent RNA polymerase homologue) mutant lines where either gene was disrupted by the insertion of rice retrotransposon Tos17 after the same treatment. DNA damage resulted in a more significant increase in cell death and a more severe inhibition of root growth in both mutant lines than in the WT. Together, these results strongly suggest that both OsRecQ1 and OsRDR1 play a pivotal role in the aRNA and qiRNA biogenesis required for the DDR and repair pathway in rice, and it may be a novel mechanism of regulation to the DDR through the production of qiRNA in plants. Introduction Chromosomal DNA damage in most organisms is caused by two major sources from exogenous factors such as ultraviolet light (UV), ionizing radiation and chemical exposure [1] , [2] , as well as through endogenous cellular processes such as cellular metabolism and replication errors [3] , [4] . Failure to repair DNA damage can lead to blockages of DNA transcription and replication, mutagenesis and cell death [5] , [6] . The mechanism of DNA damage response (DDR) and repair is essential for the maintenance of genomic integrity and survival for all organisms [7] . A variety of DNA repair pathways have been developed to fix the different kinds of DNA damage in eukaryotic cells, which mainly repair direct reversal of damage (DR), single-strand breaks (SSBs) and double-strand breaks (DSBs) [4] , [5] . The mechanisms of non-homologous end-joining (NHEJ) and homologous recombination (HR) are involved in the DSB repair pathway [8] . Many proteins acting as sensors, transducers or effectors are required for cell cycle checkpoint regulation, DNA repair and apoptosis in different DDR pathways [9] . Previous studies suggest that the RecQ family of DNA helicase is involved in the DNA repair pathway [10] , [11] . The anthrax toxin receptor (ATR) and ataxia telangiectasia mutated (ATM) protein kinases have known to be involved in a wide variety of responses to DNA damage in plants [12] , both ATM and ATR play central roles in the cellular response to DSBs by regulating DNA repair, cell-cycle arrest and apoptosis [13] , and suppressor of gamma response 1 (SOG1) participates in pathways governed by both ATR and ATM sensor kinases in plants [9] . Currently, a novel protein in mammals, RHINO (Rad9, Rad1, Hus1 interacting nuclear orphan) is shown to be required for ATR (ataxia telangiectasia and the Rad3-related) signaling and cell cycle checkpoint activation in the DDR pathway [14] , and Wolf-Hirschhorn syndrome candidate 1 (WHSC1) gene in human cells recruited to sites of DNA damage in the DDR [15] . RNA silencing is a manner of gene regulation by degrading sequence-specific RNA, which is conserved among eukaryotes including fungi, animals and plants [16] , [17] . A number of genes have been implicated in the diverse RNA silencing pathway in multiple organisms [18] , [19] , [20] . QDE-1 (RNA-dependent RNA polymerase, RDR homologue) and QDE-3 (RecQ DNA helicase homologue) in the filamentous fungus Neurospora crassa have been shown to be involved in the generation of double-stranded RNA (dsRNA) induced RNA silencing [21] , [22] . In Arabidopsis , some homologues of RDRs ( AtRDR1 , AtRDR2 and AtRDR6 ) have been shown to be responsible for RNA silencing or the antiviral pathway [23] , [24] , [25] . In rice, previous studies suggest that OsRecQ1 , a QDE-3 homologue, is thought to participated in the process of allowing inverted repeat (IR) DNA to be transcribed into dsRNA that can trigger RNA silencing [26] . OsRDR1 has been reported to be involved in virus mediated RNA silencing [27] , while rice chromomethyltransferase 3 ( OsCMT3 ) has been anticipated to be involved in the epigenetic process in affecting genome activity during abiotic stress [28] . Suppressor of gene silencing 3 ( SGS3 ) in Arabidopsis has been shown to be required for the biogenesis of trans -acting small interfering RNAs (ta-siRNAs) [24] . There are at least two copies of SGS3 [ OsSGS3a (AK064995) and OsSGS3b (AK100699)] in rice, and a recent finding showed that rice ( OsSGS3a ) interacted with a silencing suppressor, Rice stripe virus (RSV) p2 protein, which has been demonstrated to be be targets of TAS3 -derived ta-siRNAs, was up-regulated in RSV-infected rice [29] . Small RNA (sRNA), including small interfering RNA (siRNA) and microRNA (miRNA), plays an important role in the RNA silencing pathway during diverse biological processes in plants and animals [30] , [31] . Later studies have shown that siRNA and miRNA are mobile signals that control gene regulation in the RNA silencing pathway [32] , [33] . More recently, small RNA-mediated gene silencing as a new layer has been shown to modulate protein activity in the DDR pathways in Neurospora and animals [34] , [35] , [36] , but the mechanism for RNA silencing in the DDR pathway remains largely unknown in plants. In this paper, we present the role of OsRecQ1 and OsRDR1 in the small RNA regulating DDR in rice and propose a novel mechanism of gene regulation to the DDR through small RNA biogenesis in plants. Results qiRNAs Induced by DNA-damaging Agents in Rice A previous study has shown that a new class of small interfering RNAs (qiRNAs) in Neurospora crassa is involved in regulation of gene silencing in the DDR pathway [36] . It is worthwhile examining whether there is a similar mechanism of qiRNA regulation to the DDR in plants. Here, rice leaves and calli were treated by the DNA-damaging agents EMS or UV-C, respectively. Northern blot analysis with an RNA probe specific for the antisense 25S rDNA region showed that a class of small RNAs (qiRNAs) about 20–21 nucleotides (nt) in length was significantly induced after UV or EMS treatment, but it was at an undetectable level under normal conditions in WT ( Figure 1A ), and a similar result was also obtained using an RNA probe specific for the sense 25S rDNA region (data no shown), suggesting that these small RNA are double stranded. Interestingly, qiRNA accumulation was completely abolished in the OsRecQ1 mutant line (ND8004) ( Figure 1A ). These results suggest that OsRecQ1 is required for qiRNA biogenesis in the DDR pathway. 10.1371/journal.pone.0055252.g001 Figure 1 The detection of aRNAs and qiRNAs in WT and the OsRecQ1 mutant line (ND8004) after the DNA damaging agent UV or EMS treatment by northern blot and RT-qPCR analysis. (A) The results show a significant induction of qiRNAs about 20–21 nucleotides (nt) in length after UV or EMS treatments in WT, but a complete abolishment in the OsRecQ1 mutant line. An RNA probe derived from the sense 25S rDNA region was used. The arrow denotes the qiRNAs. The bottom panel of tRNAs shows equal loading control. (B) The results show a marked induction of 25S rDNA specific transcripts after UV or EMS treatments in WT, but a complete loss in the OsRecQ1 mutant line (ND8004). An RNA probe derived from sense 25S rDNA region was used. The bottom panel of rRNAs shows equal loading control. (C) A schematic diagram shows the upstream (U) and downstream (D) primers from rice rDNA regions for RT-qPCR analysis. The transcriptional start site is shown with an arrow. (D) RT-qPCR results indicating an abolishment of aRNAs from the rDNA locus induced by UV treatment in the OsRecQ1 mutant lines (ND8004). The expressing level of rice ubiquitin gene was used as the internal control. Two independent experiments are shown. Data are the mean ± SE (n = 3), *P<0.001. RT-qPCR analysis showing a loss of aRNAs from the rDNA locus induced by EMS treatment in the OsRecQ1 mutant lines (ND8004). aRNAs Required for qiRNA Biogenesis as Precursors in Rice It is generally accepted that the biogenesis of qiRNAs requires aberrant RNAs (aRNAs) as precursors [36] . To examine this possibility, the relationship between qiRNAs and aRNAs was investigated in rice by northern blot analyses and quantitative reverse transcription PCR (RT-qPCR). The results from the northern blot analyses using an RNA probe specific for the antisense 25S rDNA region show that aRNAs derived from rDNA specific transcripts with a few hundred nucleotides (nt) to 2 kilobases (kb) were markedly induced after UV or EMS treatments in WT, but were completely abolished in the OsRecQ1 mutant line ( Figure 1B ) and a similar result was also obtained using an RNA probe specific for the sense 25S rDNA region (data no shown) suggesting that these aRNAs derived from both strands of DNA for 25S rRNA. RT-qPCR analyses showed that aRNAs originated from both upstream (U) and downstream (D) regions of the transcribed 25S rDNA locus were highly induced after UV or EMS treatment, but were completely abolished in the OsRecQ1 mutant lines ( Figure 1D and E ). These results indicate that aRNAs transcribed from the rDNA locus as precursors are required for qiRNA biogenesis in the DDR in rice and that the RecQ DNA helicase, OsRecQ1 , is required for aRNA biogenesis in the DDR pathways. RNA-dependent RNA Polymerase 1 ( RDR1 ) Required for aRNA and qiRNA Biogenesis in Rice RDRs are an essential component of RNA silencing and can specifically recognize aRNAs, convert them to double-stranded RNA (dsRNA) [37] , [38] . Some other related genes such as OsCMT3 [28] and OsSGS3 [29] may participate in this biological processing of DDR. To examine this possibility in the DDR in rice, the accumulation of qiRNAs and aRNAs was investigated by northern blot analyses and RT-qPCR detection in the OsRDR1 as well as other mutant lines including OsCMT1 , OsCMT3 and OsSGS3b [24] , [28] , [29] . The results from the northern blot analyses with a RNA probe specific for the antisense 25S rDNA region show that qiRNAs and aRNAs were obviously induced after UV treatment in WT, OsCMT1 , OsCMT3 and OsSGS3b ( Figure 2A and B ). Notably, qiRNAs and aRNAs were completely abolished in both OsRDR1 mutant lines (ND2001 and ND2059) [25] but not in other mutant lines ( Figure 2A and B ), and a similar result of qiRNAs and aRNAs was also obtained using a RNA probe specific for the sense 25S rDNA region (data no shown. Furthermore, RT-qPCR analysis also showed that aRNAs derived from both the upstream (U) and downstream (D) regions of the rDNA locus were significantly induced after UV treatment in WT, OsCMT1 , OsCMT3 and OsSGS3b ( Figure 2C ), but not in the OsRDR1 mutant lines, indicating that OsRDR1 is indispensable for qiRNA and aRNA biogenesis in the DDR pathway in rice. 10.1371/journal.pone.0055252.g002 Figure 2 The detection of aRNAs and qiRNAs in WT and other mutant lines after UV treatment by Northern blot and RT-qPCR analysis. (A) The results show an obvious induction of qiRNAs after UV or EMS treatments in WT and some of the mutant lines, but a complete abolishment in both OsRDR1 mutant lines. An RNA probe derived from the sense 25S rDNA region was used. The arrow denotes the qiRNAs. The bottom panel of tRNAs shows equal loading control. OsRDR1-1 (ND2001), OsRDR1-2 (ND2059), OsCMT1 (NE7010), OsCMT3 (NC4949) and OsSGS3b (NE5050) mutant lines were used (see experimental procedures). (B) The results show a high induction of rDNA specific transcripts after UV treatment in WT and some of mutant lines, but an obvious loss in both OsRDR1 mutant lines (ND2001 and ND2059). An RNA probe derived from sense 25S rDNA region was used. The bottom panel of rRNAs shows equal loading control. OsRDR1-1 (ND2001), OsRDR1-2 (ND2059), OsCMT1 (NE7010), OsCMT3 (NC4949) and OsSGS3b (NE5050) mutant lines were used (see experimental procedures). (C) The results show an abolishment of aRNAs from the rDNA locus induced by UV treatment in both OsRDR1 mutant lines (ND2001 and ND2059). The expressing level of rice ubiquitin gene was used as the internal control. Data are the mean ± SE (n = 3), *P<0.001. OsRDR1-1 (ND2001), OsRDR1-2 (ND2059), OsCMT1 (NE7010), OsCMT3 (NC4949) and OsSGS3b (NE5050) mutant lines were used (see experimental procedures). DDR Induced by DNA-damaging Agents in Rice To investigate the DDR in the OsRecQ1 and OsRDR1 mutant lines, two-week-old seedlings were treated by UV-C irradiation for 0, 8, 16, 24 h. In Arabidopsis , Rad51A gene family is found to be involved in the DDR [4] , [5] . After the treatments, the expression of OsRad51A1 and OsRad51A2 were checked by RT-PCR analysis, and the results show that the transcription of OsRad51A2 was strongly induced by UV-C irradiation in the root tip (RT) of WT, but not in the OsRecQ1 and OsRDR1 mutant lines ( Figure 3A ), and OsRad51A2 was also strongly detected in the shoot apical meristem (SAM) after UV-C treatment, but not in the mature leaf (ML) possibly because of no proliferating stage ( Figure 3B and C ). However, the transcription of OsRad51A1 was not changed in the RT and SAM after UV-C treatment ( Figure 3B and C ). These results suggest that OsRad51A2 , not OsRad51A1, may participate in the DDR pathway induced by UV-C. 10.1371/journal.pone.0055252.g003 Figure 3 The expression of DDR genes induced by UV-C treatments in rice. (A) An increasing expression of OsRad51A2 induced by UV-C irradiation in the root tip tissues (RT) of WT, but not in the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines. Levels of the rice ubiquitin gene were used as the internal control. (B) A strongly detection of OsRad51A2 in the RT after UV-C treatment in WT, but not in the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines, and the transcription of OsRad51A1 was not changed after UV-C treatment in the RT and the mature leaf (ML). Levels of the rice ubiquitin gene were used as the internal control. (C) A highly expression of OsRad51A2 in the shoot apical meristem (SAM) and the RT after UV-C treatment in WT, but not but not in the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines, and the levels of OsRad51A1 transcription was not different before and after UV-C treatment in the SAM and the RT. Levels of the rice ubiquitin gene were used as the internal control. To further examine the DDR, the root tissues after UV-C treatment were checked for cell death by Evans blue staining. The results showed that more dead cells were stained blue under the microscope ( Figure 4A ), suggesting the higher frequency of cell death in the both OsREcQ1 and OsRDR1 mutant lines than that in the WT. 10.1371/journal.pone.0055252.g004 Figure 4 Measurement sensitivity to UV-C and EMS treatments in rice. (A) The results showing an increased number of dead cells stained by Evan's blue under the microscope in the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines. (B) UV-C irradiation resulted in a more significant increase in cell death in the root tips of OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines than in the WT. Data are the mean ± SE (n = 3). (C) Sensitivity of the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines to EMS. The EMS dosage is shown (0, 10, 20, 30 µM). Twenty seeds were tested in each assay. Three independent experiments were carried out. (D) Root growth of seedlings in the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines with a severe suppression after EMS treatments compared to the WT. Data are an average of triplicate assays and bars indicate the mean ± SE (n = 3). Meantime, the suspension cell assay was also performed by Evans blue staining for monitoring cell death estimation by spectrophotometry or by microscopic observation [39] , and both results indicate that UV irradiation resulted in a more significant increase in cell death in the OsRecQ1 and OsRDR1 mutant lines than in the WT ( Figure 4B ). Moreover, the root growth assay of seedlings [40] was carried out by liquid cultures containing 0, 10, 20, 30 µM EMS, and the results show that the root growth of the OsRecQ1 and OsRDR1 mutant seedlings was more severely suppressed compared to the WT ( Figure 4C and D ). These results indicate that OsRecQ1 and OsRDR1 play a key role in the DNA repair pathway. qiRNAs Induced by DNA-damaging Agents in Rice A previous study has shown that a new class of small interfering RNAs (qiRNAs) in Neurospora crassa is involved in regulation of gene silencing in the DDR pathway [36] . It is worthwhile examining whether there is a similar mechanism of qiRNA regulation to the DDR in plants. Here, rice leaves and calli were treated by the DNA-damaging agents EMS or UV-C, respectively. Northern blot analysis with an RNA probe specific for the antisense 25S rDNA region showed that a class of small RNAs (qiRNAs) about 20–21 nucleotides (nt) in length was significantly induced after UV or EMS treatment, but it was at an undetectable level under normal conditions in WT ( Figure 1A ), and a similar result was also obtained using an RNA probe specific for the sense 25S rDNA region (data no shown), suggesting that these small RNA are double stranded. Interestingly, qiRNA accumulation was completely abolished in the OsRecQ1 mutant line (ND8004) ( Figure 1A ). These results suggest that OsRecQ1 is required for qiRNA biogenesis in the DDR pathway. 10.1371/journal.pone.0055252.g001 Figure 1 The detection of aRNAs and qiRNAs in WT and the OsRecQ1 mutant line (ND8004) after the DNA damaging agent UV or EMS treatment by northern blot and RT-qPCR analysis. (A) The results show a significant induction of qiRNAs about 20–21 nucleotides (nt) in length after UV or EMS treatments in WT, but a complete abolishment in the OsRecQ1 mutant line. An RNA probe derived from the sense 25S rDNA region was used. The arrow denotes the qiRNAs. The bottom panel of tRNAs shows equal loading control. (B) The results show a marked induction of 25S rDNA specific transcripts after UV or EMS treatments in WT, but a complete loss in the OsRecQ1 mutant line (ND8004). An RNA probe derived from sense 25S rDNA region was used. The bottom panel of rRNAs shows equal loading control. (C) A schematic diagram shows the upstream (U) and downstream (D) primers from rice rDNA regions for RT-qPCR analysis. The transcriptional start site is shown with an arrow. (D) RT-qPCR results indicating an abolishment of aRNAs from the rDNA locus induced by UV treatment in the OsRecQ1 mutant lines (ND8004). The expressing level of rice ubiquitin gene was used as the internal control. Two independent experiments are shown. Data are the mean ± SE (n = 3), *P<0.001. RT-qPCR analysis showing a loss of aRNAs from the rDNA locus induced by EMS treatment in the OsRecQ1 mutant lines (ND8004). aRNAs Required for qiRNA Biogenesis as Precursors in Rice It is generally accepted that the biogenesis of qiRNAs requires aberrant RNAs (aRNAs) as precursors [36] . To examine this possibility, the relationship between qiRNAs and aRNAs was investigated in rice by northern blot analyses and quantitative reverse transcription PCR (RT-qPCR). The results from the northern blot analyses using an RNA probe specific for the antisense 25S rDNA region show that aRNAs derived from rDNA specific transcripts with a few hundred nucleotides (nt) to 2 kilobases (kb) were markedly induced after UV or EMS treatments in WT, but were completely abolished in the OsRecQ1 mutant line ( Figure 1B ) and a similar result was also obtained using an RNA probe specific for the sense 25S rDNA region (data no shown) suggesting that these aRNAs derived from both strands of DNA for 25S rRNA. RT-qPCR analyses showed that aRNAs originated from both upstream (U) and downstream (D) regions of the transcribed 25S rDNA locus were highly induced after UV or EMS treatment, but were completely abolished in the OsRecQ1 mutant lines ( Figure 1D and E ). These results indicate that aRNAs transcribed from the rDNA locus as precursors are required for qiRNA biogenesis in the DDR in rice and that the RecQ DNA helicase, OsRecQ1 , is required for aRNA biogenesis in the DDR pathways. RNA-dependent RNA Polymerase 1 ( RDR1 ) Required for aRNA and qiRNA Biogenesis in Rice RDRs are an essential component of RNA silencing and can specifically recognize aRNAs, convert them to double-stranded RNA (dsRNA) [37] , [38] . Some other related genes such as OsCMT3 [28] and OsSGS3 [29] may participate in this biological processing of DDR. To examine this possibility in the DDR in rice, the accumulation of qiRNAs and aRNAs was investigated by northern blot analyses and RT-qPCR detection in the OsRDR1 as well as other mutant lines including OsCMT1 , OsCMT3 and OsSGS3b [24] , [28] , [29] . The results from the northern blot analyses with a RNA probe specific for the antisense 25S rDNA region show that qiRNAs and aRNAs were obviously induced after UV treatment in WT, OsCMT1 , OsCMT3 and OsSGS3b ( Figure 2A and B ). Notably, qiRNAs and aRNAs were completely abolished in both OsRDR1 mutant lines (ND2001 and ND2059) [25] but not in other mutant lines ( Figure 2A and B ), and a similar result of qiRNAs and aRNAs was also obtained using a RNA probe specific for the sense 25S rDNA region (data no shown. Furthermore, RT-qPCR analysis also showed that aRNAs derived from both the upstream (U) and downstream (D) regions of the rDNA locus were significantly induced after UV treatment in WT, OsCMT1 , OsCMT3 and OsSGS3b ( Figure 2C ), but not in the OsRDR1 mutant lines, indicating that OsRDR1 is indispensable for qiRNA and aRNA biogenesis in the DDR pathway in rice. 10.1371/journal.pone.0055252.g002 Figure 2 The detection of aRNAs and qiRNAs in WT and other mutant lines after UV treatment by Northern blot and RT-qPCR analysis. (A) The results show an obvious induction of qiRNAs after UV or EMS treatments in WT and some of the mutant lines, but a complete abolishment in both OsRDR1 mutant lines. An RNA probe derived from the sense 25S rDNA region was used. The arrow denotes the qiRNAs. The bottom panel of tRNAs shows equal loading control. OsRDR1-1 (ND2001), OsRDR1-2 (ND2059), OsCMT1 (NE7010), OsCMT3 (NC4949) and OsSGS3b (NE5050) mutant lines were used (see experimental procedures). (B) The results show a high induction of rDNA specific transcripts after UV treatment in WT and some of mutant lines, but an obvious loss in both OsRDR1 mutant lines (ND2001 and ND2059). An RNA probe derived from sense 25S rDNA region was used. The bottom panel of rRNAs shows equal loading control. OsRDR1-1 (ND2001), OsRDR1-2 (ND2059), OsCMT1 (NE7010), OsCMT3 (NC4949) and OsSGS3b (NE5050) mutant lines were used (see experimental procedures). (C) The results show an abolishment of aRNAs from the rDNA locus induced by UV treatment in both OsRDR1 mutant lines (ND2001 and ND2059). The expressing level of rice ubiquitin gene was used as the internal control. Data are the mean ± SE (n = 3), *P<0.001. OsRDR1-1 (ND2001), OsRDR1-2 (ND2059), OsCMT1 (NE7010), OsCMT3 (NC4949) and OsSGS3b (NE5050) mutant lines were used (see experimental procedures). DDR Induced by DNA-damaging Agents in Rice To investigate the DDR in the OsRecQ1 and OsRDR1 mutant lines, two-week-old seedlings were treated by UV-C irradiation for 0, 8, 16, 24 h. In Arabidopsis , Rad51A gene family is found to be involved in the DDR [4] , [5] . After the treatments, the expression of OsRad51A1 and OsRad51A2 were checked by RT-PCR analysis, and the results show that the transcription of OsRad51A2 was strongly induced by UV-C irradiation in the root tip (RT) of WT, but not in the OsRecQ1 and OsRDR1 mutant lines ( Figure 3A ), and OsRad51A2 was also strongly detected in the shoot apical meristem (SAM) after UV-C treatment, but not in the mature leaf (ML) possibly because of no proliferating stage ( Figure 3B and C ). However, the transcription of OsRad51A1 was not changed in the RT and SAM after UV-C treatment ( Figure 3B and C ). These results suggest that OsRad51A2 , not OsRad51A1, may participate in the DDR pathway induced by UV-C. 10.1371/journal.pone.0055252.g003 Figure 3 The expression of DDR genes induced by UV-C treatments in rice. (A) An increasing expression of OsRad51A2 induced by UV-C irradiation in the root tip tissues (RT) of WT, but not in the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines. Levels of the rice ubiquitin gene were used as the internal control. (B) A strongly detection of OsRad51A2 in the RT after UV-C treatment in WT, but not in the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines, and the transcription of OsRad51A1 was not changed after UV-C treatment in the RT and the mature leaf (ML). Levels of the rice ubiquitin gene were used as the internal control. (C) A highly expression of OsRad51A2 in the shoot apical meristem (SAM) and the RT after UV-C treatment in WT, but not but not in the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines, and the levels of OsRad51A1 transcription was not different before and after UV-C treatment in the SAM and the RT. Levels of the rice ubiquitin gene were used as the internal control. To further examine the DDR, the root tissues after UV-C treatment were checked for cell death by Evans blue staining. The results showed that more dead cells were stained blue under the microscope ( Figure 4A ), suggesting the higher frequency of cell death in the both OsREcQ1 and OsRDR1 mutant lines than that in the WT. 10.1371/journal.pone.0055252.g004 Figure 4 Measurement sensitivity to UV-C and EMS treatments in rice. (A) The results showing an increased number of dead cells stained by Evan's blue under the microscope in the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines. (B) UV-C irradiation resulted in a more significant increase in cell death in the root tips of OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines than in the WT. Data are the mean ± SE (n = 3). (C) Sensitivity of the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines to EMS. The EMS dosage is shown (0, 10, 20, 30 µM). Twenty seeds were tested in each assay. Three independent experiments were carried out. (D) Root growth of seedlings in the OsRecQ1 (ND0059) and OsRDR1 (ND2001) mutant lines with a severe suppression after EMS treatments compared to the WT. Data are an average of triplicate assays and bars indicate the mean ± SE (n = 3). Meantime, the suspension cell assay was also performed by Evans blue staining for monitoring cell death estimation by spectrophotometry or by microscopic observation [39] , and both results indicate that UV irradiation resulted in a more significant increase in cell death in the OsRecQ1 and OsRDR1 mutant lines than in the WT ( Figure 4B ). Moreover, the root growth assay of seedlings [40] was carried out by liquid cultures containing 0, 10, 20, 30 µM EMS, and the results show that the root growth of the OsRecQ1 and OsRDR1 mutant seedlings was more severely suppressed compared to the WT ( Figure 4C and D ). These results indicate that OsRecQ1 and OsRDR1 play a key role in the DNA repair pathway. Discussion The dynamic state of DNA metabolism acts as replication, recombination and repair for tolerating and repairing numerous types of damage in all living organisms [5] . Although failure to repair DNA damage can lead to serious diseases in humans and animals, this situation is not a particular case in most higher plants. However, the mechanism of DNA metabolism plays a significant role in cell metabolic activity, normal growth and development in reproductive tissues of higher plants such as meristematic tissues [4] . Rad51-like genes were previously shown to be involved in HR and related repair pathways through mediating strand invasion and exchange between homologous DNA molecules [4] , [5] . The transcription of OsRadA is thought to be related to the level of cell proliferation in the meristematic tissues [41] and for meiotic homologous recombination and the repair of DSBs [42] , [43] , [44] . During the study of OsRecQ1 and OsRDR1 functions in the DDR, we observed that the expression of OsRad51A2 was significantly induced by UV-C irradiation in the RT and SAM of WT, but not in the OsRecQ1 and OsRDR1 mutant lines ( Figure 3A ). These results suggest that the transcription of OsRad51A2 is particularly relevant to the level of cell proliferation in the replicating tissues, but that of OsRad51A1 is not ( Figure 3B and C ), suggesting that OsRad51A1 may has a different role from that of OsRad51A2 in this DDR pathway. It can be thought at least that OsRecQ1 and OsRDR1 are in the upstream to OsRAD51A2 . Since these two genes are involved in qiRNA biogenesis, qiRNA might affect the transcription of OsRDA51A2 . It remains to elucidate how qiRNA (or other yet unknown OsRDR1 and OsRecQ1 involved small RNA) are directly/indirectly involved in the transcription of OsRAD51A2 , and that OsRecQ1 and OsRDR1 may play an important role in the processing of cell proliferation in the DDR pathway. It would be interesting to investigate whether this is general for other genes related to the DDR such as BRCA1 (Breast Cancer 1), MRE11 (Meiotic Recombination 11) or RECQ4 (Recombination Q4) in rice. Although the mechanisms of DNA damage and repair have been clearly established in bacteria, yeast, and mammals, it is worthwhile determining whether these mechanisms exist in higher plants [4] . As a new class of small RNA, qiRNA, has recently been shown to be involved in regulation of gene silencing in the DDR pathway in Neurospora crassa [36] . The present research focuses on a novel aspect of small RNA-mediated gene silencing in the DDR pathways in rice. The results suggest that the production of qiRNA may be a novel mechanism for the DDR in plants, which is similar to the mechanism of RNAi in the DDR pathway in Neurospora crassa . In Figure 5 , a proposed model for the RNA silencing pathway in the DDR is shown. After DNA damage, cells activate the DNA repair pathway that decides the cell's fate either to repair damage or to undergo apoptosis [34] , [36] . The DDR provokes cell-cycle progression to regulate protein levels through the small RNA-mediated gene silencing pathway, which responds to DNA damage checkpoints [36] , [45] . Our results show that both OsRecQ1 and OsRDR1 are required for aRNA and qiRNA biogenesis after DNA-damaging agent (EMS or UV) treatments, and aRNAs are required for qiRNA biogenesis as precursors. In our experiments not only qiRNA but also aRNA were found to be double-stranded because either sense or antisense RNA probe could detect their RNA bands in the northern blots. aRNA is thought to be single-stranded [46] , [47] . However, it is not the case in this study. It may possible that in DDR both sense and antisense DNA strands at the same rDNA locus could be transcribed to produce aberrant RNAs. DNA helicase and RDR may participate in this step because QDE1 showed RNA/DNA dependent RNA polymerase activity [36] and RDR6 in Arabidopsis showed polymerase activity on ssRNA as well as ssDNA in in vitro polymerase activity assay [37] . It remains to be determined whether both sense and antisense 25S rDNA regions were transcribed as a unit length or not. 10.1371/journal.pone.0055252.g005 Figure 5 A proposed model for the RNA silencing pathway in the DNA-damage response. OsRecQ1 (QDE-3 in Neurospora crassa ) and OsRDR1 (QDE-1 in Neurospora crassa ) are required for both aRNA and qiRNA biogeneses, respectively, after DNA-damaging agent (EMS or UV) treatment. qiRNAs may participate in a novel mechanism of regulation to the DDR in plants. More recently, a kind of small RNAs (DSB-induced small RNAs, diRNAs) has been reported to be involved in the DSB repair pathway. diRNAs are generated from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells [48] . In our current findings, qiRNAs are derived from rDNA repeats, which contribute to DDR by inhibiting rRNA biogenesis and regulating protein translation levels [36] . Upon exposure to DNA damaging agents, rDNA-specific small RNAs are induced that mediate DSB repair on damaged repetitive rDNAs. Therefore, qiRNAs are different kind of small RNAs in the DDR pathway from diRNAs in the DSBs repair pathway. In summary, we demonstrated that both OsRecQ1 and OsRDR1 are required for aRNA and qiRNA biogenesis in the DDR pathway, qiRNAs derived from rDNAs repeats are important for efficient DDR. It will be very exciting to have further studies on dissecting the mechanisms by which the production of small RNAs participate in the DDR pathways in plants. Materials and Methods Plant Materials and Growth Conditions The WT ( Oryza sativa L. cv. Nipponbare) and its knockout mutant lines were used in this study. OsRecQ1 (ND8004 and ND0059) and OsRDR1 (ND2001 and ND2059) mutant lines were reported previously [26] , [27] . OsSGS3b (NE5050), OsCMT1 (NE7010) and OsCMT3 (NC4949) mutant lines were used in this study. RNA silencing induction by particle bombardment was defective in these three mutants (unpublished data). The seeds of homozygous mutant lines were used to produce calli, and the plants and its calli were grown in proper conditions as previously described [26] . Plant Sensitivity Measurement to UV-C and EMS Treatments Two-week-old seedlings were irradiated under ultraviolet light (UV-C, 254 nm) using a germicidal lamp (Matsuda) for 0, 8, 16, 24 h as previously described [40] . Total RNA and mRNA were isolated from the rice tissue of ML, SAM or RT after UV-C treatment. RT-PCR analysis was performed as previously described [26] . Levels of the rice ubiquitin gene were used as the internal control. The sequences of primer pairs for RT-PCR for OsRAD51A1 (AB080262) and OsRAD51A2 (AB080264) genes were amplified by the following pairs of primers: 5′-GCTCATGCTTCCACAACAAG-3′ (OsRad51-F), 5′-GGCAGAAAACTTACTTCG-3′ (OsRad51A1-R) and 5′-AATTCTGGCTCGTCTAAC-3′ (OsRad51A2-R), respectively. For the root tissues staining by the Evan's blue and suspension cell assay, aliquots of RT were removed from treatments and performed by Evan's blue assay for monitoring cell death estimation by spectrophotometry or by microscopic observation as previously described [39] . For the root growth assay, 20 seeds of rice WT and each mutant line were grown in a Petri dish for liquid cultures containing 0, 10, 20, 30 µM EMS (Sigma-Aldrich) for two weeks with a modified version as previously described [41] . Three independent experiments were carried out. For the detection of qiRNAs and aRNAs, four leaf segments (about 6 cm) of seedlings at the two-week-old stage or one-month-old calli were used for the treatments by irradiation under UV-C light for 24 h or by liquid cultures containing 0.4% EMS (Sigma-Aldrich) for 48 h. After the treatments, northern blot and RT-qPCR analyses were performed as described below. RNA Gel Blot Analyses Total RNA was extracted from rice leaves and calli after DNA damage treatments as previously described [26] , [27] . Low and high molecular weight RNAs were used to detect qiRNAs and aRNAs with 25 and 40 µg of total RNA, respectively. Sense or antisense rRNA probes were prepared by in-vitro transcription derived from 25S rDNA regions (2035 bp fragment from 3 to 2037 region in AK119809) using a DIG RNA labeling (SP6/T7) kit (Roche) following the manufacturer's instructions. For small RNA detection, the RNA probe was hydrolyzed to an average size of 50 nt as described [49] , [50] . Prehybridization and hybridization were performed for qiRNA and aRNA detection at 42°C or 65°C, respectively. After hybridization, the membrane was washed three times with 0.1× SSC and 0.1% SDS buffer for 30 min at 50°C or 68°C and detected by a DIG detection kit (Roche) following the manufacturer's instructions. RT-qPCR Analyses Quantitative real-time PCR (RT-qPCR) was performed with a Light Cycler II system (Roche) using a previously described protocol [26] , [27] . In brief, total RNA was isolated using an RNeasy plant mini kit (QIAGEN) and treated with RNase-free DNase I (Roche), and Poly (A) + mRNA was purified by an Oligotex-dT30 mRNA purification kit (TaKaRa). Reverse transcription using random hexamers was carried out with equal amounts of mRNA (100 ng) by an AMV reverse transcriptase XL kit (TaKaRa). Synthesized cDNAs were 10-fold diluted and used for PCR by the incorporation of the fluorescent DNA dye SYBR green using the QuantiTect™ SYBR® Green PCR kit (Qiagen). Gene-specific primers were derived from the upstream (forward, 5′-AGTCCCCAGGCCTCTCTAAG-3′ and reverse, 5′-GTCCCGTCCTTGGAGTCTG-3′ 103 bp fragment from 61 to 163 region in X58275) or downstream (forward, 5′-CGATGTCGGCTCTTCCTATC-3′ and reverse, 5′-AACCTGTCTCACGACGGTC-3′ 105 bp fragment from 7892 to 7997 region in AP008245) sequence of the 25S rDNA region. Each reaction was performed in duplicate. Levels of the rice ubiquitin gene were used as the internal control [26] . Plant Materials and Growth Conditions The WT ( Oryza sativa L. cv. Nipponbare) and its knockout mutant lines were used in this study. OsRecQ1 (ND8004 and ND0059) and OsRDR1 (ND2001 and ND2059) mutant lines were reported previously [26] , [27] . OsSGS3b (NE5050), OsCMT1 (NE7010) and OsCMT3 (NC4949) mutant lines were used in this study. RNA silencing induction by particle bombardment was defective in these three mutants (unpublished data). The seeds of homozygous mutant lines were used to produce calli, and the plants and its calli were grown in proper conditions as previously described [26] . Plant Sensitivity Measurement to UV-C and EMS Treatments Two-week-old seedlings were irradiated under ultraviolet light (UV-C, 254 nm) using a germicidal lamp (Matsuda) for 0, 8, 16, 24 h as previously described [40] . Total RNA and mRNA were isolated from the rice tissue of ML, SAM or RT after UV-C treatment. RT-PCR analysis was performed as previously described [26] . Levels of the rice ubiquitin gene were used as the internal control. The sequences of primer pairs for RT-PCR for OsRAD51A1 (AB080262) and OsRAD51A2 (AB080264) genes were amplified by the following pairs of primers: 5′-GCTCATGCTTCCACAACAAG-3′ (OsRad51-F), 5′-GGCAGAAAACTTACTTCG-3′ (OsRad51A1-R) and 5′-AATTCTGGCTCGTCTAAC-3′ (OsRad51A2-R), respectively. For the root tissues staining by the Evan's blue and suspension cell assay, aliquots of RT were removed from treatments and performed by Evan's blue assay for monitoring cell death estimation by spectrophotometry or by microscopic observation as previously described [39] . For the root growth assay, 20 seeds of rice WT and each mutant line were grown in a Petri dish for liquid cultures containing 0, 10, 20, 30 µM EMS (Sigma-Aldrich) for two weeks with a modified version as previously described [41] . Three independent experiments were carried out. For the detection of qiRNAs and aRNAs, four leaf segments (about 6 cm) of seedlings at the two-week-old stage or one-month-old calli were used for the treatments by irradiation under UV-C light for 24 h or by liquid cultures containing 0.4% EMS (Sigma-Aldrich) for 48 h. After the treatments, northern blot and RT-qPCR analyses were performed as described below. RNA Gel Blot Analyses Total RNA was extracted from rice leaves and calli after DNA damage treatments as previously described [26] , [27] . Low and high molecular weight RNAs were used to detect qiRNAs and aRNAs with 25 and 40 µg of total RNA, respectively. Sense or antisense rRNA probes were prepared by in-vitro transcription derived from 25S rDNA regions (2035 bp fragment from 3 to 2037 region in AK119809) using a DIG RNA labeling (SP6/T7) kit (Roche) following the manufacturer's instructions. For small RNA detection, the RNA probe was hydrolyzed to an average size of 50 nt as described [49] , [50] . Prehybridization and hybridization were performed for qiRNA and aRNA detection at 42°C or 65°C, respectively. After hybridization, the membrane was washed three times with 0.1× SSC and 0.1% SDS buffer for 30 min at 50°C or 68°C and detected by a DIG detection kit (Roche) following the manufacturer's instructions. RT-qPCR Analyses Quantitative real-time PCR (RT-qPCR) was performed with a Light Cycler II system (Roche) using a previously described protocol [26] , [27] . In brief, total RNA was isolated using an RNeasy plant mini kit (QIAGEN) and treated with RNase-free DNase I (Roche), and Poly (A) + mRNA was purified by an Oligotex-dT30 mRNA purification kit (TaKaRa). Reverse transcription using random hexamers was carried out with equal amounts of mRNA (100 ng) by an AMV reverse transcriptase XL kit (TaKaRa). Synthesized cDNAs were 10-fold diluted and used for PCR by the incorporation of the fluorescent DNA dye SYBR green using the QuantiTect™ SYBR® Green PCR kit (Qiagen). Gene-specific primers were derived from the upstream (forward, 5′-AGTCCCCAGGCCTCTCTAAG-3′ and reverse, 5′-GTCCCGTCCTTGGAGTCTG-3′ 103 bp fragment from 61 to 163 region in X58275) or downstream (forward, 5′-CGATGTCGGCTCTTCCTATC-3′ and reverse, 5′-AACCTGTCTCACGACGGTC-3′ 105 bp fragment from 7892 to 7997 region in AP008245) sequence of the 25S rDNA region. Each reaction was performed in duplicate. Levels of the rice ubiquitin gene were used as the internal control [26] .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972862/

Sustained Specific and Cross-Reactive T Cell Responses to Zika and Dengue Virus NS3 in West Africa

ABSTRACT Recent studies on the role of T cells in Zika virus (ZIKV) infection have shown that T cell responses to Asian ZIKV infection are important for protection, and that previous dengue virus (DENV) exposure amplifies the protective T cell response to Asian ZIKV. Human T cell responses to African ZIKV infection, however, remain unexplored. Here, we utilized the modified anthrax toxin delivery system to develop a flavivirus enzyme-linked immunosorbent spot (ELISPOT) assay. Using human ZIKV and DENV samples from Senegal, West Africa, our results demonstrate specific and cross-reactive T cell responses to nonstructural protein 3 (NS3). Specifically, we found that T cell responses to NS3 protease are ZIKV and DENV specific, but responses to NS3 helicase are cross-reactive. Sequential sample analyses revealed immune responses sustained many years after infection. These results have important implications for African ZIKV/DENV vaccine development, as well as for potential flavivirus diagnostics based on T cell responses. IMPORTANCE The recent Zika virus (ZIKV) epidemic in Latin America and the associated congenital microcephaly and Guillain-Barré syndrome have raised questions as to why we have not recognized these distinct clinical diseases in Africa. The human immunologic response to ZIKV and related flaviviruses in Africa represents a research gap that may shed light on the mechanisms contributing to protection. The goal of our study was to develop an inexpensive assay to detect and characterize the T cell response to African ZIKV and DENV. Our data show long-term specific and cross-reactive human immune responses against African ZIKV and DENV, suggesting the usefulness of a diagnostic based on the T cell response. Additionally, we show that prior flavivirus exposure influences the magnitude of the T cell response. The identification of immune responses to African ZIKV and DENV is of relevance to vaccine development.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187110/

Quantification of plasma HIV RNA using chemically-engineered peptide nucleic acids

The remarkable stability of peptide nucleic acids (PNAs) toward enzymatic degradation makes this class of molecules ideal to develop as part of a diagnostic device. Here we report the development of chemically-engineered PNAs for the quantitative detection of HIV RNA at clinically relevant levels that are competitive with current PCR-based assays. Using a sandwich hybridization approach, chemical groups were systematically introduced into a surface PNA probe and a reporter PNA probe to achieve quantitative detection for HIV RNA as low as 20 copies per milliliter of plasma. For the surface PNA probe, four cyclopentane groups were incorporated to promote stronger binding to the target HIV RNA compared to PNA without the cyclopentanes. For the reporter PNA probe, 25 biotin groups were attached to promote strong signal amplification after binding to the target HIV RNA. These general approaches to engineer PNA probes may be used to detect other RNA target sequences. Introduction Diagnostic testing of HIV in both infected and non-infected patients is crucial to control the disease within the global population 1 . Moving such tests closer to the point-of-care (POC) for a patient helps doctors to quickly discern infection status and select the proper antiretroviral medication 2 . Obtaining this information more quickly and accurately promotes a test-and-treat strategy that has known benefits to limit spread of the virus 3 . HIV tests performed at the POC are typically antibody-based, qualitative, and require some type of follow-up testing to confirm infection 4 – 9 . Despite the popularity of antibody-based tests, nucleic acid testing (NAT) for HIV RNA continues to be the ultimate standard to confirm an infection and it is also the only method to quantify the viral load within infected patients 10 , 11 . Plasma HIV RNA levels correlate very well with the acute phase of infection, when the virus is most likely to be transmitted, and increases in viral load signal when the virus has developed resistance to a particular antiretroviral therapy 12 . Despite the benefits of tracking plasma HIV RNA in patients, there is no standard nucleic acid-based test for HIV that can be used at a patient's point-of-care 13 and only about one third of all public health laboratories in the U.S. are equipped to perform NAT for HIV RNA 14 . There are even fewer public health facilities in developing countries that test for HIV RNA 15 . The lack of testing for HIV RNA reflects the complications of using reverse transcription polymerase chain reaction (RT-PCR), the most common method to detect and quantify RNA 16 – 18 . The typical cost, instrumentation, and expertise needed to perform RT-PCR prohibits its implementation at most public health settings in the U.S. and abroad 10 . One approach to design NAT diagnostics without PCR amplification is to use a sandwich hybridization approach where target sequences are bound between two separate probes 19 . Normally one probe provides target segregation from the bulk solution (the surface probe), while the second probe imparts a measurable signal to the hybridization event (the reporter probe). The sandwich hybridization approach has been successfully implemented in a variety of nucleic acid sensing techniques, including: fluorescence imaging 20 – 23 , electrochemical detection 24 – 26 , template mediated fluorescence activation 27 , 28 , and surface-enhanced Raman scattering 29 . In most cases the requirement of two orthogonal binding events reduces background noise; however, the sensitivity of most sandwich assays is not as good as that achieved using a PCR-based method due to weak binding to the target or insufficient signal amplification. The aminoethylglycyl (aeg) peptide nucleic acid molecule (aegPNA) has nucleobases attached to a simple, non-natural polyamide backbone that consists of alternating ethylene diamine and glycine units ( Figure 1A ) 30 . In general, aegPNA binds to complementary DNA and RNA sequences following Watson-Crick hydrogen bond pairing rules and forms duplex structures that are often more stable than duplexes of two nucleic acids ( Figure 1B ) 31 , 32 . Since it is synthetic and does not occur in nature, aegPNA sequences are remarkably stable to degradation by proteases and nucleases 33 . Previously, we have shown that sequential introduction of cyclopentane groups into the PNA backbone (to create cyclopentane PNA) systematically enhances binding to target nucleic acid sequences ( Figure 1C ) 34 – 37 . In this manuscript we report the engineering principles to make a new type of RNA detection system that combines the accuracy of NAT with the practical convenience of ELISA methodology. Key to the success of this merger is properly designed PNAs 30 – 32 that target HIV RNA sequences. By incorporating cyclopentanes into the surface PNA probe, we demonstrate that binding to the target RNA is significantly improved over aegPNA and subsequently improves nucleic acid detection. We have also designed a signal read-out system that can be coupled to the terminal end of a reporter PNA molecule to signal binding of a target RNA sequence. In this case, a unique chemical scaffold was developed to support the multivalent display of up to 30 biotin groups. The spacing of the biotins on the scaffold has been optimized to support assembly of multiple streptavidin-conjugated horseradish peroxidase enzymes around the terminal end of a PNA reporter probe ( Figure 1D ). The assembly of this nanostructure upon PNA binding to target RNA sequences allows for dramatic signal amplification so that low levels of RNA are detectable. Proper combination of cyclopentanes in the surface PNA and multivalent biotins in the reporter PNA results in a synergistic improvement in detection for HIV RNA. Furthermore, we show that the optimized system can detect HIV RNA across a wide range of groups and clades of the virus, is compatible with detection of virus in human plasma, and that the results correlate with a current PCR standard of detection (COBAS Ampliprep v2.0) 38 . We also demonstrate that the enzymatic amplification system is robust (at least a 30 day shelf life at room temperature), that it is cost effective ($0.67 per well in a 96-well plate), and that the signal reporting on the amount of HIV can be determined either spectrophotometrically or with the naked eye. To abbreviate our detection method we have coined the term NAT-PELA, which stands for Nucleic Acid Testing-PNA Enzyme Linked Assay. Results Sandwich setup for NAT-PELA In the current study, a sandwich hybridization assay using two PNAs is used to detect HIV RNA. The basic detection scheme is outlined in Figure 2 . In this protocol, the first step is to attach a PNA surface probe (PNA-SP, 15 base sequence, see Supplementary Figure 1 for all chemical structures and Supplementary Tables 1 and 2 for characterization) covalently via an amide bond to the plastic surface of a NUNC 96-well plate (i). A PNA reporter probe (PNA-RP, 12 base sequence) is free in solution and contains biotin groups covalently attached to the C-terminal. The sequences of PNA-SP and PNA-RP are complementary to a 27-base sequence (5′-TTCTGCAGCTTCCTCATTGATGGTCTC-3′) that is part of HIV RNA in the gag region. If the target RNA is present, PNA-SP and PNA-RP both bind to their complementary sequences and a noncovalent complex of PNA-SP+PNA-RP+RNA is formed on the plastic surface (ii). The presentation of biotins from PNA-RP allows basic ELISA methodology to be used to amplify a signal for detection 39 . In this study, polyhorseradishperoxidase-streptavidin (pHRP-SA) is used in step iii to signal that the RNA target is present 40 . In this step, the streptavidin portion of pHRP-SA forms a strong complex with biotins attached to PNA-RP. Next, introduction of a substrate (tetramethylbenzidine (TMB) and peroxide) for the horseradishperoxidase enzyme part of pHRP-SA (step iv) results in oxidation of the tetramethylbenzidine to give products with blue color 41 . The enzymatic oxidation reaction can be stopped by the addition of acid to afford yellow products (step v). Both the blue and yellow products can be quantified (at 652 nm and 450 nm, respectively) to show the level of RNA originally present. If the RNA target is not present in step ii, then PNA-RP is removed in subsequent wash steps, pHRP-SA cannot attach to the plastic surface, and no signal results when the tetramethylbenzidine solution is added. Tests for the stability of pHRP-SA showed that there is no loss in enzymatic activity for 30 days at room temperature ( Supplementary Figure 2 ). Engineering, optimization and modeling of PNA-SP and PNA-RP A synthetic strand of HIV RNA with the 27-base target sequence was used to optimize the detection system. In all tests, the HIV RNA concentration in solution was varied and the data obtained at 652 nm and 450 nm were plotted as a function of absorbance vs. time and absorbance vs. concentration, respectively ( Figure 3 ). The absorbance data at 450 nm, obtained after quenching of enzymatic oxidation with H 2 SO 4 , were plotted and subsequently fit to a 4-parameter logistic curve to show the quantitative limits of detection ( Figure 3D and Supplementary Figure 3 ). To start, PNA-SP had no cyclopentane groups (PNA-SP0) ( Supplementary Figure 1a ) and PNA-RP had one biotin (PNA-RP1) ( Supplementary Figure 1f ). In this initial detection system, the lower limit of quantitative detection for the target RNA is in the hundreds of millions (10 8 ) of molecules ( Table 1 and Supplementary Figure 3a ). To improve this method so that it would be useful for clinical determination of HIV viral load, the limit of quantitative detection needed to be lowered to levels competitive with RT-PCR (which is around 20 molecules of RNA) 16 . Therefore, we explored the sequential incorporation of cyclopentane groups into PNA-SP ( Supplementary Figure 1b–e ) and the incremental addition of biotin groups to the C-terminal of PNA-RP ( Supplementary Figure 1g–m ). Additional cyclopentane groups in PNA-SP incrementally increases the binding affinity to the complementary RNA sequence ( Table 1 , SP1 to SP4, and Supplementary Table 2 ), and lowers the detection limit. Increasing the number of biotin groups on the end of PNA-RP enhances the signal intensity of the sandwich complex on the surface ( Figure 4 ). Interestingly, there is a modest decrease in the binding of PNA-RP to its complementary RNA sequence as the number of biotins attached to the end increases ( Table 1 , RP1 to RP30, and Supplementary Table 2 ). This loss in binding, however, does not diminish the stability of the sandwich complex as the cyclopentanes in PNA-SP compensate for any loss in stability ( Supplementary Table 2 ). Attempts to incorporate cyclopentane groups into PNA-RP were not successful when large numbers of biotin groups were required. Quantitative detection limits were determined for every combination of PNA-SP and PNA-RP, and the results are presented in Table 1 . Both types of modifications work together to lower the limit of RNA detection. In the optimal system, PNA-SP with four cyclopentane groups ( Figure 3A ) and PNA-RP with 25 biotin groups ( Figure 3B ) was sufficient to detect HIV RNA in a region that would be competitive with PCR diagnostics ( Figure 3C,D ), and the detection of 60 copies could also be observed visually ( Figure 3E,F ). Additional biotins did not further enhance detection (PNA-RP30, Supplementary Figure 1m and Table 1 ). Notably, these engineering principles were able to improve detection of the target RNA by about 8 orders of magnitude from the original system (PNA-SP0 and PNA-RP1). A model was developed to explain the relative contributions of adding cyclopentanes and biotins into PNA-SP and PNA-RP. As input, the log of the detection limits from Table 1 were represented on a three-dimensional plot (colored regions in Figure 4 ). Attempting to fit a plane to the detection limits resulted in a poor fit, with a root-mean-square deviation (RMSD) of 1.9. This implies a simple linear relationship between detection limits, cyclopentanes, and biotins does not exist ( Supplementary Figure 4 ). Considering that PNA-SP should retain RNA on the surface and directly influence the final concentration for detection, a simple model was developed where the detection limit is a function of the cyclopentanes plus a separable product of cyclopentane and biotin functions. This is represented by f ( x , y ) = h 1 ( x ) + h 2 ( x ) h 3 ( y ), where f ( x , y ) is the log of the detection limit, x is the number of cyclopentanes in PNA-SP, and y is the number of biotins in PNA-RP. The form of our model is especially amenable to Principle Component Analysis (PCA) 42 . In this approach, a series of orthogonal vectors are fit to the dataset with the goal of identifying a set of principle vectors that best represent the data with minimal variance. Prior to PCA, the data is mean-centered with respect to cyclopentanes, h 1 ( x ). By retaining only the largest component, the principle component vector and its associated loading vector are h 2 ( x ) and h 3 ( y ), respectively. This model accounts for 90% of the variance of the original data (RMSD 0.67). The dark lines in Figure 4 represent the model overlaid with the original data in color. This decomposition was examined to gauge the contributions of cyclopentanes and biotins to the detection limit. As shown in Supplementary Figure 5b , the monotonic increase in cyclopentane groups lowers detection ( h 2 ). For biotin there is a general trend that adding more biotins improves detection, but the functional form of improvement is more complex ( h 3 ). As shown in Supplementary Figure 5c , additional biotins can lower detection limits at specific intervals of biotins (such as those around 6 and 25 biotins) but in other ranges there is no improvement or even slight worsening of the detection limit (such as around 16 biotins). Detection of HIV in plasma The optimized PNAs (PNA-SP4 and PNA-RP25) were used to detect full length HIV RNA derived from samples where the virus was added into human plasma. For these sets of experiments, RNA extraction from the HIV positive plasma samples was performed using the commercial reagent RNAzol ® RT following the manufacturer's procedures 43 . To determine whether the PNA probes could detect different genetic variants of HIV, 7 different clades and 2 groups of the virus were tested under the same procedures. For all examples, the NAT-PELA system was able to quantify virus at levels (around 20 molecules of RNA) consistent with clinically useful levels ( Figure 5 , Supplementary Figure 6 ). Titration curve for NAT-PELA with HIV-spiked plasma samples To prepare NAT-PELA for detection of patient samples, a titration curve was determined using different concentrations of the virus. For these experiments, clade B HIV virus was spiked into human plasma and then subsequently diluted with additional human plasma to simulate a range of clinical concentrations for the virus. Each sample was then tested by both PCR (using COBAS Ampliprep v2.0) 38 and by the NAT-PELA procedure. For the PCR tests, plasma samples were directly processed by the instrumentation. For the NAT-PELA tests, RNA extraction using RNAzol®RT was performed for each plasma sample with a different HIV concentration 43 . The titration curve of NAT-PELA is shown in Supplementary Figure 7 . The quantitative values of virus in this experiment are determined by PCR. This experiment demonstrated that NAT-PELA provides clear signals over a range of HIV concentrations from 20 to 30,000 copies of HIV per mL of plasma. Detection of HIV in patient samples by NAT-PELA and PCR Finally, NAT-PELA was used to detect HIV in 20 plasma samples, each one from a different HIV-positive patient, representing a concentration range from 100 to 25,000 copies per mL of plasma. For each patient sample, there was sufficient volume for two PCR tests using the COBAS Ampliprep instrumentation and for one test by NAT-PELA. Processing of all samples was performed using the same protocol previously mentioned for the titration curve. For every HIV positive patient sample, NAT-PELA showed higher absorbance signals over the control plasma samples that were HIV negative. The variation in signal intensity for NAT-PELA changes with concentration of the virus ( Figure 6 ), with the greatest change in signal occurring at the concentrations that are below ~1,500 copies of virus per mL plasma ( Figure 6C , full data in Supplementary Figure 8 and Supplementary Table 3 ). The NAT-PELA data for the samples with low viral loads showed a strong linear correlation with change in PCR-determined viral load (coefficient of determination R 2 = 0.93, Pearson product-moment correlation p x is the signal at a fixed value of cyclopentane, averaged over all the observations of biotin. This formulation naturally leads to a decomposition with principal component analysis (PCA). This is a classic algorithmic tool to reduce multidimensional data sets into a series of orthogonal vectors designed to maximize the explained variance in each direction. Ideally, after the analysis, the data can be reproduced with a subset of these principle component vectors. In addition, if the largest principal component is relatively dominant to the others, its components can give physical insight to the contributions of individual components (in this case the addition of cyclopentane groups and biotin groups). We find that a significant fraction of the variance in Table 1 can be explained using only the largest principal component from the PCA. To perform the PCA, the first consideration is that the measured data points of Table 1 are not on a regular grid. To remove undue weight on closely centered observations, a linearly interpolated matrix at 500 regularly spaced intervals was constructed between the data range. We perform PCA on this matrix by first subtracting the mean for each row and taking the singular value decomposition of the remaining matrix F , so that: F = UDW T T = UD where D is a square diagonal matrix of singular values and the columns of U and W are the left and right singular vectors. A subset of the L largest principal components can be constructed by noting T L = U L D L = F W L . The columns of U , multiplied by the magnitude of the associated element of the diagonal matrix, u i d ii are known as the principal components, while the columns of W are known as the loading vectors. The principal components are a linear combination of the input data while the loading vectors associate a weight to each variable in this linear combination. We find that the largest principal component can explain 90% of the variance of the data. An illustration of the signal projected using only the largest component is shown in Figure 4 . The directions of the principal components and the mean values, h 1 ( x ), h 2 ( x ), h 3 ( y ) are shown in Supplementary Figure 5 . It is evident that the effect of cyclopentane gives a favorable monotonic decrease in the signal detection limit. The effect of adding more biotins, while generally favorable, shows a more complicated contribution to the detection limit. Reagents and materials All Boc- and Fmoc- peptide nucleic acid monomers were purchased from PolyOrg, Inc. (Leominster, MA); cyclopentane T and A PNA monomer was made following previously published procedures 37 . Acetonitrile (ACN), acetic anhydride (Ac 2 O), pyridine, thioanisole, bovine serum albumin (BSA), dichloromethane (DCM), N,N-diisopropylethylamine (DIEA), N,N-dimethylformamide (DMF), ethylenediamine tetraacetic acid (EDTA), diethyl ether (Et 2 O), Kaiser test reagents, m-cresol, Na 2 CO 3 , N-methyl-2-pyrolidinone (NMP), piperidine, sealing film for multiwell plates, trifluoroacetic acid (TFA), trifluoromethanesulfonic acid (TFMSA), thioanisole, polysorbate 20 (Tween 20), and single stranded salmon sperm DNA were purchased from Sigma-Aldrich (St. Louis, MO). 1-Step ™ Ultra-TMB, 96-well Nunc Immobilizer Amino plates, poly-horseradish peroxidase streptavidin (pHRP-SA) was obtained from Thermo Scientific (Fairlawn, NJ). RNA and DNA oligomers were ordered from IDT (Coralville, IA). Phosphate buffered saline (PBS) was purchased from Quality Biological (Gaithersburg, MD). High purity water (18 MΩ) was generated from a Millipore (Billerica, MA) Milli-Q water system. t-butyloxycarbonyl-8-amino-3,6-dioxaoctanoic acid (Boc-mPEG-OH), 9-fluorenylmethoxycarbonyl-8-amino-3,6-dioxaoctanoic (Fmoc-mPEG-OH), and t-butyloxycarbonyl-11-amino-3,6,9-trioxaundecanoic acid (Boc-mPEG3-OH) were purchased from Peptides International (Louisville, KY). O -benzatriazole-N,N,N′,N′-tetramethylammoniumhexafluorophosphate (HBTU), Fmoc-Lys(Boc)-OH, H-Lys-OH•HCl, methyl-benzhydrylamine (MBHA) resin were purchased from Advanced Chemtech (Louisville, KY). H 2 SO 4 was purchased from EMD Chemicals (Gibbstown, NJ). RNAzol ® RT was obtained from Molecular Research Center, Inc. (Cincinnati, OH). Resin downloading protocol MBHA resin was downloaded from 0.3 mmol/g to 0.1 mmol/g using Boc-Lys(2-Cl-Z)-OH or Boc-G(Z)-OH. The MBHA resin (1.0 g) was swelled with DCM for 1 h in a peptide synthesis vessel. The following solutions were prepared: (A) 0.4 M Boc-Lys(2-Cl-Z)-OH in NMP, or 0.4 M Boc-G(Z)-OH in NMP, (B) 0.2 M HBTU in DMF, and (C) 0.5 M DIEA in NMP. Next, 450 μL of solution A, 460 μL of solution C, and 1.59 mL NMP were combined and mixed to make solution 1. Then, 550 μL of solution B were diluted with NMP (1.95 mL) to make solution 2. Solutions 1 and 2 were combined and pre-mixed for ~30 s before adding to the drained, swelled resin. The resin/coupling mixture was agitated for 1 h before draining and washing with DMF (4 ×), DCM (4 ×), 5% DIEA in DCM (1 × ~30 s) and finally DCM (4 ×). Any remaining active sites were capped using capping cocktail (1:25:25 Ac 2 O:NMP:pyridine) for 20 min. The reaction was drained and rinsed with DMF (3 ×) and DCM (3 ×). The progress of the capping was followed by a qualitative Kaiser test. If the test was positive, the resin was resubmitted to capping. After a negative test for primary amines, the resin was washed with DCM (3 ×) and dried under vacuum for 30 – 60 min and then stored in a dessicator. General method for PNA synthesis PNAs were prepared on 5 μmol scale using either Boc- or Fmoc-solid phase peptide synthesis protocols on an Applied Biosystems 433a automated peptide synthesizer with HBTU as the amide-forming reagent. All PNA-SP were synthesized on Boc-G(Z)-OH downloaded MBHA resin, and all PNA-RP were synthesized on Boc-Lys(2-Cl-Z)-OH downloaded MBHA resin. Cleavage and recovery of crude PNA from resin The resin, in a peptide synthesis vessel, was first washed with TFA (2 × for 4 min). To the drained resin, cleavage cocktail (1.5 mL, 900 μL:300 μL:150 μL:150 μL TFA/TFMSA/thioanisole/m-cresol), cooled over ice, was added and reacted for 1 h. The cleavage mixture was collected in a glass vial using N 2 pressure to drain the vessel. The resin was resubmitted to fresh cleavage cocktail and cleaved for 1 h, and was drained into the first cleavage fraction. The volatiles were removed by flowing dry N 2 over the solution to produce a yellow-brown oil. Approximately 10 mL of Et 2 O was added to the cleavage oil to create a suspended white precipitate. The suspension was partitioned into five 2 mL microcentrifuge tubes and chilled over dry ice for 10 min. The tubes were centrifuged at 12,000 rpm for 40 s to produce a white pellet. Et 2 O was carefully decanted, leaving the white crude PNA solid. Further washing was performed by adding ~1.6 mL of Et 2 O to each tube, mixing to resuspend the precipitate, then chilling on dry ice for 5 min. Following centrifugation and decanting, the washes were repeated twice without dry ice. After the final wash, the white precipitate was dried by carefully passing a stream of dry N 2 over the crude PNA. Purification of crude PNA and characterization Purification was performed on an Agilent (Santa Clara, CA) 1200 Series RP-HPLC with automatic fraction collection using UV detection at 260 nm. Waters (Milford, MA) XBridge C18 (10 × 250 mm, 5 μm) columns were used in conjunction with Solvents A and B. Solvent A was 0.05% TFA in water and Solvent B consisted of 90% ACN in water. PNA HPLC isolates were characterized using ESI-MS on a Waters/Micromass LCT Premier ™ time-of-flight mass spectrometer. The instrument was operated in W-mode at a nominal resolution of 10,000. The electrospray capillary voltage was 2 kV and the sample cone voltage was 60 V. The desolvation temperature was 275 °C and the desolvation gas was N 2 with a flow of 300 L/h. Accurate masses were obtained using the internal reference standard method. The sample was introduced into the mass spectrometer via the direct loop injection method. Deconvolution of multiply charged ions was performed with MaxEnt I. All PNA oligomers gave molecular ions consistent with the calculated theoretical product values ( Supplementary Table 1 ). Thermal melting analysis UV concentration was determined by adding 4 μL of RNA solution to 196 μL milli-Q water. If the signal was too intense the concentration was diluted by adding 198 μL of water to 2 μL of the original RNA solution. Water was blanked against the background at 80 °C on an Agilent 8453 UV/Vis spectrometer equipped with an Agilent 89090A Peltier temperature controller and a computer interface. Then the unknown solution was added to the quartz cell (Helma) and vigorously shaken, replaced in the spectrophotometer and the absorbance was read at 260 nm. The mixing and reading was repeated 3 times. Values were converted to concentration, based on average absorbance. After initial measurement by UV, the concentration was determined based on appropriate ε 260nm (calculated on nearest neighbor approximation for PNA or provided by IDT or Thermo Scientific for oligonucleotides) and then used from that point forward for additional experiments. Thermal melting experiments were performed by preparing 2 μM oligonucleotide solution in 1 × PBS. Experiments traversed from 90 °C to 20 °C and back to 90 °C at 1 °C intervals while monitoring at 260 nm. An equilibration of 60 s at each temperature measurement step before readings was employed. Cooling and heating profiles were generated for each run with duplicates for each. The T m (melting temperature) for duplexes was determined using the maximum first derivative of the cooling and heating curves, then taken as an average of both runs. Buffers for NAT-PELA (BLB) 2% BSA, 25 mM tris(hydroxymethyl) aminomethane (Tris), 150 mM NaCl, 0.05% TWEEN 20, 0.1 mM EDTA, pH 7.4; (BLBs) BLB with 0.1 μg/mL single stranded salmon sperm DNA; (CAP) 25 mM Lys, 10 mM NaH 2 PO 4 , 100 mM NaCl, 0.1 mM EDTA, pH 8.0; (IB) immobilization buffer (100 mM Na 2 CO 3 , pH 9.6); (1 × PBS) phosphate buffered saline solution, 9.0 g/L NaCl, 144 mg/L KH 2 PO 4 , 795 mg/L Na 2 HPO 4 , pH 7.4; (1 × PBST) 1 × PBS with 0.05% Tween 20. Plate preparation PNA-SP was dissolved in IB (1.0 μM) and a volume of 100 μL was added to each well of the plate. Blank wells were left untouched throughout the process to blank against the plate. The plate was sealed with sealing tape for multi-well plates and agitated on a plate shaker (600–700 rpm) for 2 h at room temperature (RT). Then 50 μL of CAP was added to modified wells to give a final volume of 150 μL. This was again agitated at RT on a plate shaker for 30 min. Modified wells were then aspirated and washed four times with 300 μL of 1× PBST and four times with 1 × PBS. A final addition of 250 μL of 1 × PBS was used to store the wells until used in experiments. When the assay was ready to be performed, the 1 × PBS was removed by aspiration and 200 μL BLBs was added to the wells. The plate was sealed and incubated with shaking for 30 min at 37 °C, the wells were immediately aspirated and ready for sample addition. Preparation of synthetic HIV-1 gag RNA samples All samples were prepared in glass vials. Synthetic RNA was ordered from IDT. The final volume for all samples used for RNA detection was 600 μL. Each of these samples had the PNA-RP concentration fixed at 15 nM. The specific concentration of the target RNA for detection was prepared by a serial dilution of a stock 50 nM solution of the RNA target in 1 × PBS buffer prepared from the commercial RNA. The initial (and highest concentration) RNA sample for detection was the first sample made, using the desired amount of RNA from the 50 nM stock solution and diluting with a solution of the reporter probe (PNA-RP, 15 nM) in 1× PBS buffer. To dilute further from the initial RNA detection sample, a desired volume of RNA solution was removed and further diluted with the 15 nM reporter probe solution in 1 × PBS buffer. For example, to dilute an initial 450 pM RNA sample solution by three times, 200 μL of the initial RNA sample solution was removed and added to 400 μL of 15 nM reporter probe solution in 1 × PBS buffer to give a final RNA concentration of 150 pM. Once the final RNA concentration was obtained after dilution, the samples were heated to 95 °C in a sand bath for 5 min and then snap-cooled by placing the vials directly into an ice bath for 5 min. Then, the samples were directly used for RNA detection by removing 100 μL of each sample and individually adding the RNA solution to one well on the prepared 96-well plate (using a 100 μL pipettor). To construct a concentration dependent response curve, 10 different RNA concentrations were prepared and each concentration was prepared in duplicate. Two wells of each plate were dedicated to detection of one RNA concentration. After the samples were added to the wells, the plate was sealed and incubated with shaking at 37 °C for 3 h. Each well was aspirated following incubation and then washed with 300 μL of 1 × PBST four times followed by 300 μL of 1 × PBS four times. HIV-1 RNA extraction by RNAzol ® RT Analysis of patient samples was approved by the Office of Human Subjects Research at NIH. Virus was isolated from PBMC samples of HIV-1 positive individuals infected with nine different HIV-1 subtypes (Clade A, B, C, D, E, F, G, and Group N, O). The culture supernatants were harvested and spiked in Base matrix. Viral loads were measured by the Abbott RealTime HIV-1 system (Abbott Molecular Inc. Des Plaines, IL). One plasma sample from an HIV-1 negative individual was also included as a negative control. For extraction of HIV-1 RNA using RNAzol ® RT, 400 μL of plasma was mixed with 1 mL of RNAzol ® RT reagent followed by the addition of 200 μL RNAase free water and mixed well. Samples were incubated at RT for 15 mins and centrifuged at 12,000 g, 4 °C for another 15 mins. The upper layer was removed (~1500 μL) and an equal volume of isopropanol was added and mixed well by tilting tubes. The tubes were left at RT for 15 mins after which they were centrifuged at 12000 g for 15 mins, aspirated and washed with 70% ethanol twice. The pellets were dried and dissolved in 50 μL of RNAase free water. Correlation of NAT-PELA with RT-PCR HIV-1 positive plasma (Clade B) with viral loads of 1.5 × 10 9 copies/mL, stored at −80°C, was diluted to 10000, 5000, 1000, 500, 100, 50, and 10 copies/mL in HIV-1 negative plasma (basematrix). A total of 6 mL volume was prepared for each concentration, and divided into 6 tubes with 1 mL each. For each concentration, RNA extraction using RNAzol ® RT was independently performed on three 1 mL samples and the isolated RNA from each sample was subsequently used in the NAT-PELA assay. The remaining three 1 mL samples at the same concentration were subjected to the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (Roche Molecular Systems, Inc., Branchburg, NJ). HIV-1 RNA detection in patient plasma samples Twenty plasma samples collected from 20 different HIV-positive patients were each tested by NAT-PELA and RT-PCR. Each plasma sample consisted of 3 mL total volume that was divided into 3 tubes with 1 mL of plasma in each tube. For each patient sample, one of the 1 mL tubes was tested using NAT-PELA (where the RNA was extracted using RNAzol®RT), and the other two 1 mL tubes for each patient sample were tested using the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (Roche Molecular Systems, Inc., Branchburg, NJ). To exclude the possibility of biased results due to non-specific interactions, two HIV-negative plasma samples were also included. NAT-PELA protocol The wells were incubated with 200 μL of BLB. The plate was sealed and shaken at 37 °C for 30 min. Wells were then aspirated, following by addition of 100 μL 0.1 μg/mL pHRP-SA in BLB. The plate was sealed and incubated for 20 min at RT before aspirating. The wells were aspirated and followed a washing procedure that consisted of 300 μL four times of 1× PBST and four times of 1× PBS. NAT-PELA readout Following washes, 100 μL 1-Step ™ Ultra tetramethylbenzidine (TMB) solution was added via a multichannel pipette to facilitate nearly simultaneous initial starting points for all wells. The plate was immediately placed in a Molecular Devices (Sunnyvale, CA) SpectraMax M5 multi-mode microplate reader and monitored at 652 nm over the course of 30 min at RT. The plate was then removed, followed by addition of 50 μL 2 M H 2 SO 4 and mixed by hand for 10 s to quench the enzyme reaction. The plate was then returned to the plate reader and a final reading at 450 nm was performed. Plates were sealed with film following reading and placed in the refrigerator. Analysis of NAT-PELA results Analysis of absorbance (652 nm) kinetics The absorbance was monitored at 2 min intervals over 30 min (16 pts) at each concentration of target nucleic acid. The absorbance at 652 nm was plotted versus time to give the kinetic curve for each concentration using Prism 4.0 software ( Figure 3C ), from which we can clearly see the qualitative detection limit. Nonlinear regression analysis of quenched assay results The protocol employing quenched TMB product used end point absorbance values at 450 nm was used as response values and plotted with respect to target nucleic acid concentration using Prism 4.0 software. The limits of quantitation for the HIV-1 RNA were determined using the 4-PL models with Prism 4.0 software by calculating the upper limit of the zero analyte concentration parameter error using a 90% confidence interval. These values provide a limit that is distinguishable from background noise with 96% certainty (Minimal distinguishable differential concentration – MDDC). The resulting fits are graphically represented in Supplementary Figure 3 , and the quantitative detection limits are listed in Table 1 . 4-PL model equation: y = A + ( ( D - A ) / ( 1 + 10 ( Log C - x ) B ) ) , where A is the response at a concentration of zero (baseline); B is the slope factor; C is the inflection point (IC50); D is the response at infinite concentration; Y is the response; X is the analyte concentration. Analysis of absorbance (652 nm) kinetics The absorbance was monitored at 2 min intervals over 30 min (16 pts) at each concentration of target nucleic acid. The absorbance at 652 nm was plotted versus time to give the kinetic curve for each concentration using Prism 4.0 software ( Figure 3C ), from which we can clearly see the qualitative detection limit. Nonlinear regression analysis of quenched assay results The protocol employing quenched TMB product used end point absorbance values at 450 nm was used as response values and plotted with respect to target nucleic acid concentration using Prism 4.0 software. The limits of quantitation for the HIV-1 RNA were determined using the 4-PL models with Prism 4.0 software by calculating the upper limit of the zero analyte concentration parameter error using a 90% confidence interval. These values provide a limit that is distinguishable from background noise with 96% certainty (Minimal distinguishable differential concentration – MDDC). The resulting fits are graphically represented in Supplementary Figure 3 , and the quantitative detection limits are listed in Table 1 . 4-PL model equation: y = A + ( ( D - A ) / ( 1 + 10 ( Log C - x ) B ) ) , where A is the response at a concentration of zero (baseline); B is the slope factor; C is the inflection point (IC50); D is the response at infinite concentration; Y is the response; X is the analyte concentration. Enzyme stability study The pHRP-SA stability was determined by incubating the enzyme at RT for different times. 10 mL 0.1 μg/mL pHRP-SA was prepared in BLB and was incubated at RT for 0, 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 days. Aliquots were withdrawn at each time point to test the remaining activity by SP0/RP1/HIV-1 gag synthetic 27-nucleobase single-stranded DNA at standard conditions as described above, the target HIV-1 gag DNA concentration is 100 pM. The fresh prepared enzyme was considered to be a control and was assumed to have 100% activity. Principle Component Analysis model The data in Table 1 represent the detection limits as a function of the number of cyclopentanes in PNA-SP and biotins in PNA-RP. Attempts were made to develop a qualitative model that explains the dependence of the detection limits on these two variables. A simple linear response, where the dependence of the detection limit is fit to a simple plane, does not adequately explain the data ( Supplementary Figure 5 ). We next recast the data in terms of a function, f( x , y ), where f is the detection limit, x is the number of cyclopentanes in PNA-SP and y is the number of biotins in PNA-RP. We hypothesize that the interaction of the two variables is largely separable, in which case the following equation: f ( x , y ) = h 1 ( x ) + h 2 ( x ) h 3 ( y ) should be true where h 1 ( x ) = x is the signal at a fixed value of cyclopentane, averaged over all the observations of biotin. This formulation naturally leads to a decomposition with principal component analysis (PCA). This is a classic algorithmic tool to reduce multidimensional data sets into a series of orthogonal vectors designed to maximize the explained variance in each direction. Ideally, after the analysis, the data can be reproduced with a subset of these principle component vectors. In addition, if the largest principal component is relatively dominant to the others, its components can give physical insight to the contributions of individual components (in this case the addition of cyclopentane groups and biotin groups). We find that a significant fraction of the variance in Table 1 can be explained using only the largest principal component from the PCA. To perform the PCA, the first consideration is that the measured data points of Table 1 are not on a regular grid. To remove undue weight on closely centered observations, a linearly interpolated matrix at 500 regularly spaced intervals was constructed between the data range. We perform PCA on this matrix by first subtracting the mean for each row and taking the singular value decomposition of the remaining matrix F , so that: F = UDW T T = UD where D is a square diagonal matrix of singular values and the columns of U and W are the left and right singular vectors. A subset of the L largest principal components can be constructed by noting T L = U L D L = F W L . The columns of U , multiplied by the magnitude of the associated element of the diagonal matrix, u i d ii are known as the principal components, while the columns of W are known as the loading vectors. The principal components are a linear combination of the input data while the loading vectors associate a weight to each variable in this linear combination. We find that the largest principal component can explain 90% of the variance of the data. An illustration of the signal projected using only the largest component is shown in Figure 4 . The directions of the principal components and the mean values, h 1 ( x ), h 2 ( x ), h 3 ( y ) are shown in Supplementary Figure 5 . It is evident that the effect of cyclopentane gives a favorable monotonic decrease in the signal detection limit. The effect of adding more biotins, while generally favorable, shows a more complicated contribution to the detection limit. Supplementary Material 1

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427105/

Upregulation of NLRP3 Inflammasome in the Tears and Ocular Surface of Dry Eye Patients

Purpose To evaluate the mRNA and protein expressions of NLRP3 inflammasome and its downstream inflammatory factors in human dry eye. Methods We recruited 54 patients with Sjögren's syndrome dry eye (SSDE), 50 patients with non-Sjögren's syndrome dry eye (NSSDE), and 46 healthy controls. Tear film breakup time (TBUT), Schirmer I test, and fluorescein staining (FL) were performed on all subjects. Tear samples were obtained to analyze the inflammatory cytokine levels of IL-1β and IL-18 via enzyme-linked immunosorbent (ELISA). Conjunctival impression cytology (CIC) specimens were collected to detect the mRNA expression of NLRP3, caspase-1, IL-1β, and IL-18 using quantitative RT-PCR, and the protein expression of NLRP3 and caspase-1 by Western blotting. Results NLRP3 mRNA expression showed higher levels in both dry eye groups compared with controls, with a comparably significant elevation in the SSDE group (relative 2.47-fold upregulation, p <0.05). NLRP3 protein expression was also increased in SSDE group (relative1.94-fold upregulation) compared with the controls. mRNA expression of caspase-1 was significantly upregulated in both SSDE (relative 1.44-fold upregulation, p <0.05) and NSSDE (relative 1.32-fold upregulation, p <0.05). Procaspase-1 protein level was increased in SSDE (relative 1.84-fold upregulation) and NSSDE (relative 1.12-fold upregulation) versus controls; and caspase-1 protein expression was also increased in SSDE (relative 1.49-fold upregulation) and NSSDE (relative 1.17-fold upregulation) compared with the controls. The patients with SSDE and NSSDE had higher IL-1β and IL-18 mRNA values and protein expressions than the controls did. The relative mRNA expression of IL-1β upregulated 3.59-fold ( p <0.001) in SSDE and 2.13-fold ( p <0.01) in NSSDE compared with the controls. IL-1β protein level also showed significant upregulation in SSDE ( p =0.01; vs. controls groups). IL-18 mRNA expression levels were significantly upregulated in the SSDE (relative 2.97-fold upregulation, p =0.001) and NSSDE (relative 2.05-fold upregulation, p =0.001) groups compared with the controls; tear IL-18 concentrations were also significantly increased in the SSDE ( p <0.001) and NSSDE ( p <0.05) groups. Conclusions In the current study, we found that mRNA and protein expressions of NLRP3 inflammasome were upregulated in human dry eyes, especially in SSDE; the downstream inflammatory factors caspase-1, IL-1β, and IL-18 were also elevated in dry eye patients. These observations suggest the involvement of NLRP3 inflammasome in the onset and development of the inflammation in dry eye. Purpose To evaluate the mRNA and protein expressions of NLRP3 inflammasome and its downstream inflammatory factors in human dry eye. Methods We recruited 54 patients with Sjögren's syndrome dry eye (SSDE), 50 patients with non-Sjögren's syndrome dry eye (NSSDE), and 46 healthy controls. Tear film breakup time (TBUT), Schirmer I test, and fluorescein staining (FL) were performed on all subjects. Tear samples were obtained to analyze the inflammatory cytokine levels of IL-1β and IL-18 via enzyme-linked immunosorbent (ELISA). Conjunctival impression cytology (CIC) specimens were collected to detect the mRNA expression of NLRP3, caspase-1, IL-1β, and IL-18 using quantitative RT-PCR, and the protein expression of NLRP3 and caspase-1 by Western blotting. Results NLRP3 mRNA expression showed higher levels in both dry eye groups compared with controls, with a comparably significant elevation in the SSDE group (relative 2.47-fold upregulation, p <0.05). NLRP3 protein expression was also increased in SSDE group (relative1.94-fold upregulation) compared with the controls. mRNA expression of caspase-1 was significantly upregulated in both SSDE (relative 1.44-fold upregulation, p <0.05) and NSSDE (relative 1.32-fold upregulation, p <0.05). Procaspase-1 protein level was increased in SSDE (relative 1.84-fold upregulation) and NSSDE (relative 1.12-fold upregulation) versus controls; and caspase-1 protein expression was also increased in SSDE (relative 1.49-fold upregulation) and NSSDE (relative 1.17-fold upregulation) compared with the controls. The patients with SSDE and NSSDE had higher IL-1β and IL-18 mRNA values and protein expressions than the controls did. The relative mRNA expression of IL-1β upregulated 3.59-fold ( p <0.001) in SSDE and 2.13-fold ( p <0.01) in NSSDE compared with the controls. IL-1β protein level also showed significant upregulation in SSDE ( p =0.01; vs. controls groups). IL-18 mRNA expression levels were significantly upregulated in the SSDE (relative 2.97-fold upregulation, p =0.001) and NSSDE (relative 2.05-fold upregulation, p =0.001) groups compared with the controls; tear IL-18 concentrations were also significantly increased in the SSDE ( p <0.001) and NSSDE ( p <0.05) groups. Conclusions In the current study, we found that mRNA and protein expressions of NLRP3 inflammasome were upregulated in human dry eyes, especially in SSDE; the downstream inflammatory factors caspase-1, IL-1β, and IL-18 were also elevated in dry eye patients. These observations suggest the involvement of NLRP3 inflammasome in the onset and development of the inflammation in dry eye. INTRODUCTION Dry eye is one of the most common ocular surface disorders that significantly affect the quality of human life. Although the pathogenesis of dry eye has not been established clearly, there is increasing evidence that immune-based inflammation on the ocular surface might play a prominent role in the pathological damage of dry eye [ 1 , 2 ]. Numerous studies have shown increased levels of inflammatory cytokines and apoptotic modulators in the tear and conjunctival epithelium of dry eye patients and animal models, including interleukin (IL)-1α, IL-1β, IL-8, IL-6,tumor necrosis factor (TNF)-α, interferon (IFN)-α, intercellular adhesion molecule (ICAM)-1, human leukocyte antigen (HLA)-DR, cluster of differentiation (CD)-40, CD-40L, Fas, chemokine receptors CCR5 and CXCR3[ 3 – 10 ]. However, the precise mechanism of the onset and development of inflammation in dry eye remains unclear. Inflammasome plays a key role in inflammation and innate immunity. The inflammasome is an intracellular protein complex that is stimulated by pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). It can activate procaspase-1, causing cleavage of pro-IL-1β and pro-IL-18. Bioactive cytokines then initiate or amplify diverse downstream signaling pathways and drive proinflammatory processes [ 11 – 13 ]. Among different types of inflammasomes, the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is currently one of the most well-characterized subtypes. Previous studies have demonstrated critical roles for NLRP3 inflammasome activation in the immune responses of many diseases, such as asthma [ 14 ], T-cell-dependent immune complex glomerulonephritis[ 15 ], acute graft-versus-host disease (GvHD)[ 16 ], systemic lupus erythematosus (SLE)[ 17 ], metabolic disorders like type 2 diabetes[ 18 ], gout, and pseudogout [ 19 ]. Recently several reports also found NLRP3 inflammasome involved the pathology of the age-related macular degeneration [ 20 , 21 , 22 ]. However, limited information is available on the association of NLRP3 inflammasome with dry eye. A recent study reported that reactive oxygen species could trigger NLRP3 inflammasome and lead to caspase-1 auto-activation and maturation of proinflammatory cytokines IL-1β in dry eye murine models [ 23 ]. However, the alteration of NLRP3 inflammasome in the tears and ocular surface of dry eye patients has not been reported until now. In the current study, we prospectively recruited patients with SSDE and NSSDE. Tear samples and conjunctival impression cytology specimens were collected to evaluate the mRNA and protein expressions of NLRP3 inflammasome and its downstream inflammatory factors-caspase-1, IL-1β and IL-18. The results were compared with healthy control subjects. MATERIALS AND METHODS Patients Fifty-four SSDE subjects (six males, 48 females; mean age, 54.07±9.69 years) and fifty NSSDE subjects (eight males, 42 females; mean age, 48.51±11.42 years) were recruited for this prospective study and examined at the dry eye subspeciality clinic of the Eye and ENT Hospital of Fudan University. Forty-six age- and sex-matched healthy subjects were included as controls (nine males, 37 females; mean age, 48.24±13.89 years). Both eyes of all subjects were included in this study. All procedures adhered to the Declaration of Helsinki, and ethics approval was obtained from Ethics Committee of Eye and ENT Hospital of Fudan University, Shanghai, China. Written consent was obtained from all patients. All participants underwent a clinical evaluation visit to determine entry eligibility before a second visit, in which ocular samples were collected. Dry eye disease was diagnosed according to diagnostic criteria reported previously [ 24 ]. Briefly, patients with (1) dry eye-related symptoms, (2) positive staining with fluorescein, or (3) Schirmer I test results ≤5 mm or a tear film breakup time (TBUT) value <5 seconds were diagnosed as having definite dry eye. Diagnosis of SS was confirmed with the cooperation of an internist, according to the American—European Consensus Criteria (2002) [ 25 ]. All dry eye patients had used only artificial tears and topical anti-inflammatory drugs such as cyclosporine A; patients using steroids were excluded. Subjects were excluded if they wore contact lenses, had active ocular disease, or had recent eye surgery, including punctal plugs or cautery. Tear Film Breakup Time (TBUT) and Cornea Fluorescein Staining (FL) TBUT and FL were measured using prepackaged, sterile fluorescein paper strips (Jingming New Technological Development Co. Ltd, Tianjin, China). First, the fluorescein strip was wetted with 20μl saline, and then the lower tarsal conjunctiva was gently touched with the end of the strip. The patient was asked to blink a couple of times for a few seconds to assure adequate mixing of the dye. The time from the last complete blink to the appearance of the first corneal black spot in the stained-tear film was examined three times, and the mean value of the measurements was calculated. A TBUT value of less than five seconds was considered to be abnormal. After measuring the TBUT, the cornea FL score was evaluated. The FL score of the cornea ranged from 0 to 9 points; any score above 3 points was regarded as abnormal [ 26 ]. Schirmer I Test The Schirmer I test was performed using prepackaged, sterile paper strips without anesthesia (Jingming New Technological Development Co. Ltd). The rounded bulb end of the strip was folded and placed in the lateral canthus that away from the cornea and left in place for five minutes. After wetting for five minutes, readings were repeated in millimeters. Tear Sample Collection and Analysis In order to analyze inflammatory cytokine levels, tear samples were collected according to the method described previously [ 27 ]. To collect the tear samples, 30μl of phosphate-buffered saline were instilled into the inferior fornix without topical anesthetics. After a gentle blink, tear samples were taken from each eye using a 20μl capillary tube. All of the tear samples were gained from the lateral canthus, that parallel to the ocular surface, without stimulating reflex tearing, followed by immediate transfer to a 0.5ml Eppendorf tube and centrifugation at 1000 rpm for three minutes at 4°C. The supernatants then were reserved at -80°C. The amounts of tear IL-18 and IL-1β were measured using an enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, San Diego, CA) according to the manufacturer's instructions. The optical density of each well was determined at a wavelength of 450 nm. Samples were considered positive when the signal was higher than the background signal (modified Krebs solution) and was within the range of the standard curve. The experiment was repeated twice. Conjunctival Impression Cytology (CIC) Samples Collection Conjunctival impression cytology (CIC) samples were collected according to the method described previously [ 28 , 29 ]. The CIC specimens were obtained after administration of topical anesthesia with 0.4% oxybuprocaine. Two separate sterile membrane filters (0.45 μ m; Millipore, Boston, MA) that were soaked in distilled water for a few hours and dried at room temperature were applied to adjacent superior and inferior temporal bulbar conjunctiva, pressed gently by a glass rod, and then removed. The filter paper collected from the left eye was transferred immediately to a 1.5ml Eppendorf tube containing 1ml of RNA stabilization reagent (Qiagen, Germantown, Germany) for mRNA expression measurement. A CIC sample from the right eye was placed in an empty 1.5ml Eppendorf tube for western blotting to evaluate the protein expression. All samples were immediately placed on ice until transferred to −80°C for storage. RNA Isolation from CIC Samples and Reverse Transcription Tubes containing 1ml of RNA stabilization reagent and CIC samples were allowed to thaw at room temperature and then were vortexed for 2 min. Extraction of total RNA proceeded according to the manufacturer's protocol (RNeasy Mini Kit; Qiagen, Valencia, CA). The final isolation step was conducted with 35μl of RNase-free water. RNA concentration was measured by NanoDrop 2000 (Thermo Scientific, Wilmington, DE) prior to reverse transcription. The procedure of cDNA synthesis from RNA samples was performed with a thermal cycler (Thermal Technology, Santa Rosa, CA) using Oligo dT Primer and Random 6 mers according to the manufacturer's instruction (PrimeScript RT reagent Kit; Takara, Osaka, Japan). Quantitative RT-PCR of the CIC Samples NLRP3, caspase-1, IL-1β, and IL-18 mRNA expression of CIC samples were detected using a SYBR Green real-time polymerase chain reaction (PCR) kit according to the manufacturer's instructions (SYBR Premix Ex Taq; Takara, Osaka, Japan). β-actin was used as an endogenous control gene for this analysis. Sequence data for gene amplification in quantitative PCR (qPCR) is summarized in Table 1 . Real-time qPCR was performed with a ViiA 7 Real-Time PCR System (Life Technologies, Pleasanton, CA). Collected data was analyzed and fold-expression changes were calculated using the comparative CT method (2 -ΔΔCT ) of relative quantification with ViiA 7 Software (Life Technologies). 10.1371/journal.pone.0126277.t001 Table 1 Sequence Data for Gene Amplification in Quantitative RT-PCR. Gene Forward Primer Reverse Primer NLRP3 5'-TCCTCGGTACTCAGCACTAATCAG-3' 5'-GGTCGCCCAGGTCATTGTTG-3' Caspase-1 5'-AAGACCCGAGCTTTGATTGACTC-3' 5'-AAATCTCTGCCGACTTTTGTTTCC-3' IL-1β 5'-TATTACAGTGGCAATGAGG-3' 5'-ATGAAGGGAAAGAAGGTG-3' IL-18 5'-ATAGCCAGCCTAGAGGTA-3' 5'-ATCAGGAGGATTCATTTC-3' β-actin 5'-CCCTGGACTTCGAGCAAGAG -3' 5'-TCACACTTCATGATGGAGTTG-3' Western Blotting of the CIC Samples Expression of the NLRP3 and caspase-1 proteins of the CIC samples were determined by western blot analysis. CIC samples from 6 subjects were put together and lysed with radioimmunoprecipitation assay buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS) on ice for one hour. The lysates were centrifuged at 12,000 rpm at 4°C for 10 min to obtain the supernatant. Each 60μl cell sample was added, with protein loading buffer, and degenerated in a heating block for 5 min, after which the proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (0.45 μ m; Millipore, Bedford, MA). The membrane was blocked in 2% bovine serum albumin (BSA) for one hour at room temperature and incubated with the specific primary antibodies (diluted 1:1000) overnight at 4°C. After washing with phosphate buffer solution containing Tween-20 five times, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies (diluted 1:5000 by 2% BSA). In accordance with conventional methods, β-actin level was measured at the same time as the internal control. The results were quantified by analysis for grayscale using Gel-Pro Analyzer software (Media Cybernetics, Rockville, MD). The following primary antibodies were used: anti-β-actin, anti-NLRP3 and anti-caspase-1 (Abcam, Boston, MA). Statistical Analysis SPSS 19.0 (IBM Corporation, Armonk, NY) was used to evaluate significance, and GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA) was used to generate figures and tables. The Kruskal—Wallis test was used for comparisons involving tear film BUT, Schirmer I test values, ocular vital staining scores, protein and mRNA expression levels of NLRP3, caspase-1, IL-1β and IL-18. Data were shown as mean ±standard deviation(SD). P <0.05 was considered significant. Patients Fifty-four SSDE subjects (six males, 48 females; mean age, 54.07±9.69 years) and fifty NSSDE subjects (eight males, 42 females; mean age, 48.51±11.42 years) were recruited for this prospective study and examined at the dry eye subspeciality clinic of the Eye and ENT Hospital of Fudan University. Forty-six age- and sex-matched healthy subjects were included as controls (nine males, 37 females; mean age, 48.24±13.89 years). Both eyes of all subjects were included in this study. All procedures adhered to the Declaration of Helsinki, and ethics approval was obtained from Ethics Committee of Eye and ENT Hospital of Fudan University, Shanghai, China. Written consent was obtained from all patients. All participants underwent a clinical evaluation visit to determine entry eligibility before a second visit, in which ocular samples were collected. Dry eye disease was diagnosed according to diagnostic criteria reported previously [ 24 ]. Briefly, patients with (1) dry eye-related symptoms, (2) positive staining with fluorescein, or (3) Schirmer I test results ≤5 mm or a tear film breakup time (TBUT) value <5 seconds were diagnosed as having definite dry eye. Diagnosis of SS was confirmed with the cooperation of an internist, according to the American—European Consensus Criteria (2002) [ 25 ]. All dry eye patients had used only artificial tears and topical anti-inflammatory drugs such as cyclosporine A; patients using steroids were excluded. Subjects were excluded if they wore contact lenses, had active ocular disease, or had recent eye surgery, including punctal plugs or cautery. Tear Film Breakup Time (TBUT) and Cornea Fluorescein Staining (FL) TBUT and FL were measured using prepackaged, sterile fluorescein paper strips (Jingming New Technological Development Co. Ltd, Tianjin, China). First, the fluorescein strip was wetted with 20μl saline, and then the lower tarsal conjunctiva was gently touched with the end of the strip. The patient was asked to blink a couple of times for a few seconds to assure adequate mixing of the dye. The time from the last complete blink to the appearance of the first corneal black spot in the stained-tear film was examined three times, and the mean value of the measurements was calculated. A TBUT value of less than five seconds was considered to be abnormal. After measuring the TBUT, the cornea FL score was evaluated. The FL score of the cornea ranged from 0 to 9 points; any score above 3 points was regarded as abnormal [ 26 ]. Schirmer I Test The Schirmer I test was performed using prepackaged, sterile paper strips without anesthesia (Jingming New Technological Development Co. Ltd). The rounded bulb end of the strip was folded and placed in the lateral canthus that away from the cornea and left in place for five minutes. After wetting for five minutes, readings were repeated in millimeters. Tear Sample Collection and Analysis In order to analyze inflammatory cytokine levels, tear samples were collected according to the method described previously [ 27 ]. To collect the tear samples, 30μl of phosphate-buffered saline were instilled into the inferior fornix without topical anesthetics. After a gentle blink, tear samples were taken from each eye using a 20μl capillary tube. All of the tear samples were gained from the lateral canthus, that parallel to the ocular surface, without stimulating reflex tearing, followed by immediate transfer to a 0.5ml Eppendorf tube and centrifugation at 1000 rpm for three minutes at 4°C. The supernatants then were reserved at -80°C. The amounts of tear IL-18 and IL-1β were measured using an enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, San Diego, CA) according to the manufacturer's instructions. The optical density of each well was determined at a wavelength of 450 nm. Samples were considered positive when the signal was higher than the background signal (modified Krebs solution) and was within the range of the standard curve. The experiment was repeated twice. Conjunctival Impression Cytology (CIC) Samples Collection Conjunctival impression cytology (CIC) samples were collected according to the method described previously [ 28 , 29 ]. The CIC specimens were obtained after administration of topical anesthesia with 0.4% oxybuprocaine. Two separate sterile membrane filters (0.45 μ m; Millipore, Boston, MA) that were soaked in distilled water for a few hours and dried at room temperature were applied to adjacent superior and inferior temporal bulbar conjunctiva, pressed gently by a glass rod, and then removed. The filter paper collected from the left eye was transferred immediately to a 1.5ml Eppendorf tube containing 1ml of RNA stabilization reagent (Qiagen, Germantown, Germany) for mRNA expression measurement. A CIC sample from the right eye was placed in an empty 1.5ml Eppendorf tube for western blotting to evaluate the protein expression. All samples were immediately placed on ice until transferred to −80°C for storage. RNA Isolation from CIC Samples and Reverse Transcription Tubes containing 1ml of RNA stabilization reagent and CIC samples were allowed to thaw at room temperature and then were vortexed for 2 min. Extraction of total RNA proceeded according to the manufacturer's protocol (RNeasy Mini Kit; Qiagen, Valencia, CA). The final isolation step was conducted with 35μl of RNase-free water. RNA concentration was measured by NanoDrop 2000 (Thermo Scientific, Wilmington, DE) prior to reverse transcription. The procedure of cDNA synthesis from RNA samples was performed with a thermal cycler (Thermal Technology, Santa Rosa, CA) using Oligo dT Primer and Random 6 mers according to the manufacturer's instruction (PrimeScript RT reagent Kit; Takara, Osaka, Japan). Quantitative RT-PCR of the CIC Samples NLRP3, caspase-1, IL-1β, and IL-18 mRNA expression of CIC samples were detected using a SYBR Green real-time polymerase chain reaction (PCR) kit according to the manufacturer's instructions (SYBR Premix Ex Taq; Takara, Osaka, Japan). β-actin was used as an endogenous control gene for this analysis. Sequence data for gene amplification in quantitative PCR (qPCR) is summarized in Table 1 . Real-time qPCR was performed with a ViiA 7 Real-Time PCR System (Life Technologies, Pleasanton, CA). Collected data was analyzed and fold-expression changes were calculated using the comparative CT method (2 -ΔΔCT ) of relative quantification with ViiA 7 Software (Life Technologies). 10.1371/journal.pone.0126277.t001 Table 1 Sequence Data for Gene Amplification in Quantitative RT-PCR. Gene Forward Primer Reverse Primer NLRP3 5'-TCCTCGGTACTCAGCACTAATCAG-3' 5'-GGTCGCCCAGGTCATTGTTG-3' Caspase-1 5'-AAGACCCGAGCTTTGATTGACTC-3' 5'-AAATCTCTGCCGACTTTTGTTTCC-3' IL-1β 5'-TATTACAGTGGCAATGAGG-3' 5'-ATGAAGGGAAAGAAGGTG-3' IL-18 5'-ATAGCCAGCCTAGAGGTA-3' 5'-ATCAGGAGGATTCATTTC-3' β-actin 5'-CCCTGGACTTCGAGCAAGAG -3' 5'-TCACACTTCATGATGGAGTTG-3' Western Blotting of the CIC Samples Expression of the NLRP3 and caspase-1 proteins of the CIC samples were determined by western blot analysis. CIC samples from 6 subjects were put together and lysed with radioimmunoprecipitation assay buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS) on ice for one hour. The lysates were centrifuged at 12,000 rpm at 4°C for 10 min to obtain the supernatant. Each 60μl cell sample was added, with protein loading buffer, and degenerated in a heating block for 5 min, after which the proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (0.45 μ m; Millipore, Bedford, MA). The membrane was blocked in 2% bovine serum albumin (BSA) for one hour at room temperature and incubated with the specific primary antibodies (diluted 1:1000) overnight at 4°C. After washing with phosphate buffer solution containing Tween-20 five times, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies (diluted 1:5000 by 2% BSA). In accordance with conventional methods, β-actin level was measured at the same time as the internal control. The results were quantified by analysis for grayscale using Gel-Pro Analyzer software (Media Cybernetics, Rockville, MD). The following primary antibodies were used: anti-β-actin, anti-NLRP3 and anti-caspase-1 (Abcam, Boston, MA). Statistical Analysis SPSS 19.0 (IBM Corporation, Armonk, NY) was used to evaluate significance, and GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA) was used to generate figures and tables. The Kruskal—Wallis test was used for comparisons involving tear film BUT, Schirmer I test values, ocular vital staining scores, protein and mRNA expression levels of NLRP3, caspase-1, IL-1β and IL-18. Data were shown as mean ±standard deviation(SD). P <0.05 was considered significant. RESULTS Demographic Characteristics and Clinical Examination Parameters A total of 150 subjects were enrolled in this study, including 54 SSDE patients, 50 NSSDE patients, and 46 healthy sex- and age-matched controls. The baseline ocular surface and tear function parameters of the three groups are presented in Table 2 . Significant differences were found in each parameter among the three groups ( p <0.05). 10.1371/journal.pone.0126277.t002 Table 2 Summary of Diagnostic Tests for Study Groups. Group TBUT(s) FL (Points) Schirmer I(mm/5min) SSDE 2.59±1.00 a , b 4.48±2.31 a , b 3.04±2.56 a , b NSSDE 3.30±0.92 a 1.43±0.82 a 5.16±4.51 a Control 10.00 ±3.00 0.02±0.15 11.5±6.21 Abbreviations: SSDE = Sjögren's syndrome dry eye; NSSDE = non-Sjögren's syndrome dry eye; TBUT = tear break-up time; FS = fluorescein score; a p <0 . 05 , compared with normal controls, Kruskal-Wallis test. b p<0 . 05 , compared with NSSDE patients, Kruskal-Wallis test. NLRP3 mRNA and Protein Expressions in Dry Eye Since NLRP3 inflammasome has a vital function in modulating inflammation and immune responses [ 11 – 13 , 14 – 19 ], we detected its expression in human dry eye. With the use of real-time RT-PCR, the relative expression of NLRP3 mRNA showed higher levels in both the SSDE group (relative 2.47-fold upregulation) and the NSSDE group (relative 1.88-fold upregulation) compared with the controls, with a comparably significant elevation in the SSDE group ( p <0.05, Fig 1 ). 10.1371/journal.pone.0126277.g001 Fig 1 Upregulation of NLRP3 mRNA expression in the CIC samples of dry eye patients. Quantitative analysis of mRNA expression of NLRP3 showed significantly upregulation in SSDE. (* p <0.05, compared with the controls, Kruskal-Wallis test). NLRP3 protein level was detected by western blot analysis. The densitometric analyses showed that NLRP3 protein expression was clearly increased in the SSDE group (relative 1.94-fold upregulation) compared with the healthy control subjects, but no significant differences were found in NLRP3 protein expression between the NSSDE and control subjects ( Fig 2 ). 10.1371/journal.pone.0126277.g002 Fig 2 Western blot and densitometric analyses of NLRP3, procaspase-1,and caspase-1 protein expressions in the eyes of controls, NSSDE, and SSDE subjects. Expression of 120-kDa NLRP3 increased in SSDE versus controls. Expression of 45-kDa procaspase-1 and 20-kDa caspase-1 showed increased protein expressions in SSDE and NSSDE compared with controls. Caspase-1 mRNA and Protein Expressions in Dry Eye Inflammasome is a kind of multiprotein complex, after stimulated by the outside signals, it can lead to the activation of caspase-1[ 11 ]. Therefore, we further detected the expression of caspase-1. The qPCR results of caspase-1 mRNA expression showed significant upregulation in both the SSDE group (relative 1.44-fold upregulation) and the NSSDE group (relative 1.32-fold upregulation) compared with the control eyes ( p <0.05, Fig 3 ). 10.1371/journal.pone.0126277.g003 Fig 3 Upregulation of caspase-1 mRNA expression in the CIC samples of dry eyepatients. Quantitative PCR results of caspase-1 demonstrated significantly upregulation in SSDE and NSSDE groups. (* p <0.05, versus the controls, Kruskal-Wallis test). Western blot and densitometric analyses of procaspase-1 (pro-enzyme) and caspase-1 (active form) are shown in Fig 2 . An increase of the procaspase-1 protein level was found in both SSDE and NSSDE groups in comparison with the control eyes, with 1.84-fold and 1.12-fold levels, respectively. The SSDE and NSSDE groups also had 1.49-fold and 1.17-fold greater caspase-1 expressions than the control eyes. Demographic Characteristics and Clinical Examination Parameters A total of 150 subjects were enrolled in this study, including 54 SSDE patients, 50 NSSDE patients, and 46 healthy sex- and age-matched controls. The baseline ocular surface and tear function parameters of the three groups are presented in Table 2 . Significant differences were found in each parameter among the three groups ( p <0.05). 10.1371/journal.pone.0126277.t002 Table 2 Summary of Diagnostic Tests for Study Groups. Group TBUT(s) FL (Points) Schirmer I(mm/5min) SSDE 2.59±1.00 a , b 4.48±2.31 a , b 3.04±2.56 a , b NSSDE 3.30±0.92 a 1.43±0.82 a 5.16±4.51 a Control 10.00 ±3.00 0.02±0.15 11.5±6.21 Abbreviations: SSDE = Sjögren's syndrome dry eye; NSSDE = non-Sjögren's syndrome dry eye; TBUT = tear break-up time; FS = fluorescein score; a p <0 . 05 , compared with normal controls, Kruskal-Wallis test. b p<0 . 05 , compared with NSSDE patients, Kruskal-Wallis test. NLRP3 mRNA and Protein Expressions in Dry Eye Since NLRP3 inflammasome has a vital function in modulating inflammation and immune responses [ 11 – 13 , 14 – 19 ], we detected its expression in human dry eye. With the use of real-time RT-PCR, the relative expression of NLRP3 mRNA showed higher levels in both the SSDE group (relative 2.47-fold upregulation) and the NSSDE group (relative 1.88-fold upregulation) compared with the controls, with a comparably significant elevation in the SSDE group ( p <0.05, Fig 1 ). 10.1371/journal.pone.0126277.g001 Fig 1 Upregulation of NLRP3 mRNA expression in the CIC samples of dry eye patients. Quantitative analysis of mRNA expression of NLRP3 showed significantly upregulation in SSDE. (* p <0.05, compared with the controls, Kruskal-Wallis test). NLRP3 protein level was detected by western blot analysis. The densitometric analyses showed that NLRP3 protein expression was clearly increased in the SSDE group (relative 1.94-fold upregulation) compared with the healthy control subjects, but no significant differences were found in NLRP3 protein expression between the NSSDE and control subjects ( Fig 2 ). 10.1371/journal.pone.0126277.g002 Fig 2 Western blot and densitometric analyses of NLRP3, procaspase-1,and caspase-1 protein expressions in the eyes of controls, NSSDE, and SSDE subjects. Expression of 120-kDa NLRP3 increased in SSDE versus controls. Expression of 45-kDa procaspase-1 and 20-kDa caspase-1 showed increased protein expressions in SSDE and NSSDE compared with controls. Caspase-1 mRNA and Protein Expressions in Dry Eye Inflammasome is a kind of multiprotein complex, after stimulated by the outside signals, it can lead to the activation of caspase-1[ 11 ]. Therefore, we further detected the expression of caspase-1. The qPCR results of caspase-1 mRNA expression showed significant upregulation in both the SSDE group (relative 1.44-fold upregulation) and the NSSDE group (relative 1.32-fold upregulation) compared with the control eyes ( p <0.05, Fig 3 ). 10.1371/journal.pone.0126277.g003 Fig 3 Upregulation of caspase-1 mRNA expression in the CIC samples of dry eyepatients. Quantitative PCR results of caspase-1 demonstrated significantly upregulation in SSDE and NSSDE groups. (* p <0.05, versus the controls, Kruskal-Wallis test). Western blot and densitometric analyses of procaspase-1 (pro-enzyme) and caspase-1 (active form) are shown in Fig 2 . An increase of the procaspase-1 protein level was found in both SSDE and NSSDE groups in comparison with the control eyes, with 1.84-fold and 1.12-fold levels, respectively. The SSDE and NSSDE groups also had 1.49-fold and 1.17-fold greater caspase-1 expressions than the control eyes. IL-1β mRNA and Protein Expressions in Dry Eye Since the activation of caspase-1 can be processed in regulation the secretion of IL-1β and IL-18[ 30 ], we next examined the inflammatory cytokines IL-1β and IL-18. Quantitative PCR results of IL-1β mRNA expression demonstrated significant upregulation in the SSDE eyes (3.59-fold relative upregulation, p <0.001) and NSSDE eyes (2.13-fold relative upregulation, p <0.01) compared with the control eyes ( Fig 4A ). 10.1371/journal.pone.0126277.g004 Fig 4 Upregulation of IL-1β expression in the tear and CIC samples of dry eye patients. ( A ) Quantitative RT-PCR for IL-1β mRNA expression in CIC specimens showed significant upregulation in eyes with SSDE and NSSDE (*** p ≤0.001, ** p ≤0.01, compared with the controls, Kruskal-Wallis test). ( B ) Tear IL-1β protein expressions via ELISA demonstrated higher levels in SSDE group compared with controls. (** p ≤0.01, compared with the controls, Kruskal-Wallis test). Tear IL-1β levels were measured by ELISA. The mean protein levels of IL-1β in tears were 33.42±11.25 pg/mL in the control subjects, 41.33±20.34 pg/mL in patients with NSSDE, and 66.18±68.13 pg/mL in patients with SSDE. The level of IL-1β increased significantly in the tears of patients with SSDE compared with the control subjects ( p = 0.01). The NSSDE group also had a higher IL-1β protein expression level compared with the healthy control subjects ( Fig 4B ). IL-18 mRNA and Protein Expressions in Dry Eye As shown in Fig 5A , IL-18 mRNA expression in eyes with SSDE (2.97-fold relative upregulation) and NSSDE (2.05-fold relative upregulation) was significantly higher compared with the control eyes ( p = 0.001). 10.1371/journal.pone.0126277.g005 Fig 5 Upregulation of IL-18 expression in the tear and CIC samples of dry eye patients. ( A ) Significant elevation of IL-18 mRNA level was found in SSDE and NSSDE groups using quantitative RT-PCR.(*** p ≤0.001, compared with the controls, Kruskal-Wallis test). ( B ) ELISA analyses of IL-18 protein expression demonstrated tear IL-18 increased significantly in SSDE and NSSDE groups. (* p <0.05, *** p ≤0.001, compared with the controls, Kruskal-Wallis test). Protein expression of tear IL-18 was detected using ELISA. The mean levels of IL-18 in tears were 114.504±34.341pg/mL in controls, 168.364±78.018 pg/mL in patients with NSSDE, and 229.767±107.010pg/mL in patients with SSDE. A significant increase was observed in the SSDE group ( p <0.001) and NSSDE group (p<0.05) compared with the control subjects ( Fig 5B ). IL-18 mRNA and Protein Expressions in Dry Eye As shown in Fig 5A , IL-18 mRNA expression in eyes with SSDE (2.97-fold relative upregulation) and NSSDE (2.05-fold relative upregulation) was significantly higher compared with the control eyes ( p = 0.001). 10.1371/journal.pone.0126277.g005 Fig 5 Upregulation of IL-18 expression in the tear and CIC samples of dry eye patients. ( A ) Significant elevation of IL-18 mRNA level was found in SSDE and NSSDE groups using quantitative RT-PCR.(*** p ≤0.001, compared with the controls, Kruskal-Wallis test). ( B ) ELISA analyses of IL-18 protein expression demonstrated tear IL-18 increased significantly in SSDE and NSSDE groups. (* p <0.05, *** p ≤0.001, compared with the controls, Kruskal-Wallis test). Protein expression of tear IL-18 was detected using ELISA. The mean levels of IL-18 in tears were 114.504±34.341pg/mL in controls, 168.364±78.018 pg/mL in patients with NSSDE, and 229.767±107.010pg/mL in patients with SSDE. A significant increase was observed in the SSDE group ( p <0.001) and NSSDE group (p<0.05) compared with the control subjects ( Fig 5B ). DISCUSSION NLRP3 inflammasome plays a vital role in modulating innate or adaptive immune responses. Much valuable work has implicated its importance in the onset and development of many inflammatory related diseases [ 14 – 19 ]. Recently, Baldini et al reported increased expressions of the P2X 7 receptor-NLRP3 inflammasome, caspase-1 and IL-18 in the salivary gland of Sjögren's syndrome patients. Those results suggested the involvement of NLRP3 inflammasome-capase-1-IL-18 axis in the development of primary Sjögren's syndrome exocrinopathy [ 31 ]. Immune-based inflammation on the ocular surface has been acknowledged as playing a prominent role in the pathological damage of dry eye [ 1 , 2 ]. However, the exact mechanism of inflammation development and activation of various inflammatory cytokines in dry eye is still unclear. We speculated that NLPR3 inflammasome may be involved in the development of dry eye ocular surface inflammation. To study the potential involvement of the NLRP3 inflammasome in dry eye patients, we prospectively recruited patients with SSDE and NSSDE, and collected tear and impression cytology samples to evaluate mRNA and protein expression of NLRP3 inflammasome and its downstream inflammatory factors-caspase-1, IL-1β, and IL-18. Our results showed a significant increase of NLRP3 mRNA and protein expressions in SSDE subjects versus the controls. Subjects with NSSDE also display a higher level of NLRP3 mRNA expression, although a statistically significant difference was not found in comparison with the controls. These findings indicate that NLRP3 inflammasome may be associated with the development of dry eye ocular surface inflammation. In our study, the baseline tear film BUT, FL and Schirmer test values all were significantly worse in SSDE group than the data in NSSDE group. We speculate the level of NLRP3 expression in SSDE and NSSDE may be associated with the severity of the dry eye disease. In the study of Baldini et al [ 31 ], they found increased expression of NLRP3 in the salivary gland of SS patients but not in the salivary gland of NSSDE patients. Pathological alterations are noted in the salivary gland in SS, but this is not the case in NSSDE. However, the ocular surface is affected in both SSDE and NSSDE. In our findings, we found not only a statistical increase in the NLRP3 expression on the ocular surface of SSDE but also a higher expression level of NLRP3 in NSSDE. These findings also suggest that inflammation levels on the ocular surface vary in different types of dry eye. As previous studies have confirmed that NLRP3 leads up to the activation of caspase-1 in many inflammatory diseases[ 11 , 14 – 19 ], we found significant upregulation in the levels of caspase-1 mRNA and proteins in both SSDE and NSSDE subjects compared with the control subjects. These findings were consistent with the NLRP3 inflammasome upregulation that we observed. These findings suggest that increased NLRP3 expression may be activating caspase-1 to develop further inflammation in patients with dry eye. The best-characterized consequence of the caspase-1 activation of NLRP3 inflammasome is secretion of the pro-inflammatory cytokines IL-1β and IL-18 [ 30 ]. Once released, these cytokines initiate an inflammatory cascade that leads to the recruitment of innate immune cells and can regulate the subsequent adaptive immune response [ 32 , 33 ]. The current study confirmed a significant increase in IL-1β and IL-18 mRNA and protein expressions in tears and on the ocular surface of dry eyes. These findings suggested that the increased IL-1β and IL-18 expressions might be regulated by NLRP3 inflammasome.IL-1β has been found to decrease tear production via neuronal and hormonal effects [ 34 ]. IL-18 has been reported to potentially cause serious damage in the lacrimal and salivary glands of Sjögren's syndrome patients [ 10 , 35 ]. But the function of IL-18 in dry eye is still not well understood. In summary, based on tear samples and conjunctival impression cytology specimens of dry eye patients, we found that the expressions of NLRP3 inflammasome and its downstream inflammatory factors-caspase-1, IL-1β and IL-18 are upregulated in dry eye patients, especially in SSDE. These findings suggest the involvement of NLRP3 inflammasome and its downstream inflammatory factors-caspase-1, IL-1β, and IL-18 in the development of ocular surface inflammation in dry eye. To our knowledge, this is the first study to investigate the changes in NLRP3 inflammasome in the tear and ocular surface of dry eye patients. Inflammasome activation could be triggered by many exogenous and endogenous noninfectious stimuli [ 36 , 37 ]. Risk factors for the development of dry eye such as aging, dry environments and contact lens wear [ 38 ] may trigger the expression of inflammasome. Higher levels of inflammasome can in turn activate caspase-1 and the secretion of IL-1β and IL-18, which then induce the inflammatory damage of dry eye. In a recent study, Zheng et al [ 23 ] found that reactive oxygen species could trigger NLRP3 inflammasome in a dry eye murine model. Understanding the expression of NLRP3 inflammasome associated with the dry eye pathology may clarify factors involved in the progression of the disease and enhance the development of targeted therapies. Further research should focus on the regulatory mechanism of NLRP3 inflammasome in dry eye inflammation; the function of IL-18 in dry eye; and whether inhibition of inflammasome components can serve as a viable target for therapeutic development in dry eye.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438843/

High-Throughput Quantitative Real-Time Polymerase Chain Reaction Array for Absolute and Relative Quantification of Rhesus Macaque Types I, II, and III Interferon and Their Subtypes

Rhesus macaques provide a valuable research and preclinical model for cancer and infectious diseases, as nonhuman primates share immune pathways with humans. Interferons (IFNs) are key cytokines in both innate and adaptive immunity, so a detailed analysis of gene expression in peripheral blood and tissues may shed insight into immune responses. Macaques have 18 IFN genes, of which 14 encode for 13 distinct IFN-α subtypes, and one for IFN-β. Here, we developed a high-throughput array to evaluate each of the IFN-α subtypes, as well as IFN-β, IFN-γ and 2 subtypes of IFN-λ. With this array, expression of each IFN species may be quantified as relative to a reference (housekeeping) gene (ΔCq) or fitted to its own 4-point standard curve for absolute quantification (copy number per mass unit RNA). After validating the assay with IFN complementary DNA, we determined the IFN expression profile of peripheral blood mononuclear cells from 3 rhesus macaques in response to TLR agonists, and demonstrated that the profiles are consistent among animals. Furthermore, because the IFN expression profiles differ depending on the TLR stimuli, they suggest different biological functions for many of the IFN species measured, including individual subtypes of IFN-α.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010738/

Endoplasmic reticulum stress induces spatial memory deficits by activating GSK ‐3

Abstract Endoplasmic reticulum ( ER ) stress is involved in Alzheimer's disease ( AD ), but the mechanism is not fully understood. Here, we injected tunicamycin ( TM ), a recognized ER stress inducer, into the brain ventricle of Sprague‐Dawley ( SD ) rats to induce the unfolded protein response ( UPR ), demonstrated by the enhanced phosphorylation of pancreatic ER kinase ( PERK ), inositol‐requiring enzyme‐1 ( IRE ‐1) and activating transcription factor‐6 ( ATF ‐6). We observed that UPR induced spatial memory deficits and impairments of synaptic plasticity in the rats. After TM treatment, GSK ‐3β was activated and phosphorylation of cAMP response element binding protein at Ser129 ( pS 129‐ CREB ) was increased with an increased nuclear co‐localization of pY 126‐ GSK ‐3β and pS 129‐ CREB . Simultaneous inhibition of GSK ‐3β by hippocampal infusion of SB 216763 ( SB ) attenuated TM ‐induced UPR and spatial memory impairment with restoration of pS 129‐ CREB and synaptic plasticity. We concluded that UPR induces AD ‐like spatial memory deficits with mechanisms involving GSK ‐3β/ pS 129‐CREB pathway. 1 INTRODUCTION Alzheimer's disease (AD) is a chronic neurodegenerative disorder with progressive impairment of memory and other cognitive functions. Apart from the loss of synapses, AD is histopathologically characterized by the accumulation of numerous intracellular neurofibrillary tangles (NFTs) and extracellular plaques. 1 , 2 Previous studies have shown that the amount of NFTs, mainly composed of the hyperphosphorylated tau, 3 , 4 in the AD brains is correlated with the degree of dementia. 5 , 6 Studies also showed that β‐amyloid (Aβ), forming toxic oligomers that aggregate into amyloid plaques, was associated with age‐related memory impairment. 7 , 8 Our previous studies indicate that tunicamycin (TM) could induce AD‐like tau hyperphosphorylation and reduction in some synapse‐related proteins in temporal cortex, frontal cortex and hippocampus. 9 However, whether TM treatment affects learning and memory and the molecular mechanisms are unknown. Endoplasmic reticulum (ER) is an important cellular organelle, responsible for the posttranslational processing of newly synthesized proteins and ensuring proper protein folding and assembly. 10 ER stress is an important form of ER dysfunction, and ER stress has been observed in several neurological conditions, such as AD, Parkinson's disease, Amyotrophic lateral sclerosis and so on. Some studies have showed neurons are constantly exposed to ER stress in the AD brains. ER stress could be expressed by chaperone proteins and trigger many rescuer responses, including unfolded protein response (UPR) and ER‐associated degradation. 11 , 12 , 13 The ER chaperone binding immunoglobulin protein (Bip) is physiologically bound to 3 important proteins in the ER membrane, pancreatic ER kinase (PERK), inositol‐requiring enzyme‐1 (IRE‐1) and activating transcription factor‐6 (ATF‐6). When UPR is induced, Bip is attracted to bind to the unfolded proteins accumulated in the ER to keep the correct protein folding and is thereby released from PERK, IRE‐1 and ATF‐6, which are consequently phosphorylated and activated. 14 , 15 , 16 Although the initial UPR protects the cell from the toxicity of misfolded proteins in the ER, prolonged UPR activation may participate in the pathogenesis of protein misfolding diseases, such as AD. 17 , 18 , 19 , 20 Recently, several reports have shown that the UPR is activated in the AD brain. Bip, an ER stress marker, is increased in the temporal cortex and the hippocampus of AD cases compared with no demented control cases. 21 The phosphorylated PERK (pPERK), an UPR activation marker, is most abundant in neurons with diffuse localization of the phosphorylated tau protein in the brain of AD patients. 22 Our previous report has also shown that TM treatment induces tau hyperphosphorylation in frontal cortex, temporal cortex and hippocampus in rats with an increased level of Bip and reduction in some synapse‐related proteins, 9 while overexpressing SIL1 rescued Bip elevation‐related Tau hyperphosphorylation in ER stress. 23 Glycogen synthase kinase‐3 (GSK‐3) is highly expressed in the central nervous system (CNS) 24 and plays an important role in AD. Studies showed that the activated form of GSK‐3 was elevated in the AD brains. 25 GSK‐3 could not only phosphorylate tau at most of the AD sites 26 , 27 , 28 but also induce Aβ overproduction. 29 Activation or overexpression of GSK‐3 induces memory deficit, 30 , 31 , 32 whereas inhibition of GSK‐3 reverses this effect. 31 , 33 However, the mechanism by which GSK‐3 regulates learning and memory is only partly understood. Recent study has found that overexpression of GSK‐3β could cause memory deficits by inhibiting long‐term potentiation which is accompanied by prominent impairment of synapses. 34 , 35 An in vitro study also show that GSK‐3β could be activated during ER stress 36 , 37 and induce tau hyperphosphorylation 23 , 38 that be involved in memory impairment. However, it is still not understood whether and how GSK‐3β plays an in vivo role in ER stress‐induced spatial cognitive alterations. The cAMP response element binding protein (CREB), named by Montminy, 39 regulates transcription of multiple genes in eukaryotic nuclei. CREB plays an important role in increasing long‐term potentiation (LTP), synaptic plasticity, development, differentiation and survival of neurons. Phosphorylation of CREB at Ser133 increases CREB activity, whereas phosphorylation at Ser129 and Ser142 inhibits its activity. GSK‐3β is an important kinase regulating the transcription of CREB. 40 In this study, we established a rat model with activated UPR by brain injection of TM. We found that TM infusion induces spatial memory deficits in rats with ER stresses, shown by the increased level of phosphorylated PERK, IRE‐1, ATF6, CREB at Ser129, GSK‐3β at Tyr216 and impairment of synapses. Simultaneous inhibition of GSK‐3 rescues the UPR‐induced spatial memory impairments with restoration of ER stress and the associated dysfunction. 2 MATERIALS AND METHODS 2.1 Antibodies and chemicals The antibodies used in this study are listed in Table 1 . TM was from Alexis Biochemical (San Diego, CA, USA). TM was dissolved in DMSO at concentration of 12.5 mmol/L and stored at −20°C. SB216763 (SB) was from Tocris Bioscience (Bristol, UK) and freshly dissolved in DMSO from light before use. Bicinchoninic acid (BCA) protein detection kit was from Pierce Chemical Company (Rockford, IL, USA). Enhanced chemiluminescence was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Table 1 A list of antibodies and their epitopes on the molecule of protein used in this study Antibody Epitopes Type Dilution Source GSK‐3β Total‐GSK‐3β Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) pS9‐GSK‐3β Phospho‐GSK‐3β at Ser9 Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) Tyr216‐GSK‐3β Phospho‐GSK‐3β at Tyr279/Tyr216 Mono‐ 1:1000 for WB 1:200 for IFC Millipore (Billerica, MA, USA) PERK Total PERK Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) pPERK Phospho‐PERK(Thr980) Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) IRE1 Total IRE1 Poly‐ 1:1000 for WB Abcam (Cambridge, UK) P‐IRE1 Phospho‐IRE1(Ser724) Poly‐ 1:1000 for WB Abcam (Cambridge, UK) ATF6 Total ATF6 Poly‐ 1:1000 for WB Abcam (Cambridge, UK) P‐ATF6 Phospho‐ATF6 Poly‐ 1:1000 for WB Abcam (Cambridge, UK) NR2A Total NR2A Poly‐ 1:1000 for WB Abcam (Cambridge, UK) NR2B Total NR2B Poly‐ 1:1000 for WB Abcam (Cambridge, UK) PSD95 Total PSD95 Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) Synapsin 1 Total Synapsin 1 Poly‐ 1:1000 for WB Millipore (Billerica, MA, USA) CREB Total CREB Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) pS129‐CREB Phospho‐CREB at Ser129 Poly‐ 1:1000 for WB 1:200 for IHC 1:200 for IFC Sigma (NY, USA) pS133‐CREB Phospho‐CREB at Ser133 Poly‐ 1:1000 for WB 1:200 for IHC 1:200 for IFC Cell Signalling (Danvers, MA, USA) GAPDH Full‐length GDPDH Mono‐ 1:1000 for WB Abcam (Cambridge, UK) Histone 3 (H3) Total histone 3 protein Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) DM1A Alpha‐tublin Mono‐ 1:2000 for WB Abcam (Cambridge, UK) IHC, immunohistochemistry staining; IFC, immunofluorescence staining; Mono‐, monoclonal; Poly‐, polyclonal; WB, Western blotting. John Wiley & Sons, Ltd 2.2 Drug administration Three‐month‐old (250 ± 20 g) male Sprague‐Dawley rats were supplied by the Experimental Animal Central of Tongji Medical College. All experimental procedures were approved by the Animal Care and Use Committee at the Huazhong University of Science and Technology and were performed in compliance with National Institutes of Health guidelines on the ethical use of animals. Rats were kept in cages under a 12‐hour light: 12‐hour dark (L/D) cycle with the light on from 7:00 am to 7:00 pm . The rats (rats for each group were used in this study are listed in Table 2 ) were anaesthetized with 6% chloral hydrate (400 mg/kg) and placed in a Jiangwan‐II stereotaxic instrument (Jiangwan Medical Instrument Co. Shanghai, China). 41 The skull was cleaned, and the hole (diameter 1.0 mm) was made for the infusion after the scalp was incised (5.0‐8.0 mm). For the lateral ventricular infusion, the coordinate of AP‐0.8, L‐1.5, V‐4.0 (in mm from bregma and dura, flat skull) was selected according to the stereotaxic atlas of Franklin and Paxinos. A sterilized needle connected to a Hamilton syringe was used to deliver TM or in combination with SB into the lateral ventricle (10 μL). Equal volume of DMSO with 0.9% NaCl was infused as vehicle controls. Table 2 A list of rats used in this study Groups Nor DMSO TM (μm) TM + SB 25 50 75 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h Western blotting 6 6 6 6 6 6 6 6 6 6 6 Behavioural test 20 20 a 20 20 a 20 20 a a Six rats of the group were used for Nissl and Immunofluorescence staining, 3 other rats were used for Golgi staining. John Wiley & Sons, Ltd 2.3 Behavioural test Spatial memory was measured by Morris water maze test. 42 The Morris water maze apparatus was the same as previously described. 43 In brief, it consisted of a circular pool, 150 cm in diameter and 50 cm in height, with the interior painted black. The escape platform was made of clear plexiglass, 14 cm in diameter and 27 cm in height and was located 1 cm below the surface of the water. Visual cues were visible to the rats, including several geometric shapes that measured at least 20 cm in height and were positioned so that they were 15 cm above water level and remained distal and constant to the rats at all times. The water was maintained at 24 ± 2°C and was made opaque by the addition of non‐toxic black ink that generated the obvious contrast with the white skin of rats to record their movements. The swimming pathways and the latencies of the rats to find the hidden platform were recorded each day by a video camera fixed to the ceiling of the room, 1.5 m from the water surface. The camera was connected to a digital‐tracking device attached to an IBM computer loaded with the water maze software. The less time a rat spent in finding the platform, the better it scored the spatial learning and memory. For spatial learning, rats were trained in water maze to remember the hidden platform for 7 consecutive days. For the first day, the rats only proceeded with one trial that started from the first quadrant. And for the following 5 days, 3 trials were performed for each rat every day, starting from the first, the second and the fourth quadrant, respectively. For the last training day (the 7 day), only one trial that started from the first quadrant's movements were performed, and total 20 trials were finished for 7 days' training. On each trial, the rat started from each quadrant by facing the wall of the pool and ended when the animal climbed on the platform. The rats were not allowed to search for the platform more than 60 seconds, after which they were guided to the platform. Through these training sessions, rats acquired spatial memory about location of the safe platform, and rats that could find the platform after training from quadrant 1‐4 in turn for 24 trials within 20 seconds were selected and randomly divided into 3 groups for the brain lateral ventricle injections, respectively, with DMSO (10 μL) or TM (50 μmol/L, 10 μL) or TM + SB (50 μmol/L, 10 μL) as mentioned above. At 24 and 48 hours after the injection, the spatial memory retention of the rats was tested in the same water maze with the searching time extended to 90 seconds. After the behaviour test, the rats were killed for the rest studies. Memory was also measured by step‐down avoidance tests which was made of electrically conductive metal fence at the bottom, around with open transparent plastic box (length × width × height: 23 × 23 × 40 cm). A wooden platform was placed on the bottom of the metal fence (length × width × height: 3.5 × 3.5 × 2.5 cm). Put the rats into the experimental device to make them be familiar with the surrounding environment for 5 minutes, respectively. And then began the stage of learning to put the rats gently on the platform, when animals jumping off the platform to the wire fence, immediately gave them electric shock (0.5 mA, for 5 seconds). After that put them back to the platform and began to record duration, if the duration reached 60 seconds that indicated rats have learned how to avoid electric shock. If the duration on the platform was <60 seconds, gave the rats electric shock again when they jumped off the platform until they could stay on the platform at least 60 seconds. Record the number of rats received shocks (mistake number) and the total time (learning time for the first time), the whole period was 5 minutes. The laboratory equipment should be cleaned with 75% alcohol after each rat was tested. When all rats were trained, following start the formal test to check the memory retention at 24 and 48 hours after DMSO or TM being injected. The rats were put on platform in this phase experiment, and the duration period and numbers of wrong were recorded within 5 minutes. 2.4 Cytoplasm–nucleus protein extraction The 250‐mg fresh rats brain or cryopreserved tissue in −80°C were taken immediately into the glass homogenizer with ice pre‐cooling, add 500 μL cytosol extraction reagent (CER, the proportion of brain tissue with CER is 1:2) in it. Triturate the brain tissue with pestle and then fluctuate homogenate them by manual operation for 20 times. Ice bath for 10 minutes and then fluctuate homogenate them for 7 times again. Take 500‐μL lysate, transfer it to the new centrifugal tube, centrifuge for 5 minutes with 800 g in 4°C circumstance. The crude product of nucleus precipitates at the end of the pipe and the supernatant is the mixture of cell membrane and cytoplasm. Transfer the supernatant to the new centrifugal tube and add membrane extraction reagent (MER, 1/10 volume of the supernatant fluid) into it. Ice bath for 5 minutes, centrifuge for 30 minutes with 10956.4 g in 4°C circumstance. Take the supernatant into the new centrifugal tube which is the cytoplasm components and the precipitation is cell membrane components including cell membranes and organelles fragments which can be suspended again with 50‐100 μL suspension buffer. Add 500 μL nuclear extraction reagent (NER) into the crude product of nucleus obtained such as the above and vibrate it to suspend again. 4000 g , 4°C centrifuge for 5 minutes, abandon the supernatant and then add 500 μL NER into it to suspend it again. Repeat the above centrifugal steps and clear the centrifugal supernatant. Add 50‐100 μL suspension buffer to suspend the precipitation again to obtain the nuclei. 2.5 Western blotting For brain samples, the hippocampus taken immediately after Morris water maze test were homogenized in buffer containing 10 mmol/L Tris–Cl, pH 7.6, 50 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L edetic acid, 1 mmol/L benzamidine, 1 mmol/L PMSF and a mixture of aprotinin, leupeptin and pepstatin A (10 μg/mL each) or obtain the cytoplasm–nucleus protein extraction as the above way. Three volumes of the homogenate for brain samples were added to one volume of the extracting buffer containing 200 mmol/L Tris–Cl, pH 7.6, 8% SDS, 40% glycerol, and the samples were boiled in water bath for 10 minutes and then followed by sonication for 15 seconds on ice. After measurement of protein concentration in the extracts using BCA kit (Pierce, Rockford, IL), a final concentration of 10% β‐mercaptoethanol and 0.05% bromophenol blue was added. The proteins in the extracts were separated by 10% SDS‐PAGE and transferred to nitrocellulose membrane. The membranes were blocked with 5% non‐fat milk dissolved in TBS‐Tween‐20 (50 mmol/L Tris–HCl, pH 7.6, 150 mmol/L NaCl, 0.2% Tween‐20) for 1 hour and probed with primary antibody (see Table 1 for detail) at 4°for overnight. Then, the blots were incubated with anti‐mouse or anti‐rabbit IgG conjugated to horseradish peroxidase (1:15 000) for 1 hour at room temperature and scanned after being washed with TBS‐Tween‐20,and the greyscale was analysed with odyssey system. 2.6 Nissl staining Nissl staining was established in 1892 by Franna Nissl, the German pathologist, with alkaline dye to discover Nissl body, and this method was widely used. The picked brain slices were pasted on the glass slide disposed by gelatine in PBS liquid, dustproof atmospheric drying. And then, 1% Toluidine blue was dropped on the brain slices, keeping 5‐10 minutes, 95% alcohol was used to differentiate and observed under a microscope at the same time until the background was clean and the Nissl body was clear. Then dehydrated with 100% alcohol for 5 minutes × 2, transparented with xylene for 5 minutes × 2 and sealed with neutral gummi, and finally analysed under a microscope and collected images. 2.7 Golgi staining After the rats (n = 3 for each group) being anaesthetized with 6% chloral hydrate (400 mg/kg), the aorta of the rat was inserted into with blunt infusion needle and perfused with 350‐500 mL (37°C) physiological saline containing 0.5% sodium nitrite into the systemic circulation quickly. When the liver became pale and the rinse become clear, continued to perfuse with fixed liquid containing 4% formaldehyde (500 mL) for about 1‐2 hours until the rat body was stiff. Then replaced the fixed liquid with mordant dyeing (500 mL, containing 5% chloral hydrate, 5% potassium dichromate, 4% formaldehyde), continued to pursue quickly for 5‐10 minutes to replace the systemic circulation stationary liquid. When flowing out thick orange liquid, slowed down the speed to 25 drop/minutes and the perfusion process of mordant dyeing liquid lasted about 3‐4 hours. The brain tissue after perfusion was taken and divided the brain into 2 parts along with the midline incision, kept the tissue containing hippocampus to 5 mm thick. Then dipped the tissue into fresh mordant dye, avoided light for 4 days and replaced the mordant dye every day. Next permutated the mordant dye with 1.5% silver nitrate solution for 3 days and avoided light and replaced a fresh silver nitrate solution daily. The brain slice (35 μm) was prepared by oscillation microtome (Germany, VT1000S, Leica). The slices were soaked in 2% potassium dichromate solution for 20 minutes and then rinsed with steaming water to clear. Then pasted the slices in 1.5% gelatine solution, dried dustproof air and then dehydrated with gradient alcohol, transparent with xylene and sealed piece with neutral gum. Finally, images were collected under a microscope to analyse. The spine numbers were measured by Image J software, and the different types of spines were analysed according to the schematic structure of the dendritic spines. 44 2.8 Immunofluorescence staining The brain sections were obtained as the immunohistochemistry staining described. The sections were incubated for 48 hours at 4°C with the first primary antibodies after being ruptured membrane and blocked with 5% BSA and then washed with PBS and incubated the second primary antibodies like described above. The sections were subsequently incubated with Rhodamine Green 488 or Red 546‐conjugated secondary antibodies (1:500) for 1 hour at 37°C. Pasted the brain sections to the glass slide and sealed them with 50% glycerine after being washed with PBS. The images were observed by a laser confocal microscope (LSM710, Zeiss, Germany), and the fluorescence images were analysed by the software affiliated. 45 , 46 2.9 Statistical analysis Data were expressed as means ± SEM and analysed using SPSS 12.0 statistical software (SPSS Inc., Chicago, Illinois, USA). Means were compared by one‐way analysis of variance (ANOVA) procedure followed by LSD's post hoc Bonferroni's tests. P values <.05 were considered as significant. 2.1 Antibodies and chemicals The antibodies used in this study are listed in Table 1 . TM was from Alexis Biochemical (San Diego, CA, USA). TM was dissolved in DMSO at concentration of 12.5 mmol/L and stored at −20°C. SB216763 (SB) was from Tocris Bioscience (Bristol, UK) and freshly dissolved in DMSO from light before use. Bicinchoninic acid (BCA) protein detection kit was from Pierce Chemical Company (Rockford, IL, USA). Enhanced chemiluminescence was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Table 1 A list of antibodies and their epitopes on the molecule of protein used in this study Antibody Epitopes Type Dilution Source GSK‐3β Total‐GSK‐3β Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) pS9‐GSK‐3β Phospho‐GSK‐3β at Ser9 Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) Tyr216‐GSK‐3β Phospho‐GSK‐3β at Tyr279/Tyr216 Mono‐ 1:1000 for WB 1:200 for IFC Millipore (Billerica, MA, USA) PERK Total PERK Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) pPERK Phospho‐PERK(Thr980) Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) IRE1 Total IRE1 Poly‐ 1:1000 for WB Abcam (Cambridge, UK) P‐IRE1 Phospho‐IRE1(Ser724) Poly‐ 1:1000 for WB Abcam (Cambridge, UK) ATF6 Total ATF6 Poly‐ 1:1000 for WB Abcam (Cambridge, UK) P‐ATF6 Phospho‐ATF6 Poly‐ 1:1000 for WB Abcam (Cambridge, UK) NR2A Total NR2A Poly‐ 1:1000 for WB Abcam (Cambridge, UK) NR2B Total NR2B Poly‐ 1:1000 for WB Abcam (Cambridge, UK) PSD95 Total PSD95 Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) Synapsin 1 Total Synapsin 1 Poly‐ 1:1000 for WB Millipore (Billerica, MA, USA) CREB Total CREB Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) pS129‐CREB Phospho‐CREB at Ser129 Poly‐ 1:1000 for WB 1:200 for IHC 1:200 for IFC Sigma (NY, USA) pS133‐CREB Phospho‐CREB at Ser133 Poly‐ 1:1000 for WB 1:200 for IHC 1:200 for IFC Cell Signalling (Danvers, MA, USA) GAPDH Full‐length GDPDH Mono‐ 1:1000 for WB Abcam (Cambridge, UK) Histone 3 (H3) Total histone 3 protein Poly‐ 1:1000 for WB Cell Signalling (Danvers, MA, USA) DM1A Alpha‐tublin Mono‐ 1:2000 for WB Abcam (Cambridge, UK) IHC, immunohistochemistry staining; IFC, immunofluorescence staining; Mono‐, monoclonal; Poly‐, polyclonal; WB, Western blotting. John Wiley & Sons, Ltd 2.2 Drug administration Three‐month‐old (250 ± 20 g) male Sprague‐Dawley rats were supplied by the Experimental Animal Central of Tongji Medical College. All experimental procedures were approved by the Animal Care and Use Committee at the Huazhong University of Science and Technology and were performed in compliance with National Institutes of Health guidelines on the ethical use of animals. Rats were kept in cages under a 12‐hour light: 12‐hour dark (L/D) cycle with the light on from 7:00 am to 7:00 pm . The rats (rats for each group were used in this study are listed in Table 2 ) were anaesthetized with 6% chloral hydrate (400 mg/kg) and placed in a Jiangwan‐II stereotaxic instrument (Jiangwan Medical Instrument Co. Shanghai, China). 41 The skull was cleaned, and the hole (diameter 1.0 mm) was made for the infusion after the scalp was incised (5.0‐8.0 mm). For the lateral ventricular infusion, the coordinate of AP‐0.8, L‐1.5, V‐4.0 (in mm from bregma and dura, flat skull) was selected according to the stereotaxic atlas of Franklin and Paxinos. A sterilized needle connected to a Hamilton syringe was used to deliver TM or in combination with SB into the lateral ventricle (10 μL). Equal volume of DMSO with 0.9% NaCl was infused as vehicle controls. Table 2 A list of rats used in this study Groups Nor DMSO TM (μm) TM + SB 25 50 75 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h Western blotting 6 6 6 6 6 6 6 6 6 6 6 Behavioural test 20 20 a 20 20 a 20 20 a a Six rats of the group were used for Nissl and Immunofluorescence staining, 3 other rats were used for Golgi staining. John Wiley & Sons, Ltd 2.3 Behavioural test Spatial memory was measured by Morris water maze test. 42 The Morris water maze apparatus was the same as previously described. 43 In brief, it consisted of a circular pool, 150 cm in diameter and 50 cm in height, with the interior painted black. The escape platform was made of clear plexiglass, 14 cm in diameter and 27 cm in height and was located 1 cm below the surface of the water. Visual cues were visible to the rats, including several geometric shapes that measured at least 20 cm in height and were positioned so that they were 15 cm above water level and remained distal and constant to the rats at all times. The water was maintained at 24 ± 2°C and was made opaque by the addition of non‐toxic black ink that generated the obvious contrast with the white skin of rats to record their movements. The swimming pathways and the latencies of the rats to find the hidden platform were recorded each day by a video camera fixed to the ceiling of the room, 1.5 m from the water surface. The camera was connected to a digital‐tracking device attached to an IBM computer loaded with the water maze software. The less time a rat spent in finding the platform, the better it scored the spatial learning and memory. For spatial learning, rats were trained in water maze to remember the hidden platform for 7 consecutive days. For the first day, the rats only proceeded with one trial that started from the first quadrant. And for the following 5 days, 3 trials were performed for each rat every day, starting from the first, the second and the fourth quadrant, respectively. For the last training day (the 7 day), only one trial that started from the first quadrant's movements were performed, and total 20 trials were finished for 7 days' training. On each trial, the rat started from each quadrant by facing the wall of the pool and ended when the animal climbed on the platform. The rats were not allowed to search for the platform more than 60 seconds, after which they were guided to the platform. Through these training sessions, rats acquired spatial memory about location of the safe platform, and rats that could find the platform after training from quadrant 1‐4 in turn for 24 trials within 20 seconds were selected and randomly divided into 3 groups for the brain lateral ventricle injections, respectively, with DMSO (10 μL) or TM (50 μmol/L, 10 μL) or TM + SB (50 μmol/L, 10 μL) as mentioned above. At 24 and 48 hours after the injection, the spatial memory retention of the rats was tested in the same water maze with the searching time extended to 90 seconds. After the behaviour test, the rats were killed for the rest studies. Memory was also measured by step‐down avoidance tests which was made of electrically conductive metal fence at the bottom, around with open transparent plastic box (length × width × height: 23 × 23 × 40 cm). A wooden platform was placed on the bottom of the metal fence (length × width × height: 3.5 × 3.5 × 2.5 cm). Put the rats into the experimental device to make them be familiar with the surrounding environment for 5 minutes, respectively. And then began the stage of learning to put the rats gently on the platform, when animals jumping off the platform to the wire fence, immediately gave them electric shock (0.5 mA, for 5 seconds). After that put them back to the platform and began to record duration, if the duration reached 60 seconds that indicated rats have learned how to avoid electric shock. If the duration on the platform was <60 seconds, gave the rats electric shock again when they jumped off the platform until they could stay on the platform at least 60 seconds. Record the number of rats received shocks (mistake number) and the total time (learning time for the first time), the whole period was 5 minutes. The laboratory equipment should be cleaned with 75% alcohol after each rat was tested. When all rats were trained, following start the formal test to check the memory retention at 24 and 48 hours after DMSO or TM being injected. The rats were put on platform in this phase experiment, and the duration period and numbers of wrong were recorded within 5 minutes. 2.4 Cytoplasm–nucleus protein extraction The 250‐mg fresh rats brain or cryopreserved tissue in −80°C were taken immediately into the glass homogenizer with ice pre‐cooling, add 500 μL cytosol extraction reagent (CER, the proportion of brain tissue with CER is 1:2) in it. Triturate the brain tissue with pestle and then fluctuate homogenate them by manual operation for 20 times. Ice bath for 10 minutes and then fluctuate homogenate them for 7 times again. Take 500‐μL lysate, transfer it to the new centrifugal tube, centrifuge for 5 minutes with 800 g in 4°C circumstance. The crude product of nucleus precipitates at the end of the pipe and the supernatant is the mixture of cell membrane and cytoplasm. Transfer the supernatant to the new centrifugal tube and add membrane extraction reagent (MER, 1/10 volume of the supernatant fluid) into it. Ice bath for 5 minutes, centrifuge for 30 minutes with 10956.4 g in 4°C circumstance. Take the supernatant into the new centrifugal tube which is the cytoplasm components and the precipitation is cell membrane components including cell membranes and organelles fragments which can be suspended again with 50‐100 μL suspension buffer. Add 500 μL nuclear extraction reagent (NER) into the crude product of nucleus obtained such as the above and vibrate it to suspend again. 4000 g , 4°C centrifuge for 5 minutes, abandon the supernatant and then add 500 μL NER into it to suspend it again. Repeat the above centrifugal steps and clear the centrifugal supernatant. Add 50‐100 μL suspension buffer to suspend the precipitation again to obtain the nuclei. 2.5 Western blotting For brain samples, the hippocampus taken immediately after Morris water maze test were homogenized in buffer containing 10 mmol/L Tris–Cl, pH 7.6, 50 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L edetic acid, 1 mmol/L benzamidine, 1 mmol/L PMSF and a mixture of aprotinin, leupeptin and pepstatin A (10 μg/mL each) or obtain the cytoplasm–nucleus protein extraction as the above way. Three volumes of the homogenate for brain samples were added to one volume of the extracting buffer containing 200 mmol/L Tris–Cl, pH 7.6, 8% SDS, 40% glycerol, and the samples were boiled in water bath for 10 minutes and then followed by sonication for 15 seconds on ice. After measurement of protein concentration in the extracts using BCA kit (Pierce, Rockford, IL), a final concentration of 10% β‐mercaptoethanol and 0.05% bromophenol blue was added. The proteins in the extracts were separated by 10% SDS‐PAGE and transferred to nitrocellulose membrane. The membranes were blocked with 5% non‐fat milk dissolved in TBS‐Tween‐20 (50 mmol/L Tris–HCl, pH 7.6, 150 mmol/L NaCl, 0.2% Tween‐20) for 1 hour and probed with primary antibody (see Table 1 for detail) at 4°for overnight. Then, the blots were incubated with anti‐mouse or anti‐rabbit IgG conjugated to horseradish peroxidase (1:15 000) for 1 hour at room temperature and scanned after being washed with TBS‐Tween‐20,and the greyscale was analysed with odyssey system. 2.6 Nissl staining Nissl staining was established in 1892 by Franna Nissl, the German pathologist, with alkaline dye to discover Nissl body, and this method was widely used. The picked brain slices were pasted on the glass slide disposed by gelatine in PBS liquid, dustproof atmospheric drying. And then, 1% Toluidine blue was dropped on the brain slices, keeping 5‐10 minutes, 95% alcohol was used to differentiate and observed under a microscope at the same time until the background was clean and the Nissl body was clear. Then dehydrated with 100% alcohol for 5 minutes × 2, transparented with xylene for 5 minutes × 2 and sealed with neutral gummi, and finally analysed under a microscope and collected images. 2.7 Golgi staining After the rats (n = 3 for each group) being anaesthetized with 6% chloral hydrate (400 mg/kg), the aorta of the rat was inserted into with blunt infusion needle and perfused with 350‐500 mL (37°C) physiological saline containing 0.5% sodium nitrite into the systemic circulation quickly. When the liver became pale and the rinse become clear, continued to perfuse with fixed liquid containing 4% formaldehyde (500 mL) for about 1‐2 hours until the rat body was stiff. Then replaced the fixed liquid with mordant dyeing (500 mL, containing 5% chloral hydrate, 5% potassium dichromate, 4% formaldehyde), continued to pursue quickly for 5‐10 minutes to replace the systemic circulation stationary liquid. When flowing out thick orange liquid, slowed down the speed to 25 drop/minutes and the perfusion process of mordant dyeing liquid lasted about 3‐4 hours. The brain tissue after perfusion was taken and divided the brain into 2 parts along with the midline incision, kept the tissue containing hippocampus to 5 mm thick. Then dipped the tissue into fresh mordant dye, avoided light for 4 days and replaced the mordant dye every day. Next permutated the mordant dye with 1.5% silver nitrate solution for 3 days and avoided light and replaced a fresh silver nitrate solution daily. The brain slice (35 μm) was prepared by oscillation microtome (Germany, VT1000S, Leica). The slices were soaked in 2% potassium dichromate solution for 20 minutes and then rinsed with steaming water to clear. Then pasted the slices in 1.5% gelatine solution, dried dustproof air and then dehydrated with gradient alcohol, transparent with xylene and sealed piece with neutral gum. Finally, images were collected under a microscope to analyse. The spine numbers were measured by Image J software, and the different types of spines were analysed according to the schematic structure of the dendritic spines. 44 2.8 Immunofluorescence staining The brain sections were obtained as the immunohistochemistry staining described. The sections were incubated for 48 hours at 4°C with the first primary antibodies after being ruptured membrane and blocked with 5% BSA and then washed with PBS and incubated the second primary antibodies like described above. The sections were subsequently incubated with Rhodamine Green 488 or Red 546‐conjugated secondary antibodies (1:500) for 1 hour at 37°C. Pasted the brain sections to the glass slide and sealed them with 50% glycerine after being washed with PBS. The images were observed by a laser confocal microscope (LSM710, Zeiss, Germany), and the fluorescence images were analysed by the software affiliated. 45 , 46 2.9 Statistical analysis Data were expressed as means ± SEM and analysed using SPSS 12.0 statistical software (SPSS Inc., Chicago, Illinois, USA). Means were compared by one‐way analysis of variance (ANOVA) procedure followed by LSD's post hoc Bonferroni's tests. P values <.05 were considered as significant. 3 RESULTS 3.1 TM induces UPR independent of GSK‐3 activation and causes tau hyperphosphorylation with spatial memory deficits in rats To produce an in vivo UPR model, we infused different concentrations of ER stressor, TM, into the lateral ventricle of rats and measured the alterations of ER transmembrane protein, phosphorylated PERK (pPERK). We observed that infusion of TM at 25 μmol/L, 50 μmol/L and 75 μmol/L increased the protein level of pPERK, an ER stress marker (Figure 1 A,B). Simultaneously, we found that level of Bip, an important ER‐associated chaperon, significantly increased by TM at 50 μmol/L and 75 μmol/L but not at 25 μmol/L (Figure 1 C,D). Then, we infused the rats with 50 μmol/L of TM and measured the UPR, including pPERK, phosphorylated IRE‐1 (pIRE‐1) and phosphorylated ATF‐6 (pATF‐6) at different time‐points. The increased levels of pPERK, pIRE‐1 and pATF‐6 were detected at both 24 hours and 48 hours after the infusion (Figure 1 E,F). In our previous study, we observed that TM could activate GSK‐3β. Therefore, we studied whether simultaneous inhibition of GSK‐3 by SB216763 (SB) affects UPR. The results showed that application of SB did not rescue UPR (Figure 1 E,F). These results suggest that ventricular infusion of TM can induce UPR in rat brain independent of GSK‐3 activation. Figure 1 Tunicamycin induces UPR independent of GSK ‐3 in rats. The male SD rats (4 m old) received ventricular infusion of 25, 50 or 75 μmol/L tunicamycin ( TM , 10 μL) for 24 h (A,C), or infused with 50 μmol/L SB for 24 h and 48 h (E). The same volume of DMSO was infused as vehicle control, and the normal group (Nor) was killed without any treatment. The hippocampal extract was used for Western blotting (A,C,E) and quantitative analysis (B,D,F). The levels of the phosphorylated ER stress marker proteins as labelled except Bip were normalized against the total level, the latter and Bip were normalized against tubulin probed by DM 1A. The data were expressed as means ± SD (n = 6). ** P < .01 vs Nor, ## P < .01 vs DMSO in B; * P < .05 vs DMSO , # P < .05 vs TM (25 μm) in D; ** P < .01 vs DMSO ‐24 h, ## P < .01 vs DMSO ‐48 h in F In our previous studies, we found that TM treatment increased phosphorylated level of tau at Thr205, Thr231 and Ser396. 9 We observed the similar alternation of tau proteins in this study and SB attenuated tau phosphorylation (Figure 2 A,B). To measure the effects of UPR in spatial memory, we trained the rats for 7 consecutive days to allow remembering the hidden platform in water maze (Figure 2 C), then we injected 50 μmol/L TM (10 μL) or isasteric DMSO or TM plus SB (50 μmol/L) into the rats lateral ventricle, after 24 or 48 hours, the hippocampus‐dependent spatial memory was measured by removed the platform. Compared with the DMSO‐injected control rats that could find the platform within 20 seconds by a direct searching strategy, while injection of TM increased the latency to about 60 seconds (Figure 2 D,E). Learning and memory of the rats were also measured by step‐down avoidance tests. Compared with the DMSO vehicle control, TM treatment showed no difference of the number of errors in the training period. In the detection period during step‐down avoidance test, all the rats could not successfully avoid the risk of electric shock at 24 hours and there were no difference of latency period at 48 hours, but increased the number of errors both at 24 and 48 hours after TM injection (Figure 2 F‐I). SB rescued TM‐induced memory deficits shown by the significantly decreased latency to find the hidden platform in MWM test and decreased the number of errors in step‐down avoidance tests (D‐I). These data suggest that TM can induce memory deficits of rats. Figure 2 SB attenuates tau hyperphosphorylation and memory deficits induced by tunicamycin in rats. The rats were randomly divided into 3 groups infused, respectively, through ventricle with 50 μmol/L TM or DMSO or TM plus SB (50 μmol/L). The rats were trained in Morris water maze ( MWM ) for 7 days before DMSO , TM and TM + SB injection (C). After 24 or 48 h, the brain extract from hippocampal regions ( HP ) was used to measure the alterations of tau proteins by Western blotting (A) and quantitative analysis (B). The levels of unphosphorylated tau at Tau1 epitope and the phosphorylation level of tau at Ser396 epitope as labelled on the blot were normalized against total tau probed by Tau5 which was normalized against DM 1A (n = 6). SB could more obviously rescue the decreased Tau1 and the increased phosphorylation level of tau at Ser396 epitope after TM being injected for 48 h. Simultaneously, the MWM and step‐down avoidance tests were used to assess learning and memory capacities (D‐I). The rats had same cognitive levels during 7 days training before TM treatment (C), while injection of TM for 24 or 48 h induced memory deficits shown by the increased latency to find the hidden platform in MWM test (D,E). TM ‐injected rats used more time to learn to protect themselves from the risk of electric shock in the training period during step‐down avoidance test measured at 24 and 48 h after the injection (F). TM ‐injected rats showed no difference of the number of errors compared with the control group rats (G). TM ‐injected rats showed no difference of latency period but increased number of errors in the detection period during step‐down avoidance test measured at 24 and 48 h after the injection. SB rescued TM ‐induced memory deficits shown by the significantly decreased latency to find the hidden platform in MWM test (E). The data were expressed as mean ± SD (n = 10). * P < .05, ** P < .01 vs DMSO ‐24 h; # P < .05, ## P < .01 vs DMSO ‐ 48 h; ■P < .05 vs TM ‐24 h, ▲ P < .05 vs TM ‐48 h in B; * P < .05, ** P < .01 vs DMSO ; # P < .05 vs TM in E‐I 3.2 TM inhibits mushroom spine formation and expression of several synaptic proteins To explore the mechanisms underlying the TM‐induced spatial memory deficits, we measured spine morphology and synapse‐associated proteins. We found that number of mushroom‐type spines significantly decreased in DG (5.95 ± 1.02 vs 3.45 ± 0.98) and CA3 (3.85 ± 1.01 vs 2.42 ± 0.85) subsets but not in CA1 of the TM‐treated group, and no significant change of thin spines was detected (Figure 3 A‐F). SB could reverse the decreased mushroom‐type spines in DG and CA3 subsets. We also measured the levels of synapse‐associated proteins. The results showed that levels of synapsin 1, a synaptic vesicle protein regulating pre‐synaptic release of glutamate, and the postsynaptic associated proteins, PSD95 significantly decreased in TM group, but GluN2A and GluN2B were no obvious alteration after TM injection (Figure 3 G,H). These data suggest that TM induces impairments in hippocampal synaptic maturation. SB treatment restored PSD95 but not synapsin 1, instead it reduced level of synapsin 1. By Nissle's staining, we observed that cell number in hippocampal CA1 significantly decreased in TM group compared with control group, the decrease was not seen in DG subset, suggesting that TM induces cell death in CA1 subset and SB could reverse the cell death in CA1 subset (Figure 4 A‐D). Figure 3 Tunicamycin inhibits mushroom spine formation and expression of synaptic protein and attenuation by SB . The representative images of dendritic spines in rat hippocampal CA 1, CA 3 and DG at 48 h after TM injection (A,B). TM decreased numbers of mushroom‐type spines significantly in DG and CA 3 subsets but not in CA 1 of the TM ‐treated group, and no significant change of thin‐spines was detected (B‐F). SB could reverse the above phenomenon. The levels of synapse‐associated proteins were measured by Western blotting and quantitative analysis, normalized against tubulin probed by DM 1A (G,H). The data were expressed as mean ± SD (n = 3 for A and B, bar = 50 μm for A, bar = 2 μm for B; n = 6 for G). * P < .05, ** P < .05 vs DMSO , # P < .05 vs TM Figure 4 Tunicamycin induces cell loss in hippocampal CA 1 subset and attenuation by SB . The representative Nissl staining analysis shows temporal cortex ( TC ), frontal cortex ( FC ) and hippocampus ( HP ) after TM injection for 48 h (A). The neuronal numbers in hippocampal CA 1, CA 3, CA 4 and DG were analysed (bar = 500 μm for TC , FC and HP ; bar = 50 μm for CA 1, CA 3, CA 4 and DG ) (B,C). The data were expressed as mean ± SD (n = 6). * P < .05 vs DMSO , # P < .05 vs TM 3.3 TM treatment affects CREB phosphorylation with involvement of GSK‐3 To understand the mechanisms underlying the TM‐induced spatial memory deficit and altered synapse protein levels, we measured CREB, a crucial protein in regulating gene transcription. It was reported that GSK‐3 may be involved in CREB phosphorylation 47 ; therefore, we measured GSK‐3β using activity‐dependent antibodies. We found that the active phosphorylation of GSK‐3β at Tyr216 (pY216‐GSK‐3β) increased and the inhibitory phosphorylation of GSK‐3β at Ser9 (pS9‐GSK‐3β) decreased at 24 and 48 hours after infusion of TM (Figure 5 A,B), suggesting activation of GSK‐3β by TM. We observed that level of phosphorylated CREB at Ser133 (pS133‐CREB) was decreased in hippocampus treated with TM, while the level of phosphorylated CREB at Ser129 (pSer129‐CREB) was increased (Figure 5 C,D). These alterations could be reversed by ventricular infusion of SB, the inhibitor of GSK‐3, especially at 48 hours (Figure 5 A‐D). These data suggest that the alteration of CREB phosphorylation induced by TM may be related to the activation of GSK‐3. Figure 5 Tunicamycin treatment affects GSK ‐3 and CREB phosphorylation. After ventricular infusion of DMSO , TM at concentration of 50 μmol/L or TM plus SB ( SB , 50 μmol/L) for 24 and 48 h, GSK ‐3β levels of the hippocampal extract were measured by Western blotting (A) and quantitative analysis (B). SB treatment rescued the increased Y216‐ GSK ‐3β and decreased S9‐ GSK ‐3β induced by TM especially at 48 h. And for 48 h, SB treatment restored the levels of pS er133‐ CREB and pS er129‐ CREB measured by Western blotting (C) and quantitative analysis (D). The data were expressed as means ± SD (n = 6) * P < .05, ** P < .01 vs DMSO ‐24 h, # P < .05 vs DMSO ‐48 h, ■P < .05 vs TM ‐24 h, ▲ P < .05 vs TM ‐48 h, & P < .05 vs TM + SB ‐24 h in B. * P < .05 vs DMSO , # P < .05 vs TM in D To verify the role of GSK‐3β and CREB in TM‐induced spatial memory deficit and synapse impairments, we checked the levels of GSK‐3β and CREB both in cytoplasm fraction and in nuclear of hippocampus treated with TM. We observed that level of phosphorylated CREB at Ser133 (pS133‐CREB) was decreased in nuclear fraction and increased in cytoplasm of hippocampus treated with TM, while the level of phosphorylated CREB at Ser129 (pSer129‐CREB) in nucleus fraction was increased. Meanwhile, we observed that level of phosphorylated Y216‐GSK‐3β increased and level of phosphorylated S9‐GSK‐3β decreased in cytoplasm of hippocampus treated with TM (Figure 6 A‐C). Immunofluorescence staining data also showed that level of pSer129‐CREB significantly increased in the nucleus of cortex and hippocampus and decreased in cytoplasm, while the level of pSer133‐CREB decreased in nucleus of hippocampus and increased in cytoplasm. Simultaneous inhibition of GSK‐3 by ventricular infusion of SB216763 rescued the alteration of CREB at pSer129‐CREB and pSer133‐CREB in nucleus fraction and cytoplasm of cortex and hippocampus treated with TM. An increased co‐localization of pY216‐GSK‐3β with pSer129‐CREB was also detected in the cytoplasm fraction (Figure 6 D‐G). These data suggest that change of CREB phosphorylation induced by TM may be related to the activation of GSK‐3. Figure 6 Tunicamycin increases nuclear co‐localization of Y216‐ GSK ‐3β and pS er129‐ CREB with reduced pS er133‐ CREB . Rats were treated with TM or DMSO for 48 h and then the cytoplasmic and nuclear fractions of the hippocampus were analysed by Western blotting and quantitative analysis (A‐C). The representative immunofluorescence images show phosphorylated CREB probed by pS er133 and pS er129 (green), and phosphorylated GSK ‐3β by Y216‐ GSK ‐3β (red) in cortex and hippocampus (bar = 50 μm) (D,E). SB treatment restored the nuclear internal and external metastasis of pS er133‐ CREB and pS er129‐ CREB . The co‐localization of pY 216‐ GSK ‐3β with p‐S129‐ CREB and p‐S133‐ CREB in hippocampus were statistically measured. The data were expressed as means ± SD. The tendency of pY 216‐ GSK ‐3β with p‐S129‐ CREB and p‐S133‐ CREB in cortex was similar that in hippocampus (F,G) and the statistical graph not be showed. (n = 6) * P < .05, ** P < .01, *** P < .001 vs DMSO in B and C. * P < .05 vs DMSO , # P < .05 vs TM in F and G 3.1 TM induces UPR independent of GSK‐3 activation and causes tau hyperphosphorylation with spatial memory deficits in rats To produce an in vivo UPR model, we infused different concentrations of ER stressor, TM, into the lateral ventricle of rats and measured the alterations of ER transmembrane protein, phosphorylated PERK (pPERK). We observed that infusion of TM at 25 μmol/L, 50 μmol/L and 75 μmol/L increased the protein level of pPERK, an ER stress marker (Figure 1 A,B). Simultaneously, we found that level of Bip, an important ER‐associated chaperon, significantly increased by TM at 50 μmol/L and 75 μmol/L but not at 25 μmol/L (Figure 1 C,D). Then, we infused the rats with 50 μmol/L of TM and measured the UPR, including pPERK, phosphorylated IRE‐1 (pIRE‐1) and phosphorylated ATF‐6 (pATF‐6) at different time‐points. The increased levels of pPERK, pIRE‐1 and pATF‐6 were detected at both 24 hours and 48 hours after the infusion (Figure 1 E,F). In our previous study, we observed that TM could activate GSK‐3β. Therefore, we studied whether simultaneous inhibition of GSK‐3 by SB216763 (SB) affects UPR. The results showed that application of SB did not rescue UPR (Figure 1 E,F). These results suggest that ventricular infusion of TM can induce UPR in rat brain independent of GSK‐3 activation. Figure 1 Tunicamycin induces UPR independent of GSK ‐3 in rats. The male SD rats (4 m old) received ventricular infusion of 25, 50 or 75 μmol/L tunicamycin ( TM , 10 μL) for 24 h (A,C), or infused with 50 μmol/L SB for 24 h and 48 h (E). The same volume of DMSO was infused as vehicle control, and the normal group (Nor) was killed without any treatment. The hippocampal extract was used for Western blotting (A,C,E) and quantitative analysis (B,D,F). The levels of the phosphorylated ER stress marker proteins as labelled except Bip were normalized against the total level, the latter and Bip were normalized against tubulin probed by DM 1A. The data were expressed as means ± SD (n = 6). ** P < .01 vs Nor, ## P < .01 vs DMSO in B; * P < .05 vs DMSO , # P < .05 vs TM (25 μm) in D; ** P < .01 vs DMSO ‐24 h, ## P < .01 vs DMSO ‐48 h in F In our previous studies, we found that TM treatment increased phosphorylated level of tau at Thr205, Thr231 and Ser396. 9 We observed the similar alternation of tau proteins in this study and SB attenuated tau phosphorylation (Figure 2 A,B). To measure the effects of UPR in spatial memory, we trained the rats for 7 consecutive days to allow remembering the hidden platform in water maze (Figure 2 C), then we injected 50 μmol/L TM (10 μL) or isasteric DMSO or TM plus SB (50 μmol/L) into the rats lateral ventricle, after 24 or 48 hours, the hippocampus‐dependent spatial memory was measured by removed the platform. Compared with the DMSO‐injected control rats that could find the platform within 20 seconds by a direct searching strategy, while injection of TM increased the latency to about 60 seconds (Figure 2 D,E). Learning and memory of the rats were also measured by step‐down avoidance tests. Compared with the DMSO vehicle control, TM treatment showed no difference of the number of errors in the training period. In the detection period during step‐down avoidance test, all the rats could not successfully avoid the risk of electric shock at 24 hours and there were no difference of latency period at 48 hours, but increased the number of errors both at 24 and 48 hours after TM injection (Figure 2 F‐I). SB rescued TM‐induced memory deficits shown by the significantly decreased latency to find the hidden platform in MWM test and decreased the number of errors in step‐down avoidance tests (D‐I). These data suggest that TM can induce memory deficits of rats. Figure 2 SB attenuates tau hyperphosphorylation and memory deficits induced by tunicamycin in rats. The rats were randomly divided into 3 groups infused, respectively, through ventricle with 50 μmol/L TM or DMSO or TM plus SB (50 μmol/L). The rats were trained in Morris water maze ( MWM ) for 7 days before DMSO , TM and TM + SB injection (C). After 24 or 48 h, the brain extract from hippocampal regions ( HP ) was used to measure the alterations of tau proteins by Western blotting (A) and quantitative analysis (B). The levels of unphosphorylated tau at Tau1 epitope and the phosphorylation level of tau at Ser396 epitope as labelled on the blot were normalized against total tau probed by Tau5 which was normalized against DM 1A (n = 6). SB could more obviously rescue the decreased Tau1 and the increased phosphorylation level of tau at Ser396 epitope after TM being injected for 48 h. Simultaneously, the MWM and step‐down avoidance tests were used to assess learning and memory capacities (D‐I). The rats had same cognitive levels during 7 days training before TM treatment (C), while injection of TM for 24 or 48 h induced memory deficits shown by the increased latency to find the hidden platform in MWM test (D,E). TM ‐injected rats used more time to learn to protect themselves from the risk of electric shock in the training period during step‐down avoidance test measured at 24 and 48 h after the injection (F). TM ‐injected rats showed no difference of the number of errors compared with the control group rats (G). TM ‐injected rats showed no difference of latency period but increased number of errors in the detection period during step‐down avoidance test measured at 24 and 48 h after the injection. SB rescued TM ‐induced memory deficits shown by the significantly decreased latency to find the hidden platform in MWM test (E). The data were expressed as mean ± SD (n = 10). * P < .05, ** P < .01 vs DMSO ‐24 h; # P < .05, ## P < .01 vs DMSO ‐ 48 h; ■P < .05 vs TM ‐24 h, ▲ P < .05 vs TM ‐48 h in B; * P < .05, ** P < .01 vs DMSO ; # P < .05 vs TM in E‐I 3.2 TM inhibits mushroom spine formation and expression of several synaptic proteins To explore the mechanisms underlying the TM‐induced spatial memory deficits, we measured spine morphology and synapse‐associated proteins. We found that number of mushroom‐type spines significantly decreased in DG (5.95 ± 1.02 vs 3.45 ± 0.98) and CA3 (3.85 ± 1.01 vs 2.42 ± 0.85) subsets but not in CA1 of the TM‐treated group, and no significant change of thin spines was detected (Figure 3 A‐F). SB could reverse the decreased mushroom‐type spines in DG and CA3 subsets. We also measured the levels of synapse‐associated proteins. The results showed that levels of synapsin 1, a synaptic vesicle protein regulating pre‐synaptic release of glutamate, and the postsynaptic associated proteins, PSD95 significantly decreased in TM group, but GluN2A and GluN2B were no obvious alteration after TM injection (Figure 3 G,H). These data suggest that TM induces impairments in hippocampal synaptic maturation. SB treatment restored PSD95 but not synapsin 1, instead it reduced level of synapsin 1. By Nissle's staining, we observed that cell number in hippocampal CA1 significantly decreased in TM group compared with control group, the decrease was not seen in DG subset, suggesting that TM induces cell death in CA1 subset and SB could reverse the cell death in CA1 subset (Figure 4 A‐D). Figure 3 Tunicamycin inhibits mushroom spine formation and expression of synaptic protein and attenuation by SB . The representative images of dendritic spines in rat hippocampal CA 1, CA 3 and DG at 48 h after TM injection (A,B). TM decreased numbers of mushroom‐type spines significantly in DG and CA 3 subsets but not in CA 1 of the TM ‐treated group, and no significant change of thin‐spines was detected (B‐F). SB could reverse the above phenomenon. The levels of synapse‐associated proteins were measured by Western blotting and quantitative analysis, normalized against tubulin probed by DM 1A (G,H). The data were expressed as mean ± SD (n = 3 for A and B, bar = 50 μm for A, bar = 2 μm for B; n = 6 for G). * P < .05, ** P < .05 vs DMSO , # P < .05 vs TM Figure 4 Tunicamycin induces cell loss in hippocampal CA 1 subset and attenuation by SB . The representative Nissl staining analysis shows temporal cortex ( TC ), frontal cortex ( FC ) and hippocampus ( HP ) after TM injection for 48 h (A). The neuronal numbers in hippocampal CA 1, CA 3, CA 4 and DG were analysed (bar = 500 μm for TC , FC and HP ; bar = 50 μm for CA 1, CA 3, CA 4 and DG ) (B,C). The data were expressed as mean ± SD (n = 6). * P < .05 vs DMSO , # P < .05 vs TM 3.3 TM treatment affects CREB phosphorylation with involvement of GSK‐3 To understand the mechanisms underlying the TM‐induced spatial memory deficit and altered synapse protein levels, we measured CREB, a crucial protein in regulating gene transcription. It was reported that GSK‐3 may be involved in CREB phosphorylation 47 ; therefore, we measured GSK‐3β using activity‐dependent antibodies. We found that the active phosphorylation of GSK‐3β at Tyr216 (pY216‐GSK‐3β) increased and the inhibitory phosphorylation of GSK‐3β at Ser9 (pS9‐GSK‐3β) decreased at 24 and 48 hours after infusion of TM (Figure 5 A,B), suggesting activation of GSK‐3β by TM. We observed that level of phosphorylated CREB at Ser133 (pS133‐CREB) was decreased in hippocampus treated with TM, while the level of phosphorylated CREB at Ser129 (pSer129‐CREB) was increased (Figure 5 C,D). These alterations could be reversed by ventricular infusion of SB, the inhibitor of GSK‐3, especially at 48 hours (Figure 5 A‐D). These data suggest that the alteration of CREB phosphorylation induced by TM may be related to the activation of GSK‐3. Figure 5 Tunicamycin treatment affects GSK ‐3 and CREB phosphorylation. After ventricular infusion of DMSO , TM at concentration of 50 μmol/L or TM plus SB ( SB , 50 μmol/L) for 24 and 48 h, GSK ‐3β levels of the hippocampal extract were measured by Western blotting (A) and quantitative analysis (B). SB treatment rescued the increased Y216‐ GSK ‐3β and decreased S9‐ GSK ‐3β induced by TM especially at 48 h. And for 48 h, SB treatment restored the levels of pS er133‐ CREB and pS er129‐ CREB measured by Western blotting (C) and quantitative analysis (D). The data were expressed as means ± SD (n = 6) * P < .05, ** P < .01 vs DMSO ‐24 h, # P < .05 vs DMSO ‐48 h, ■P < .05 vs TM ‐24 h, ▲ P < .05 vs TM ‐48 h, & P < .05 vs TM + SB ‐24 h in B. * P < .05 vs DMSO , # P < .05 vs TM in D To verify the role of GSK‐3β and CREB in TM‐induced spatial memory deficit and synapse impairments, we checked the levels of GSK‐3β and CREB both in cytoplasm fraction and in nuclear of hippocampus treated with TM. We observed that level of phosphorylated CREB at Ser133 (pS133‐CREB) was decreased in nuclear fraction and increased in cytoplasm of hippocampus treated with TM, while the level of phosphorylated CREB at Ser129 (pSer129‐CREB) in nucleus fraction was increased. Meanwhile, we observed that level of phosphorylated Y216‐GSK‐3β increased and level of phosphorylated S9‐GSK‐3β decreased in cytoplasm of hippocampus treated with TM (Figure 6 A‐C). Immunofluorescence staining data also showed that level of pSer129‐CREB significantly increased in the nucleus of cortex and hippocampus and decreased in cytoplasm, while the level of pSer133‐CREB decreased in nucleus of hippocampus and increased in cytoplasm. Simultaneous inhibition of GSK‐3 by ventricular infusion of SB216763 rescued the alteration of CREB at pSer129‐CREB and pSer133‐CREB in nucleus fraction and cytoplasm of cortex and hippocampus treated with TM. An increased co‐localization of pY216‐GSK‐3β with pSer129‐CREB was also detected in the cytoplasm fraction (Figure 6 D‐G). These data suggest that change of CREB phosphorylation induced by TM may be related to the activation of GSK‐3. Figure 6 Tunicamycin increases nuclear co‐localization of Y216‐ GSK ‐3β and pS er129‐ CREB with reduced pS er133‐ CREB . Rats were treated with TM or DMSO for 48 h and then the cytoplasmic and nuclear fractions of the hippocampus were analysed by Western blotting and quantitative analysis (A‐C). The representative immunofluorescence images show phosphorylated CREB probed by pS er133 and pS er129 (green), and phosphorylated GSK ‐3β by Y216‐ GSK ‐3β (red) in cortex and hippocampus (bar = 50 μm) (D,E). SB treatment restored the nuclear internal and external metastasis of pS er133‐ CREB and pS er129‐ CREB . The co‐localization of pY 216‐ GSK ‐3β with p‐S129‐ CREB and p‐S133‐ CREB in hippocampus were statistically measured. The data were expressed as means ± SD. The tendency of pY 216‐ GSK ‐3β with p‐S129‐ CREB and p‐S133‐ CREB in cortex was similar that in hippocampus (F,G) and the statistical graph not be showed. (n = 6) * P < .05, ** P < .01, *** P < .001 vs DMSO in B and C. * P < .05 vs DMSO , # P < .05 vs TM in F and G 4 DISCUSSION In the AD brains, the immunoreactivity of the ER stress markers, such as pPERK, eIF2α and IRE‐1α, was observed in hippocampal neurons associated with granulovacuolar degeneration, and the pPERK‐immunoreactive neurons were increased. 21 Moreover, ER stress features are prominent in the brain of AD patients but not in Prion diseases, 22 suggesting a specific role of ER stress in the pathophysiological process of AD. Many other evidence also suggests that ER dysfunction is closely related to AD. For instance, pPERK immunoreactivity was most abundant in the neurons with diffuse localization of the phosphorylated tau proteins. 22 Exogenous Aβ can induce ER stress signalling pathways directly through Bip in cell culture. Furthermore, mutation in presenilin‐1 (PS1) gene is one of the most important factors of familial AD and the mutation of PS1 appears as unfolding protein in the ER. In addition, PS2 can be up‐regulated in sporadic familial AD which can inhibit UPR. These studies suggest a specific role of ER stress in the pathological process of AD. In our recent studies, we observed that constant illumination could induce tau hyperphosphorylation, memory deficits and imbalance of kinases/phosphatases with ER damage. 48 Rats brain treated with TM, an ER stress inducer, could significantly increase the phosphorylated tau. 9 , 38 These studies indicate a crucial role of ER stress in the AD‐like tau pathology and behavioural abnormalities. However, we are puzzled that whether and how ER stresses induce behavioural abnormalities. Whether the level of memory‐related kinase or other molecules, such as GSK‐3β or CREB, is also changed and what is the possible relation between them? Which parameter(s) is activated by ER stress and is responsible for the behavioural abnormalities? To address these questions, we firstly produced an in vivo ER stress model by brain ventricular infusion of TM at different concentrations by measuring the increased ER transmembrane protein pPERK and Bip. Then, 50 μmol/L TM was selected to infuse rats for 24 and 48 hours, and elevation of 3 ER transmembrane proteins, including pPERK, pIRE‐1 and pATF‐6, was observed and the elevation was associated with memory deficits, suggesting that UPR could induce memory deficits in rats. When treated with TM plus SB, the levels of pPERK, pIRE‐1 and pATF‐6 had no alteration compared with the group treated with TM. These data indicate that inhibition of GSK‐3 by ventricular infusion of SB does not significantly affect TM‐induced UPR. Although TM treatment can induce ER stress with AD‐like tau hyperphosphorylation, we have to note that ER stress seen in the AD brains is a chronic process while TM treatment used in the current study is acute. Tau is a major microtubule‐associated protein which stabilizes the neuronal cytoskeleton. Hyperphosphorylated tau which is incompetent in microtubules binding and stabilizing has reported to aggregate into filaments and ultimately lead to dysfunction of synapses, degeneration of neurons and cognitive impairment. Tau reduction that can block Aβ‐ and excitotoxin‐induced neuronal dysfunction has been represented to be an effective strategy for treating Alzheimer's disease and related conditions. 49 In our previous study, we also demonstrated that UPR could induce increasing tau hyperphosphorylation in different brain regions. 9 So we speculated the spatial memory deficit induced by TM was related to hyperphosphorylated tau. Simultaneously, we observed the number of neural cell in CA1 decreased after TM treatment, which suggests that the decrease of the neuron number in hippocampus CA1 may contribute to the learning and memory impairments induced by TM. Previous study has suggested that neurons in CA1 were more vulnerable to the stresses. 50 Meanwhile, the dendritic spines in DG and CA3 subsets also decreased accompanied with the mushroom type of dendritic spines of hippocampus. The mushroom type of dendritic spines has been reported to be closely related to memory, and its plastic was limited. Moreover, the postsynaptic associated proteins, PSD95 and synapsin 1 significantly decreased after TM injection and SB could rescue the decrease in PSD95 but not synapsin 1; furthermore, it decreased the level of synapsin 1. The mechanism may deserve further investigation. GSK‐3β is a key kinase that plays a crucial role in AD‐like tau hyperphosphorylation. 51 , 52 GSK‐3β activation or conditionally overexpressed GSK‐3β has been previously reported to cause spatial memory deficits in animals and inhibiting GSK‐3β could revise AD‐like cognitive deficit. 43 , 53 , 54 An in vitro study also shows that GSK‐3β is activated during ER stress. 38 However, it is still not understood whether and how GSK‐3β plays an in vivo role in ER stress‐induced tau phosphorylation and cognitive alterations. In this and our previous studies, we showed that UPR induced by TM and activated GSK‐3β that resulted in tau hyperphosphorylation in vivo and impaired spatial memory of rats. Inhibiting GSK‐3β by GSK‐3β inhibitor SB216763 could reverse the spatial memory detentions induced by ventricle brain injection TM. In addition, studies proved that GSK‐3β could mediate phosphorylation of CREB, but its function and the mechanism was still not clear. Jason L. et al found that inhibitor of GSK‐3 could reduce the expression of fluorescein of RAW‐CRE cell treated with water toxin, which mediated by CREB. Simultaneously, they found water toxin did not activate the level of PKA‐dependent phosphorylation of CREB at Ser133 but increased the level of phosphorylation of CREB at Ser129. 55 Some studies have shown that GSK‐3β could make CREB phosphorylate at Ser129. But there were conflicting views on CREB phosphorylation at Ser129. Some believe that phosphorylation at Ser129 causes the trans‐activation of GAL4‐CREB fusion protein and then promote the expression of syntrophic transcription factor and gene. 56 , 57 Some others reported that activating GSK‐3 and/or increasing the level of pS129‐CREB inhibit the transcription activity of CREB by decreasing the bonding affinity of CREB and DNA. 58 , 59 , 60 In the present study, we found that TM could active GSK‐3β and increase the level of CREB phosphorylation at Ser129 in hippocampus, consequently resulted in a fall in learning and memory ability relevant CREB. At the same time, we found that TM could induce the increasing CREB phosphorylation at Ser129 locus in nucleus and CREB phosphorylation at Ser133 locus in cytoplasm of cortex and hippocampus, which maybe relate to CREB transfer both inside and outside the nuclear induced by TM and then regulate its downstream target genes. Taken together, we find in the present study that TM‐induced UPR causes spatial memory deficits and synapse impairments with activation of GSK‐3. Simultaneous inhibition of GSK‐3 improves spatial memory and synaptic plasticity with mechanisms involving CREB phosphorylation at Ser129 and Ser133.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743019/

Corruption of Innate Immunity by Bacterial Proteases

The innate immune system of the human body has developed numerous mechanisms to control endogenous and exogenous bacteria and thus prevent infections by these microorganisms. These mechanisms range from physical barriers such as the skin or mucosal epithelium to a sophisticated array of molecules and cells that function to suppress or prevent bacterial infection. Many bacteria express a variety of proteases, ranging from nonspecific and powerful enzymes that degrade many proteins involved in innate immunity to proteases that are extremely precise and specific in their mode of action. Here we have assembled a comprehensive picture of how bacterial proteases affect the host's innate immune system to gain advantage and cause infection. This picture is far from being complete since the numbers of mechanisms utilized are as astonishing as they are diverse, ranging from degradation of molecules vital to innate immune mechanisms to subversion of the mechanisms to allow the bacterium to hide from the system or take advantage of it. It is vital that such mechanisms are elucidated to allow strategies to be developed to aid the innate immune system in controlling bacterial infections.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595644/

Acinetobacter baumannii can use multiple siderophores for iron acquisition, but only acinetobactin is required for virulence

Acinetobacter baumannii is an emerging pathogen that poses a global health threat due to a lack of therapeutic options for treating drug-resistant strains. In addition to acquiring resistance to last-resort antibiotics, the success of A . baumannii is partially due to its ability to effectively compete with the host for essential metals. Iron is fundamental in shaping host-pathogen interactions, where the host restricts availability of this nutrient in an effort to curtail bacterial proliferation. To circumvent restriction, pathogens possess numerous mechanisms to obtain iron, including through the use of iron-scavenging siderophores. A . baumannii elaborates up to ten distinct siderophores, encoded from three different loci: acinetobactin and pre-acinetobactin (collectively, acinetobactin), baumannoferrins A and B, and fimsbactins A-F. The expression of multiple siderophores is common amongst bacterial pathogens and often linked to virulence, yet the collective contribution of these siderophores to A . baumannii survival and pathogenesis has not been investigated. Here we begin dissecting functional redundancy in the siderophore-based iron acquisition pathways of A . baumannii . Excess iron inhibits overall siderophore production by the bacterium, and the siderophore-associated loci are uniformly upregulated during iron restriction in vitro and in vivo . Further, disrupting all of the siderophore biosynthetic pathways is necessary to drastically reduce total siderophore production by A . baumannii , together suggesting a high degree of functional redundancy between the metabolites. By contrast, inactivation of acinetobactin biosynthesis alone impairs growth on human serum, transferrin, and lactoferrin, and severely attenuates survival of A . baumannii in a murine bacteremia model. These results suggest that whilst A . baumannii synthesizes multiple iron chelators, acinetobactin is critical to supporting growth of the pathogen on host iron sources. Given the acinetobactin locus is highly conserved and required for virulence of A . baumannii , designing therapeutics targeting the biosynthesis and/or transport of this siderophore may represent an effective means of combating this pathogen. Introduction Acinetobacter baumannii is an opportunistic pathogen that is capable of causing a wide variety of diseases ranging from burn, wound, and urinary tract infections to more serious conditions such as ventilator-associated pneumonia and sepsis [ 1 ]. Initially regarded as a relatively innocuous nosocomial pathogen, A . baumannii has gained increased notoriety through the appearance of severe community-acquired infections [ 2 – 4 ], and concerningly, through the rapid emergence of multidrug-resistance [ 5 – 8 ]. As a result, the World Health Organization has placed A . baumannii at the top of its list of bacteria urgently requiring research and development into novel therapeutic approaches, designating it as a priority "critical" pathogen [ 9 ]. More recently, the Centers for Disease Control increased the threat level categorization of carbapenem-resistant A . baumannii from serious to urgent, due in part to a lack of drugs in the development pipeline [ 10 ]. Unfortunately, little is known about the factors that contribute to the survival and proliferation of A . baumannii within the host, and this knowledge is necessary for the informed design of new treatment options. The success of A . baumannii as an emerging pathogen is not thought to be due to the evolution of traditional virulence factors, like toxins, but rather through a strategy referred to as "persist and resist" [ 11 ]. In addition to avoiding antibiotic-mediated killing, A . baumannii endures a wide variety of environmental insults including desiccation [ 12 – 14 ], oxidative stress [ 15 – 18 ], and micronutrient limitation [ 19 – 21 ]. Iron is one trace element that is essential to the vertebrate host and nearly all bacterial pathogens [ 22 ]. However, the concentration of free iron at the host-pathogen interface falls orders of magnitude below what is required to support microbial growth, due both to the formation of insoluble ferric oxyhydroxide precipitates under physiological conditions, and to the host maintaining stringent control over the availability of iron [ 23 ]. The withholding of metals, such as iron, to curtail bacterial growth in the host is a facet of the innate immune response referred to as "nutritional immunity" [ 24 – 26 ]. Host mechanisms of iron-withholding include maintaining it in complex with heme in hemoproteins, reducing ferroportin expression to limit its release from macrophages, and sequestering it within glycoproteins such as transferrin and lactoferrin, found predominantly in the blood and bodily secretions, respectively [ 27 ]. As such, accessing iron is perhaps one of the most formidable challenges faced by invading pathogens and is required for replication within the host. To overcome the barrier imposed by nutritional immunity, bacteria express a multitude of strategies to obtain metals. Broadly, the mechanisms of bacterial iron acquisition include capturing and extracting heme-iron from hemoproteins, expressing transporters for the uptake of free ferrous iron, and stealing iron from transferrin and lactoferrin by way of surface-bound receptor proteins and/or through secretion of small iron-binding molecules known as siderophores [ 22 ]. Many pathogens produce more than one type of siderophore, which are characterized based on the moieties used to coordinate ferric iron: catecholate, hydroxamate, and α-hydroxycarboxylate [ 28 , 29 ]. A fourth class includes siderophores possessing more than one of the aforementioned iron-coordinating groups and thus is referred to as "mixed-type" [ 28 ]. At least eight gene clusters have been identified to function in iron acquisition in A . baumannii , with the number of clusters present often differing between strains [ 30 – 32 ]. These clusters include a conserved ferrous iron acquisition system ( feoABC ) [ 33 , 34 ], two heme uptake loci (heme uptake cluster 1, and hemO cluster) [ 35 ], and five clusters involved in endogenous siderophore biosynthesis and utilization [ 30 , 31 , 36 , 37 ]. Of the siderophore biosynthetic clusters, one consists of two genes, entA and entB , which are used in the synthesis of 2,3-dihydroxybenzoic acid, a siderophore precursor molecule and weak iron chelator [ 31 ]. Another cluster, found only in A . baumannii strain 8399 is involved in the synthesis of an uncharacterized catecholate siderophore [ 36 , 37 ]. The remaining clusters have been more extensively investigated and encode for as many as ten structurally distinct siderophores, expressed from three loci and include acinetobactin and pre-acinetobactin [ 38 – 41 ], baumannoferrins A and B [ 42 ], and fimsbactins A through F [ 43 ] ( Fig 1 ). 10.1371/journal.ppat.1008995.g001 Fig 1 Genetic loci associated with siderophore biosynthesis and transport in A . baumannii ATCC 17978. Gene clusters for acinetobactin (A), baumannoferrin (B), and fimsbactins (C) are shown. Genes for the reductive release of iron from the siderophore, biosynthesis, import, export, and regulation are shown in the colors indicated in the corresponding legend. Putative Fur boxes are represented by black arrows, and the scale bar for each panel represents 1 kb. Key biosynthetic genes that were disrupted to elucidate siderophore function in this study are highlighted with thick black borders. A schematic highlighting the known and predicted siderophore biosynthetic and transport proteins in A . baumannii ATCC 17978, using the same color scheme as in A-C (D). Acinetobactin is often referred to as being the major siderophore produced by A . baumannii , largely due to the high degree of conservation of the encoding locus amongst Acinetobacter clinical isolates [ 30 , 31 , 44 ]. The siderophore exists as two pH-dependent isomers, pre-acinetobactin which is released by the bacteria and is prevalent under acidic conditions, and acinetobactin which is favored under neutral and basic conditions [ 38 , 39 , 45 ]. All of the genes required for the biosynthesis ( basA-J ), efflux ( barAB ) and uptake ( bauA-E ) of pre-acinetobactin and acinetobactin (herein collectively referred to as acinetobactin) are encoded from the same locus, with the exception of an entA homologue, found elsewhere in the chromosome ( Fig 1A and 1D ; S1 and S3 Tables [ 41 ]). Expression of acinetobactin is required for virulence of A . baumannii strain ATCC 19606 in vertebrate and invertebrate models of infection [ 41 , 46 ]. It is important to note, however, that acinetobactin-mediated iron acquisition is the only high affinity iron uptake system that is functional in ATCC 19606 [ 41 , 47 ], and thus the importance of this siderophore in the context of multiple iron utilization systems is unknown. Indeed, the prevalence of at least two siderophore-encoding loci amongst clinical A . baumannii isolates highlights the importance of elucidating their individual contributions to infection [ 30 ]. In addition to acinetobactin, in silico analyses predict that most A . baumannii strains also express two hydroxamate-type siderophores, baumannoferrins A and B [ 30 , 31 , 42 ]. Twelve genes are thought to be involved in the biosynthesis ( bfnA , B , D , E , G , I and L ), transport ( bfnC , H , J , and K ), and utilization ( bfnF ) of the baumannoferrins and they are confined to a single locus ( Fig 1B and 1D ; S2 Table [ 42 ]). The bfn gene cluster bears homology to other loci associated with the nonribosomal peptide synthase (NRPS)-independent assembly of hydroxamate or carboxylate-type siderophores [ 42 , 48 ]. Purification and characterization of the baumannoferrins was performed using A . baumannii strain AYE [ 42 ], a commonly used clinical isolate which does not produce acinetobactin or other siderophores [ 41 , 49 ]. Structural elucidation revealed that baumannoferrins A and B differ only by one double-bond, but the significance of this is unknown [ 42 ]. Both baumannoferrins are able to chelate iron and facilitate growth of A . baumannii when supplied as an exogenous iron source [ 42 , 50 ]. Notably, A . baumannii strain AYE proliferates robustly under iron limitation [ 42 ], and is virulent in Galleria mellonella and mouse models of infection [ 51 , 52 ], suggesting that the expression of the baumannoferrins may play an important but underappreciated role in the survival and pathogenicity of A . baumannii . Unlike with acinetobactin and baumannoferrin, whose loci are broadly represented in A . baumannii strains, the locus for fimsbactins biosynthesis and transport only appears to be present in a small fraction of sequenced A . baumannii isolates (~2%) [ 30 , 31 , 43 , 53 ]. Although the siderophores were initially isolated from the non-pathogenic species Acinetobacter baylyi [ 43 ], the fimsbactins cluster has been identified in pathogenic Acinetobacter isolates, including A . baumannii strain ATCC 17978 which is a commonly used laboratory strain that was initially isolated from a case of fatal meningitis [ 54 ], and AbPK1 which was identified as the causative agent of a deadly outbreak of pneumonia in sheep [ 53 ]. In the aforementioned species, the fimsbactins locus (( fbsA-Q ) Fig 1C ; S3 Table ) is flanked by transposases, suggesting that it may have been acquired by horizontal gene transfer [ 31 ]. Like acinetobactin, the fimsbactins A-F are mixed catechol-hydroxamate type siderophores, where fimsbactin A represents the most abundant product of synthesis pathway, and fimsbactins B through F are likely biosynthetic intermediates or shunt products [ 43 ]. Given their structural similarity, it is perhaps not surprising that genes within the fimsbactins pathway ( fbsB , fbsC , and fbsH ) appear to be functionally redundant with those in the acinetobactin pathway (( basJ , basF , and basE , respectively) Fig 1D ; S1 and S3 Tables [ 41 ]). Further, the sole entA homologue in A . baumannii strain ATCC 17978, required for both acinetobactin and fimsbactins production, appears to be located in the fimsbactins biosynthetic cluster ( fbsD ), and the siderophores are thought to compete for the same periplasmic binding protein [ 41 , 55 ]. The high degree of functional redundancy not only between acinetobactin and fimsbactins biosynthesis, but between the siderophore biosynthetic pathways overall in A . baumannii has confounded the ability to elucidate the individual function of the siderophores. Indeed, whilst purified fimsbactins A and B are capable of supporting the iron-dependent growth of A . baumannii [ 55 , 56 ], it is not known how endogenous expression of this siderophore influences survival of the pathogen in vitro or in vivo . The expression of multiple pathways for the synthesis and utilization of siderophores is a common feature of bacterial pathogens, although the evolutionary reasons for having these seemingly redundant iron acquisition systems is not always known. With A . baumannii , the functional characterization of siderophores has historically been assessed using strains that inherently express only one siderophore, where determinations of overall importance to A . baumannii survival and pathogenesis can be difficult to make. In this study, we sought to address the complexity of A . baumannii siderophore-based iron acquisition systems by using a strain that encodes for acinetobactin, baumannoferrin, and fimsbactins [ 30 , 31 ], but does not utilize heme as an iron source [ 35 ]. A panel of A . baumannii ATCC 17978 mutants was generated where one, two, or all three siderophore biosynthetic pathways were genetically inactivated, such that the contributions of the siderophores could be assessed, both independently and in combination. The siderophores were found to be largely functionally redundant in their iron chelation ability in vitro , where disrupting all three biosynthetic pathways was required to substantially reduce total siderophore activity and to abolish growth under iron restriction. These findings were consistent with the observation that all three pathways are upregulated during iron-restriction in vitro and in the metal-restricted host. By contrast, disruption to acinetobactin biosynthesis alone was sufficient to attenuate growth on human serum, transferrin, or lactoferrin as a sole iron source. Strikingly, the acinetobactin biosynthetic mutant was severely attenuated for survival in the murine host, where reduced bacterial burdens were recovered from every major organ during infection. However, in the same model of murine bacteremia, the baumannoferrin and fimsbactins biosynthetic mutants did not exhibit a defect in survival, and a siderophore-deficient strain was no more attenuated for virulence than the acinetobactin mutant alone. Together these results suggest that whilst all three siderophores contribute to iron acquisition by A . baumannii , acinetobactin is the major siderophore required by this pathogen in vivo due to its ability to mobilize iron from host sources and its requirement for survival in a murine model of bacteremia. Results Acinetobacter baumannii siderophore biosynthesis and transport are upregulated in response to iron limitation A . baumannii strain ATCC 17978 encodes for ten structurally distinct siderophores encoded from three different biosynthetic loci ( Fig 1 ; S1 , S2 and S3 Tables). We previously observed that expression of the loci encoding for the biosynthesis and transport of acinetobactin, baumannoferrin, and fimsbactins is upregulated in the presence of the multi-metal sequestering innate immune protein, calprotectin [ 57 ]. Given the apparent functional redundancies in the siderophore pathways of A . baumannii , we hypothesized that these systems are differentially regulated, thus allowing for expression in response to distinct environmental cues, such as differences in the availability of essential transition metals over time. To address this, wild-type (WT) A . baumannii was grown in metal-chelated Tris minimal succinate media (chelex-treated; cTMS) with and without the addition of exogenous iron or zinc. Following 4 or 12 hours (h) growth, RNA was extracted and the expression of various siderophore-associated transcripts from each of the three loci was assessed by quantitative reverse transcription PCR (qRT-PCR). Consistent with identification of putative Fur boxes upstream of many genes or operons within these loci ( Fig 1A and 1B, and 1C and reference [ 31 ]), key biosynthetic, transport, and regulatory genes in all three siderophore-associated loci are strongly upregulated in iron-deplete versus replete conditions ( Fig 2A and 2B ). By contrast, and with the exception of bfnF , which encodes a putative oxidoreductase thought to release iron from the siderophore, the change in gene expression in zinc-deplete versus -replete conditions is 10 to 1000-fold lower than that observed with iron ( Fig 2C and 2D ), suggesting that the expression of genes encoding for siderophore biosynthesis and transport in A . baumannii is predominantly iron-regulated. Notably, gene expression under zinc limitation increased between 4 and 12 h, suggesting that zinc may play a role in the regulation of the siderophore-associated loci during prolonged metal restriction. To determine if these genes are directly regulated by the zinc uptake regulator, Zur, we performed qRT-PCR on a representative gene from each locus in WT A . baumannii and a Δ zur mutant [ 58 ]. Additionally, we assessed whether Zur can directly regulate fur by similarly looking at expression of this transcriptional regulator in the zur mutant. We found that although the expression of fur was unaltered in Δ zur , basA and bfnA were slightly but significantly upregulated in this background ( S1 Fig ). Together these results suggest that whilst Zur does not appear to directly regulate fur in A . baumannii , it may play a small but unappreciated role in the expression of known fur -regulated genes. 10.1371/journal.ppat.1008995.g002 Fig 2 Siderophore biosynthetic and transport genes are upregulated in metal deplete conditions. WT A . baumannii was grown in metal-restricted media with or without the addition of exogenous iron or zinc. RNA was extracted and transcriptional changes in the expression of siderophore-associated genes were assessed by qRT-PCR in iron-deplete vs. replete conditions at 4 (A) and 12 h (B), and zinc-deplete vs. replete at 4 (C) and 12 (D) conditions, where expression was normalized to the expression of rpoB . * p < 0.05 and ** p < 0.01, as determined by Student's t test relative to a hypothetical value of 1. Data are the means combined from two independent experiments, each with three biological replicates. To determine if the transcriptional changes observed above translate to overall differences in siderophore production by A . baumannii , WT bacteria were grown in cTMS with and without the addition of exogenous metals. Following 12 h growth, assessment of siderophore activity in the A . baumannii supernatants was assessed by Chrome Azurol S (CAS) assay. CAS is an iron-binding dye where mobilization of iron from CAS to a chelator within the media can be detected through a colorimetric change of the dye from blue to orange [ 59 , 60 ]. Consistent with the impact of iron on gene transcription, the addition of iron to A . baumannii cultures strongly represses overall siderophore production by the bacteria ( Fig 3 ), whereas the addition of zinc does not appear to impact this activity. These observations indicate that derepression of siderophore-associated genes during zinc limitation may not lead to increased siderophore production overall, and that acinetobactin, baumannoferrin, and fimsbactins biosynthesis and utilization in A . baumannii is primarily influenced by iron availability. It is possible, however, that the semi-quantitative nature of assessing total siderophore activity by CAS assay is not sensitive enough to detect subtle changes in siderophore production upon zinc limitation over time, as the accumulation of siderophores and/or siderophore biosynthetic enzymes may mask these effects. 10.1371/journal.ppat.1008995.g003 Fig 3 Iron, but not zinc, represses the overall siderophore activity of A . baumannii . Chrome Azurol S (CAS) assays to assess for total siderophore activity were performed on the spent culture supernatants of WT A . baumannii grown in cTMS media for 12 h with or without the addition of exogenous metals, as indicated. The colorimetric changes observed in the CAS assay are shown, where blue indicates an absence of siderophore activity and orange indicates that an iron chelator capable of mobilizing iron from CAS is present. Total siderophore activity is expressed as the percent activity of WT A . baumannii grown in the absence of exogenously added metals. **** p < 0.0001, as determined by one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons test. Data are the means of three biological replicates and are representative of three independent assays. A . baumannii siderophores exhibit functional redundancy in vitro To begin identifying the contribution of acinetobactin, baumannoferrin, and fimsbactins production to A . baumannii iron acquisition, key genes in each of the siderophore biosynthetic pathways were disrupted using recombineering [ 61 ]. The details of all strains employed in this study can be found in Materials and Methods, and the genes disrupted are highlighted in Fig 1 . Genes targeted for inactivation were selected based on predicted function, as well as a lack of any potentially redundant homologues present elsewhere in the A . baumannii ATCC 17978 genome. The disrupted genes include basG , encoding a putative histidine decarboxylase predicted to synthesize histamine as an essential precursor molecule to acinetobactin biosynthesis [ 44 , 62 ], bfnL , encoding a putative acetyltransferase predicted to facilitate the conversion of N 3 -hydroxy-1,3-diaminopropane to N 3 -decanoyl-hydroxy-1,3-diaminopropane as an early step in baumannoferrin biosynthesis [ 42 ], and fbsE , encoding a putative nonribosomal peptide synthase (NRPS) and the first part of the multi-modular unit required to assemble fimsbactins [ 43 ]. To determine if disruption to any one siderophore biosynthetic pathway impacts the ability of A . baumannii to chelate iron, WT A . baumannii and the strains inactivated for acinetobactin (Δ basG ), baumannoferrin (Δ bfnL ), or fimsbactins (Δ fbsE ) biosynthesis were grown in cTMS for 12 h and CAS assays were performed on the culture supernatants. Aside from a small but reproducible reduction in siderophore activity in the Δ fbsE mutant, inactivating one siderophore pathway in A . baumannii does not drastically impact total siderophore activity ( Fig 4 ). These results suggest that functional redundancy exists between the siderophore-based iron chelation activities of A . baumannii in vitro . 10.1371/journal.ppat.1008995.g004 Fig 4 Disrupting a single siderophore biosynthetic pathway does not drastically impact overall siderophore activity in A . baumannii . Wild-type (WT) A . baumannii and its isogenic acinetobactin (Δ basG ), baumannoferrin (Δ bfnL) and fimsbactins (Δ fbsE ) biosynthetic mutants were grown for 12 h in cTMS, and CAS assays to assess overall siderophore activity were performed on the spent culture supernatants. Total siderophore activity is expressed as the percent activity of WT A . baumannii , where data are the means of three biological replicates and are representative of three independent assays. ** p < 0.01, as determined by one-way analysis ANOVA with Dunnett's multiple comparisons test. Acinetobactin is required for maximal growth on host iron sources In addition to sequestering small amounts of free ferric iron from the extracellular milieu, siderophores are capable of pirating iron from host glycoproteins such as transferrin and lactoferrin. To determine if acinetobactin, baumannoferrin, or fimsbactins are essential for iron acquisition from host iron sources, WT A . baumannii and the three siderophore biosynthetic mutants, Δ basG , Δ bfnL , and Δ fbsE , were grown in cTMS media with 20% human serum as the sole iron source. Bacterial growth was monitored by optical density (OD 6oonm ) over 20 hours. In contrast to the results of the CAS assay indicating that none of the mutations strongly impact overall siderophore production, the Δ basG mutant was found to be impaired for growth in serum relative to WT A . baumannii and the Δ bfnL and Δ fbsE mutants ( Fig 5A ). The defect in growth of Δ basG in serum is likely due to reduced acquisition of iron from transferrin, as this acinetobactin-deficient mutant is similarly impaired for growth on human transferrin as a sole iron source ( Fig 5B ). In addition to growing to a lower maximal OD 6oonm than WT when grown under iron restriction ( Fig 5 and S2 Fig ), further analysis of the growth kinetics of the basG -deficient strain revealed that that it also exhibits a statistically significant decrease in growth rate, and an increase in lag time under these conditions ( S2 Fig ). Although the Δ fbsE mutant exhibits a slight lag and decrease in growth rate in serum, it ultimately reaches an OD 6oonm comparable to WT and the Δ bfnL mutant ( Fig 5A and S2 Fig). These effects were confirmed to be iron-dependent, as none of the strains are capable of robust growth in cTMS without an exogenous iron source ( Fig 5C ) but grow equivalently in the same media in the presence of excess free ferric iron ( Fig 5D ). The aforementioned phenotypes could be complemented by expression of basG in trans ( S3 Fig ), and are not specific to A . baumannii ATCC 17978, as similar results were observed for a basG -deficient strain of AB5075 ( S4 Fig ), a multidrug-resistant isolate that possesses the loci to express both acinetobactin and baumannoferrins [ 63 ]. Additionally, the basG -deficient strain also exhibited a similar growth defect when lactoferrin was provided as the sole iron source ( S5 Fig ). Together these findings show that iron is essential to the proliferation of A . baumannii , and that acinetobactin is required for maximal acquisition of iron from host sources such as serum transferrin, and lactoferrin. 10.1371/journal.ppat.1008995.g005 Fig 5 Acinetobactin biosynthetic mutants are impaired for growth under iron restriction. Wild-type (WT) A . baumannii and its isogenic acinetobactin (Δ basG ), baumannoferrin (Δ bfnL) and fimsbactins (Δ fbsE ) biosynthetic mutants were grown in cTMS media with 20% human serum (A), 180 mg/dL human transferrin (B), no added iron source (C), or 30 μM FeCl 3 (D). Bacterial growth was assessed by determining the optical density at 600 nm (OD 600 ), at the time points indicated. Data are representative of three independent experiments, and error bars represent the standard error of the mean. Where error bars are not visible, they are shorter than the height of the symbol. Statistical analysis is given for the endpoint growth, as performed by repeated measures two-way ANOVA, where *p < 0.05 and *** p < 0.001. Additional analyses of growth kinetics for these data can be found in S2 Fig . Expression of a single siderophore biosynthetic pathway is sufficient to promote WT siderophore activity To ascertain the role of each individual siderophore in A . baumannii iron acquisition, it was necessary to generate strains with only one siderophore biosynthetic pathway intact. To this end, double mutants were generated, such that a panel of strains capable of synthesizing only acinetobactin (Δ bfnL fbsE ), baumannoferrin (Δ basG fbsE ), or fimsbactins (Δ basG bfnL ) was available. To assess the overall role of siderophore production on A . baumannii growth and pathogenesis, a strain with disruptions in each of the three biosynthetic pathways (Δ basG bfnL fbsE ) was produced. With these strains in hand, the CAS assays were revisited, assessing overall siderophore activity in the culture supernatants of WT A . baumannii and its isogenic mutants grown in cTMS for 12 h. Interestingly, whilst the Δ basG fbsE and Δ bfnL fbsE mutants exhibited a small but reproducible reduction in overall siderophore activity relative to the WT, none of the double mutants were drastically impaired overall ( Fig 6 ). In fact, the Δ basG bfnL mutant consistently exhibited increased CAS activity relative to the WT, suggesting that fimsbactins may be overexpressed in the absence of the other two siderophores, although the difference at 12 h was not statistically significant. We did find that genes within the fimsbactins-encoding locus are upregulated approximately 6.6 to 10-fold under the same conditions ( S6 Fig ), indicating that the loss of acinetobactin and baumannoferrin may induce further iron starvation in the bacteria leading to further transcriptional derepression of fimsbactins. In contrast to the robust siderophore production by the double mutants, when all three siderophore pathways were disrupted in the Δ basG bfnL fbsE mutant, siderophore activity was severely attenuated ( Fig 6 ). Overall, these findings demonstrate that acinetobactin, baumannoferrin, and fimsbactins appear to be the predominant iron chelators produced by A . baumannii , and that expression of just one of these siderophores is sufficient to confer robust iron chelating activity in vitro . 10.1371/journal.ppat.1008995.g006 Fig 6 Disruption to all three siderophore biosynthetic pathways in A . baumannii is required to severely attenuate overall siderophore activity. Wild-type (WT) A . baumannii and its isogenic combinatorial mutants were grown for 12 h in cTMS, and CAS assays to assess overall siderophore production were performed on the spent culture supernatants. Total siderophore activity is expressed as the percent activity of WT A . baumannii , where data are the means of three biological replicates and are representative of three independent assays. **** p < 0.0001, as determined by one-way ANOVA with Dunnett's multiple comparisons test. Siderophores are essential for A . baumannii growth when using serum transferrin as a sole iron source Given that the Δ basG mutant alone was impaired for growth on serum and transferrin, we next sought to determine if more than just acinetobactin contributes to the acquisition of iron from these sources. To reveal the overall contribution of individual siderophores to A . baumannii growth under iron restriction, WT and the combinatorial mutant strains were grown in cTMS media with 20% human serum as the sole iron source. As previously described, growth was assessed by determining the OD 600nm over 20 h. Under these conditions, it was observed that all three mutants were capable of growing on serum as a sole iron source ( Fig 7A ), with varying degrees of impairment relative to the WT. The strains expressing baumannoferrin (Δ basG fbsE ) or acinetobactin alone (Δ bfnL fbsE ) exhibited a substantial growth lag relative to WT, while the strain expressing fimsbactins alone (Δ basG bfnL ) grew comparably ( Fig 7A and S7 Fig ). Although it is unclear how a strain expressing fimsbactins alone is capable of growing on serum as a sole iron source, possible explanations include increased expression of fimsbactins biosynthesis ( S6 Fig ) or the utilization of other weak chelators such as siderophore biosynthetic intermediates or shunt products ( Fig 6 ). Notably, the complete siderophore mutant was abolished for growth on 20% serum as a sole iron source ( Fig 7A ). Similar results were observed when 180 mg/dL human transferrin was provided as the iron source ( Fig 7B ), except that the Δ basG bfnL mutant grew poorly under these conditions, indicating that the fimsbactins or another chelator may scavenge residual iron from serum, but are less efficient at acquiring iron from unsaturated human transferrin. Again, all of the strains grew poorly in the absence of an iron source ( Fig 7C ), but growth was restored in the double mutants with the addition of exogenous iron ( Fig 7D ). Even with excess iron added, the siderophore-deficient strain exhibited a reproducible growth lag and reduced final biomass relative to WT ( Fig 7 and S7 Fig ), likely due to the absence of a high affinity means of facilitating iron uptake. Together these results highlight a high degree of functional redundancy between acinetobactin, baumannoferrins, and fimsbactins, and show that siderophore expression overall is essential to growth of A . baumannii on host iron-binding glycoproteins. 10.1371/journal.ppat.1008995.g007 Fig 7 Siderophores are required for A . baumannii to utilize human serum and transferrin as iron sources to support growth. Wild-type (WT) A . baumannii and its isogenic combinatorial mutants were grown in cTMS media with 20% human serum (A), 180 mg/dL human transferrin (B), no added iron source (C), or 30 μM FeCl 3 (D). Bacterial growth was assessed by determining OD 600nm , at the time points indicated. Data are representative of three independent experiments, and error bars represent the standard error of the mean. Statistical analysis is given for the endpoint growth, as performed by repeated measures two-way ANOVA, where *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Additional analyses of growth kinetics for these data can be found in S7 Fig . Acinetobactin, baumannoferrin, and fimsbactins biosynthesis and transport genes are upregulated during infection To begin dissecting the potential functional redundancy of acinetobactin, baumannoferrin, and fimsbactins in vivo , a murine model of systemic infection was employed to assess the expression of the siderophore biosynthesis and utilization pathways during infection using NanoString technology. NanoString is a digital, amplification-free means of quantifying targeted transcripts using color-coded molecular probes [ 64 , 65 ], and has been shown to reliably detect pathogen gene expression in complex samples [ 66 , 67 ]. In preparation for NanoString, female C57BL6/J mice were infected systemically with 2 x 10 8 to 5 x 10 8 colony forming units (CFUs) of WT A . baumannii by retroorbital injection. After 24 h, mice were humanely euthanized, and the organs were harvested. Following homogenization, RNA was extracted from the infected tissues, hybridized to the custom oligonucleotide probe set, and analyzed as per manufacturer instructions. As the in vitro comparator, RNA was similarly extracted and analyzed from bacteria grown in the same manner as used to prepare the inoculum for infection. Consistent with the in vitro qRT-PCR results ( Fig 2A ), all three siderophore loci were found to be broadly upregulated in the heart ( Fig 8A ) and lungs ( Fig 8B ) of the A . baumannii infected host, relative to bacteria grown in vitro . Similar results were observed in the kidneys, liver, and spleen ( S8 Fig ). Although the overall magnitude of gene expression varied between organs, likely due to the bioavailability of iron to the pathogen, no other major differences were observed in the expression of the three siderophore loci. These results suggest that the siderophore pathways are upregulated overall in the iron-restricted host and that the presence of functionally redundant siderophores cannot be readily explained by differential expression to facilitate growth in specific host niches. 10.1371/journal.ppat.1008995.g008 Fig 8 Siderophore biosynthetic and transport genes are upregulated in vivo . Mice were systemically infected with WT A . baumannii and sacrificed at 24 h post-infection. Organs were harvested, RNA extracted, and gene expression changes relative to growth in vitro were determined in the heart (A) and lungs (B) using NanoString technology. Genes are clustered by known or predicted function, as indicated. Acinetobactin biosynthesis is essential for the survival and proliferation of A . baumannii within the host Since all three siderophore pathways were found to be upregulated during infection, it remained unclear as to what system, if any, is important to A . baumannii pathogenesis and whether the siderophores exhibit differential utility in specific host tissues. To elucidate the function of the individual siderophores in vivo , the same murine model of infection described above was employed, but instead mice were infected with WT A . baumannii or one of the single or combinatorial biosynthetic mutants. At 24 h post-infection the mice were humanely euthanized, the kidneys, hearts, livers, spleens, lungs, and blood were harvested, and the bacterial burdens of each were determined. In support of the results demonstrating that acinetobactin is required for acquisition of iron from host sources such as serum transferrin and lactoferrin, the Δ basG mutant was found to be severely attenuated in vivo . Relative to WT, the acinetobactin-deficient Δ basG mutant had reduced bacterial burdens recovered from every organ and the blood, in the range of 2.8 to 5.0 log 10 ( Fig 9 ). Conversely, the Δ bfnL and Δ fbsE mutants did not exhibit a statistically significant reduction in burdens in any organ or in the blood relative to WT. Although the strain producing fimsbactins alone was capable of supporting growth of A . baumannii in serum ( Fig 7A ), neither this siderophore nor baumannoferrin were required for survival during A . baumannii bacteremia, as both Δ basG bfnL and Δ basG fbsE were recovered in similar numbers to the Δ basG mutant alone. Conversely, the strain producing acinetobactin alone (Δ bfnL fbsE ) was not statistically different from WT. Lastly, disrupting all three siderophore biosynthetic pathways in the Δ basG bfnL fbsE mutant did not appreciably decrease the survival of the bacteria in vivo relative to Δ basG . Together, these results suggest that acinetobactin alone is required for the survival and pathogenesis of A . baumannii during bacteremia, whilst the function of the baumannoferrins and fimsbactins in vivo remains unclear. 10.1371/journal.ppat.1008995.g009 Fig 9 Acinetobactin biosynthetic mutant is severely attenuated for survival and proliferation within the host. Mice were systemically infected with WT A . baumannii or its isogenic siderophore biosynthetic mutants, as indicated. After 24 h, mice were sacrificed and the bacterial burdens of the kidneys, heart, liver, spleen, lungs, and blood were determined by plating for viable cell counts on lysogeny agar. Each symbol represents the A . baumannii count in the corresponding organ of one animal. Data are compiled from three independent experiments. Statistical significance was determined by Kruskal-Wallis with Dunn's multiple comparisons test, where *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Acinetobacter baumannii siderophore biosynthesis and transport are upregulated in response to iron limitation A . baumannii strain ATCC 17978 encodes for ten structurally distinct siderophores encoded from three different biosynthetic loci ( Fig 1 ; S1 , S2 and S3 Tables). We previously observed that expression of the loci encoding for the biosynthesis and transport of acinetobactin, baumannoferrin, and fimsbactins is upregulated in the presence of the multi-metal sequestering innate immune protein, calprotectin [ 57 ]. Given the apparent functional redundancies in the siderophore pathways of A . baumannii , we hypothesized that these systems are differentially regulated, thus allowing for expression in response to distinct environmental cues, such as differences in the availability of essential transition metals over time. To address this, wild-type (WT) A . baumannii was grown in metal-chelated Tris minimal succinate media (chelex-treated; cTMS) with and without the addition of exogenous iron or zinc. Following 4 or 12 hours (h) growth, RNA was extracted and the expression of various siderophore-associated transcripts from each of the three loci was assessed by quantitative reverse transcription PCR (qRT-PCR). Consistent with identification of putative Fur boxes upstream of many genes or operons within these loci ( Fig 1A and 1B, and 1C and reference [ 31 ]), key biosynthetic, transport, and regulatory genes in all three siderophore-associated loci are strongly upregulated in iron-deplete versus replete conditions ( Fig 2A and 2B ). By contrast, and with the exception of bfnF , which encodes a putative oxidoreductase thought to release iron from the siderophore, the change in gene expression in zinc-deplete versus -replete conditions is 10 to 1000-fold lower than that observed with iron ( Fig 2C and 2D ), suggesting that the expression of genes encoding for siderophore biosynthesis and transport in A . baumannii is predominantly iron-regulated. Notably, gene expression under zinc limitation increased between 4 and 12 h, suggesting that zinc may play a role in the regulation of the siderophore-associated loci during prolonged metal restriction. To determine if these genes are directly regulated by the zinc uptake regulator, Zur, we performed qRT-PCR on a representative gene from each locus in WT A . baumannii and a Δ zur mutant [ 58 ]. Additionally, we assessed whether Zur can directly regulate fur by similarly looking at expression of this transcriptional regulator in the zur mutant. We found that although the expression of fur was unaltered in Δ zur , basA and bfnA were slightly but significantly upregulated in this background ( S1 Fig ). Together these results suggest that whilst Zur does not appear to directly regulate fur in A . baumannii , it may play a small but unappreciated role in the expression of known fur -regulated genes. 10.1371/journal.ppat.1008995.g002 Fig 2 Siderophore biosynthetic and transport genes are upregulated in metal deplete conditions. WT A . baumannii was grown in metal-restricted media with or without the addition of exogenous iron or zinc. RNA was extracted and transcriptional changes in the expression of siderophore-associated genes were assessed by qRT-PCR in iron-deplete vs. replete conditions at 4 (A) and 12 h (B), and zinc-deplete vs. replete at 4 (C) and 12 (D) conditions, where expression was normalized to the expression of rpoB . * p < 0.05 and ** p < 0.01, as determined by Student's t test relative to a hypothetical value of 1. Data are the means combined from two independent experiments, each with three biological replicates. To determine if the transcriptional changes observed above translate to overall differences in siderophore production by A . baumannii , WT bacteria were grown in cTMS with and without the addition of exogenous metals. Following 12 h growth, assessment of siderophore activity in the A . baumannii supernatants was assessed by Chrome Azurol S (CAS) assay. CAS is an iron-binding dye where mobilization of iron from CAS to a chelator within the media can be detected through a colorimetric change of the dye from blue to orange [ 59 , 60 ]. Consistent with the impact of iron on gene transcription, the addition of iron to A . baumannii cultures strongly represses overall siderophore production by the bacteria ( Fig 3 ), whereas the addition of zinc does not appear to impact this activity. These observations indicate that derepression of siderophore-associated genes during zinc limitation may not lead to increased siderophore production overall, and that acinetobactin, baumannoferrin, and fimsbactins biosynthesis and utilization in A . baumannii is primarily influenced by iron availability. It is possible, however, that the semi-quantitative nature of assessing total siderophore activity by CAS assay is not sensitive enough to detect subtle changes in siderophore production upon zinc limitation over time, as the accumulation of siderophores and/or siderophore biosynthetic enzymes may mask these effects. 10.1371/journal.ppat.1008995.g003 Fig 3 Iron, but not zinc, represses the overall siderophore activity of A . baumannii . Chrome Azurol S (CAS) assays to assess for total siderophore activity were performed on the spent culture supernatants of WT A . baumannii grown in cTMS media for 12 h with or without the addition of exogenous metals, as indicated. The colorimetric changes observed in the CAS assay are shown, where blue indicates an absence of siderophore activity and orange indicates that an iron chelator capable of mobilizing iron from CAS is present. Total siderophore activity is expressed as the percent activity of WT A . baumannii grown in the absence of exogenously added metals. **** p < 0.0001, as determined by one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons test. Data are the means of three biological replicates and are representative of three independent assays. A . baumannii siderophores exhibit functional redundancy in vitro To begin identifying the contribution of acinetobactin, baumannoferrin, and fimsbactins production to A . baumannii iron acquisition, key genes in each of the siderophore biosynthetic pathways were disrupted using recombineering [ 61 ]. The details of all strains employed in this study can be found in Materials and Methods, and the genes disrupted are highlighted in Fig 1 . Genes targeted for inactivation were selected based on predicted function, as well as a lack of any potentially redundant homologues present elsewhere in the A . baumannii ATCC 17978 genome. The disrupted genes include basG , encoding a putative histidine decarboxylase predicted to synthesize histamine as an essential precursor molecule to acinetobactin biosynthesis [ 44 , 62 ], bfnL , encoding a putative acetyltransferase predicted to facilitate the conversion of N 3 -hydroxy-1,3-diaminopropane to N 3 -decanoyl-hydroxy-1,3-diaminopropane as an early step in baumannoferrin biosynthesis [ 42 ], and fbsE , encoding a putative nonribosomal peptide synthase (NRPS) and the first part of the multi-modular unit required to assemble fimsbactins [ 43 ]. To determine if disruption to any one siderophore biosynthetic pathway impacts the ability of A . baumannii to chelate iron, WT A . baumannii and the strains inactivated for acinetobactin (Δ basG ), baumannoferrin (Δ bfnL ), or fimsbactins (Δ fbsE ) biosynthesis were grown in cTMS for 12 h and CAS assays were performed on the culture supernatants. Aside from a small but reproducible reduction in siderophore activity in the Δ fbsE mutant, inactivating one siderophore pathway in A . baumannii does not drastically impact total siderophore activity ( Fig 4 ). These results suggest that functional redundancy exists between the siderophore-based iron chelation activities of A . baumannii in vitro . 10.1371/journal.ppat.1008995.g004 Fig 4 Disrupting a single siderophore biosynthetic pathway does not drastically impact overall siderophore activity in A . baumannii . Wild-type (WT) A . baumannii and its isogenic acinetobactin (Δ basG ), baumannoferrin (Δ bfnL) and fimsbactins (Δ fbsE ) biosynthetic mutants were grown for 12 h in cTMS, and CAS assays to assess overall siderophore activity were performed on the spent culture supernatants. Total siderophore activity is expressed as the percent activity of WT A . baumannii , where data are the means of three biological replicates and are representative of three independent assays. ** p < 0.01, as determined by one-way analysis ANOVA with Dunnett's multiple comparisons test. Acinetobactin is required for maximal growth on host iron sources In addition to sequestering small amounts of free ferric iron from the extracellular milieu, siderophores are capable of pirating iron from host glycoproteins such as transferrin and lactoferrin. To determine if acinetobactin, baumannoferrin, or fimsbactins are essential for iron acquisition from host iron sources, WT A . baumannii and the three siderophore biosynthetic mutants, Δ basG , Δ bfnL , and Δ fbsE , were grown in cTMS media with 20% human serum as the sole iron source. Bacterial growth was monitored by optical density (OD 6oonm ) over 20 hours. In contrast to the results of the CAS assay indicating that none of the mutations strongly impact overall siderophore production, the Δ basG mutant was found to be impaired for growth in serum relative to WT A . baumannii and the Δ bfnL and Δ fbsE mutants ( Fig 5A ). The defect in growth of Δ basG in serum is likely due to reduced acquisition of iron from transferrin, as this acinetobactin-deficient mutant is similarly impaired for growth on human transferrin as a sole iron source ( Fig 5B ). In addition to growing to a lower maximal OD 6oonm than WT when grown under iron restriction ( Fig 5 and S2 Fig ), further analysis of the growth kinetics of the basG -deficient strain revealed that that it also exhibits a statistically significant decrease in growth rate, and an increase in lag time under these conditions ( S2 Fig ). Although the Δ fbsE mutant exhibits a slight lag and decrease in growth rate in serum, it ultimately reaches an OD 6oonm comparable to WT and the Δ bfnL mutant ( Fig 5A and S2 Fig). These effects were confirmed to be iron-dependent, as none of the strains are capable of robust growth in cTMS without an exogenous iron source ( Fig 5C ) but grow equivalently in the same media in the presence of excess free ferric iron ( Fig 5D ). The aforementioned phenotypes could be complemented by expression of basG in trans ( S3 Fig ), and are not specific to A . baumannii ATCC 17978, as similar results were observed for a basG -deficient strain of AB5075 ( S4 Fig ), a multidrug-resistant isolate that possesses the loci to express both acinetobactin and baumannoferrins [ 63 ]. Additionally, the basG -deficient strain also exhibited a similar growth defect when lactoferrin was provided as the sole iron source ( S5 Fig ). Together these findings show that iron is essential to the proliferation of A . baumannii , and that acinetobactin is required for maximal acquisition of iron from host sources such as serum transferrin, and lactoferrin. 10.1371/journal.ppat.1008995.g005 Fig 5 Acinetobactin biosynthetic mutants are impaired for growth under iron restriction. Wild-type (WT) A . baumannii and its isogenic acinetobactin (Δ basG ), baumannoferrin (Δ bfnL) and fimsbactins (Δ fbsE ) biosynthetic mutants were grown in cTMS media with 20% human serum (A), 180 mg/dL human transferrin (B), no added iron source (C), or 30 μM FeCl 3 (D). Bacterial growth was assessed by determining the optical density at 600 nm (OD 600 ), at the time points indicated. Data are representative of three independent experiments, and error bars represent the standard error of the mean. Where error bars are not visible, they are shorter than the height of the symbol. Statistical analysis is given for the endpoint growth, as performed by repeated measures two-way ANOVA, where *p < 0.05 and *** p < 0.001. Additional analyses of growth kinetics for these data can be found in S2 Fig . Expression of a single siderophore biosynthetic pathway is sufficient to promote WT siderophore activity To ascertain the role of each individual siderophore in A . baumannii iron acquisition, it was necessary to generate strains with only one siderophore biosynthetic pathway intact. To this end, double mutants were generated, such that a panel of strains capable of synthesizing only acinetobactin (Δ bfnL fbsE ), baumannoferrin (Δ basG fbsE ), or fimsbactins (Δ basG bfnL ) was available. To assess the overall role of siderophore production on A . baumannii growth and pathogenesis, a strain with disruptions in each of the three biosynthetic pathways (Δ basG bfnL fbsE ) was produced. With these strains in hand, the CAS assays were revisited, assessing overall siderophore activity in the culture supernatants of WT A . baumannii and its isogenic mutants grown in cTMS for 12 h. Interestingly, whilst the Δ basG fbsE and Δ bfnL fbsE mutants exhibited a small but reproducible reduction in overall siderophore activity relative to the WT, none of the double mutants were drastically impaired overall ( Fig 6 ). In fact, the Δ basG bfnL mutant consistently exhibited increased CAS activity relative to the WT, suggesting that fimsbactins may be overexpressed in the absence of the other two siderophores, although the difference at 12 h was not statistically significant. We did find that genes within the fimsbactins-encoding locus are upregulated approximately 6.6 to 10-fold under the same conditions ( S6 Fig ), indicating that the loss of acinetobactin and baumannoferrin may induce further iron starvation in the bacteria leading to further transcriptional derepression of fimsbactins. In contrast to the robust siderophore production by the double mutants, when all three siderophore pathways were disrupted in the Δ basG bfnL fbsE mutant, siderophore activity was severely attenuated ( Fig 6 ). Overall, these findings demonstrate that acinetobactin, baumannoferrin, and fimsbactins appear to be the predominant iron chelators produced by A . baumannii , and that expression of just one of these siderophores is sufficient to confer robust iron chelating activity in vitro . 10.1371/journal.ppat.1008995.g006 Fig 6 Disruption to all three siderophore biosynthetic pathways in A . baumannii is required to severely attenuate overall siderophore activity. Wild-type (WT) A . baumannii and its isogenic combinatorial mutants were grown for 12 h in cTMS, and CAS assays to assess overall siderophore production were performed on the spent culture supernatants. Total siderophore activity is expressed as the percent activity of WT A . baumannii , where data are the means of three biological replicates and are representative of three independent assays. **** p < 0.0001, as determined by one-way ANOVA with Dunnett's multiple comparisons test. Siderophores are essential for A . baumannii growth when using serum transferrin as a sole iron source Given that the Δ basG mutant alone was impaired for growth on serum and transferrin, we next sought to determine if more than just acinetobactin contributes to the acquisition of iron from these sources. To reveal the overall contribution of individual siderophores to A . baumannii growth under iron restriction, WT and the combinatorial mutant strains were grown in cTMS media with 20% human serum as the sole iron source. As previously described, growth was assessed by determining the OD 600nm over 20 h. Under these conditions, it was observed that all three mutants were capable of growing on serum as a sole iron source ( Fig 7A ), with varying degrees of impairment relative to the WT. The strains expressing baumannoferrin (Δ basG fbsE ) or acinetobactin alone (Δ bfnL fbsE ) exhibited a substantial growth lag relative to WT, while the strain expressing fimsbactins alone (Δ basG bfnL ) grew comparably ( Fig 7A and S7 Fig ). Although it is unclear how a strain expressing fimsbactins alone is capable of growing on serum as a sole iron source, possible explanations include increased expression of fimsbactins biosynthesis ( S6 Fig ) or the utilization of other weak chelators such as siderophore biosynthetic intermediates or shunt products ( Fig 6 ). Notably, the complete siderophore mutant was abolished for growth on 20% serum as a sole iron source ( Fig 7A ). Similar results were observed when 180 mg/dL human transferrin was provided as the iron source ( Fig 7B ), except that the Δ basG bfnL mutant grew poorly under these conditions, indicating that the fimsbactins or another chelator may scavenge residual iron from serum, but are less efficient at acquiring iron from unsaturated human transferrin. Again, all of the strains grew poorly in the absence of an iron source ( Fig 7C ), but growth was restored in the double mutants with the addition of exogenous iron ( Fig 7D ). Even with excess iron added, the siderophore-deficient strain exhibited a reproducible growth lag and reduced final biomass relative to WT ( Fig 7 and S7 Fig ), likely due to the absence of a high affinity means of facilitating iron uptake. Together these results highlight a high degree of functional redundancy between acinetobactin, baumannoferrins, and fimsbactins, and show that siderophore expression overall is essential to growth of A . baumannii on host iron-binding glycoproteins. 10.1371/journal.ppat.1008995.g007 Fig 7 Siderophores are required for A . baumannii to utilize human serum and transferrin as iron sources to support growth. Wild-type (WT) A . baumannii and its isogenic combinatorial mutants were grown in cTMS media with 20% human serum (A), 180 mg/dL human transferrin (B), no added iron source (C), or 30 μM FeCl 3 (D). Bacterial growth was assessed by determining OD 600nm , at the time points indicated. Data are representative of three independent experiments, and error bars represent the standard error of the mean. Statistical analysis is given for the endpoint growth, as performed by repeated measures two-way ANOVA, where *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Additional analyses of growth kinetics for these data can be found in S7 Fig . Acinetobactin, baumannoferrin, and fimsbactins biosynthesis and transport genes are upregulated during infection To begin dissecting the potential functional redundancy of acinetobactin, baumannoferrin, and fimsbactins in vivo , a murine model of systemic infection was employed to assess the expression of the siderophore biosynthesis and utilization pathways during infection using NanoString technology. NanoString is a digital, amplification-free means of quantifying targeted transcripts using color-coded molecular probes [ 64 , 65 ], and has been shown to reliably detect pathogen gene expression in complex samples [ 66 , 67 ]. In preparation for NanoString, female C57BL6/J mice were infected systemically with 2 x 10 8 to 5 x 10 8 colony forming units (CFUs) of WT A . baumannii by retroorbital injection. After 24 h, mice were humanely euthanized, and the organs were harvested. Following homogenization, RNA was extracted from the infected tissues, hybridized to the custom oligonucleotide probe set, and analyzed as per manufacturer instructions. As the in vitro comparator, RNA was similarly extracted and analyzed from bacteria grown in the same manner as used to prepare the inoculum for infection. Consistent with the in vitro qRT-PCR results ( Fig 2A ), all three siderophore loci were found to be broadly upregulated in the heart ( Fig 8A ) and lungs ( Fig 8B ) of the A . baumannii infected host, relative to bacteria grown in vitro . Similar results were observed in the kidneys, liver, and spleen ( S8 Fig ). Although the overall magnitude of gene expression varied between organs, likely due to the bioavailability of iron to the pathogen, no other major differences were observed in the expression of the three siderophore loci. These results suggest that the siderophore pathways are upregulated overall in the iron-restricted host and that the presence of functionally redundant siderophores cannot be readily explained by differential expression to facilitate growth in specific host niches. 10.1371/journal.ppat.1008995.g008 Fig 8 Siderophore biosynthetic and transport genes are upregulated in vivo . Mice were systemically infected with WT A . baumannii and sacrificed at 24 h post-infection. Organs were harvested, RNA extracted, and gene expression changes relative to growth in vitro were determined in the heart (A) and lungs (B) using NanoString technology. Genes are clustered by known or predicted function, as indicated. Acinetobactin biosynthesis is essential for the survival and proliferation of A . baumannii within the host Since all three siderophore pathways were found to be upregulated during infection, it remained unclear as to what system, if any, is important to A . baumannii pathogenesis and whether the siderophores exhibit differential utility in specific host tissues. To elucidate the function of the individual siderophores in vivo , the same murine model of infection described above was employed, but instead mice were infected with WT A . baumannii or one of the single or combinatorial biosynthetic mutants. At 24 h post-infection the mice were humanely euthanized, the kidneys, hearts, livers, spleens, lungs, and blood were harvested, and the bacterial burdens of each were determined. In support of the results demonstrating that acinetobactin is required for acquisition of iron from host sources such as serum transferrin and lactoferrin, the Δ basG mutant was found to be severely attenuated in vivo . Relative to WT, the acinetobactin-deficient Δ basG mutant had reduced bacterial burdens recovered from every organ and the blood, in the range of 2.8 to 5.0 log 10 ( Fig 9 ). Conversely, the Δ bfnL and Δ fbsE mutants did not exhibit a statistically significant reduction in burdens in any organ or in the blood relative to WT. Although the strain producing fimsbactins alone was capable of supporting growth of A . baumannii in serum ( Fig 7A ), neither this siderophore nor baumannoferrin were required for survival during A . baumannii bacteremia, as both Δ basG bfnL and Δ basG fbsE were recovered in similar numbers to the Δ basG mutant alone. Conversely, the strain producing acinetobactin alone (Δ bfnL fbsE ) was not statistically different from WT. Lastly, disrupting all three siderophore biosynthetic pathways in the Δ basG bfnL fbsE mutant did not appreciably decrease the survival of the bacteria in vivo relative to Δ basG . Together, these results suggest that acinetobactin alone is required for the survival and pathogenesis of A . baumannii during bacteremia, whilst the function of the baumannoferrins and fimsbactins in vivo remains unclear. 10.1371/journal.ppat.1008995.g009 Fig 9 Acinetobactin biosynthetic mutant is severely attenuated for survival and proliferation within the host. Mice were systemically infected with WT A . baumannii or its isogenic siderophore biosynthetic mutants, as indicated. After 24 h, mice were sacrificed and the bacterial burdens of the kidneys, heart, liver, spleen, lungs, and blood were determined by plating for viable cell counts on lysogeny agar. Each symbol represents the A . baumannii count in the corresponding organ of one animal. Data are compiled from three independent experiments. Statistical significance was determined by Kruskal-Wallis with Dunn's multiple comparisons test, where *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Discussion The expression of multiple, structurally distinct but functionally similar siderophores is a common feature of many bacteria [ 29 ]. For bacterial pathogens, the utilization of more than one siderophore class may fulfill a number of purposes, such as avoiding siderophore piracy by other bacteria and promoting competitiveness, facilitating iron acquisition under different physiological conditions and/or iron source composition, promoting uptake of metals other than iron, and most importantly, helping to evade nutritional immunity [ 29 , 68 ]. Although most A . baumannii clinical isolates possess the loci for acinetobactin and baumannoferrin biosynthesis and utilization, the individual contributions of these siderophores in vitro and in vivo have not been fully elucidated. Furthermore, although the synthesis pathway and structures of the fimsbactins have been characterized [ 43 , 55 , 56 ], their function in A . baumannii growth and survival has never been interrogated using strains that are specifically inactivated for fimsbactins biosynthesis. Here we investigate the function of the individual A . baumannii siderophores using a series of single and combinatorial biosynthetic mutants. We demonstrate that all three siderophore biosynthetic pathways contribute to iron chelation in vitro and functional redundancy exists in their ability to facilitate iron mobilization from host sources. However, acinetobactin is essential to the efficient use of iron from host iron-binding glycoproteins, and to the survival and dissemination of A . baumannii in a murine model of bacteremia. These findings support an often stated, but not fully substantiated assertion that acinetobactin is the major, or most important, siderophore produced by A . baumannii [ 44 ]. In the context of A . baumannii bacteremia, it remains unclear why acinetobactin biosynthesis is indispensable to colonization and survival, especially with the availability of multiple alternative mechanisms for iron acquisition. Acinetobactin has been proposed to function as a "two-for-one" siderophore allowing for iron chelation over an expanded pH range, with pre-acinetobactin predominating under acidic conditions, and acinetobactin under neutral and basic conditions [ 38 ]. Thus, the critical importance of acinetobactin may be explained by its ability to bind iron under different physiological conditions. As acinetobactin biosynthesis is required for efficient utilization of transferrin and lactoferrin as sole iron sources ( Fig 5 and S5 Fig , respectively), it is also possible that this siderophore has a higher affinity for iron than the baumannoferrins and fimsbactins, and is therefore better able to appropriate iron from the host. However, whilst the affinity of the baumannoferrins for iron has not been elucidated, pre-acinetobactin binds iron with a comparable affinity to fimsbactins A (log K Fe = 27.1 ± 0.2 versus 27.4 ± 0.2) and acinetobactin binds iron with a lower affinity (log K Fe = 26.2 ± 0.2) [ 38 , 55 ], suggesting that iron affinity alone does not readily explain the importance of acinetobactin. Alternatively, ferric-acinetobactin may be captured and utilized more efficiently than the other two siderophores, supported partly by the observation that the receptor protein for acinetobactin (BauA) binds a heterotrimeric acinetobactin:preacinetobactin:Fe(III) complex or a preacinetobactin 2 :Fe(III) complex with nanomolar affinity prior to uptake of the siderophore [ 69 ]. The specificity and affinity of the predicted baumannoferrin (BfnH) and fimsbactins (FbsN) receptors have not been defined, and thus further research is required to determine if efficacy in siderophore transport contributes to the utility of the siderophores. In addition to intrinsic factors controlling the uptake of siderophores, it is also known that during infection neutrophils release siderocalin (also known as neutrophil gelatinase associated lipocalin (NGAL), 24p3, and lipocalin-2), an acute phase protein that is capable of binding and sequestering extracellular ferric catecholate and carboxymycobactin-type siderophores, thus preventing their capture by invading pathogens [ 70 – 72 ]. To circumvent the effects of siderocalin, some bacteria secrete structurally modified siderophores that are incompatible with the binding site of the protein, and thus are referred to as "stealth siderophores" [ 73 ]. The interactions between A . baumannii siderophores and siderocalin has not been interrogated, but it is possible their redundancy may be explained by differing susceptibilities to sequestration by the vertebrate host. Indeed, hydroxamate siderophores such as baumannoferrins A and B are unlikely to be bound by siderocalin and thus would be free to facilitate iron acquisition in vivo , although our findings suggest that baumannoferrin expression is dispensable to pathogenesis in a murine bacteremia model ( Fig 9 ). Notably, the baumannoferrins possess a lipophilic decanoic side chain, which may anchor them to the cell envelope [ 55 , 74 ] and potentially minimize their role in the capture of iron from the extracellular milieu. Unlike the baumannoferrins, the fimsbactins are mixed ligand, bis-catecholate monohydroxamate siderophores that are also reminiscent of tris-catecholate siderophores such as enterobactin and vibriobactin, known ligands of siderocalin [ 55 , 70 , 75 , 76 ]. Although not experimentally validated, the fimsbactins are predicted to bind siderocalin [ 55 ], which would effectively inactivate them in vivo and would be consistent with our findings that disruption to fimsbactins biosynthesis does not significantly impact the outcome of infection ( Fig 9 ). Research by the Wencewicz group has revealed that apo-fimsbactins and holo-acinetobactin compete for the same periplasmic binding protein (BauB; Fig 1D ), and hence are antagonistic in vitro [ 77 ]. Reductive release of iron from fimsbactins is thought to be facilitated by a putative periplasmic ferric iron reductase, FbsP, allowing for accumulation of the apo-siderophore in this compartment whilst free ferrous iron is transported into the cytoplasm by FeoABC ( Fig 1D ) [ 55 ]. It is possible that if the fimsbactins are sequestered by siderocalin in vivo that this competition is alleviated, and there may be a trade-off between an in vivo benefit and an in vitro detriment to the bacteria. However, further research is required to determine what, if any, interaction exists between the fimsbactins and siderocalin. Lastly, the essentiality of acinetobactin to A . baumannii pathogenesis suggests that this siderophore is capable of evading or overwhelming siderocalin to facilitate iron acquisition by the bacteria within the host. In assessing the role of metals on the expression of siderophore biosynthetic clusters, our results are consistent with previous findings that the acinetobactin, baumannoferrins, and fimsbactins-encoding loci are all upregulated during iron restriction [ 31 ]. Zinc starvation could also induce expression of siderophore-associated genes ( Fig 2 ), but these transcriptional changes were 10 to 1000-fold lower than that seen with iron. It is unclear if the influence of zinc on the expression of siderophore-associated transcripts is biologically relevant, and could be explained by similarities between the Fur and Zur box consensus sequences leading to accidental crosstalk between the two regulators [ 20 , 31 ]. Alternatively, if Fur binds structural zinc in A . baumannii as has been reported in other bacteria [ 78 ], prolonged zinc starvation could result in dissociation of the regulator from its cognate DNA, resulting in derepression of the locus. Whilst our results strongly support that iron limitation induces the expression of acinetobactin, baumannoferrin, and fimsbactins, the role for zinc remains unknown. Consistent with a previous study demonstrating that all three siderophore biosynthetic clusters are upregulated in the blood [ 79 ], we found each locus to be upregulated in the heart, lungs, kidneys, liver and spleen of mice during A . baumannii bacteremia ( Fig 8 and S8 Fig ). Notably, we did not detect niche-dependent variation in transcription of the different siderophore loci in distinct organs, although by assessing expression in whole organ homogenates, these assays may not have had the resolution required to observe these differences. Additionally, it is possible that whilst the genes for baumannoferrin and fimsbactins biosynthesis were expressed, the siderophores were not produced. Recently, high-performance matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was used to reveal that Staphylococcus aureus exhibits differential production of its siderophores, staphyloferrin A and staphyloferrin B, between abscesses within the same infected tissue [ 80 ]. Utilization of higher resolution techniques such as MALDI-IMS that incorporate spatial detection may help confirm not only the production and relative abundances of acinetobactin, baumannoferrins, and fimsbactins but may also be used to reveal niche-specific expression of the siderophores in vivo . Notably, in assessing total mass recovery following the in vitro purification of acinetobactin and fimsbactins, Bohac et al observed greater mass production of the former, whilst the baumannoferrins were not detected using the methods employed [ 55 ]. The aforementioned results suggest that there may indeed be differences in overall siderophore abundances that may help to explain why acinetobactin is critically important to infection. Whilst the use of mass spectrometry to identify and quantify the production of each of the siderophores was outside the scope of this study, use of these technologies represent powerful tools going forward. Together, the conservation of the acinetobactin locus amongst clinical isolates of A . baumannii and essentiality to survival of the pathogen A . baumannii in vivo [ 31 , 46 , 51 ], even when other siderophores are expressed, leads us to propose that developing novel therapeutics that specifically target acinetobactin biosynthesis or transport may represent a viable strategy to combat this extensively drug-resistant pathogen. Several approaches exist for targeting iron homeostasis pathways in bacterial pathogens, but two main methods include developing vaccines against iron-regulated surface-exposed antigens and coopting iron acquisition systems to deliver a toxic payload to the bacterium [ 22 ]. Antibodies raised against iron-regulated TonB-dependent transporters (TBDTs) often function both to promote osponophagocytosis and to inhibit iron uptake [ 81 ], and indeed the receptor proteins for both acinetobactin and baumannoferrins, BauA and BfnH, respectively, have shown promise as antigens for vaccine development against A . baumannii [ 82 – 84 ]. Although the expression of bfnH was not specifically assessed by NanoString in this study, both bauA and fbsN are upregulated during infection, with the latter possessing some of the highest transcript abundances in most organs ( Fig 8 and S8 Fig ), again providing support for the use of TBDTs as vaccine antigens. A potential limitation to vaccination against A . baumannii , is that patients afflicted by the disease are often critically ill and immunocompromised and thus unable to mount a sufficient antibody response. Passive immunization, using monoclonal antibodies, therefore represents a potential option for protecting patients in high risk environments, such as intensive care units. An alternative strategy for exploiting iron acquisition systems to the detriment of bacterial pathogens is to covalently link either a natural or synthetic siderophore to an antibiotic and utilize the siderophore uptake systems of the bacteria to help internalize this compound, known as a sideromycin. This "Trojan horse" approach to treating bacterial infections recently gained traction with the development of cefiderocol (Fetroja), a catechol-cephalosporin conjugate that recently became the first sideromycin-type antibiotic approved by the United States Food and Drug Administration (FDA) [ 85 ]. Indicated for the treatment of complicated urinary tract infections with few or no other treatment options, cefiderocol is active against Gram-negative, multidrug-resistant pathogens such as A . baumannii [ 86 , 87 ]. As with other sideromycins, cefiderocol is actively transported into the cell by coopting a siderophore-TBDT [ 88 ], and its minimum inhibitory concentration decreases under iron restriction [ 86 ]. Additionally, fimsbactins A and fimsbactins-like analogues conjugated with daptomycin or cephalosporins are highly effective and selective in killing A . baumannii in vitro , showing that antibiotics typically reserved for Gram-positives may be repurposed by using a siderophore delivery mechanism to gain access to the periplasm of Gram-negative bacteria [ 89 , 90 ]. These findings highlight that sideromycins can be effective as antibiotics against A . baumannii . Importantly, the essential features required for iron coordination by, and cellular uptake of acinetobactin have been identified, along with a promising sites for the conjugation of an antibiotic [ 38 , 44 , 91 ]. Interestingly, in assessing the development of resistance to another experimental sideromycin (BAL30072), A . baumannii developed mutations in genes encoding the TonB machinery required to energize siderophore uptake systems, tonB3 and exbD3 [ 33 , 88 ]. Although these mutants had decreased sensitivity to the sideromycin, the tonB3 and exbD3 mutants exhibited decreased growth under iron restriction, suggesting that should these mutations evolve in the iron-restricted host, the bacteria may still be compromised for survival [ 88 ]. Indeed, Runci et al . have found that tonB3 is required for maximal virulence of A . baumannii ATCC 19606 in insect and murine models of infection [ 33 ]. The results of this study have revealed that acinetobactin is essential for iron acquisition in vivo , whereas other siderophores, the baumannoferrins and fimsbactins, are dispensable. Moreover, we have demonstrated that the genes for acinetobactin biosynthesis and transport are broadly upregulated in a systemic murine infection model. Collectively these data, coupled with the observation that the acinetobactin locus is highly conserved amongst clinical isolates, presents the targeting of acinetobactin biosynthesis or transport as an attractive candidate for the development of novel therapeutics to combat multidrug-resistant A . baumannii . Materials and methods Bacterial strains and growth conditions All bacterial strains and plasmids employed in this study can be found in Table 1 . Unless otherwise indicated, experiments were performed with A . baumannii strain ATCC 17978 and its isogenic mutants. For routine cultivation and genetic manipulation, A . baumannii was cultured in lysogeny broth (LB) or on LB with 1.5% w/v agar (LBA). Antibiotics, when required for selection of recombinants or maintenance of plasmids in A . baumannii , were supplied at the following concentrations: ampicillin, 500 μg/mL; carbenicillin, 75 μg/mL; kanamycin, 15 μg/mL; sulfamethoxazole, 100 μg/mL; as indicated. For selection and maintenance of plasmids in E . coli , 100 μg/mL of ampicillin was used. Plasmid gene expression was induced using 1–2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), as detailed in the text. 10.1371/journal.ppat.1008995.t001 Table 1 Strains and plasmids employed in this study. Strain Description Reference A . baumannii ATCC 17978 WT A . baumannii fatal meningitis isolate from 1951 American Type Culture Collection (ATCC) strain 5377 [ 54 ] WT/pWH1266 WT A . baumannii ATCC 17978 with empty pWH1266 plasmid This study A . baumannii AB5075-UW Multidrug-resistant WT A . baumannii AB5075 isolate from a wound infection [ 92 , 93 ] Δ basG Acinetobactin biosynthetic mutant where basG (A1S_2379) was replaced by a kanamycin resistance determinant, and then the cassette was excised, to leave a markerless mutant This study basG 102::Tn26 Transposon mutant of basG in A . baumannii AB5075-UW from three-allele mutant library; identifier tnab1_kr121128p06q102 [ 92 ] basG 142::Tn26 Transposon mutant of basG in A . baumannii AB5075-UW from three-allele mutant library; identifier tnab1_kr130909p02q142 [ 92 ] Δ basG /pWH1266:: basG basG mutant complemented with pWH1266 expressing basG from its native promoter This study Δ bfnL Baumannoferrin biosynthetic mutant where bfnL (A1S_1657) was replaced by a kanamycin resistance determinant, and then the cassette was excised, to leave a markerless mutant This study Δ fbsE Fimsbactins biosynthetic mutant where fbsE (A1S_2578) was replaced by a kanamycin resistance determinant; Kan R This study Δ basG bfnL Acinetobactin and baumannoferrin biosynthetic mutant. Proficient in fimsbactins biosynthesis; Kan R This study Δ basG fbsE Acinetobactin and fimsbactins biosynthetic mutant. Proficient in baumannoferrin biosynthesis; Kan R This study Δ bfnL fbsE Baumannoferrin and fimsbactins biosynthetic mutant. Proficient in acinetobactin biosynthesis; Kan R This study Δ basG bfnL fbsE Complete siderophore biosynthetic mutant with disruptions in acinetobactin, baumannoferrin, and fimsbactins production; Kan R This study Δz ur Zur mutant; Kan R [ 58 ] Plasmid pKD4 Template for amplification of the FRT-flanked kanamycin resistance determinant; Kan R , Amp R [ 94 ] pAT02 A . baumannii recombinase expressing plasmid; Carb R /Amp R [ 61 ] pAT03 Expresses an IPTG-inducible flipase to remove the kanamycin resistance determinant; Carb R /Amp R [ 61 ] pWH1266 A . baumannii / E . coli shuttle and complementation vector; Carb R /Amp R [ 95 ] pWH1266:: basG pWH1266 expressing basG from its native promoter; Carb R /Amp R This study For bacterial growth under metal-restriction, media were prepared in sterile polypropylene vessels or in glassware that was washed overnight with 0.1 M HCl and then subsequently rinsed extensively with ultrapure water to remove contaminating iron. Media and all components were prepared using water purified with a Nanopure Diamond filtration system (Thermo Scientific). When required, base media and additives were Chelex-treated using 5% w/v Chelex-100 resin (Sigma-Aldrich) stirring overnight at 4°C. Tris Minimal Succinate (TMS) media were employed in zinc and iron-restriction studies, prepared as previously described [ 96 ]. For growth curves, TMS was treated with Chelex-100 (cTMS) to remove residual iron. RNA extraction and quantitative reverse transcription PCR To assess in vitro gene expression, WT A . baumannii or its isogenic mutants were grown overnight in TMS media prior to subculturing 1:50 in 10 mL cTMS media with or without the addition of 30 μM ZnCl 2 or FeCl 3 . Bacteria were grown until exponential (4 h) or stationary phase (12 h), pelleted by centrifugation, and siderophore activity was confirmed in metal-deplete culture supernatants (see "Determination of siderophore production by Chrome Azurol S assay"). Bacterial pellets were stored in a 1:1 solution of acetone and ethanol at -80°C until RNA extraction. Prior to extraction, the acetate:ethanol was removed by aspiration following centrifugation for 15 min at maximum speed. The bacterial pellets were resuspended in 750 μL of LETS buffer (0.1 M LiCl, 10 mM EDTA, 10 mM Tris HCl (pH 7.4), 1% sodium dodecyl sulphate) and lysed in Lysing Matrix B tubes using a FastPrep-24 bead beater (MP Biomedicals) with two 45 s pulses at 6 M/s separated by a 5-min rest. Following incubation at 55°C for 5 min, the samples were centrifuged at 21,130 x g for 10 min and then transferred to new 2 mL RNAase-free tubes. RNA was solubilized by mixing lysates with 1 mL TRI reagent (Sigma-Aldrich) and incubated at room temperature for 5 min. To each sample, 0.2 mL of chloroform was added and mixed by rapid inversion for 15 s. Following incubation at room temperature for 2–3 min, the samples were centrifuged at 21,130 x g for 10 min, and the upper aqueous phase was transferred to a new tube and mixed with 1 mL of isopropanol. RNA was precipitated at room temperature for 10 min, pelleted by centrifugation, washed with 200 μL of 70% ethanol, and dried at room temperature for 1 min. The RNA pellet was resuspended in 100 μL of nuclease-free water and treated with RNA Qualified (RQ1) RNase-Free DNase for 2 h at 37°C (Promega). RNA was then purified further using an RNeasy miniprep kit, as per instructions from the manufacturer (Qiagen). RNA quality and quantity were assessed using a NanoDrop 8000 Spectrophotometer (Fisher Scientific). cDNA was synthesized from 1 μg RNA using random primers and M-MLV reverse transcriptase, using instructions from the supplier (Promega). The cDNA was diluted 1:50 and used in quantitative reverse transcription PCR (qRT-PCR) using the primer pairs denoted in Table 2 and iQ SYBR Green Supermix, as directed (Bio-Rad). qRT-PCR was performed using a two-step melt curve program on a CFX96 Touch Real-Time PCR detection system (Bio-Rad). Target gene expression was normalized to the expression of rpoB and presented as the fold change (2 -ΔΔCT ) relative to expression under metal-replete conditions, or to WT when gene expression was assessed in the Δ zur or Δ basG bfnL mutants. 10.1371/journal.ppat.1008995.t002 Table 2 Primers employed in this study. Designation a , b Sequence Function basE -F RT CGCGCATTTTAACGGTAGGG basE qRT-PCR basE -R RT CCCTTGCCCAAGCCTTTTTC basE qRT-PCR basD -F RT GGCAACGCAACTTTAGGTGG basD qRT-PCR basD -R RT AGCCATGATGTTTGCGATGC basD qRT-PCR bauD -F RT GTATCGGGTTGGCGGTATGT bauD qRT-PCR bauD -R RT ATCATCTTGCTCAGCGTGCT bauD qRT-PCR basB -F RT CAAAATGCCAAAGGTCGCCA basB qRT-PCR basB -R RT GCTTTCCAGTTTGGGGCTTG basB qRT-PCR basA- F RT TATGGATTCTCCGCCATCGC basA qRT-PCR basA -R RT AGCCGGACGTCTGTTGATTT basA qRT-PCR bauF -F RT ATTGTCTCGATCCAGACGCC bauF qRT-PCR bauF -R RT TTGCCAAATTGGGAGCTTGC bauF qRT-PCR bfnA -F RT TCGGTTTACGTGGATGGCAA bfnA qRT-PCR bfnA -R RT TCGCCACGATGACACTCTTT bfnA qRT-PCR bfnF -F RT AGTCAAAAGCTGCCGAAAGT bfnF qRT-PCR bfnF -R RT ACGGCATAAGGTCTGCTCAG bfnF qRT-PCR bfnL -F RT TACACCGCTGGATGCATGAG bfnL qRT-PCR bfnL -R RT AATTTCGGCATACCCCACGT bfnL qRT-PCR fbsA -F RT TCACCTCTCCCCAACCATCA fbsA qRT-PCR fbsA -R RT GGCGACTCAGCACTCATCTT fbsA qRT-PCR fbsB -F RT AGCCTTGGCATTGTCTCTCC fbsB qRT-PCR fbsB -R RT ATCCATCCACCCGACCAAAC fbsB qRT-PCR fbsM -F RT GTGTTCCCTCTGCAGGTAGC fbsM qRT-PCR fbsM -R RT CTTTCGTCCATGACCAGGGT fbsM qRT-PCR fbsN -F RT ACCGTTATTTGGGTTCGGCT fbsN qRT-PCR fbsN -R RT AATCGCACCACGTGTTTTGG fbsN qRT-PCR basG -F RT AGCGCAAATCGGAATCATGC basG qRT-PCR basG -R RT TGGCCAGACACACAAATCGA basG qRT-PCR fur- F RT TGCGCAAAGCTGGACTTAAA fur qRT-PCR fur- R RT TCGCAAGTCCGACATCTTCC fur qRT-PCR zur- F RT GTCACCCACGTGAAGGTCAT zur qRT-PCR zur- R RT CTGTGTTGTGCTGCGAAATC zur qRT-PCR zigA -F RT GATCAGGCTCAGCAAGACCAG zigA qRT-PCR zigA -R RT GTGCTTGGACAGCTTCATCA zigA qRT-PCR rpoB -F RT ATGCCGCCTGAAAAAGTAAC rpoB qRT-PCR (housekeeping) rpoB -R RT TCCGCACGTAAAGTAGGAAC rpoB qRT-PCR (housekeeping) gyrA -F RT GACGACGGTACCGGTTTACA gyrA qRT-PCR (housekeeping) gyrA -R RT ACCGCGGCCATTATCTGAAA gyrA qRT-PCR (housekeeping) basG -F CAGGGTAGAGGGTTGCCATCATAAGGCATACACAAATCCTGGTCGCTAAATTTATACATAAAATTTATATCTTTGATAGCGACTCCTTAGACGACTTGTAATCGTCATATTAACGGAGCAAAAA GTGTAGGCTGGAGCTGCTTC Recombineering to replace basG with Km R basG -R AGTATTTTTTTAAAAACTTTAATTGCATCAAAAACTTCCTAACCACCCTATCAAAATATTTTTAGATCATTTTCTAGACTGTAAAAATTAATATGGATTTGTCTAAATCATCTTGGTTGATATGA CATATGAATATC CTCCTTAG Recombineering to replace basG with Km R Full basG -F GTATCTTCACGTTGCGGTCAGGTC Checking recombineering of basG Full basG -R AATGCTGGCTTGAGTGCAGGT Checking recombineering of basG bfnL -F GGGGAAAGCTGAATTTTTTGTCCATGATTTATGCAATAAAAAAAATAGTAAAAATAAGCTTGCATCTTAAATAAAAATGATTATCATTATCAATTATAGGTTATGTCATAGCAAGGACCGTTATA GTGTAGGCTGGAGCTGCTTC Recombineering to replace bfnL with Km R bfnL -R AACTCCTGCCCAATGAGTTCTTGAGATGCCTGTTGCAATGTTTGAAACCGCAACCGTTCAGTATCAAACATTGTCCCATCCATATCGAAGATAGCTCCATGAACAGGTTTTCCATGAAAAATAAG CATATGAATATCCTCCTTAG Recombineering to replace bfnL with Km R Full bfnL -F TGTCGATCTGGCGCTCATAC Checking recombineering of bfnL Full bfnL -R GGTTCCTGTATCTCTGGCGT Checking recombineering of bfnL fbsE -F ATGCAATTACAACAAGAAATTACCGCAGACATTCACCATGTTCTGCAAATTGATAGCATTCAAATTCAACCTGAAGATAATTTGATTGAACATGGGCTGCATTCATTGGCGATCATGCAGTTAGT GTGTAGGCTGGAGCTGCTTC Recombineering to replace fbsE with Km R fbsE -R ATCACTTGCATCAATTGCATTTCATCGATGGTGGTACGTAATGCAGGATATAATGCAATCAATTGAGTCAAACGCTGAGTCAGTGTTTCAACATCCATTCGCCCATGAAATTCTTGAAAGTCATG CATATGAATATCCTCCTTAG Recombineering to replace fbsE with Km R Full fbsE -F AACTGTGGGGTTGGAACTCG Checking recombineering of fbsE Full fbsE -R TGAATGGTCGCTTCCATGCT Checking recombineering of fbsE basG Gibson-F gcgaccacacccgtcctgtgTTGGACTCATTACGAATTATG Complementation of basG basG Gibson-R aaggctctcaagggcatcggCTAAAAGCCAACTGTACG Complementation of basG a Underlined areas denote regions of homology with the kanamycin resistance determinant b Lower case letters denote regions of homology with pWH1266 Generation of A . baumannii siderophore biosynthetic mutants Oligonucleotides used in generating, confirming, or complementing the siderophore biosynthetic mutants detailed below can be found in Table 2 . All mutants were generated using recombineering, as previously described [ 61 ]. In brief, the FRT-flanked kanamycin resistance determinant of pKD4 [ 94 ] was amplified using primers bearing 120 bp of homology to the region flanking the gene of interest ( basG , bfnL , or fbsE , using primers basG- F/R, bfnL -F/R, and fbsE- F/R, respectively). The PCR product was purified and subsequently concentrated to ~1–2 μg/μL. Approximately 5 μg of linear recombineering product was introduced to 100 μL (~10 8 colony forming units (CFU)) of competent A . baumannii containing the recombinase-expressing plasmid pAT02 [ 61 ] by electroporation at 1800 V. Cells were recovered immediately in pre-warmed LB, and recombinase expression was induced through the addition of 2 mM of IPTG to the media. After 4 h of growth at 37°C, the transformants were plated to LBA with kanamycin. The putative Δ basG ::km, Δ bfnL ::km or Δ fbsE ::km colonies that arose on the transformation plates were screened by PCR using primer pairs Full basG- F/R, Full bfnL- F/R, or Full fbsE- F/R, respectively, each of which flank the site of recombination. Colonies were screened for the loss of pAT02 through patching to LBA supplemented with carbenicillin, and maintenance of the native plasmid pAB3 was confirmed through resistance to sulfamethoxazole [ 97 ]. The basG mutants obtained from the A . baumannii AB5075-UW three-allele mutant library [ 92 ] were confirmed through PCR using primer pairs Full basG -F/R. In order to generate combinatorial mutants, it was necessary to excise the kanamycin cassette from the single siderophore biosynthetic mutants to yield unmarked mutations. The strains Δ basG ::km and Δ bfnL ::km were transformed with the Flp recombinase plasmid, pAT03 [ 61 ]. Expression of the flippase was induced by growth of Δ basG ::km/pAT03 and Δ bfnL ::km/pAT03 in LB with 1 mM IPTG for 4 h. The cells were harvested by centrifugation, plated to LBA containing 1 mM IPTG, and incubated overnight at 37°C. Biomass was scraped from the induction plates and streaked for isolated colonies on LBA without antibiotics. Colonies were screened for excision of the kanamycin cassette and simultaneous loss of the pAT03 plasmid through identifying isolates that were both kanamycin and carbenicillin sensitive. Excision of the kanamycin cassette was confirmed through colony PCR using primer pairs Full basG- F/R or Full bfnL- F/R. Upon generation of the markerless strains, combinatorial mutants were generated in the Δ basG and Δ bfnL backgrounds. The procedure was performed as described above, except that the linear DNA fragment bearing homology to fbsE was introduced to both Δ basG /pAT02 and Δ bfnL /pAT02, and the linear DNA fragment bearing homology to bfnL was also introduced to Δ basG /pAT02. Screening and confirmation of the Δ basG bfnL ::km, Δ basG fbsE ::km, and Δ bfnL fbsE ::km mutants was performed as detailed above. A complete siderophore biosynthetic mutant was generated by excising the kanamycin cassette from Δ basG bfnL ::km, and subsequently disrupting fbsE . All mutations were confirmed through PCR and maintenance of pAB3 was assured through resistance to sulfamethoxazole. Complementation of basG Complementation of basG was performed in trans using the vector pWH1266 [ 95 ]. The basG gene and its native promoter were amplified using the primers basG Gibson-F/R ( Table 2 ). pWH1266 was digested with BamHI and SalI and the PCR product and plasmid were joined using NEB Builder HiFi Assembly (New England BioLabs) at 50°C for 1 h. The resulting assembly was transformed into chemically competent DH5 α by heat shock, and successful transformants were selected for on LBA with ampicillin following overnight growth at 37°C. The pWH1266:: basG construct was confirmed by PCR and sequencing prior to introduction of both this vector, as well as the native empty vector, to Δ basG by electroporation. Successful transformants were selected on LBA with carbenicillin. Determination of siderophore production by Chrome Azurol S assay To assess for overall siderophore production, Chrome Azurol S (CAS) assays were performed. In preparation for the assays, bacteria were grown in biological triplicate overnight in TMS media, pelleted by centrifugation at 1073 x g and washed three times with 1X phosphate buffered saline (PBS). The OD 600 of each resuspension was adjusted to 1.0 and used to inoculate 10 mL of cTMS media to an OD 600 of 0.005 in 50 mL conical tubes. The cultures were grown for 12 h at 37°C with shaking at 180 rpm. Following growth, the OD 600nm of each culture was determined and bacteria were pelleted by centrifugation. The supernatants were removed and sterilized using a 0.2 μm filter prior to use in the CAS assays. CAS reagent was prepared, and the assays performed, essentially as described previously [ 59 , 60 ]. In brief, CAS is an iron-binding dye complex that detects mobilization of iron from the dye to a chelator through a colorimetric change from blue (negative reaction) to pink/orange (positive reaction) and/or through a decrease in absorbance at 630 nm (A 630 ). Prepared CAS reagent was mixed 1:1 with the filtered culture supernatants and incubated in the dark at room temperature for 30 min. The A 630 was determined, each value was normalized to the final OD 600nm of the corresponding culture, and siderophore activity was expressed as a percentage of WT A . baumannii . Assessing the ability of A . baumannii to utilize host iron sources under iron-restricted growth To assess growth of A . baumannii WT and the siderophore biosynthetic mutants under iron-restriction, single-isolated colonies of bacteria grown on LBA were used to inoculate 3 mL of TMS media and incubated overnight at 37°C with shaking at 180 rpm. The overnight cultures were pelleted by centrifugation at 604 x g for 15 min and washed once with sterile 1x PBS. The pellets were resuspended in PBS and subcultured 1:100 in cTMS media. Cultures were grown to an OD 600nm of ~1, pelleted by centrifugation, washed three times with sterile 1 x PBS and resuspended to an OD 600nm of 0.1. Growth curves were set-up in cTMS in 96-well plates using 20% v/v heat inactivated and filter sterilized human serum (Sigma-Aldrich; H4522), 180 mg/dL partially saturated human transferrin (Sigma-Aldrich; T3309), or 15 mg/mL lactoferrin from human milk (Sigma-Aldrich; L0520). Concentrations were selected to be physiologically relevant and to support growth of WT A . baumannii in the absence of another iron source. For zinc and iron-replete conditions, 30 μM of ZnCl 2 or FeCl 3 were added, respectively. Carbenicillin was added when required to maintain pWH1266 or its derivatives. Plates were inoculated using 5 μL of the prepared cells in 200 μL of media and grown in an Epoch 2 microplate reader (BioTek) at 37°C with medium amplitude linear shaking. Cell density was assessed by OD 600nm every 30 min, but for graphical clarity only data collected at 4 h intervals is shown. Estimations of the asymptote of the maximal OD 600nm (A) , maximum specific growth rate (μ m ) and lag time (λ) were modeled using single Gompertz growth curve fitting and open source code available at https://scott-h-saunders.shinyapps.io/gompertz_fitting_0v2/ [ 98 – 100 ]. Murine model of A . baumannii bacteremia All infection experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee and are in compliance with guidelines set by the Animal Welfare Act, the National Institutes of Health, and the American Veterinary Medical Association. Six-to-eight- week old female C57BL/6J mice (Jackson Laboratories) were injected retro-orbitally with 100 μL of bacterial cell suspensions containing ~2 x 10 8 to 5 x 10 8 colony forming units (CFU) of WT A . baumannii or its isogenic mutants in PBS. In preparation for infection, overnight cultures of bacteria were subcultured 1:100 in fresh LB and grown to mid-to-late log phase (OD 600 = 2.0–2.5) at 37°C with shaking. The bacteria were pelleted by centrifugation, washed twice with PBS, and normalized to an OD 6oonm of 0.35 at a 1:20 dilution. Following injection, the mice were humanely euthanized at 24 h, and the kidneys, heart, liver, spleen, lungs, and blood of each mouse were harvested. The organs were homogenized in PBS using a Bullet Blender (Next Advance), and all samples were serially diluted in PBS prior to plating on LBA to determine the bacterial burdens. RNA extraction and in vivo gene expression analysis by NanoString For assessing in vivo gene expression, mice were infected as detailed above. At 24 h, mice were humanely euthanized, and the harvested organs were placed in RNAase-free navy bullet blender tubes (Next Advance) containing 600 μL of RLT buffer (Qiagen) with 1% (v/v) β-mercaptoethanol. Following homogenization in the bullet blender for 2 x 5 min (speed 8 for livers, speed 10 for all other organs; Next Advance), the homogenate was transferred to a fresh bullet blender tube containing 600 μL of phenol:chloroform:isoamyl alcohol (25:24:1, Fisher Scientific). After an additional 5 min homogenization cycle, the samples were centrifuged at 21,139 x g for 10 min. The upper aqueous phase was transferred to a 2-mL nuclease-free tube containing 600 μL of 70% ethanol and inverted until a visible mass of RNA formed. The extracted RNA was subsequently purified using an RNeasy kit (Qiagen), as per manufacturer's instructions. As an in vitro comparator, WT A . baumannii was grown in LB using the same methodology used to prepare for infection (see section "Murine model of A . baumannii bacteremia"). RNA was extracted from in vitro grown bacteria using the method described in "RNA extraction and quantitative reverse transcription PCR". RNA quality and concentration were determined using a NanoDrop 8000 Spectrophotometer (Fisher Scientific). In preparation for NanoString, 100 ng of RNA in 5 μL of nuclease-free water was aliquoted to 12-well tube strips and mixed with 8 μL of the Reporter Probe Set in hybridization buffer (NanoString). To each well, 2 μL of the Capture Probe Set (NanoString) were added and the samples were hybridized at 65°C in a preheated thermocycler for 18 h. Following hybridization, samples were processed by Vanderbilt Technologies for Advanced Genomics (VANTAGE) on an nCounter FLEX analysis system (NanoString). Data were analyzed using nSolver Analysis software (NanoString), where A . baumannii gene expression was normalized to the expression of housekeeping genes rpsA . Using negative control probes included by the manufacturer in the Reporter Probe Set and designed not to interact with biological samples (NanoString), the minimum threshold for gene expression was determined and all target genes included in the analysis exhibited expression above background. Expression changes are given as the fold change of in vivo A . baumannii gene expression versus gene expression in in vitro grown bacteria. Bacterial strains and growth conditions All bacterial strains and plasmids employed in this study can be found in Table 1 . Unless otherwise indicated, experiments were performed with A . baumannii strain ATCC 17978 and its isogenic mutants. For routine cultivation and genetic manipulation, A . baumannii was cultured in lysogeny broth (LB) or on LB with 1.5% w/v agar (LBA). Antibiotics, when required for selection of recombinants or maintenance of plasmids in A . baumannii , were supplied at the following concentrations: ampicillin, 500 μg/mL; carbenicillin, 75 μg/mL; kanamycin, 15 μg/mL; sulfamethoxazole, 100 μg/mL; as indicated. For selection and maintenance of plasmids in E . coli , 100 μg/mL of ampicillin was used. Plasmid gene expression was induced using 1–2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), as detailed in the text. 10.1371/journal.ppat.1008995.t001 Table 1 Strains and plasmids employed in this study. Strain Description Reference A . baumannii ATCC 17978 WT A . baumannii fatal meningitis isolate from 1951 American Type Culture Collection (ATCC) strain 5377 [ 54 ] WT/pWH1266 WT A . baumannii ATCC 17978 with empty pWH1266 plasmid This study A . baumannii AB5075-UW Multidrug-resistant WT A . baumannii AB5075 isolate from a wound infection [ 92 , 93 ] Δ basG Acinetobactin biosynthetic mutant where basG (A1S_2379) was replaced by a kanamycin resistance determinant, and then the cassette was excised, to leave a markerless mutant This study basG 102::Tn26 Transposon mutant of basG in A . baumannii AB5075-UW from three-allele mutant library; identifier tnab1_kr121128p06q102 [ 92 ] basG 142::Tn26 Transposon mutant of basG in A . baumannii AB5075-UW from three-allele mutant library; identifier tnab1_kr130909p02q142 [ 92 ] Δ basG /pWH1266:: basG basG mutant complemented with pWH1266 expressing basG from its native promoter This study Δ bfnL Baumannoferrin biosynthetic mutant where bfnL (A1S_1657) was replaced by a kanamycin resistance determinant, and then the cassette was excised, to leave a markerless mutant This study Δ fbsE Fimsbactins biosynthetic mutant where fbsE (A1S_2578) was replaced by a kanamycin resistance determinant; Kan R This study Δ basG bfnL Acinetobactin and baumannoferrin biosynthetic mutant. Proficient in fimsbactins biosynthesis; Kan R This study Δ basG fbsE Acinetobactin and fimsbactins biosynthetic mutant. Proficient in baumannoferrin biosynthesis; Kan R This study Δ bfnL fbsE Baumannoferrin and fimsbactins biosynthetic mutant. Proficient in acinetobactin biosynthesis; Kan R This study Δ basG bfnL fbsE Complete siderophore biosynthetic mutant with disruptions in acinetobactin, baumannoferrin, and fimsbactins production; Kan R This study Δz ur Zur mutant; Kan R [ 58 ] Plasmid pKD4 Template for amplification of the FRT-flanked kanamycin resistance determinant; Kan R , Amp R [ 94 ] pAT02 A . baumannii recombinase expressing plasmid; Carb R /Amp R [ 61 ] pAT03 Expresses an IPTG-inducible flipase to remove the kanamycin resistance determinant; Carb R /Amp R [ 61 ] pWH1266 A . baumannii / E . coli shuttle and complementation vector; Carb R /Amp R [ 95 ] pWH1266:: basG pWH1266 expressing basG from its native promoter; Carb R /Amp R This study For bacterial growth under metal-restriction, media were prepared in sterile polypropylene vessels or in glassware that was washed overnight with 0.1 M HCl and then subsequently rinsed extensively with ultrapure water to remove contaminating iron. Media and all components were prepared using water purified with a Nanopure Diamond filtration system (Thermo Scientific). When required, base media and additives were Chelex-treated using 5% w/v Chelex-100 resin (Sigma-Aldrich) stirring overnight at 4°C. Tris Minimal Succinate (TMS) media were employed in zinc and iron-restriction studies, prepared as previously described [ 96 ]. For growth curves, TMS was treated with Chelex-100 (cTMS) to remove residual iron. RNA extraction and quantitative reverse transcription PCR To assess in vitro gene expression, WT A . baumannii or its isogenic mutants were grown overnight in TMS media prior to subculturing 1:50 in 10 mL cTMS media with or without the addition of 30 μM ZnCl 2 or FeCl 3 . Bacteria were grown until exponential (4 h) or stationary phase (12 h), pelleted by centrifugation, and siderophore activity was confirmed in metal-deplete culture supernatants (see "Determination of siderophore production by Chrome Azurol S assay"). Bacterial pellets were stored in a 1:1 solution of acetone and ethanol at -80°C until RNA extraction. Prior to extraction, the acetate:ethanol was removed by aspiration following centrifugation for 15 min at maximum speed. The bacterial pellets were resuspended in 750 μL of LETS buffer (0.1 M LiCl, 10 mM EDTA, 10 mM Tris HCl (pH 7.4), 1% sodium dodecyl sulphate) and lysed in Lysing Matrix B tubes using a FastPrep-24 bead beater (MP Biomedicals) with two 45 s pulses at 6 M/s separated by a 5-min rest. Following incubation at 55°C for 5 min, the samples were centrifuged at 21,130 x g for 10 min and then transferred to new 2 mL RNAase-free tubes. RNA was solubilized by mixing lysates with 1 mL TRI reagent (Sigma-Aldrich) and incubated at room temperature for 5 min. To each sample, 0.2 mL of chloroform was added and mixed by rapid inversion for 15 s. Following incubation at room temperature for 2–3 min, the samples were centrifuged at 21,130 x g for 10 min, and the upper aqueous phase was transferred to a new tube and mixed with 1 mL of isopropanol. RNA was precipitated at room temperature for 10 min, pelleted by centrifugation, washed with 200 μL of 70% ethanol, and dried at room temperature for 1 min. The RNA pellet was resuspended in 100 μL of nuclease-free water and treated with RNA Qualified (RQ1) RNase-Free DNase for 2 h at 37°C (Promega). RNA was then purified further using an RNeasy miniprep kit, as per instructions from the manufacturer (Qiagen). RNA quality and quantity were assessed using a NanoDrop 8000 Spectrophotometer (Fisher Scientific). cDNA was synthesized from 1 μg RNA using random primers and M-MLV reverse transcriptase, using instructions from the supplier (Promega). The cDNA was diluted 1:50 and used in quantitative reverse transcription PCR (qRT-PCR) using the primer pairs denoted in Table 2 and iQ SYBR Green Supermix, as directed (Bio-Rad). qRT-PCR was performed using a two-step melt curve program on a CFX96 Touch Real-Time PCR detection system (Bio-Rad). Target gene expression was normalized to the expression of rpoB and presented as the fold change (2 -ΔΔCT ) relative to expression under metal-replete conditions, or to WT when gene expression was assessed in the Δ zur or Δ basG bfnL mutants. 10.1371/journal.ppat.1008995.t002 Table 2 Primers employed in this study. Designation a , b Sequence Function basE -F RT CGCGCATTTTAACGGTAGGG basE qRT-PCR basE -R RT CCCTTGCCCAAGCCTTTTTC basE qRT-PCR basD -F RT GGCAACGCAACTTTAGGTGG basD qRT-PCR basD -R RT AGCCATGATGTTTGCGATGC basD qRT-PCR bauD -F RT GTATCGGGTTGGCGGTATGT bauD qRT-PCR bauD -R RT ATCATCTTGCTCAGCGTGCT bauD qRT-PCR basB -F RT CAAAATGCCAAAGGTCGCCA basB qRT-PCR basB -R RT GCTTTCCAGTTTGGGGCTTG basB qRT-PCR basA- F RT TATGGATTCTCCGCCATCGC basA qRT-PCR basA -R RT AGCCGGACGTCTGTTGATTT basA qRT-PCR bauF -F RT ATTGTCTCGATCCAGACGCC bauF qRT-PCR bauF -R RT TTGCCAAATTGGGAGCTTGC bauF qRT-PCR bfnA -F RT TCGGTTTACGTGGATGGCAA bfnA qRT-PCR bfnA -R RT TCGCCACGATGACACTCTTT bfnA qRT-PCR bfnF -F RT AGTCAAAAGCTGCCGAAAGT bfnF qRT-PCR bfnF -R RT ACGGCATAAGGTCTGCTCAG bfnF qRT-PCR bfnL -F RT TACACCGCTGGATGCATGAG bfnL qRT-PCR bfnL -R RT AATTTCGGCATACCCCACGT bfnL qRT-PCR fbsA -F RT TCACCTCTCCCCAACCATCA fbsA qRT-PCR fbsA -R RT GGCGACTCAGCACTCATCTT fbsA qRT-PCR fbsB -F RT AGCCTTGGCATTGTCTCTCC fbsB qRT-PCR fbsB -R RT ATCCATCCACCCGACCAAAC fbsB qRT-PCR fbsM -F RT GTGTTCCCTCTGCAGGTAGC fbsM qRT-PCR fbsM -R RT CTTTCGTCCATGACCAGGGT fbsM qRT-PCR fbsN -F RT ACCGTTATTTGGGTTCGGCT fbsN qRT-PCR fbsN -R RT AATCGCACCACGTGTTTTGG fbsN qRT-PCR basG -F RT AGCGCAAATCGGAATCATGC basG qRT-PCR basG -R RT TGGCCAGACACACAAATCGA basG qRT-PCR fur- F RT TGCGCAAAGCTGGACTTAAA fur qRT-PCR fur- R RT TCGCAAGTCCGACATCTTCC fur qRT-PCR zur- F RT GTCACCCACGTGAAGGTCAT zur qRT-PCR zur- R RT CTGTGTTGTGCTGCGAAATC zur qRT-PCR zigA -F RT GATCAGGCTCAGCAAGACCAG zigA qRT-PCR zigA -R RT GTGCTTGGACAGCTTCATCA zigA qRT-PCR rpoB -F RT ATGCCGCCTGAAAAAGTAAC rpoB qRT-PCR (housekeeping) rpoB -R RT TCCGCACGTAAAGTAGGAAC rpoB qRT-PCR (housekeeping) gyrA -F RT GACGACGGTACCGGTTTACA gyrA qRT-PCR (housekeeping) gyrA -R RT ACCGCGGCCATTATCTGAAA gyrA qRT-PCR (housekeeping) basG -F CAGGGTAGAGGGTTGCCATCATAAGGCATACACAAATCCTGGTCGCTAAATTTATACATAAAATTTATATCTTTGATAGCGACTCCTTAGACGACTTGTAATCGTCATATTAACGGAGCAAAAA GTGTAGGCTGGAGCTGCTTC Recombineering to replace basG with Km R basG -R AGTATTTTTTTAAAAACTTTAATTGCATCAAAAACTTCCTAACCACCCTATCAAAATATTTTTAGATCATTTTCTAGACTGTAAAAATTAATATGGATTTGTCTAAATCATCTTGGTTGATATGA CATATGAATATC CTCCTTAG Recombineering to replace basG with Km R Full basG -F GTATCTTCACGTTGCGGTCAGGTC Checking recombineering of basG Full basG -R AATGCTGGCTTGAGTGCAGGT Checking recombineering of basG bfnL -F GGGGAAAGCTGAATTTTTTGTCCATGATTTATGCAATAAAAAAAATAGTAAAAATAAGCTTGCATCTTAAATAAAAATGATTATCATTATCAATTATAGGTTATGTCATAGCAAGGACCGTTATA GTGTAGGCTGGAGCTGCTTC Recombineering to replace bfnL with Km R bfnL -R AACTCCTGCCCAATGAGTTCTTGAGATGCCTGTTGCAATGTTTGAAACCGCAACCGTTCAGTATCAAACATTGTCCCATCCATATCGAAGATAGCTCCATGAACAGGTTTTCCATGAAAAATAAG CATATGAATATCCTCCTTAG Recombineering to replace bfnL with Km R Full bfnL -F TGTCGATCTGGCGCTCATAC Checking recombineering of bfnL Full bfnL -R GGTTCCTGTATCTCTGGCGT Checking recombineering of bfnL fbsE -F ATGCAATTACAACAAGAAATTACCGCAGACATTCACCATGTTCTGCAAATTGATAGCATTCAAATTCAACCTGAAGATAATTTGATTGAACATGGGCTGCATTCATTGGCGATCATGCAGTTAGT GTGTAGGCTGGAGCTGCTTC Recombineering to replace fbsE with Km R fbsE -R ATCACTTGCATCAATTGCATTTCATCGATGGTGGTACGTAATGCAGGATATAATGCAATCAATTGAGTCAAACGCTGAGTCAGTGTTTCAACATCCATTCGCCCATGAAATTCTTGAAAGTCATG CATATGAATATCCTCCTTAG Recombineering to replace fbsE with Km R Full fbsE -F AACTGTGGGGTTGGAACTCG Checking recombineering of fbsE Full fbsE -R TGAATGGTCGCTTCCATGCT Checking recombineering of fbsE basG Gibson-F gcgaccacacccgtcctgtgTTGGACTCATTACGAATTATG Complementation of basG basG Gibson-R aaggctctcaagggcatcggCTAAAAGCCAACTGTACG Complementation of basG a Underlined areas denote regions of homology with the kanamycin resistance determinant b Lower case letters denote regions of homology with pWH1266 Generation of A . baumannii siderophore biosynthetic mutants Oligonucleotides used in generating, confirming, or complementing the siderophore biosynthetic mutants detailed below can be found in Table 2 . All mutants were generated using recombineering, as previously described [ 61 ]. In brief, the FRT-flanked kanamycin resistance determinant of pKD4 [ 94 ] was amplified using primers bearing 120 bp of homology to the region flanking the gene of interest ( basG , bfnL , or fbsE , using primers basG- F/R, bfnL -F/R, and fbsE- F/R, respectively). The PCR product was purified and subsequently concentrated to ~1–2 μg/μL. Approximately 5 μg of linear recombineering product was introduced to 100 μL (~10 8 colony forming units (CFU)) of competent A . baumannii containing the recombinase-expressing plasmid pAT02 [ 61 ] by electroporation at 1800 V. Cells were recovered immediately in pre-warmed LB, and recombinase expression was induced through the addition of 2 mM of IPTG to the media. After 4 h of growth at 37°C, the transformants were plated to LBA with kanamycin. The putative Δ basG ::km, Δ bfnL ::km or Δ fbsE ::km colonies that arose on the transformation plates were screened by PCR using primer pairs Full basG- F/R, Full bfnL- F/R, or Full fbsE- F/R, respectively, each of which flank the site of recombination. Colonies were screened for the loss of pAT02 through patching to LBA supplemented with carbenicillin, and maintenance of the native plasmid pAB3 was confirmed through resistance to sulfamethoxazole [ 97 ]. The basG mutants obtained from the A . baumannii AB5075-UW three-allele mutant library [ 92 ] were confirmed through PCR using primer pairs Full basG -F/R. In order to generate combinatorial mutants, it was necessary to excise the kanamycin cassette from the single siderophore biosynthetic mutants to yield unmarked mutations. The strains Δ basG ::km and Δ bfnL ::km were transformed with the Flp recombinase plasmid, pAT03 [ 61 ]. Expression of the flippase was induced by growth of Δ basG ::km/pAT03 and Δ bfnL ::km/pAT03 in LB with 1 mM IPTG for 4 h. The cells were harvested by centrifugation, plated to LBA containing 1 mM IPTG, and incubated overnight at 37°C. Biomass was scraped from the induction plates and streaked for isolated colonies on LBA without antibiotics. Colonies were screened for excision of the kanamycin cassette and simultaneous loss of the pAT03 plasmid through identifying isolates that were both kanamycin and carbenicillin sensitive. Excision of the kanamycin cassette was confirmed through colony PCR using primer pairs Full basG- F/R or Full bfnL- F/R. Upon generation of the markerless strains, combinatorial mutants were generated in the Δ basG and Δ bfnL backgrounds. The procedure was performed as described above, except that the linear DNA fragment bearing homology to fbsE was introduced to both Δ basG /pAT02 and Δ bfnL /pAT02, and the linear DNA fragment bearing homology to bfnL was also introduced to Δ basG /pAT02. Screening and confirmation of the Δ basG bfnL ::km, Δ basG fbsE ::km, and Δ bfnL fbsE ::km mutants was performed as detailed above. A complete siderophore biosynthetic mutant was generated by excising the kanamycin cassette from Δ basG bfnL ::km, and subsequently disrupting fbsE . All mutations were confirmed through PCR and maintenance of pAB3 was assured through resistance to sulfamethoxazole. Complementation of basG Complementation of basG was performed in trans using the vector pWH1266 [ 95 ]. The basG gene and its native promoter were amplified using the primers basG Gibson-F/R ( Table 2 ). pWH1266 was digested with BamHI and SalI and the PCR product and plasmid were joined using NEB Builder HiFi Assembly (New England BioLabs) at 50°C for 1 h. The resulting assembly was transformed into chemically competent DH5 α by heat shock, and successful transformants were selected for on LBA with ampicillin following overnight growth at 37°C. The pWH1266:: basG construct was confirmed by PCR and sequencing prior to introduction of both this vector, as well as the native empty vector, to Δ basG by electroporation. Successful transformants were selected on LBA with carbenicillin. Determination of siderophore production by Chrome Azurol S assay To assess for overall siderophore production, Chrome Azurol S (CAS) assays were performed. In preparation for the assays, bacteria were grown in biological triplicate overnight in TMS media, pelleted by centrifugation at 1073 x g and washed three times with 1X phosphate buffered saline (PBS). The OD 600 of each resuspension was adjusted to 1.0 and used to inoculate 10 mL of cTMS media to an OD 600 of 0.005 in 50 mL conical tubes. The cultures were grown for 12 h at 37°C with shaking at 180 rpm. Following growth, the OD 600nm of each culture was determined and bacteria were pelleted by centrifugation. The supernatants were removed and sterilized using a 0.2 μm filter prior to use in the CAS assays. CAS reagent was prepared, and the assays performed, essentially as described previously [ 59 , 60 ]. In brief, CAS is an iron-binding dye complex that detects mobilization of iron from the dye to a chelator through a colorimetric change from blue (negative reaction) to pink/orange (positive reaction) and/or through a decrease in absorbance at 630 nm (A 630 ). Prepared CAS reagent was mixed 1:1 with the filtered culture supernatants and incubated in the dark at room temperature for 30 min. The A 630 was determined, each value was normalized to the final OD 600nm of the corresponding culture, and siderophore activity was expressed as a percentage of WT A . baumannii . Assessing the ability of A . baumannii to utilize host iron sources under iron-restricted growth To assess growth of A . baumannii WT and the siderophore biosynthetic mutants under iron-restriction, single-isolated colonies of bacteria grown on LBA were used to inoculate 3 mL of TMS media and incubated overnight at 37°C with shaking at 180 rpm. The overnight cultures were pelleted by centrifugation at 604 x g for 15 min and washed once with sterile 1x PBS. The pellets were resuspended in PBS and subcultured 1:100 in cTMS media. Cultures were grown to an OD 600nm of ~1, pelleted by centrifugation, washed three times with sterile 1 x PBS and resuspended to an OD 600nm of 0.1. Growth curves were set-up in cTMS in 96-well plates using 20% v/v heat inactivated and filter sterilized human serum (Sigma-Aldrich; H4522), 180 mg/dL partially saturated human transferrin (Sigma-Aldrich; T3309), or 15 mg/mL lactoferrin from human milk (Sigma-Aldrich; L0520). Concentrations were selected to be physiologically relevant and to support growth of WT A . baumannii in the absence of another iron source. For zinc and iron-replete conditions, 30 μM of ZnCl 2 or FeCl 3 were added, respectively. Carbenicillin was added when required to maintain pWH1266 or its derivatives. Plates were inoculated using 5 μL of the prepared cells in 200 μL of media and grown in an Epoch 2 microplate reader (BioTek) at 37°C with medium amplitude linear shaking. Cell density was assessed by OD 600nm every 30 min, but for graphical clarity only data collected at 4 h intervals is shown. Estimations of the asymptote of the maximal OD 600nm (A) , maximum specific growth rate (μ m ) and lag time (λ) were modeled using single Gompertz growth curve fitting and open source code available at https://scott-h-saunders.shinyapps.io/gompertz_fitting_0v2/ [ 98 – 100 ]. Murine model of A . baumannii bacteremia All infection experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee and are in compliance with guidelines set by the Animal Welfare Act, the National Institutes of Health, and the American Veterinary Medical Association. Six-to-eight- week old female C57BL/6J mice (Jackson Laboratories) were injected retro-orbitally with 100 μL of bacterial cell suspensions containing ~2 x 10 8 to 5 x 10 8 colony forming units (CFU) of WT A . baumannii or its isogenic mutants in PBS. In preparation for infection, overnight cultures of bacteria were subcultured 1:100 in fresh LB and grown to mid-to-late log phase (OD 600 = 2.0–2.5) at 37°C with shaking. The bacteria were pelleted by centrifugation, washed twice with PBS, and normalized to an OD 6oonm of 0.35 at a 1:20 dilution. Following injection, the mice were humanely euthanized at 24 h, and the kidneys, heart, liver, spleen, lungs, and blood of each mouse were harvested. The organs were homogenized in PBS using a Bullet Blender (Next Advance), and all samples were serially diluted in PBS prior to plating on LBA to determine the bacterial burdens. RNA extraction and in vivo gene expression analysis by NanoString For assessing in vivo gene expression, mice were infected as detailed above. At 24 h, mice were humanely euthanized, and the harvested organs were placed in RNAase-free navy bullet blender tubes (Next Advance) containing 600 μL of RLT buffer (Qiagen) with 1% (v/v) β-mercaptoethanol. Following homogenization in the bullet blender for 2 x 5 min (speed 8 for livers, speed 10 for all other organs; Next Advance), the homogenate was transferred to a fresh bullet blender tube containing 600 μL of phenol:chloroform:isoamyl alcohol (25:24:1, Fisher Scientific). After an additional 5 min homogenization cycle, the samples were centrifuged at 21,139 x g for 10 min. The upper aqueous phase was transferred to a 2-mL nuclease-free tube containing 600 μL of 70% ethanol and inverted until a visible mass of RNA formed. The extracted RNA was subsequently purified using an RNeasy kit (Qiagen), as per manufacturer's instructions. As an in vitro comparator, WT A . baumannii was grown in LB using the same methodology used to prepare for infection (see section "Murine model of A . baumannii bacteremia"). RNA was extracted from in vitro grown bacteria using the method described in "RNA extraction and quantitative reverse transcription PCR". RNA quality and concentration were determined using a NanoDrop 8000 Spectrophotometer (Fisher Scientific). In preparation for NanoString, 100 ng of RNA in 5 μL of nuclease-free water was aliquoted to 12-well tube strips and mixed with 8 μL of the Reporter Probe Set in hybridization buffer (NanoString). To each well, 2 μL of the Capture Probe Set (NanoString) were added and the samples were hybridized at 65°C in a preheated thermocycler for 18 h. Following hybridization, samples were processed by Vanderbilt Technologies for Advanced Genomics (VANTAGE) on an nCounter FLEX analysis system (NanoString). Data were analyzed using nSolver Analysis software (NanoString), where A . baumannii gene expression was normalized to the expression of housekeeping genes rpsA . Using negative control probes included by the manufacturer in the Reporter Probe Set and designed not to interact with biological samples (NanoString), the minimum threshold for gene expression was determined and all target genes included in the analysis exhibited expression above background. Expression changes are given as the fold change of in vivo A . baumannii gene expression versus gene expression in in vitro grown bacteria. Supporting information S1 Table Genes involved in acinetobactin biosynthesis and utilization in A . baumannii ATCC 17978. (DOCX) Click here for additional data file. S2 Table Genes involved in baumannoferrin biosynthesis and utilization in A . baumannii ATCC 17978. (DOCX) Click here for additional data file. S3 Table Genes involved in fimsbactins biosynthesis and utilization in A . baumannii ATCC 17978. (DOCX) Click here for additional data file. S1 Fig Zinc may play a minor role in the regulation of siderophore-associated genes in A . baumannii but does not appear to be involved in the direct transcriptional regulation of fur . WT A . baumannii and its isogenic Δ zur mutant were grown in metal-restricted media for 12 h. RNA was extracted and transcriptional changes in the expression of siderophore-associated genes and fur were assessed by qRT-PCR and normalized to the expression of rpoB . Expression of a known zur -regulated gene, zigA , was assessed as a positive control, whereas zur was run as a negative control. * p < 0.05, *** p < 0.001, and **** p < 0.0001 as determined by Student's t test relative to a hypothetical value of 1. Data are representative of two experiments performed in biological quadruplicate. (TIFF) Click here for additional data file. S2 Fig Acinetobactin biosynthetic mutants exhibit decreased maximal growth and growth rates, as well as an increased lag time relative to WT A . baumannii ATCC 179789 when grown under iron restriction. Growth kinetics of WT A . baumannii ATCC 17978 and its isogenic acinetobactin ( ΔbasG ), baumannoferrin (Δ bfnL) and fimsbactins (Δ fbsE ) biosynthetic mutants were analyzed from the data presented in Fig 5 . Estimates of the maximal OD 600 (asymptote (A) A-D), maximum specific growth rate (μ m , E-H) and lag time (λ, I-L) are given where *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (TIFF) Click here for additional data file. S3 Fig Expression of basG in trans restores growth of A . baumannii 17978 on human serum. WT A . baumannii ATCC 17978 with empty vector (WT/pWH1266), the acinetobactin-deficient mutant with empty vector (Δ basG /pWH1266), and a mutant complemented with basG expressed from pWH1266 (Δ basG /pWH1266:: basG ) were grown in cTMS media with human serum added at the concentrations indicated. Bacterial growth was assessed by determining the optical density at 600 nm (OD 600nm ) at the timepoints indicated. Data are the average of technical triplicates and represent the results of two independent experiments. (TIFF) Click here for additional data file. S4 Fig Acinetobactin biosynthetic mutants in A . baumannii AB5075-UW are deficient for growth in human serum. WT A . baumannii AB5075-UW and two unique basG transposon mutants were grown in cTMS media with human serum added at the concentration indicated. Bacterial growth was assessed by determining the OD 600nm at the timepoints indicated. Data are the average of technical triplicates and represent the results of two independent experiments. Where error bars are not visible, they are shorter than the height of the symbol. (TIFF) Click here for additional data file. S5 Fig Acinetobactin biosynthetic mutants are impaired for growth on lactoferrin as a sole iron source. Wild-type (WT) A . baumannii and its isogenic acinetobactin (Δ basG ), baumannoferrin (Δ bfnL) and fimsbactins (Δ fbsE ) biosynthetic mutants were grown in cTMS media with lactoferrin, no added iron source, or 30 μM FeCl 3 , as indicated. Bacterial growth was assessed by determining the OD 600nm , at the time points indicated. Data are representative of two independent experiments, and error bars represent the standard error of the mean. Where error bars are not visible, they are shorter than the height of the symbol. (TIFF) Click here for additional data file. S6 Fig Fimsbactins biosynthetic, regulatory, and transport genes are upregulated in a Δ basG bfnL mutant. WT A . baumannii and its isogenic Δ basG bfnL mutant were grown in metal-restricted media for 12 h. RNA was extracted and transcriptional changes in genes of the fimsbactins locus were assessed by qRT-PCR and normalized to the expression of rpoB . Expression of bfnL was used as a negative control. * p < 0.05, ** p < 0.01 and **** p < 0.0001, as determined by Student's t test relative to a hypothetical value of 1. Data are representative of two experiments performed in biological quadruplicate. (TIFF) Click here for additional data file. S7 Fig Siderophore production impacts the growth kinetics of A . baumannii and is required for growth on human serum and transferrin as sole iron sources. Growth kinetics of WT A . baumannii ATCC 17978 and its isogenic siderophore biosynthetic mutants, as indicated, were analyzed from the data presented in Fig 7 . Estimates of the maximal OD 600 (asymptote (A) A-D), maximum specific growth rate (μ m , E-H) and lag time (λ, I-L) are given for the conditions listed where are given where *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. When growth was insufficient to calculate the parameter, no data is given (ND). (TIFF) Click here for additional data file. S8 Fig Metal responsive genes are upregulated in the A . baumannii infected host. Mice were systemically infected with WT A . baumannii and sacrificed at 24 h post-infection. Organs were harvested, RNA extracted, and gene expression changes relative to growth in vitro were determined in the kidney (A), liver (B), and spleen (C) using NanoString technology. Genes are clustered by known or predicted function, as indicated. (TIFF) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150179/

Clinical Trials of Vaccines for Biodefense and Emerging and Neglected Diseases

The development of safe and effective vaccines for the prevention and control of emerging and neglected infectious diseases is an international priority, as is the development of similar control measures for bioterrorist threats. International standards have been established to ensure the protection of human subjects and the scientific integrity of clinical trial study design and data collection. A general overview of the history of the regulatory guidelines and ethical principles underpinning the vaccine development process is provided, with an emphasis on clinical trials conducted in the United States. The contemporary vaccine development process involves sequential assessment of safety, immunogenicity, and efficacy in phase1, 2, and 3 clinical trials. The process is not always linear or straightforward, and adaptive flexible clinical trial designs can increase the likelihood of a successful outcome. Because the efficacy of vaccines for a number of the diseases discussed in this volume cannot be tested directly in humans, alternative approaches have been developed to address this challenge. The major elements of typical clinical trial protocol are discussed, and a checklist of essential documents supporting the trial is provided. As novel vaccine approaches and technologies emerge, the regulatory and ethical considerations will need to be revisited and adapted to respond to the ever-changing landscape. Introduction Vaccines are considered among the most valuable and cost-effective tools for the control of infectious diseases; indeed, universal immunization of infants and children against a variety of pathogens was considered one of the top ten achievements of the 20th century ( CDC, 1999 ). Major scientific, technical, ethical, and regulatory principles and/or processes underpinning the vaccine development and evaluation process have been defined; however, challenges posed by the threats of bioterrorism and emerging infectious diseases, as well as technological advances in the composition, formulation, and delivery of vaccines, expose gaps in policy and practice that require ongoing consideration and refinement of approaches to the evaluation of new vaccines. The purpose of this chapter is to provide a brief overview of the approach to the evaluation of candidate vaccines in humans, with a particular attention to issues related to vaccines for biodefense and emerging and neglected diseases. The major focus will be on clinical trials and protocol development in the US, although many of the principles, regulations, and practices have been harmonized across the globe. A brief overview of related regulatory issues and preclinical vaccine development is provided; however, detailed discussions of these topics can be found elsewhere in this volume. Historical Considerations In the US, vaccines are regulated as biologicals, although vaccines are legally defined as drugs under the Food, Drug and Cosmetic Act (FDCA). The ethical principles, and the regulatory agencies and requirements that guide product development and clinical research in the 21st century evolved progressively over the 20th century. In many cases, the development of codified guidelines and the establishment of a regulatory authority were spurred by a tragic accident or a clear ethical breech (for reviews of Historical Considerations, see Baylor and Midthun, 2004 ; Borchers et al., 2007 ). Regulatory Issues In 1901, a number of children became ill and died after treatment with diphtheria antitoxin contaminated with tetanus toxin. This episode resulted in the first legislation designed to regulate the purity and potency of biologicals—the Biologics Control Act (BCA) of 1902. In 1938, over 100 people died after ingesting an elixir of sulfanilamide containing diethy­lene glycol (antifreeze), leading to the enactment of the FDCA ( Ballentine, 1981 ). Provisions of the FDCA required manufacturers to submit safety data to the Food and Drug Administration (FDA) prior to registration. The 1962 Kefauver–Harris Amendments to the FDCA required that efficacy data also be submitted. The1944 Public Health Service Act incorporated the BCA into section 351 of the US Code of Federal Regulations (CFR), which gave the federal government the authority to license biologicals and manufacturing facilities. In 1955, incomplete inactivation of a poliovirus vaccine resulted in the development of polio in a number of children (the "Cutter Incident"); this led to the establishment of the Division of Biologics Standards (DBS) within the NIH ( Offit, 2005 ). The authority vested in the DBS was transferred to the FDA in 1972. The organization that is currently responsible for regulating biologics is the Center for Biologics Evaluation and Research (CBER) at the FDA. Title 21 of the CFR contains regulations pertaining to biologicals, including product standards, manufacturing, labeling, licensing, advertising, investigational use, and protection of human subjects (informed consent, nonclinical laboratory studies, and Institutional Review Boards, or IRBs). Part 312 of Title 21 CFR contains regulations related to the Investigational New Drug Application (IND). The CFR is updated each year to reflect changes in policies and procedures. Additional guidance documents related to vaccines are published by the FDA, as appropriate. During the 1990s, an international group comprised of scientists, regulators, ethicists, and pharmaceutical representatives convened to discuss standards for designing, implementing, documenting and reporting clinical trials. The International Conference on Harmonisation of Technical Requirements for Regis­tration of Pharmaceuticals for Human Use (ICH) established guidelines for the conduct of clinical research (Good Clinical Practice, or GCP), many of which have been adopted by the US FDA ( FDA, 1996 ). The ICH GCP provides a unified standard to ensure the quality of clinical trial data and the protection of human subjects for the European Union, Japan, and the US for clinical trials that will support licensure of new vaccines and drugs. For research related to the evaluation of vaccines for emerging and neglected diseases, implementation of GCP in developing countries may pose unique challenges. Acosta et al. (2007) conducted a multinational clinical trial of Vi polysaccharide typhoid fever vaccine in Asia among 200,000 individuals, during which implementation of GCP required adaptations in order to comply with the goals of the guidelines. Ethical Issues The revelation that Nazi physicians tortured and experimented on prisoners during World War II culminated in the publication of the Nuremberg Code in 1947 ( Macrae, 2007 ). The Code is recognized as the first set of ethical guidelines for human research to be recognized by the international community. In 1965, the Declaration of Helsinki articulated additional responsibilities of investigators to research subjects, and paved the way for the development of IRBs. Continued violations of informed consent were summarized in a New England Journal of Medicine article written by Dr. Henry Beecher (1966) . This article and the subsequent expose of the ethical violations perpetrated during the Tuskeege Syphilis Study led to the 1974 National Research Act, which established IRBs and the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. After considering the principles and practices of human research in the US, the commission published the Belmont Report in 1979. This report defined the three major ethical principles (and their related clinical trial applications) pertaining to human research: respect for persons (informed consent); beneficence (risk-benefit assessment); and justice (equitable subject selection). Recommendations of the National Bioethics Commission from 1981 related to research involving human subjects were adopted into federal law in 1991. The so-called Common Rule (45 CFR 46 Subpart A) articulated the federal policy for protection of human subjects who are participating in clinical trials. Additional protections for pregnant women and fetuses, prisoners, and children are outlined in Subparts B, C, and D of 45 CFR 46. The Council for International Organizations and Medical Sciences, formed in 1949, recently published updated international ethical guidelines for research involving human subjects ( CIOMS, 2002 ). It is abundantly clear that the ethical considerations and regulatory guidelines for the conduct of clinical research are inextricably intertwined: ethical considerations drive the need to regulate the conduct of clinical trials to ensure the protection of human subjects and the quality and integrity of the data generated. Seven ethical requirements that should be fulfilled in the conduct of clinical research have been proposed by Emanuel et al. (2000) : the research should provide information that will advance science and knowledge (value); scientific validity; fair subject selection; favorable risk-benefit ratio; independent review; informed consent; and respect for enrolled subjects. The increasing complexity and redundancy of the oversight of contemporary clinical trials reflects the commitment to accomplish these goals. A simplified prototypical organizational structure for a vaccine research clinical trial is shown in Fig. 12.1 . Further details regarding the roles and responsibilities of the participants in the clinical trials process are provided below. Figure 12.1 Organizational structure of a typical single-center vaccine clinical trial. Regulatory Issues In 1901, a number of children became ill and died after treatment with diphtheria antitoxin contaminated with tetanus toxin. This episode resulted in the first legislation designed to regulate the purity and potency of biologicals—the Biologics Control Act (BCA) of 1902. In 1938, over 100 people died after ingesting an elixir of sulfanilamide containing diethy­lene glycol (antifreeze), leading to the enactment of the FDCA ( Ballentine, 1981 ). Provisions of the FDCA required manufacturers to submit safety data to the Food and Drug Administration (FDA) prior to registration. The 1962 Kefauver–Harris Amendments to the FDCA required that efficacy data also be submitted. The1944 Public Health Service Act incorporated the BCA into section 351 of the US Code of Federal Regulations (CFR), which gave the federal government the authority to license biologicals and manufacturing facilities. In 1955, incomplete inactivation of a poliovirus vaccine resulted in the development of polio in a number of children (the "Cutter Incident"); this led to the establishment of the Division of Biologics Standards (DBS) within the NIH ( Offit, 2005 ). The authority vested in the DBS was transferred to the FDA in 1972. The organization that is currently responsible for regulating biologics is the Center for Biologics Evaluation and Research (CBER) at the FDA. Title 21 of the CFR contains regulations pertaining to biologicals, including product standards, manufacturing, labeling, licensing, advertising, investigational use, and protection of human subjects (informed consent, nonclinical laboratory studies, and Institutional Review Boards, or IRBs). Part 312 of Title 21 CFR contains regulations related to the Investigational New Drug Application (IND). The CFR is updated each year to reflect changes in policies and procedures. Additional guidance documents related to vaccines are published by the FDA, as appropriate. During the 1990s, an international group comprised of scientists, regulators, ethicists, and pharmaceutical representatives convened to discuss standards for designing, implementing, documenting and reporting clinical trials. The International Conference on Harmonisation of Technical Requirements for Regis­tration of Pharmaceuticals for Human Use (ICH) established guidelines for the conduct of clinical research (Good Clinical Practice, or GCP), many of which have been adopted by the US FDA ( FDA, 1996 ). The ICH GCP provides a unified standard to ensure the quality of clinical trial data and the protection of human subjects for the European Union, Japan, and the US for clinical trials that will support licensure of new vaccines and drugs. For research related to the evaluation of vaccines for emerging and neglected diseases, implementation of GCP in developing countries may pose unique challenges. Acosta et al. (2007) conducted a multinational clinical trial of Vi polysaccharide typhoid fever vaccine in Asia among 200,000 individuals, during which implementation of GCP required adaptations in order to comply with the goals of the guidelines. Ethical Issues The revelation that Nazi physicians tortured and experimented on prisoners during World War II culminated in the publication of the Nuremberg Code in 1947 ( Macrae, 2007 ). The Code is recognized as the first set of ethical guidelines for human research to be recognized by the international community. In 1965, the Declaration of Helsinki articulated additional responsibilities of investigators to research subjects, and paved the way for the development of IRBs. Continued violations of informed consent were summarized in a New England Journal of Medicine article written by Dr. Henry Beecher (1966) . This article and the subsequent expose of the ethical violations perpetrated during the Tuskeege Syphilis Study led to the 1974 National Research Act, which established IRBs and the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. After considering the principles and practices of human research in the US, the commission published the Belmont Report in 1979. This report defined the three major ethical principles (and their related clinical trial applications) pertaining to human research: respect for persons (informed consent); beneficence (risk-benefit assessment); and justice (equitable subject selection). Recommendations of the National Bioethics Commission from 1981 related to research involving human subjects were adopted into federal law in 1991. The so-called Common Rule (45 CFR 46 Subpart A) articulated the federal policy for protection of human subjects who are participating in clinical trials. Additional protections for pregnant women and fetuses, prisoners, and children are outlined in Subparts B, C, and D of 45 CFR 46. The Council for International Organizations and Medical Sciences, formed in 1949, recently published updated international ethical guidelines for research involving human subjects ( CIOMS, 2002 ). It is abundantly clear that the ethical considerations and regulatory guidelines for the conduct of clinical research are inextricably intertwined: ethical considerations drive the need to regulate the conduct of clinical trials to ensure the protection of human subjects and the quality and integrity of the data generated. Seven ethical requirements that should be fulfilled in the conduct of clinical research have been proposed by Emanuel et al. (2000) : the research should provide information that will advance science and knowledge (value); scientific validity; fair subject selection; favorable risk-benefit ratio; independent review; informed consent; and respect for enrolled subjects. The increasing complexity and redundancy of the oversight of contemporary clinical trials reflects the commitment to accomplish these goals. A simplified prototypical organizational structure for a vaccine research clinical trial is shown in Fig. 12.1 . Further details regarding the roles and responsibilities of the participants in the clinical trials process are provided below. Figure 12.1 Organizational structure of a typical single-center vaccine clinical trial. Stages of Vaccine Development Overview The major stages of vaccine development are outlined in Fig. 12.2 . The initial stage is referred to as the pre-IND (investigational new drug) stage. Activities conducted in this stage culminate in the production of a vaccine that can be evaluated in humans—the IND stage. Vaccines that are shown to be safe, pure, and effective in humans may then be licensed for use (licensing and postmarketing stage). Figure 12.2 Overview of the major stages of vaccine development; IND, investigational new drug. Pre-IND Stage Once a public health need for control of a disease by means of vaccination has been identified, detailed studies designed to understand the pathogenesis of infection and to identify immune responses associated with protection are conducted. Studies may involve assessment of infection and immunity in humans if the disease occurs naturally at a frequency high enough to permit their evaluation (interpandemic influenza, malaria, tuberculosis, and others); however, detailed evaluations of other rare and lethal diseases—particularly those included in the Centers for Disease Control and Prevention (CDC) Category A biothreat list—must be studied in animal models that might predict the pathology and immune responses that occur in humans (smallpox, tularemia, inhalational anthrax, hemorrhagic fever viruses, etc.). Candidate vaccine formulations containing or expressing epitopes that can elicit immune responses associated with protection are then designed and evaluated in vitro and in animals, where their safety and immunogenicity profiles are established. A critical part of the pre-IND stage is the development and validation of a manufacturing process. IND Stage Candidate vaccines typically undergo a sequential series of evaluations in humans. Three major phases are identified, as summarized in Table 12.1 . In practice, the scheme is not so straightforward: phase 1 and phase 2 clinical trials may overlap or be combined, and multiple phase 1 and/or phase 2 evaluations may be necessary for adjustment of dosage, change in regimen, and evaluation in other age and/or risk groups, even after pivotal phase 3 studies have demonstrated efficacy. Close and ongoing communication between the sponsor, regulatory agencies, investigators, and IRBs is critical throughout the IND stage, including pre-IND meetings, end-of-phase 2 meeting, pre-Biologic Licensing Application (BLA), and BLA meetings to facilitate the timely progression through the clinical trials process. Table 12.1 Phases of clinical vaccine development Clinical trial phase Major objectives Typical number of subjects Type of subjects Comments I Safety and tolerability; preliminary immunogenicity 20–80 Healthy adults First introduction of a candidate vaccine into humans II Immunogenicity; safety Hundreds Healthy adults, followed by target populations Immunization regimen defined (dosage levels, number of doses) III Efficacy; safety Hundreds to thousands At-risk population(s) May be limited to immune response determinations a IV Safety; duration of protection; efficacy in other groups or for other indications Thousands to millions Vaccine recipients Postmarketing studies a If efficacy cannot be assessed in humans because disease does not occur naturally or at a high enough frequency (smallpox, inhalational anthrax, etc.), then efficacy may be established in animal models (see text) and correlates of protection identified in humans. Phase 1 Clinical Trials The first evaluation of a candidate vaccine in humans is referred to as a phase 1 clinical trial. Typicallyless than 100 healthy young adult subjects are enrolled in phase 1 trials. In fact, the number may be considerably smaller for novel products, in which case investigators may elect to inoculate only a handful of subjects to exclude the possibility of unexpected reactogenicity or toxicity ( Keitel et al., 1993a ). The major goal of the phase 1 trial is to assess safety and tolerability; however, preliminary immunogenicity assessments and dose-ranging information can provide valuable information regarding dosage selection for subsequent evaluations. Although the design of phase 1 clinical trials of drugs often is open-label, many contemporary phase 1 clinical trials of candidate vaccines are randomized, double-blind, placebo-controlled trials. The phase 1 trial may be performed in stages, where a small cohort of subjects is vaccinated and observed for a period of 1–4 or more weeks before the remainder of the study cohort is vaccinated in stage 2. This approach reduces the exposure of subjects to the occurrence of unexpected toxicity. For vaccines that are produced in the US whose ultimate use will be in other countries (such as malaria vaccines), the initial evaluation occurs in the US (phase 1a), followed by phase 1b testing in a healthy population in the target country. Inclusion of placebos in this and other phases of development reduces bias in the assessment of adverse events (AEs) and serious AEs (SAEs), and provides internal controls for laboratory assessments of immune responses following immunization. Because the number of subjects enrolled in a phase 1 trial is small, these studies lack statistical power to detect AEs that occur at a low rate. Nevertheless, small, carefully monitored phase 1 trials can identify unexpected and/or unacceptable toxicities that require reformulation or reevaluation of a potential candidate ( Keitel et al., 1999 ; Edelman et al., 2002 ). In some circumstances, specific monitoring of subjects in phase 1 or phase 2 trials for evidence of infection after immunization is relevant, particularly if there are concerns that the vaccine might elicit immunopathologic responses when the vaccinated subject is naturally infected. Immunopatholgy resulting in severe atypical infection and/or death occurred among persons immunized with an inactivated measles vaccine and in infants given an inactivated vaccine to prevent respiratory syncytial virus (RSV) vaccine ( Polack, 2007 ). Similar concerns exist regarding the potential for dengue virus and SARS coronavirus vaccines to elicit immunopathologic responses ( Edelman et al., 2003 ; Deming et al., 2006 ). Although not specifically relevant for this chapter, an exploratory IND study may precede a typical phase 1 evaluation for the assessment of drugs and therapeutic biologicals ( FDA, 2006 ). The exploratory IND study option is characterized as a small early phase 1 trial that has no therapeutic or diagnostic intent; rather, the goal is to assess feasibility for further development of a drug or biological. Goals of this type of study may include determination of the mechanism of action of a drug in humans; evaluation of the pharmacokinetic profile of the agent; selection of one from a group of promising candidates; and exploring biodistribution characteristics using imaging techniques. Because these studies pose lower potential risk to subjects, exploratory IND studies require less or different preclinical support than typical phase 1 studies. Phase 2 Clinical Trials Vaccine candidates that have favorable safety and immunogenicity profiles in phase 1 trials may progress to expanded phase 2 trials. Several hundred healthy subjects may be enrolled into phase 2 trials. The major goals of phase 2 trials are to assess safety and to develop optimal regimens for immunization. Typically several dosage levels are evaluated; different immunization regimens (number of doses and interval between doses) also may be explored. Most phase 2 trials are randomized, double-blind, placebo-controlled trials. Although larger numbers of subjects are evaluated, phase 2 trials still lack statistical power to detect events that occur in a low proportion of subjects. Initial phase 2 trials are usually conducted among persons who are at low risk for acquiring the target disease; therefore, additional phase 1 and phase 2 trials may be necessary to assess safety and immunogenicity among populations who are at high risk for the disease, in which pivotal efficacy trials will be conducted. For several emerging and neglected diseases, the preliminary efficacy of a vaccine can be assessed in a human challenge model, including malaria, influenza, and cholera ( Couch et al., 1971 ; Epstein et al., 2007 ; Tacket et al., 1999 ). These carefully controlled experimental inoculations of subjects with the target pathogen can provide proof of concept that immunization with a vaccine candidate confers protection prior to large phase 3 clinical trials. In the pediatric population, rechallenge of vaccinated subjects with homologous or related live attenuated influenza virus vaccines (LAIV) provided supportive evidence that intranasal immunization with LAIV would confer protection against subsequent infection with wild-type influenza viruses ( Belshe et al., 2000 ). Phase 2 trials may also incorporate plans to assess the concomitant administration of other vaccines, biologicals, or medications. For example, administration of a new vaccine to infants in the context of the increasingly complicated childhood immunization schedule poses complex problems; evidence that simultaneous administration of licensed and experimental vaccines does not interfere with protective responses to components in either product is necessary ( Rennels et al., 2000 ). Phase 3 Clinical Trials Once a vaccine candidate has been shown to be safe and to possess acceptable reactogenicity and immunogenicity profiles, assessment of the efficacy can be undertaken in phase 3 clinical trials. The type and size of the population to be studied will depend on epidemiology of the target disease (population at risk for disease and the disease attack rate) and the level of protection conferred via immunization. A phase 3 clinical trial for prevention of disease that occurs at a low incidence may require hundreds of thousands of subjects, such as the US field trial of inactivated poliovirus vaccine in the 1950s ( Francis, 1955 ); whereas phase 3 trials of vaccines for prevention of high-incidence infections (such as RSV in infants) theoretically would require no more than hundreds of subjects, particularly if the vaccine was expected to be highly efficacious. The assessment of efficacy of vaccines against agents of bioterrorism, as well as vaccines for candidate pandemic influenza, may be problematic. For example, smallpox, inhalational anthrax, hemorrhagic fever viruses, tularemia, plague, and influenza A/H5N1 do not occur naturally at a frequency high enough to permit controlled evaluation of clinical vaccine efficacy prior to licensure of vaccines. The FDA final rule entitled "New Drug and Biological Drug Products: Evidence Needed to Demonstrate Effectiveness of New Drugs When Human Efficacy Studies Are Not Ethical or Feasible" (otherwise referred to as the "Animal Rule") was published to address this circumstance ( FDA, 2002 ). The rule permits use of animal efficacy data when collection of human efficacy data is not feasible. Safety and immunogenicity data in humans and animal efficacy data can be used to support licensure when several conditions are met, as follows: (1) the pathophysiology of the infection is reasonably well understood; (2) the pathogenesis and immune responses in one or two animal species are expected to predict these responses in humans; (3) the immunogenicity endpoint(s) correlated with protection in the animal(s) are related to human immune responses; and (4) immunogenicity endpoints in animals and humans are sufficiently well understood to permit selection of a regimen that would be expected to predict protection in humans. For other diseases where correlates of protection are reasonably well defined (for example, serum HAI antibody level against influenza viruses), surrogate markers can be used to support licensure ( FDA, 2007a ). Finally, in order to facilitate the approval of vaccines for such severe, life-threatening illnesses, additional mechanisms for expedited review and accelerated approval have been developed by the FDA. Phase 4 Clinical Trials Regulatory authorities are increasingly requiring additional studies to be conducted after market approval. Phase 4 studies may also be initiated by the sponsor for a variety of reasons. While a vaccine appears safe after it has been studied in thousands of individuals, rare adverse events may only be observed after hundreds of thousands or even millions of people have been vaccinated, as occurred with the first-generation live attenuated rotavirus vaccine ( Murphy et al., 2001 ). The long-term safety or continued efficacy of a vaccine may be unknown at the time of licensure, and the benefit of immunization of special populations that may have been underrepresented or not studied in the IND stage may be of interest. Unfortunately, phase 4 clinical trials typically are not randomized, and they may be uncontrolled. Nevertheless, several safety surveillance systems have been established to facilitate the early detection of potential rare, serious reactions to vaccines, including the Vaccine Adverse Event Reporting System (VAERS), the Vaccine Safety Datalink (VSD), and the American Academy of Pediatrics Practice Research Office Settings (PROS) ( Ellenberg et al., 2005 ). Design options for phase 4 studies include case-control studies and cohort studies. Overview The major stages of vaccine development are outlined in Fig. 12.2 . The initial stage is referred to as the pre-IND (investigational new drug) stage. Activities conducted in this stage culminate in the production of a vaccine that can be evaluated in humans—the IND stage. Vaccines that are shown to be safe, pure, and effective in humans may then be licensed for use (licensing and postmarketing stage). Figure 12.2 Overview of the major stages of vaccine development; IND, investigational new drug. Pre-IND Stage Once a public health need for control of a disease by means of vaccination has been identified, detailed studies designed to understand the pathogenesis of infection and to identify immune responses associated with protection are conducted. Studies may involve assessment of infection and immunity in humans if the disease occurs naturally at a frequency high enough to permit their evaluation (interpandemic influenza, malaria, tuberculosis, and others); however, detailed evaluations of other rare and lethal diseases—particularly those included in the Centers for Disease Control and Prevention (CDC) Category A biothreat list—must be studied in animal models that might predict the pathology and immune responses that occur in humans (smallpox, tularemia, inhalational anthrax, hemorrhagic fever viruses, etc.). Candidate vaccine formulations containing or expressing epitopes that can elicit immune responses associated with protection are then designed and evaluated in vitro and in animals, where their safety and immunogenicity profiles are established. A critical part of the pre-IND stage is the development and validation of a manufacturing process. IND Stage Candidate vaccines typically undergo a sequential series of evaluations in humans. Three major phases are identified, as summarized in Table 12.1 . In practice, the scheme is not so straightforward: phase 1 and phase 2 clinical trials may overlap or be combined, and multiple phase 1 and/or phase 2 evaluations may be necessary for adjustment of dosage, change in regimen, and evaluation in other age and/or risk groups, even after pivotal phase 3 studies have demonstrated efficacy. Close and ongoing communication between the sponsor, regulatory agencies, investigators, and IRBs is critical throughout the IND stage, including pre-IND meetings, end-of-phase 2 meeting, pre-Biologic Licensing Application (BLA), and BLA meetings to facilitate the timely progression through the clinical trials process. Table 12.1 Phases of clinical vaccine development Clinical trial phase Major objectives Typical number of subjects Type of subjects Comments I Safety and tolerability; preliminary immunogenicity 20–80 Healthy adults First introduction of a candidate vaccine into humans II Immunogenicity; safety Hundreds Healthy adults, followed by target populations Immunization regimen defined (dosage levels, number of doses) III Efficacy; safety Hundreds to thousands At-risk population(s) May be limited to immune response determinations a IV Safety; duration of protection; efficacy in other groups or for other indications Thousands to millions Vaccine recipients Postmarketing studies a If efficacy cannot be assessed in humans because disease does not occur naturally or at a high enough frequency (smallpox, inhalational anthrax, etc.), then efficacy may be established in animal models (see text) and correlates of protection identified in humans. Phase 1 Clinical Trials The first evaluation of a candidate vaccine in humans is referred to as a phase 1 clinical trial. Typicallyless than 100 healthy young adult subjects are enrolled in phase 1 trials. In fact, the number may be considerably smaller for novel products, in which case investigators may elect to inoculate only a handful of subjects to exclude the possibility of unexpected reactogenicity or toxicity ( Keitel et al., 1993a ). The major goal of the phase 1 trial is to assess safety and tolerability; however, preliminary immunogenicity assessments and dose-ranging information can provide valuable information regarding dosage selection for subsequent evaluations. Although the design of phase 1 clinical trials of drugs often is open-label, many contemporary phase 1 clinical trials of candidate vaccines are randomized, double-blind, placebo-controlled trials. The phase 1 trial may be performed in stages, where a small cohort of subjects is vaccinated and observed for a period of 1–4 or more weeks before the remainder of the study cohort is vaccinated in stage 2. This approach reduces the exposure of subjects to the occurrence of unexpected toxicity. For vaccines that are produced in the US whose ultimate use will be in other countries (such as malaria vaccines), the initial evaluation occurs in the US (phase 1a), followed by phase 1b testing in a healthy population in the target country. Inclusion of placebos in this and other phases of development reduces bias in the assessment of adverse events (AEs) and serious AEs (SAEs), and provides internal controls for laboratory assessments of immune responses following immunization. Because the number of subjects enrolled in a phase 1 trial is small, these studies lack statistical power to detect AEs that occur at a low rate. Nevertheless, small, carefully monitored phase 1 trials can identify unexpected and/or unacceptable toxicities that require reformulation or reevaluation of a potential candidate ( Keitel et al., 1999 ; Edelman et al., 2002 ). In some circumstances, specific monitoring of subjects in phase 1 or phase 2 trials for evidence of infection after immunization is relevant, particularly if there are concerns that the vaccine might elicit immunopathologic responses when the vaccinated subject is naturally infected. Immunopatholgy resulting in severe atypical infection and/or death occurred among persons immunized with an inactivated measles vaccine and in infants given an inactivated vaccine to prevent respiratory syncytial virus (RSV) vaccine ( Polack, 2007 ). Similar concerns exist regarding the potential for dengue virus and SARS coronavirus vaccines to elicit immunopathologic responses ( Edelman et al., 2003 ; Deming et al., 2006 ). Although not specifically relevant for this chapter, an exploratory IND study may precede a typical phase 1 evaluation for the assessment of drugs and therapeutic biologicals ( FDA, 2006 ). The exploratory IND study option is characterized as a small early phase 1 trial that has no therapeutic or diagnostic intent; rather, the goal is to assess feasibility for further development of a drug or biological. Goals of this type of study may include determination of the mechanism of action of a drug in humans; evaluation of the pharmacokinetic profile of the agent; selection of one from a group of promising candidates; and exploring biodistribution characteristics using imaging techniques. Because these studies pose lower potential risk to subjects, exploratory IND studies require less or different preclinical support than typical phase 1 studies. Phase 2 Clinical Trials Vaccine candidates that have favorable safety and immunogenicity profiles in phase 1 trials may progress to expanded phase 2 trials. Several hundred healthy subjects may be enrolled into phase 2 trials. The major goals of phase 2 trials are to assess safety and to develop optimal regimens for immunization. Typically several dosage levels are evaluated; different immunization regimens (number of doses and interval between doses) also may be explored. Most phase 2 trials are randomized, double-blind, placebo-controlled trials. Although larger numbers of subjects are evaluated, phase 2 trials still lack statistical power to detect events that occur in a low proportion of subjects. Initial phase 2 trials are usually conducted among persons who are at low risk for acquiring the target disease; therefore, additional phase 1 and phase 2 trials may be necessary to assess safety and immunogenicity among populations who are at high risk for the disease, in which pivotal efficacy trials will be conducted. For several emerging and neglected diseases, the preliminary efficacy of a vaccine can be assessed in a human challenge model, including malaria, influenza, and cholera ( Couch et al., 1971 ; Epstein et al., 2007 ; Tacket et al., 1999 ). These carefully controlled experimental inoculations of subjects with the target pathogen can provide proof of concept that immunization with a vaccine candidate confers protection prior to large phase 3 clinical trials. In the pediatric population, rechallenge of vaccinated subjects with homologous or related live attenuated influenza virus vaccines (LAIV) provided supportive evidence that intranasal immunization with LAIV would confer protection against subsequent infection with wild-type influenza viruses ( Belshe et al., 2000 ). Phase 2 trials may also incorporate plans to assess the concomitant administration of other vaccines, biologicals, or medications. For example, administration of a new vaccine to infants in the context of the increasingly complicated childhood immunization schedule poses complex problems; evidence that simultaneous administration of licensed and experimental vaccines does not interfere with protective responses to components in either product is necessary ( Rennels et al., 2000 ). Phase 3 Clinical Trials Once a vaccine candidate has been shown to be safe and to possess acceptable reactogenicity and immunogenicity profiles, assessment of the efficacy can be undertaken in phase 3 clinical trials. The type and size of the population to be studied will depend on epidemiology of the target disease (population at risk for disease and the disease attack rate) and the level of protection conferred via immunization. A phase 3 clinical trial for prevention of disease that occurs at a low incidence may require hundreds of thousands of subjects, such as the US field trial of inactivated poliovirus vaccine in the 1950s ( Francis, 1955 ); whereas phase 3 trials of vaccines for prevention of high-incidence infections (such as RSV in infants) theoretically would require no more than hundreds of subjects, particularly if the vaccine was expected to be highly efficacious. The assessment of efficacy of vaccines against agents of bioterrorism, as well as vaccines for candidate pandemic influenza, may be problematic. For example, smallpox, inhalational anthrax, hemorrhagic fever viruses, tularemia, plague, and influenza A/H5N1 do not occur naturally at a frequency high enough to permit controlled evaluation of clinical vaccine efficacy prior to licensure of vaccines. The FDA final rule entitled "New Drug and Biological Drug Products: Evidence Needed to Demonstrate Effectiveness of New Drugs When Human Efficacy Studies Are Not Ethical or Feasible" (otherwise referred to as the "Animal Rule") was published to address this circumstance ( FDA, 2002 ). The rule permits use of animal efficacy data when collection of human efficacy data is not feasible. Safety and immunogenicity data in humans and animal efficacy data can be used to support licensure when several conditions are met, as follows: (1) the pathophysiology of the infection is reasonably well understood; (2) the pathogenesis and immune responses in one or two animal species are expected to predict these responses in humans; (3) the immunogenicity endpoint(s) correlated with protection in the animal(s) are related to human immune responses; and (4) immunogenicity endpoints in animals and humans are sufficiently well understood to permit selection of a regimen that would be expected to predict protection in humans. For other diseases where correlates of protection are reasonably well defined (for example, serum HAI antibody level against influenza viruses), surrogate markers can be used to support licensure ( FDA, 2007a ). Finally, in order to facilitate the approval of vaccines for such severe, life-threatening illnesses, additional mechanisms for expedited review and accelerated approval have been developed by the FDA. Phase 4 Clinical Trials Regulatory authorities are increasingly requiring additional studies to be conducted after market approval. Phase 4 studies may also be initiated by the sponsor for a variety of reasons. While a vaccine appears safe after it has been studied in thousands of individuals, rare adverse events may only be observed after hundreds of thousands or even millions of people have been vaccinated, as occurred with the first-generation live attenuated rotavirus vaccine ( Murphy et al., 2001 ). The long-term safety or continued efficacy of a vaccine may be unknown at the time of licensure, and the benefit of immunization of special populations that may have been underrepresented or not studied in the IND stage may be of interest. Unfortunately, phase 4 clinical trials typically are not randomized, and they may be uncontrolled. Nevertheless, several safety surveillance systems have been established to facilitate the early detection of potential rare, serious reactions to vaccines, including the Vaccine Adverse Event Reporting System (VAERS), the Vaccine Safety Datalink (VSD), and the American Academy of Pediatrics Practice Research Office Settings (PROS) ( Ellenberg et al., 2005 ). Design options for phase 4 studies include case-control studies and cohort studies. Clinical Trial Protocol Development The design of protocols for the evaluation of candidate vaccines requires consideration of the pathogen, the disease pathogenesis and immune response profile, and the characteristics of the vaccine candidate itself. The success of a clinical trial will depend on implementation of a well-designed protocol, followed by careful monitoring and retention of subjects, close adherence to the protocol, and accurate reporting and documentation of observations made during the trial ( for Comprehensive discussions, see Chow and Liu, 2004 ; Meinert, 1986 ; Wang and Bakhai, 2006 ). A brief outline of essential elements for inclusion in clinical trial protocols is shown in Table 12.2 . A detailed template for protocol development has been prepared by the National Institute of Allergy and Infectious Diseases that can be used to guide protocol development ( NIAID, 2006 ). Novel constructs such as live, attenuated vaccines may require additional discussion of risks and special facilities for isolation, such as the evaluation of live attenuated vaccines based on potential pandemic influenza viruses. Note that the basic protocol elements are mirrored in the Informed Consent document. Table 12.2 Checklist of essential clinical trial protocol elements Title page Statement of compliance Signature page Protocol summary Background and rationale Purpose and objectives Study design and endpoints Study population: Description, inclusion/exclusion criteria; recruitment and retention Study agent/interventions Study procedures/evaluations Study schedule Assessment of safety: Safety parameters, reporting requirements, halting rules Clinical monitoring structure: Site/safety monitoring plan, and safety reviews Statistical consideration: Sample size; data analyses, etc. Quality control and quality assurance Ethics/protection of human subjects: IRB, informed consent; confidentiality, etc. Data management Appendices: Personnel roster; table of procedures; lab processing flow sheet, etc. Source : Adapted from NIAID protocol template guidance ( NIAID, 2006). Courtesy : NIAID. Background and Rationale The background and rationale should provide information regarding the current understanding of the disease epidemiology, pathogenesis, and immune responses relevant for protection against infection; and need for development of control measures. The scientific rationale for selection of the vaccine candidate should be discussed. A concise description of the study agent should be provided, including summaries of preclinical studies and relevant clinical studies. Finally, potential risks and benefits of immunization with the investigational agent should be delineated. Objectives and Purpose The objectives and purpose of the trial should be clearly and explicitly stated. For phase 1 trials, the primary objectives will be to assess the safety, tolerability, and reactogenicity of a vaccine, whereas assessment of immunogenicity is a secondary objective. For combined phase 1/2 and phase 2 clinical trials, safety and immunogenicity may be coprimary endpoints. Exploratory endpoints may also be included, such as the effect of age, race, or gender on immune responses ( Keitel et al., 2006 ). Combined phase 1/2 trials may be proposed when the vaccine candidate represents a variant of a previously licensed construct, such as a subvirion influenza vaccine for prevention of avian influenza ( Treanor et al., 2006 ). Efficacy and safety typically are the primary endpoints for phase 3 trial. Study Design The study design then should be described. For clinical trials of candidate vaccines, the study design typically is a randomized, double-blind, controlled clinical trial—one example of a parallel group design. Phase 1 safety and tolerability studies often utilize a titration design, where ascending dosages of the experimental agent are sequentially administered to new cohorts of subjects ( Gorse et al., 2006 ). Cluster-randomized clinical trial design is occasionally employed to assess vaccine efficacy. In this circumstance, larger groups of individuals (such as nursing homes or schools) are randomized to an intervention, rather than individual subjects, and the clinical endpoints are ascertained for vaccinated subjects ( Rodrigues et al., 2005 ), or for a subset of the cluster, such as the contacts of healthcare workers in a closed setting ( Hayward et al., 2006 ). Phase 1 clinical trials historically were open-label; however, in recent years most phase 1 clinical trials of vaccines have been randomized and blinded. The value of a placebo control in clinical trials has been described; however, a licensed control vaccine may be used rather than a placebo, particularly for phase 3 clinical trials in children. In a recent phase 3 clinical trial of pneumococcal conjugate vaccine in infants, a meningococcal type C vaccine served as the control vaccine ( Black et al., 2000 ). In this case, the meningococcal control vaccine provided potential benefits to the study participants. Flexible adaptive design methods frequently are employed in the development of vaccines; these incorporate plans for modifications of the clinical trial design that are made before or during the conduct of the research ( Chow and Chang, 2007 ). Adaptations to ongoing trials may include prospective adaptations, such as interim analysis, stopping rules for early termination due to futility/safety concerns/efficacy, or sample size re-estimation; ad hoc adaptations such as changes in inclusion and exclusion criteria, dosage or regimen alteration, and trial duration; or retrospective adaptations at the end of the study but before unblinding, including changing the study endpoint or altering the statistical hypothesis (superiority to noninferiority). The goal of this approach is to permit modification based on accumulated evidence to alter trial design to increase the probability of success without undermining the validity of the trial ( Gallo et al., 2006 ). Such adaptations may require modifications of the study hypotheses, protocol amendments, and sample size recalculations. Clinical trials are also classified as single-center or multicenter studies. Phase 1 studies often are single-center studies; however, multicenter trial design may be used for any phase of clinical vaccine development. Multicenter study design provides several advantages: enrollment of subjects is expedited, and the results of the trial are likely to be more generalizable. Study endpoints need to be clearly identified. For phase 1 trials, safety, tolerability, and reactogenicity primary endpoints may include the frequencies and severities of injection site reactions (pain, tenderness, redness, and swelling) and systemic reactions (fever, chills, headache, myalgia, arthralgia, etc.), as well a laboratory evidence of adverse reactions (hematologic, biochemical, and other). For phase 2 clinical trials, specific immune responses at defined time points after immunization typically characterize the primary endpoints; safety and reactogenicity may be primary or secondary endpoints. For phase 3 clinical trials, protection against laboratory-confirmed infection and/or disease is the primary endpoint, and the major safety assessment may be the frequency of SAEs associated with administration of the investigational agent. Study Population A detailed description of the proposed study population and the number of subjects to be studied must be provided; specifically, characteristics (age range, health status, ability to provide informed consent, etc.) of potentially eligible persons (Inclusion Criteria) and factors that would render an individual ineligible (Exclusion Criteria) should be explicitly enumerated. For some phase 1 trials, screening for eligibility may include medical history, physical examination, and laboratory screening for evidence of good health (normal hematologic and biochemical parameters, and no evidence of active hepatitis B, hepatitis C, or HIV infection). Information regarding serosusceptibility to the candidate pathogen may be necessary. For example, a phase 1 clinical trial of a dengue virus vaccine may require evidence of no prior infections caused by these viruses ( Edelman et al., 2003 ), and a phase 1 or 2 clinical trial assessing the immunogenicity of LAIV may focus on persons with low or absent levels of preexisting immunity to the candidate vaccine ( Keitel et al., 1993b ). For phase 3 clinical trials, it is necessary to identify a population in which the infection or disease occurs at a high enough frequency to assess the ability of a vaccine to protect. For example, pivotal phase 3 trials of an inactivated hepatitis A vaccine were conducted in specific US communities where the rate of hepatitis A infections in children was high ( Werzberger et al., 1992 ). Human subjects considerations may include a description of certain behaviors and/or concomitant medications that would exclude a subject. For most clinical trials of vaccines, women who are capable of bearing children must consent to certain birth control measures. The US Department of Health and Human Services (DHHS) has published a guidance regarding research in pregnant women: for research conducted in this population, there must be direct benefit to the woman or her fetus or there must be only minimal risk to the fetus, and information cannot be obtained any other way. For many phase 1 clinical trials, use of prescription medications is not permitted. Clinical trials of vaccines that potentially could be transmitted to others in the community raise special concerns. Recent reevaluations of smallpox vaccines posed concerns with regard to transmission of the vaccine virus from subjects to their contacts ( Frey et al., 2002 ). In this case, persons who had household or other significant contacts with young infants, people with eczema, pregnant women, and immunocompromised individuals were excluded from participation. The methods for test article allocation should also be described. For most phase 1 and phase 2 clinical trials of vaccines, the subjects are randomized to receive one of several dosage levels of vaccine or placebo. Ideally, randomization should not occur until the subjects have been qualified for participation. Typically the randomization occurs in blocks of a prespecified number that represents a multiple of the number of test articles. For example, if there were four dosage levels of vaccine and a placebo, then the block size might be 5, 10, or 15. If the block size chosen were 5, then the subjects would be randomized 1:1:1:1:1. Block randomization can reduce the risk of unequal group sizes. In some circumstances the probability of receiving one product differs from the probability of receiving another. For example, in an efficacy study to be conducted in children, an investigator may wish to reduce the number of subjects randomized to receive the placebo, and the randomization scheme selected may be 2:1 (vaccine:placebo). The vaccine group assignments for subjects should be concealed from the subjects and from investigators to reduce bias in the assessments performed after vaccination (so-called double blinding). Additional measures can be taken to reduce the potential for imbalances in baseline characteristics of enrolled subjects, such as stratification of subjects according to age, prior receipt of a related vaccine, etc., prior to randomization. Study Agent/Interventions The clinical trial protocol should contain basic information regarding the characterization of the vaccine formulations—including dosage(s), packaging, labeling, storage; preparation, administration, dosing, and accountability methods for each study product, including placebo and/or control preparations. More complete descriptions of study vaccines, including manufacturing information, preclinical and clinical safety, immunogenicity, and efficacy should be provided separately in the Investigators' Brochure (IB). Information regarding the use of concomitant medications, including prohibited medications, should be detailed. For example, during phase 1 clinical trials concomitant use of prescription medications may be prohibited. During phase 2 clinical trials, concomitant use of certain medications may be allowed, such as antihypertensive medications or antidepressants. In general, concomitant use of immunosuppressive, immunomodulatory, or cytotoxic drugs would be prohibited in any clinical trials of live attenuated vaccines. Study Procedures and Evaluations A description of the proposed clinical evaluations then follows. In phase 1 or 2 clinical trials, detailed and frequent physical assessments of the injection site and systemic responses may be indicated, as well as review of subject records of clinical responses following immunization. The intensity of study assessments will vary according to the nature of the study product: more frequent and detailed assessments would be indicated for novel products whose safety profile is undefined. Periodic collection of blood, nasal, fecal, or other samples to assess for the occurrence of toxicity, or to determine the frequency, magnitude, and/or duration of shedding of a live vaccine candidate may also be indicated ( Piedra et al., 1993 ; Taylor et al., 1997 ). These laboratory assessments should be tailored to the particular needs of the protocol, and should be based on the pathogenesis of the disease, the vaccine under evaluation, and information collected in the pre-IND stage. Brief descriptions of the type(s) of specimens to be collected, methods for specimen collection, preparation, handling, storing, and shipping (if applicable) should be outlined; detailed procedures should be provided in a separate Manual of Procedures (MOP) for each study. For phase 3 trials, clinical follow-up is specifically targeted at ascertaining whether the vaccine prevents infection or disease and capturing the occurrence of SAEs; however, limited prospective safety assessments may be included (perhaps only in a subset of subjects) to expand the safety database ( Oxman et al., 2005 ). Study Schedule Once the specific clinical and laboratory procedures for assessing safety, immunogenicity, and/or efficacy have been described, a detailed study schedule indicating the timing of various assessments/procedures/interventions should be outlined, including those that will be conducted at screening, enrollment, and each follow-up visit. Clinical assessments and laboratory procedures that should be completed in the event the subject's participation in the trial is terminated early should be described, as should those for women who become pregnant during the study. Finally, a description of clinical and laboratory assessments to be performed in the event the subject returns for an unscheduled visit (for example, for a severe vaccine reaction) may be appropriate. A summary table or figure outlining study procedures is particularly useful. Assessment of Safety As described above, the clinical trial protocol should detail the specific safety parameters that will be monitored during the course of the trial, along with the methods and timing for their assessment and reporting. Standardized definitions for AEs and SAEs have been formulated ( FDA, 1996 ): an AE is defined as any untoward medical event that occurs in a subject who has received a study agent. SAEs include death, life-threatening events, hospitalization or prolongation of hospitalization, congenital anomalies, and events that result in permanent disability. Each AE and SAE that is recorded should be assessed for its severity and relationship to vaccination. The grading of AEs is usually based on the interference with activity, where 1=mild, does not interfere with usual activities; 2=moderate, interferes somewhat with usual activities; and 3=severe, incapacitating. The severity grading system of laboratory abnormalities should be established before the initiation of the trial. The FDA has suggested guidelines for toxicity grading scale for healthy adults and adolescents enrolled in vaccine clinical trials ( FDA, 2007b ). Contemporary standards for assessing the relationship of the AE to immunization have narrowed the choices to "associated" and "not associated." All AEs that are temporally related to administration of the investigational agent and have no alternative etiologies to explain the event are considered associated. SAEs should be reported promptly to the sponsor; in turn, the Sponsor must notify the FDA in a timely fashion. Prospective guidelines for discontinuation of individuals from further vaccinations, as well as halting rules for the clinical trial should be outlined. Typical circumstances for discontinuation of an individual include severe and/or hypersensitivity reactions following immunization, development of an Exclusion Criterion during the trial, and failure or inability of the subject to comply with study procedures. Note that every attempt should be made to continue to follow these subjects for safety assessments. Rules for halting a phase 1 clinical trial may include the occurrence of one or two hypersensitivity reactions or SAEs associated with the investigational agent or the occurrence of moderate or severe reactogenicity among a predefined proportion of subjects. Finally, the safety oversight plan should be described. For single-center trials or other small clinical trials, a Safety Monitoring Committee (SMC) consisting an Independent Safety Monitor (ISM) from each site and an independent member with expertize relevant to the protocol has the responsibility to review trial results periodically and on an ad hoc basis, and to make recommendations to the Sponsor regarding the conduct of the clinical trial. For larger, multicenter trials, a Data and Safety Monitoring Board (DSMB) consisting of site ISMs and individuals with clinical and statistical expertize relevant to the protocol is constituted to review study progress and advise the Sponsor. The SMC or DSMB may recommend terminating a trial because of unexpected or severe toxicity; continuation of a trial after review of AEs that triggered halting rules; alteration of sample size based on interim analyses; or any other protocol modifications that are deemed necessary to complete the trial successfully. Clinical Monitoring A clear plan for monitoring the conduct of the clinical trial site(s) should be outlined in the study protocol. Specific objectives of site-monitoring visits are to review all study documentation to ensure protection of human subjects; compliance with GCP, clinical and laboratory procedures, test article administration and accountability guidelines; and accurate and complete data collection and documentation. The Sponsor may conduct monitoring visits, and may also designate an independent Contract Research Organization (CRO) to conduct monitoring visits on a regular basis throughout the trial. Early monitoring visits can be particularly valuable in order to identify systematic, unintentional deviations from the protocol. Statistical Considerations A detailed Statistical Analysis Plan (SAP) should be prepared that restates the study hypotheses, objective and endpoints, describes the statistical basis for the sample size selected, and outlines the methods that will be used to analyze safety and efficacy. If interim analyses are planned, then the statistical issues related to this should be discussed. Quality Management The clinical trial site is responsible for protocol compliance and accurate and complete data collection and recording. In a separate document, site-specific Standard Operating Procedures (SOPs) should outline the methods that will be used to ensure that these activities are accomplished, as well as methods to ensure appropriate training of the study staff. The overall quality management plan should be described in the protocol. Ethics/Protection of Human Subjects A description of the ethical standards that will be followed to ensure protection of human subjects should be described: in the US, compliance with 45 CFR Part 46 and ICH E6 GCP is expected. The protocol should indicate that no research (including screening) will begin until the protocol and the consent form have been IRB-approved. The consent process should be described, as should the provisions for subject confidentiality. The Health Insurance Portability and Accountability Act of 1996 (HIPAA) was enacted to improve portability and continuity of health insurance coverage; nevertheless, it contains regulations that have direct relevance for clinical research ( DHHS, 2007 ). For example, informed consent documents are required to include extensive information on how the participant's protected health information (PHI) will be kept private. Administrative, physical, and technical safeguards must be adopted to ensure the security of electronic PHI. Finally, special issues related to exclusion of special populations (women, minorities, and children) should be addressed. Data Management and Record Keeping The goals of data management are to ensure the accuracy, completeness, and timeliness of clinical trial data collection. While the primary responsibilities for data collection rest with the clinical trial site, additional oversight and data management responsibilities (quality review, analysis, and reporting) may be shared with the sponsor and a data coordinating center (DCC). The organizational structure of data management should be described in this part of the protocol. Data capture methods and internal quality checks, types of data, timing of reports, study records retention, and identification of protocol deviations and corrective actions should be addressed here, as well as in greater detail in the MOP. Other Considerations In addition to the major protocol elements described above, a full protocol should include a title page, a statement of compliance with GCP, and other regulatory guidelines, a signature page, a table of contents, a list of abbreviations; a protocol summary; a list of key personnel and their roles, a description of unique facilities, if applicable, a list of references, publication policy; and appropriate appendices. A list of essential documents that should be on file before the trial starts is shown in Table 12.3 ; additional documents should be added to the trial documentation as new information becomes available (protocol amendments, updates, IRB approvals, training certificates, CVs, screening and enrollment logs, test article accountability and shipment logs, monitoring reports, consent forms, source documents, CRFs, communications with the sponsor, etc.). Table 12.3 Checklist of essential documents on file at the clinical trial site before the clinical trial begins Signed full clinical trial protocol, and amendments, if applicable Sample case report forms (CRFs) IRB-approved informed consent document Investigators' brochure Manual of procedures (MOP) Information that will be given to subjects Recruiting materials (text of advertisements, flyers, etc.) IRB approval letter Copy of IRB/IEC Federal Wide Assurance Composition of IRB FDA Form 1572 (Principal Investigator Responsibilities) Curriculum vitae of participating investigators Financial disclosures; other clinical trial agreements Copy of the principal investigator's medical license Laboratory credentials/certifications Laboratory reference ranges Sample labels for investigational product Instructions for handling investigational product and other trial materials Shipping records for trial-related materials Clinical trial site initiation monitoring report Source : Adapted from ICH E6 GCP guidance ( FDA, 1996 ). Background and Rationale The background and rationale should provide information regarding the current understanding of the disease epidemiology, pathogenesis, and immune responses relevant for protection against infection; and need for development of control measures. The scientific rationale for selection of the vaccine candidate should be discussed. A concise description of the study agent should be provided, including summaries of preclinical studies and relevant clinical studies. Finally, potential risks and benefits of immunization with the investigational agent should be delineated. Objectives and Purpose The objectives and purpose of the trial should be clearly and explicitly stated. For phase 1 trials, the primary objectives will be to assess the safety, tolerability, and reactogenicity of a vaccine, whereas assessment of immunogenicity is a secondary objective. For combined phase 1/2 and phase 2 clinical trials, safety and immunogenicity may be coprimary endpoints. Exploratory endpoints may also be included, such as the effect of age, race, or gender on immune responses ( Keitel et al., 2006 ). Combined phase 1/2 trials may be proposed when the vaccine candidate represents a variant of a previously licensed construct, such as a subvirion influenza vaccine for prevention of avian influenza ( Treanor et al., 2006 ). Efficacy and safety typically are the primary endpoints for phase 3 trial. Study Design The study design then should be described. For clinical trials of candidate vaccines, the study design typically is a randomized, double-blind, controlled clinical trial—one example of a parallel group design. Phase 1 safety and tolerability studies often utilize a titration design, where ascending dosages of the experimental agent are sequentially administered to new cohorts of subjects ( Gorse et al., 2006 ). Cluster-randomized clinical trial design is occasionally employed to assess vaccine efficacy. In this circumstance, larger groups of individuals (such as nursing homes or schools) are randomized to an intervention, rather than individual subjects, and the clinical endpoints are ascertained for vaccinated subjects ( Rodrigues et al., 2005 ), or for a subset of the cluster, such as the contacts of healthcare workers in a closed setting ( Hayward et al., 2006 ). Phase 1 clinical trials historically were open-label; however, in recent years most phase 1 clinical trials of vaccines have been randomized and blinded. The value of a placebo control in clinical trials has been described; however, a licensed control vaccine may be used rather than a placebo, particularly for phase 3 clinical trials in children. In a recent phase 3 clinical trial of pneumococcal conjugate vaccine in infants, a meningococcal type C vaccine served as the control vaccine ( Black et al., 2000 ). In this case, the meningococcal control vaccine provided potential benefits to the study participants. Flexible adaptive design methods frequently are employed in the development of vaccines; these incorporate plans for modifications of the clinical trial design that are made before or during the conduct of the research ( Chow and Chang, 2007 ). Adaptations to ongoing trials may include prospective adaptations, such as interim analysis, stopping rules for early termination due to futility/safety concerns/efficacy, or sample size re-estimation; ad hoc adaptations such as changes in inclusion and exclusion criteria, dosage or regimen alteration, and trial duration; or retrospective adaptations at the end of the study but before unblinding, including changing the study endpoint or altering the statistical hypothesis (superiority to noninferiority). The goal of this approach is to permit modification based on accumulated evidence to alter trial design to increase the probability of success without undermining the validity of the trial ( Gallo et al., 2006 ). Such adaptations may require modifications of the study hypotheses, protocol amendments, and sample size recalculations. Clinical trials are also classified as single-center or multicenter studies. Phase 1 studies often are single-center studies; however, multicenter trial design may be used for any phase of clinical vaccine development. Multicenter study design provides several advantages: enrollment of subjects is expedited, and the results of the trial are likely to be more generalizable. Study endpoints need to be clearly identified. For phase 1 trials, safety, tolerability, and reactogenicity primary endpoints may include the frequencies and severities of injection site reactions (pain, tenderness, redness, and swelling) and systemic reactions (fever, chills, headache, myalgia, arthralgia, etc.), as well a laboratory evidence of adverse reactions (hematologic, biochemical, and other). For phase 2 clinical trials, specific immune responses at defined time points after immunization typically characterize the primary endpoints; safety and reactogenicity may be primary or secondary endpoints. For phase 3 clinical trials, protection against laboratory-confirmed infection and/or disease is the primary endpoint, and the major safety assessment may be the frequency of SAEs associated with administration of the investigational agent. Study Population A detailed description of the proposed study population and the number of subjects to be studied must be provided; specifically, characteristics (age range, health status, ability to provide informed consent, etc.) of potentially eligible persons (Inclusion Criteria) and factors that would render an individual ineligible (Exclusion Criteria) should be explicitly enumerated. For some phase 1 trials, screening for eligibility may include medical history, physical examination, and laboratory screening for evidence of good health (normal hematologic and biochemical parameters, and no evidence of active hepatitis B, hepatitis C, or HIV infection). Information regarding serosusceptibility to the candidate pathogen may be necessary. For example, a phase 1 clinical trial of a dengue virus vaccine may require evidence of no prior infections caused by these viruses ( Edelman et al., 2003 ), and a phase 1 or 2 clinical trial assessing the immunogenicity of LAIV may focus on persons with low or absent levels of preexisting immunity to the candidate vaccine ( Keitel et al., 1993b ). For phase 3 clinical trials, it is necessary to identify a population in which the infection or disease occurs at a high enough frequency to assess the ability of a vaccine to protect. For example, pivotal phase 3 trials of an inactivated hepatitis A vaccine were conducted in specific US communities where the rate of hepatitis A infections in children was high ( Werzberger et al., 1992 ). Human subjects considerations may include a description of certain behaviors and/or concomitant medications that would exclude a subject. For most clinical trials of vaccines, women who are capable of bearing children must consent to certain birth control measures. The US Department of Health and Human Services (DHHS) has published a guidance regarding research in pregnant women: for research conducted in this population, there must be direct benefit to the woman or her fetus or there must be only minimal risk to the fetus, and information cannot be obtained any other way. For many phase 1 clinical trials, use of prescription medications is not permitted. Clinical trials of vaccines that potentially could be transmitted to others in the community raise special concerns. Recent reevaluations of smallpox vaccines posed concerns with regard to transmission of the vaccine virus from subjects to their contacts ( Frey et al., 2002 ). In this case, persons who had household or other significant contacts with young infants, people with eczema, pregnant women, and immunocompromised individuals were excluded from participation. The methods for test article allocation should also be described. For most phase 1 and phase 2 clinical trials of vaccines, the subjects are randomized to receive one of several dosage levels of vaccine or placebo. Ideally, randomization should not occur until the subjects have been qualified for participation. Typically the randomization occurs in blocks of a prespecified number that represents a multiple of the number of test articles. For example, if there were four dosage levels of vaccine and a placebo, then the block size might be 5, 10, or 15. If the block size chosen were 5, then the subjects would be randomized 1:1:1:1:1. Block randomization can reduce the risk of unequal group sizes. In some circumstances the probability of receiving one product differs from the probability of receiving another. For example, in an efficacy study to be conducted in children, an investigator may wish to reduce the number of subjects randomized to receive the placebo, and the randomization scheme selected may be 2:1 (vaccine:placebo). The vaccine group assignments for subjects should be concealed from the subjects and from investigators to reduce bias in the assessments performed after vaccination (so-called double blinding). Additional measures can be taken to reduce the potential for imbalances in baseline characteristics of enrolled subjects, such as stratification of subjects according to age, prior receipt of a related vaccine, etc., prior to randomization. Study Agent/Interventions The clinical trial protocol should contain basic information regarding the characterization of the vaccine formulations—including dosage(s), packaging, labeling, storage; preparation, administration, dosing, and accountability methods for each study product, including placebo and/or control preparations. More complete descriptions of study vaccines, including manufacturing information, preclinical and clinical safety, immunogenicity, and efficacy should be provided separately in the Investigators' Brochure (IB). Information regarding the use of concomitant medications, including prohibited medications, should be detailed. For example, during phase 1 clinical trials concomitant use of prescription medications may be prohibited. During phase 2 clinical trials, concomitant use of certain medications may be allowed, such as antihypertensive medications or antidepressants. In general, concomitant use of immunosuppressive, immunomodulatory, or cytotoxic drugs would be prohibited in any clinical trials of live attenuated vaccines. Study Procedures and Evaluations A description of the proposed clinical evaluations then follows. In phase 1 or 2 clinical trials, detailed and frequent physical assessments of the injection site and systemic responses may be indicated, as well as review of subject records of clinical responses following immunization. The intensity of study assessments will vary according to the nature of the study product: more frequent and detailed assessments would be indicated for novel products whose safety profile is undefined. Periodic collection of blood, nasal, fecal, or other samples to assess for the occurrence of toxicity, or to determine the frequency, magnitude, and/or duration of shedding of a live vaccine candidate may also be indicated ( Piedra et al., 1993 ; Taylor et al., 1997 ). These laboratory assessments should be tailored to the particular needs of the protocol, and should be based on the pathogenesis of the disease, the vaccine under evaluation, and information collected in the pre-IND stage. Brief descriptions of the type(s) of specimens to be collected, methods for specimen collection, preparation, handling, storing, and shipping (if applicable) should be outlined; detailed procedures should be provided in a separate Manual of Procedures (MOP) for each study. For phase 3 trials, clinical follow-up is specifically targeted at ascertaining whether the vaccine prevents infection or disease and capturing the occurrence of SAEs; however, limited prospective safety assessments may be included (perhaps only in a subset of subjects) to expand the safety database ( Oxman et al., 2005 ). Study Schedule Once the specific clinical and laboratory procedures for assessing safety, immunogenicity, and/or efficacy have been described, a detailed study schedule indicating the timing of various assessments/procedures/interventions should be outlined, including those that will be conducted at screening, enrollment, and each follow-up visit. Clinical assessments and laboratory procedures that should be completed in the event the subject's participation in the trial is terminated early should be described, as should those for women who become pregnant during the study. Finally, a description of clinical and laboratory assessments to be performed in the event the subject returns for an unscheduled visit (for example, for a severe vaccine reaction) may be appropriate. A summary table or figure outlining study procedures is particularly useful. Assessment of Safety As described above, the clinical trial protocol should detail the specific safety parameters that will be monitored during the course of the trial, along with the methods and timing for their assessment and reporting. Standardized definitions for AEs and SAEs have been formulated ( FDA, 1996 ): an AE is defined as any untoward medical event that occurs in a subject who has received a study agent. SAEs include death, life-threatening events, hospitalization or prolongation of hospitalization, congenital anomalies, and events that result in permanent disability. Each AE and SAE that is recorded should be assessed for its severity and relationship to vaccination. The grading of AEs is usually based on the interference with activity, where 1=mild, does not interfere with usual activities; 2=moderate, interferes somewhat with usual activities; and 3=severe, incapacitating. The severity grading system of laboratory abnormalities should be established before the initiation of the trial. The FDA has suggested guidelines for toxicity grading scale for healthy adults and adolescents enrolled in vaccine clinical trials ( FDA, 2007b ). Contemporary standards for assessing the relationship of the AE to immunization have narrowed the choices to "associated" and "not associated." All AEs that are temporally related to administration of the investigational agent and have no alternative etiologies to explain the event are considered associated. SAEs should be reported promptly to the sponsor; in turn, the Sponsor must notify the FDA in a timely fashion. Prospective guidelines for discontinuation of individuals from further vaccinations, as well as halting rules for the clinical trial should be outlined. Typical circumstances for discontinuation of an individual include severe and/or hypersensitivity reactions following immunization, development of an Exclusion Criterion during the trial, and failure or inability of the subject to comply with study procedures. Note that every attempt should be made to continue to follow these subjects for safety assessments. Rules for halting a phase 1 clinical trial may include the occurrence of one or two hypersensitivity reactions or SAEs associated with the investigational agent or the occurrence of moderate or severe reactogenicity among a predefined proportion of subjects. Finally, the safety oversight plan should be described. For single-center trials or other small clinical trials, a Safety Monitoring Committee (SMC) consisting an Independent Safety Monitor (ISM) from each site and an independent member with expertize relevant to the protocol has the responsibility to review trial results periodically and on an ad hoc basis, and to make recommendations to the Sponsor regarding the conduct of the clinical trial. For larger, multicenter trials, a Data and Safety Monitoring Board (DSMB) consisting of site ISMs and individuals with clinical and statistical expertize relevant to the protocol is constituted to review study progress and advise the Sponsor. The SMC or DSMB may recommend terminating a trial because of unexpected or severe toxicity; continuation of a trial after review of AEs that triggered halting rules; alteration of sample size based on interim analyses; or any other protocol modifications that are deemed necessary to complete the trial successfully. Clinical Monitoring A clear plan for monitoring the conduct of the clinical trial site(s) should be outlined in the study protocol. Specific objectives of site-monitoring visits are to review all study documentation to ensure protection of human subjects; compliance with GCP, clinical and laboratory procedures, test article administration and accountability guidelines; and accurate and complete data collection and documentation. The Sponsor may conduct monitoring visits, and may also designate an independent Contract Research Organization (CRO) to conduct monitoring visits on a regular basis throughout the trial. Early monitoring visits can be particularly valuable in order to identify systematic, unintentional deviations from the protocol. Statistical Considerations A detailed Statistical Analysis Plan (SAP) should be prepared that restates the study hypotheses, objective and endpoints, describes the statistical basis for the sample size selected, and outlines the methods that will be used to analyze safety and efficacy. If interim analyses are planned, then the statistical issues related to this should be discussed. Quality Management The clinical trial site is responsible for protocol compliance and accurate and complete data collection and recording. In a separate document, site-specific Standard Operating Procedures (SOPs) should outline the methods that will be used to ensure that these activities are accomplished, as well as methods to ensure appropriate training of the study staff. The overall quality management plan should be described in the protocol. Ethics/Protection of Human Subjects A description of the ethical standards that will be followed to ensure protection of human subjects should be described: in the US, compliance with 45 CFR Part 46 and ICH E6 GCP is expected. The protocol should indicate that no research (including screening) will begin until the protocol and the consent form have been IRB-approved. The consent process should be described, as should the provisions for subject confidentiality. The Health Insurance Portability and Accountability Act of 1996 (HIPAA) was enacted to improve portability and continuity of health insurance coverage; nevertheless, it contains regulations that have direct relevance for clinical research ( DHHS, 2007 ). For example, informed consent documents are required to include extensive information on how the participant's protected health information (PHI) will be kept private. Administrative, physical, and technical safeguards must be adopted to ensure the security of electronic PHI. Finally, special issues related to exclusion of special populations (women, minorities, and children) should be addressed. Data Management and Record Keeping The goals of data management are to ensure the accuracy, completeness, and timeliness of clinical trial data collection. While the primary responsibilities for data collection rest with the clinical trial site, additional oversight and data management responsibilities (quality review, analysis, and reporting) may be shared with the sponsor and a data coordinating center (DCC). The organizational structure of data management should be described in this part of the protocol. Data capture methods and internal quality checks, types of data, timing of reports, study records retention, and identification of protocol deviations and corrective actions should be addressed here, as well as in greater detail in the MOP. Other Considerations In addition to the major protocol elements described above, a full protocol should include a title page, a statement of compliance with GCP, and other regulatory guidelines, a signature page, a table of contents, a list of abbreviations; a protocol summary; a list of key personnel and their roles, a description of unique facilities, if applicable, a list of references, publication policy; and appropriate appendices. A list of essential documents that should be on file before the trial starts is shown in Table 12.3 ; additional documents should be added to the trial documentation as new information becomes available (protocol amendments, updates, IRB approvals, training certificates, CVs, screening and enrollment logs, test article accountability and shipment logs, monitoring reports, consent forms, source documents, CRFs, communications with the sponsor, etc.). Table 12.3 Checklist of essential documents on file at the clinical trial site before the clinical trial begins Signed full clinical trial protocol, and amendments, if applicable Sample case report forms (CRFs) IRB-approved informed consent document Investigators' brochure Manual of procedures (MOP) Information that will be given to subjects Recruiting materials (text of advertisements, flyers, etc.) IRB approval letter Copy of IRB/IEC Federal Wide Assurance Composition of IRB FDA Form 1572 (Principal Investigator Responsibilities) Curriculum vitae of participating investigators Financial disclosures; other clinical trial agreements Copy of the principal investigator's medical license Laboratory credentials/certifications Laboratory reference ranges Sample labels for investigational product Instructions for handling investigational product and other trial materials Shipping records for trial-related materials Clinical trial site initiation monitoring report Source : Adapted from ICH E6 GCP guidance ( FDA, 1996 ). Conclusions The success of any clinical trial hinges on the development of a carefully designed protocol. Although discussion of clinical trial implementation is beyond the scope of this chapter, it is clear that the protection of human subjects and scientific integrity of the trial design and documentation are the overarching goals of any clinical research protocol. The clinical trial protocol must document the processes that will be used to ensure that these goals are attained. Conscientious supervision of clinical trial activities is a shared responsibility of the investigators, the IRB, the sponsor, the CRO, the DCC, the safety oversight committee, and all other partners who are participating in the trial. While meticulous attention to detail and strict protocol compliance are essential, the entire study team must be flexible and prepared to respond in a timely fashion to unexpected findings. Novel approaches to the development of vaccines for biodefense and emerging and neglected diseases will continue to evolve and will require ongoing reconsideration of the ethical and regulatory principles and practices that guide the conduct of clinical trials. Acknowledgments I gratefully acknowledge Dr. Robert Atmar and Connie Rangel, R.N., for their thoughtful reading of the manuscript, and Yvette Rugeley and Michelle Thomas-Sturm for assistance with manuscript preparation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147860/

HDAC8 Activates AKT through Upregulating PLCB1 and Suppressing DESC1 Expression in MEK1/2 Inhibition-Resistant Cells

Inhibition of the RAF-MEK1/2-ERK signaling pathway is an ideal strategy for treating cancers with NRAS or BRAF mutations. However, the development of resistance due to incomplete inhibition of the pathway and activation of compensatory cell proliferation pathways is a major impediment of the targeted therapy. The anthrax lethal toxin (LT), which cleaves and inactivates MEKs, is a modifiable biomolecule that can be delivered selectively to tumor cells and potently kills various tumor cells. However, resistance to LT and the mechanism involved are yet to be explored. Here, we show that LT, through inhibiting MEK1/2-ERK activation, inhibits the proliferation of cancer cells with NRAS/BRAF mutations. Among them, the human colorectal tumor HT-29 and murine melanoma B16-BL6 cells developed resistance to LT in 2 to 3 days of treatment. These resistant cells activated AKT through a histone deacetylase (HDAC) 8-dependent pathway. Using an Affymetrix microarray, followed by qPCR validation, we identified that the differential expression of the phospholipase C-β1 (PLCB1) and squamous cell carcinoma-1 (DESC1) played an important role in HDAC8-mediated AKT activation and resistance to MEK1/2-ERK inhibition. By using inhibitors, small interference RNAs and/or expression vectors, we found that the inhibition of HDAC8 suppressed PLCB1 expression and induced DESC1 expression in the resistant cells, which led to the inhibition of AKT and re-sensitization to LT and MEK1/2 inhibition. These results suggest that targeting PLCB1 and DESC1 is a novel strategy for inhibiting the resistance to MEK1/2 inhibition. 1. Introduction Hyperactivation of the MEK1/2-ERK signaling axis due to mutations in NRAS and BRAF drives oncogenesis in ~30% of human cancers, and targeting RAF and MEK can be a curative therapy for these cancers [ 1 ]. However, the development of resistance often prompts clinical relapse and therapeutic failure. Among various causes, incomplete inhibition of the MEK1/2-ERK pathway contributes to be an intrinsic and acquired resistance to these inhibitors [ 2 , 3 ]. Indeed, combinatory therapies using both RAF and MEK1/2 inhibitors provide a better prognosis and are the current standard-of-care in certain cancers [ 4 , 5 ]. The anthrax lethal toxin (LT), which potently inhibits MEK1/2-ERK activation and can be modified to selectively target cancers, is a promising biomolecule [ 6 , 7 , 8 ], likely with less of a chance of resistance development. LT, which is composed of a carrier protective antigen (PA) and protease lethal factor (LF), selectively cleaves the N-termini of all MEKs, except MEK5 [ 9 , 10 ], and induces cell cycle arrest and cell death [ 11 , 12 ]. However, we showed that macrophages adaptively respond to LT and become resistant to LT-induced cell cycle arrest through activating the phosphatidylinositol 3-kinase (PI3K)/AKT signaling cascade [ 13 , 14 ]. Similarly, in certain tumor cells, resistance to RAF/MEK inhibitors is attributed to activation of the PI3K-AKT signaling axis caused by a loss of phosphatase and tensin homology (PTEN) or adaptive stress responses [ 15 , 16 , 17 ]. However, the mechanisms that activate the PI3K-AKT signaling pathway in resistant cancer cells are yet to be fully delineated. As one of the potential mechanisms, we previously showed that histone deacetylase 8 (HDAC8), which is a member of the class I HDAC family, is involved in the resistance to LT in macrophages [ 14 ]. HDAC8 was also shown to mediate the resistance to RAF inhibitors in melanoma [ 18 ]. In these cells, HDAC8 deacetylates and activates the c-JUN transcription factor, resulting in the increased expression of receptor tyrosine kinases and ERK activation. Therefore, HDAC8 may induce a resistance to RAF-MEK inhibition in different pathways, depending on the cell type. To further delineate the mechanisms of HDAC8 in resistance to MEK1/2-ERK inhibition, we examined whether LT induces resistance and, if so, then what mechanisms are involved in cancer cell types with known mutations in the RAS-RAF-MEK signaling axis. We found that HDAC8 was required for a resistance to LT and the MEK1/2 inhibitor U0126 in the human colorectal tumor cell line HT-29 and murine melanoma B16-BL6 cells. HDAC8 induced AKT activation in these resistant cells, in part, through inducing PLCB1 expression. The inhibition of HDAC8 suppressed PLCB1 expression but enhanced DESC1 expression, both of which were involved in preventing the compensatory activation of AKT and resistance to MEK1/2 inhibition. 2. Materials and Methods Reagents—Protective antigen (PA) and lethal factor (LF) were purchased from the List Biological Laboratories (Campbell, CA, USA). The ERK inhibitor U0126, p38 MAPK inhibitor SB203580, AKT inhibitor, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were obtained from APExBIO Technology (Houston, TX, USA), Selleck Chemicals (Houston, TX, USA), Calbiochem (San Diego, CA, USA), and Sigma-Aldrich (St. Louis, MO, USA), respectively. HDAC8 inhibitor PCI-34051, edelfosine, and 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) were obtained Cayman Chemical (Ann Arbor, MI, USA). The HDAC8 and PLCB1 antibodies were obtained from AB clonal Technology (Woburn, MA, USA). Antibodies for phospho-AKT (Ser-473), MEK1 (N-terminal 12 amino acids), and β-actin were purchased from Cell Signaling (Danvers, MA, USA), Stressgen Biotechnologies (Cat# KAP-MA010; Ann Arbor, MI, USA), and Rockland Inc. (Gilvertsville, PA, USA), respectively. The cOmpleteTM EDTA-free protease inhibitor cocktail and phosphatase inhibitor cocktail (phosSTOP) tablets were obtained through Thermo Scientific (Roche; Indianapolis, IN, USA). DESC1 (vector ID; VB170123-1118ntk, hTMPRSS1 (ORF023752)) plasmid was constructed through Cyagen (Vector Builder; Chicago, IL, USA). Cell culture—Mouse B16-BL6 melanoma, human colorectal tumor HT-29 cells, and human melanoma MDA-435 and SK-MEL-5 cells were maintained in complete RPMI 1640 or DMEM, supplemented with 10% heated-inactivated fetal bovine serum (WISENT; Saint-Jean-Baptiste, QC, Canada, 10-mM MEM nonessential amino acid solution, 100-U/mL penicillin G sodium, 100-μg/mL streptomycin sulfate, and 1-mM sodium pyruvate. Cell viability and proliferation assay—Cell viability/proliferation was measured by the MTT analysis, as previously described [ 13 ]. Briefly, cells were seeded in 96-well plates and cultured in the presence or absence of LT (LF and PA) and/or chemical inhibitors for the time indicated. MTT at a final concentration of 0.5 mg/mL was added and incubated 2–4 h before stopping the experiments by replacing the cell culture media with 100 µL of dimethyl sulfoxide to dissolve the crystals. For cells in suspension, the experiments were ended by adding 100 µL of 0.04-N HCl in isopropanol for 30 min in a shaker at room temperature. Optical densities of each well were analyzed using an automatic microplate reader (Synergy H4 Hybrid Reader, BioTek; Winooski, VT, USA) at a wavelength of 570 nm. The % of cell survival was calculated based on cell numbers in comparison with those of nontreated cells. The % of cell proliferation was based on the cell numbers in comparison with those of nontreated cells 24 h after seeding cells. All cell numbers were estimated based on the standard curve generated by optical densities of known cell numbers. Gene expression microarray—HT-29 (3 × 10 6 ) cells were cultured with or without LT (500 ng/mL of each PA and LF) or LT+ PCI-34051 (PCI: 5 μM) for 48 h. Total cellular RNAs were prepared using TRIzol TM (Ambion Inc.; Carlsbad, CA, USA), and the quantity and quality of the total RNAs were verified through an Agilent 2100 Bioanalyzer. Total RNAs (100 ng) were then amplified and labeled to prepare complementary RNAs, 5.5 µg of which was loaded onto the array, following the manufacturer's guidelines (Affymetrix, Santa Clara, CA, USA). Gene array was performed using the GeneChip™ Human Genome U133 Plus 2.0 Array kit in the London Regional Genomics Centre at Western University, London, ON, Canada. CEL files were then imported to Partek TM Genomics Suite TM for differential gene expression (with 2-fold change cut-off) and gene ontology enrichment analyses. Immunoblotting—Total cell lysate preparation and Immunoblotting were conducted as previously described [ 13 ]. Briefly, cells were lysed in ice-cold lysis buffer (20-mM MOPS, 2-mM EGTA, 5-mM EDTA, 1-mM Na3VO4, 40-mM β-glycerophosphate, 30-mM sodium fluoride, 20-mM sodium pyrophosphate, 0.1% SDS, and 1% Triton X-100, pH 7.2) containing a cOmplete TM EDTA-free protease inhibitor and phosphatase inhibitor (phosSTOP), and the cells were incubated on ice for 10 min. Whole lysates were centrifuged at 12,500 rpm for 15 min at 4 °C. Proteins in supernatants were separated by SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% ( w / v ) skim milk for 1 h at room temperature and exposed to primary antibodies overnight at room temperature and then washed three times with 1 × TBST (20-mM Tris and 150-mM NaCl, pH 7.5) containing 0.05% Tween 20. The membranes were incubated with the secondary antibody for 60 min at room temperature, and immunoreacting bends were developed using an Enhanced Chemiluminescence detection system (ECL; Thermo Scientific; Rockford, IL, USA) or Luminata TM Forte (Millipore, Billerica, MA, USA). Quantitative real-time PCR—Quantitative real-time PCR (qPCR) was carried out as previously described [ 13 ]. Briefly, the isolation of total cellular RNAs and reverse transcribing were performed using TRIzol TM (Ambion Inc.) and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (New England Biotechnology; Ipswich, MA, USA), according to the manufacturer's instructions. The qPCR analyses were processed using a Rotor-Gene RG3000 quantitative multiplex PCR instrument (Montreal Biotech Inc.; Dorval, QC, Canada) and Power UP TM SYBR Green Master Mix (Applied Biosystems, Life Technologies; Foster City, CA, USA). Data were normalized to the levels of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene. Human primers used for qPCR were: for GAPDH, 5′-ACCCACTCCTCCACCTTTG-3′ (5′ primer) and 5′-CTCTTGTGCTCTTGCTGGG-3′ (3′ primer); for HDAC8, 5′-ATTCTCTACGTGGATTTGGATC-3′ (5′ primer) and 5′-ATGCCATCCTGAATGGGCACA-3′ (3′ primer); for PLCB1, 5′-GGTGCAGTATATCAAGAGGCTAGA-3′ (5′ primer) and 5′- TGGTCACCACTTGAGAGCTT-3′ (3′ primer); and for DESC1, 5′-AGAGTTTGTTGGGAACCCTGG-3′ (5′ primer) and 5′-AAGCCTCTCTGCCAAACTCAG-3′ (3′ primer). Mouse PLCB1 and DESC1 mRNA expression were analyzed using primers GAPDH, 5′-GCATTGTGGAAGGGCTCATG-3′ (5′ primer) and 5′-TTGCTGTTGAAGTCGCAGGAG-3′ (3′ primer); PLCB1, 5′-GGTGCAGTATATCAAGAGGCTAGA-3′ (5′ primer) and 5′-TGGTCACCACTTGAGAGCTT-3′ (3′ primer); and DESC1, 5′-AGAGTTTGTTGGGAACCCTGG-3′ (5′ primer) and 5′-AAGCCTCTCTGCCAAACTCAG-3′ (3′ primer). Transfection of small interfering (si)RNAs and plasmids—HT-29 cells were transfected with human HDAC8-targeting siRNA [(Invitrogen, Life Technologies; Carlsbad, CA, USA) catalog No. 10620318-19, HDAC8HSS125194)] or human PLCB1-targeting siRNA (Invitrogen, Life Technologies, Cat No. 10620318-329608F08 and 10620319-329974A06, PLCB1VHS41619) at 64 nM for 18 h using Lipofectamine RNAiMAX (Invitrogen, Life Technologies), according to the manufacturer's instructions. Cells were then replated to 6-well or 96-well plates, and, after incubation for an additional 6 h, cells were treated with LT or U0126 for the time indicated. Plasmid transfection was carried out using Lipofectamine 2000 or 3000 (Invitrogen, Life Technologies) according to the manufacturer's instructions. Briefly, 1.5 × 10 6 cells were plated on 6-well plates at 6–8 h prior to transfection and transfected with plasmids for 4 h. Cells were further incubated for 16–18 h with an additional cell culture medium. Cells were then replated and treated with or without LT or U0126 for the time indicated. Statistical analysis—Data were analyzed using GraphPad Prism Version 4.0 software, and the results were presented as the mean ± SD of two or three independent repeats. Statistical significance was defined as p < 0.05 (*). 3. Results 3.1. Murine Melanoma B16-BL6 and Human Colorectal HT-29 Cells Develop Resistance to LT in an HDAC8-Dependent Pathway We first examined the cytotoxic effect of LT in four cancer cell lines with known mutations in the signaling axis of RAS-RAF-MEK1/2: murine melanoma B16-BL6, human colorectal cancer HT-29, and human melanoma MDA-MB-435 (MDA) [ 19 ] and SK-MEL-5 (SK-MEL) cell lines [ 6 , 20 , 21 , 22 , 23 ]. LT was able to decrease cell survival within 24 h in these cells ( Figure 1 A). To further examine whether the decreased cell survival was due to cell death or cell cycle arrest, small numbers of these cells were plated and monitored for cell proliferation over 96 h in the presence of LT. During the 48 h of LT treatment, no apparent changes in live cell numbers were detected ( Figure 1 B). In 72 h of LT treatment, the live cell numbers of B16-BL6 and HT-29 cells started increasing, whereas those of MDA and SK-MEL cells did not. These results suggest that B16-BL6 and HT-29 cells became resistant to LT and started proliferating in the presence of LT. Since HDAC8 plays a key role in the resistance to LT cytotoxicity in macrophages [ 14 ], we examined if HDAC8 was also involved in the resistance in B16-BL6 and HT-29 cells. As in macrophages, both B16-BL6 and HT-29 cells failed to proliferate in the presence of LT when the cells were treated together with the HDAC8-specific inhibitor PCI34051 (PCI; Figure 1 B). PCI alone had no apparent effect on the cell proliferation. To further confirm that the proliferating cells in the presence of LT are resistant cells, rather than cells delayed in cell proliferation, these cells were replated and rechallenged with LT and examined for cell survival. Unlike in parental cells, these resistant cells did not show any defects in cell survival/proliferation ( Figure 1 C). To further confirm the role of HDAC8, HDAC8 was knocked down by small interference (si)RNAs in HT-29 cells. As in macrophages [ 14 ], LT induced HDAC8 expression in both mRNA and the protein levels ( Figure 1 D). HDAC8-targeting siRNAs (si-HDAC8), but not random siRNAs (si-Random), significantly decreased the mRNA ( Figure 1 D, left panel) and protein ( Figure 1 D, right panel) levels of HDAC8 in both the parental and LT-resistant cells. Indeed, like PCI, si-HDAC8 prevented cell proliferation in LT-exposed HT-29 cells ( Figure 1 E). 3.2. LT-Induced Cell Cycle Arrest and Resistance Are Manifested by MEK1/2 Inhibition LT inactivates both the MEK1/2-ERK and MEK3/6-p38 signaling pathways by cleaving MEK1-7, except MEK5 [ 24 ]. Therefore, we examined if these two signaling pathways were involved in preventing cell proliferation in LT-treated cells. In both B16-BL6 and HT-29 cells, the MEK1/2 inhibitor U0126, but not the p38 inhibitor SB203580, decreased cell survival to the similar extent observed by LT ( Figure 1 A and Figure 2 A). In both B16-BL6 and HT-29 cells, the U0126 treatment also transiently inhibited cell proliferation for ~48 h, and cells started proliferating after 72 h of the treatment ( Figure 2 B). As in LT-resistant cells, when U0126-resistant cells were rechallenged with U0126, no defects in cell survival/proliferation were detected within 48 h ( Figure 2 C). PCI also prevented a resistance to U0126 in both HT-29 and B16-BL6 cells. Similar to LT-resistant cells, knocking down HDAC8 also prevented cell proliferation in U0126-exposed HT-29 cells ( Figure 2 D). These results suggest that the transient inhibition of cell proliferation and development of resistance induced by LT was mainly mediated by MEK1/2 inhibition. 3.3. Activation of AKT Is Mediated by HDAC8 and Required for Cell Proliferation in LT- and U0126-Resistant Cells In the absence of ERK activation, cell cycles can be proceeded by activating the AKT pathway [ 25 , 26 ]. Therefore, we examined whether LT and U0126 induced AKT activation and whether it was mediated by HDAC8. HT-29 and B16-BL6 cells were treated with LT or U0126 for 72 h, and the activation of AKT was examined by Western blots using an antibody specific for phosphorylated AKT at Ser-473 [ 27 ]. We first examined whether the MEK1/2-ERK signaling axis was inhibited in cells treated with LT for 72 h. Western blots using antibodies raised against the N-terminus (the first 12 amino acids) of MEK1 and phospho-specific ERK readily detected MEK1 and activated ERK in control and PCI-treated cells but not in LT- and LT + PCI-treated HT-29 and B16-BL6 cells ( Figure 3 A, upper panel). These data suggest that the MEK1/2-ERK signaling axis was inactivated in cells treated with LT for 72 h. In these cells, AKT was highly phosphorylated, which was prevented by PCI. Similarly, U0126-exposed cells showed a robust activation of AKT but not in PCI-exposed cells ( Figure 3 A, lower panels). Consistent with PCI, knocking down HDAC8 by siRNA also prevented LT- and U0126-induced AKT activation in HT-29 cells ( Figure 3 B). In addition, the inhibition of AKT by the AKT inhibitor (AKTi) prevented cell proliferation in U0126-resistant HT-29 and B16-BL6 cells ( Figure 3 C), suggesting that AKT activation was required for a resistance to cell cycle arrest. Altogether, these results suggest that HDAC8-mediated AKT activation is required for a resistance to LT and MEK1/2 inhibition. 3.4. HDAC8 is Involved in Regulating Expression of PLCB1 and DESC1 in MEK1/2 Inhibition-Resistant Cells To examine the mechanism of HDAC8 in activating AKT, HT-29 cells were treated with LT in the presence or absence of PCI for 48 h, and the expression of over 47,000 transcripts were first examined using the Affymetrix microarray with GeneChip™ Human Genome U133 Plus 2.0 Array, followed by a gene ontology enrichment analysis using the Partek™ Genomics Suite TM . The microarray found ~1500 transcripts changed in expression more than two-fold by the LT or LT + PCI treatments. The top gene ontology enrichment was a cell cycle progress, followed by mitotic cell cycle process ( Supplemental Table S1 ). A total of 141 cell cycle progress protein-coding genes were changed in expression by LT and/or LT + PCI ( Supplemental Table S2 ). We expected that the genes involved in the LT resistance phenotype were likely upregulated in LT-resistant (LT-treated) cells but suppressed in re-sensitized (LT + PCI-treated) cells. Among the 141 genes, nine genes were upregulated in LT-resistant cells. Among the nine induced genes, PLCB1 (phospholipase C β1; also known as phosphatidylinositol-specific phospholipase C), which is involved in AKT activation [ 28 ], was downregulated in re-sensitized cells. We also examined the tumor suppressors that could be involved in the regulation of AKT activation and LT resistance. A total of 24 tumor-suppressor protein-coding genes were changed more than two-fold by LT or LT + PCI ( Supplemental Table S3 ). We expected that the tumor suppressors involved in AKT inhibition were likely downregulated in LT-resistant cells but upregulated in LT re-sensitized cells. Among the 24 tumor suppressors, DESC1 (differentially expressed in squamous cell carcinoma 1), which is involved in the inhibition of AKT [ 29 ], was upregulated in LT re-sensitized cells. Therefore, we decided to further examine the roles of PLCB1 and DESC1 in AKT activation and resistance and re-sensitization to MEK1/2 inhibition. We confirmed that LT and U0126 greatly enhanced the PLCB1 mRNA expression, which was significantly inhibited by PCI through quantitative (q)PCR analysis ( Figure 4 A, left panel). Consistent with the mRNA levels, the Western blot analysis also showed that U0126 alone induced PLCB1 expression, which was inhibited by PCI ( Figure 4 B, left panel). Furthermore, HDAC8-targeting siRNAs also recapitulated the effects of PCI, inhibiting and enhancing the expression of PLCB1 in U0126-exposed cells ( Figure 4 C, left panel). Similarly, LT and U0126, together with PCI, greatly enhanced the DESC1 expression in the mRNA ( Figure 4 A, right panel) and protein ( Figure 4 B, right panel) levels. Of note, U0126 alone slightly induced DESC1 in the protein levels but not in the mRNA levels. The discrepancy could be due to the transient upregulation and/or short half-life of DESC1 mRNAs. Additionally, HDAC8-targeting siRNAs had similar effects as PCI in inducing DESC1 expression in U0126-treated cells ( Figure 4 C, right panel). Like HT-29 cells, B16-BL6 cells also induced the expression of PLCB1 in response to U0126, which was inhibited by PCI, and the expression of DESC1 in response to U0126 + PCI in both the mRNA ( Figure 4 D) and protein ( Figure 4 E) levels. These results suggest that HDAC8 is involved in positively and negatively regulating the expression of PLCB1 and DESC1, respectively, in LT- and U0126-resistant cells. 3.5. Inhibition of PLCB1 Prevents Resistance to LT and MEK1/2 Inhibition in HT-29 Cells To examine the role of PLCB1 in AKT activation and resistance to MEK1/2 inhibition, the effects of the PLCB1 inhibitor edelfosine [ 30 ] and PLCB1-targeting siRNAs were examined in LT- and/or U0126-resistant cells. Edelfosine at a noncytotoxic dose of 1.25 µM ( Figure 5 A) significantly inhibited cell proliferation in U0126-exposed cells ( Figure 5 B). Similarly, siRNA-targeting PLCB1 (si-PLCB1), which reduced the PLCB1 expression by 50% ( Figure 5 C), prevented cell proliferation in LT- and U0126-resistant HT-29 cells ( Figure 5 D,E). In line with these data, si-PLCB1 prevented AKT activation, which was induced by LT and U0126 ( Figure 5 F). These results suggest that PLCB1 is required for AKT activation and recovery from cell cycle arrest in LT- and U0126-exposed HT-29 cells. 3.6. DESC1 Prevents Resistance to LT and MEK1/2 Inhibition in HT-29 Cells To examine the role of DESC1 in resistance to LT and MEK1/2 inhibition, we first examined the effects of the broad-spectrum serine protease inhibitor AEBSF, which inhibits cell membrane-associated proteases, including DESC1 [ 31 ]. AEBSF was able to reverse the PCI effect on the resistance to U0126 ( Figure 6 A), suggesting that the increase of DESC1 expression was involved in preventing the resistance to MEK1/2 inhibition. To further confirm its role in resistance, we ectopically expressed DESC1 and examined the resistance to LT and U0126. As expected, the overexpression of DESC1 mimicked the effect of PCI and inhibited the resistance to LT and U0126 in HT-29 cells ( Figure 6 B). Additionally, the overexpression of DESC1 inhibited AKT activation in U0126-resistant cells ( Figure 6 C). Altogether, these results suggest that the upregulation of DESC1 by PCI in U0126-resistant cells inhibits AKT activation and re-sensitized cells to MEK1/2 inhibition. 3.1. Murine Melanoma B16-BL6 and Human Colorectal HT-29 Cells Develop Resistance to LT in an HDAC8-Dependent Pathway We first examined the cytotoxic effect of LT in four cancer cell lines with known mutations in the signaling axis of RAS-RAF-MEK1/2: murine melanoma B16-BL6, human colorectal cancer HT-29, and human melanoma MDA-MB-435 (MDA) [ 19 ] and SK-MEL-5 (SK-MEL) cell lines [ 6 , 20 , 21 , 22 , 23 ]. LT was able to decrease cell survival within 24 h in these cells ( Figure 1 A). To further examine whether the decreased cell survival was due to cell death or cell cycle arrest, small numbers of these cells were plated and monitored for cell proliferation over 96 h in the presence of LT. During the 48 h of LT treatment, no apparent changes in live cell numbers were detected ( Figure 1 B). In 72 h of LT treatment, the live cell numbers of B16-BL6 and HT-29 cells started increasing, whereas those of MDA and SK-MEL cells did not. These results suggest that B16-BL6 and HT-29 cells became resistant to LT and started proliferating in the presence of LT. Since HDAC8 plays a key role in the resistance to LT cytotoxicity in macrophages [ 14 ], we examined if HDAC8 was also involved in the resistance in B16-BL6 and HT-29 cells. As in macrophages, both B16-BL6 and HT-29 cells failed to proliferate in the presence of LT when the cells were treated together with the HDAC8-specific inhibitor PCI34051 (PCI; Figure 1 B). PCI alone had no apparent effect on the cell proliferation. To further confirm that the proliferating cells in the presence of LT are resistant cells, rather than cells delayed in cell proliferation, these cells were replated and rechallenged with LT and examined for cell survival. Unlike in parental cells, these resistant cells did not show any defects in cell survival/proliferation ( Figure 1 C). To further confirm the role of HDAC8, HDAC8 was knocked down by small interference (si)RNAs in HT-29 cells. As in macrophages [ 14 ], LT induced HDAC8 expression in both mRNA and the protein levels ( Figure 1 D). HDAC8-targeting siRNAs (si-HDAC8), but not random siRNAs (si-Random), significantly decreased the mRNA ( Figure 1 D, left panel) and protein ( Figure 1 D, right panel) levels of HDAC8 in both the parental and LT-resistant cells. Indeed, like PCI, si-HDAC8 prevented cell proliferation in LT-exposed HT-29 cells ( Figure 1 E). 3.2. LT-Induced Cell Cycle Arrest and Resistance Are Manifested by MEK1/2 Inhibition LT inactivates both the MEK1/2-ERK and MEK3/6-p38 signaling pathways by cleaving MEK1-7, except MEK5 [ 24 ]. Therefore, we examined if these two signaling pathways were involved in preventing cell proliferation in LT-treated cells. In both B16-BL6 and HT-29 cells, the MEK1/2 inhibitor U0126, but not the p38 inhibitor SB203580, decreased cell survival to the similar extent observed by LT ( Figure 1 A and Figure 2 A). In both B16-BL6 and HT-29 cells, the U0126 treatment also transiently inhibited cell proliferation for ~48 h, and cells started proliferating after 72 h of the treatment ( Figure 2 B). As in LT-resistant cells, when U0126-resistant cells were rechallenged with U0126, no defects in cell survival/proliferation were detected within 48 h ( Figure 2 C). PCI also prevented a resistance to U0126 in both HT-29 and B16-BL6 cells. Similar to LT-resistant cells, knocking down HDAC8 also prevented cell proliferation in U0126-exposed HT-29 cells ( Figure 2 D). These results suggest that the transient inhibition of cell proliferation and development of resistance induced by LT was mainly mediated by MEK1/2 inhibition. 3.3. Activation of AKT Is Mediated by HDAC8 and Required for Cell Proliferation in LT- and U0126-Resistant Cells In the absence of ERK activation, cell cycles can be proceeded by activating the AKT pathway [ 25 , 26 ]. Therefore, we examined whether LT and U0126 induced AKT activation and whether it was mediated by HDAC8. HT-29 and B16-BL6 cells were treated with LT or U0126 for 72 h, and the activation of AKT was examined by Western blots using an antibody specific for phosphorylated AKT at Ser-473 [ 27 ]. We first examined whether the MEK1/2-ERK signaling axis was inhibited in cells treated with LT for 72 h. Western blots using antibodies raised against the N-terminus (the first 12 amino acids) of MEK1 and phospho-specific ERK readily detected MEK1 and activated ERK in control and PCI-treated cells but not in LT- and LT + PCI-treated HT-29 and B16-BL6 cells ( Figure 3 A, upper panel). These data suggest that the MEK1/2-ERK signaling axis was inactivated in cells treated with LT for 72 h. In these cells, AKT was highly phosphorylated, which was prevented by PCI. Similarly, U0126-exposed cells showed a robust activation of AKT but not in PCI-exposed cells ( Figure 3 A, lower panels). Consistent with PCI, knocking down HDAC8 by siRNA also prevented LT- and U0126-induced AKT activation in HT-29 cells ( Figure 3 B). In addition, the inhibition of AKT by the AKT inhibitor (AKTi) prevented cell proliferation in U0126-resistant HT-29 and B16-BL6 cells ( Figure 3 C), suggesting that AKT activation was required for a resistance to cell cycle arrest. Altogether, these results suggest that HDAC8-mediated AKT activation is required for a resistance to LT and MEK1/2 inhibition. 3.4. HDAC8 is Involved in Regulating Expression of PLCB1 and DESC1 in MEK1/2 Inhibition-Resistant Cells To examine the mechanism of HDAC8 in activating AKT, HT-29 cells were treated with LT in the presence or absence of PCI for 48 h, and the expression of over 47,000 transcripts were first examined using the Affymetrix microarray with GeneChip™ Human Genome U133 Plus 2.0 Array, followed by a gene ontology enrichment analysis using the Partek™ Genomics Suite TM . The microarray found ~1500 transcripts changed in expression more than two-fold by the LT or LT + PCI treatments. The top gene ontology enrichment was a cell cycle progress, followed by mitotic cell cycle process ( Supplemental Table S1 ). A total of 141 cell cycle progress protein-coding genes were changed in expression by LT and/or LT + PCI ( Supplemental Table S2 ). We expected that the genes involved in the LT resistance phenotype were likely upregulated in LT-resistant (LT-treated) cells but suppressed in re-sensitized (LT + PCI-treated) cells. Among the 141 genes, nine genes were upregulated in LT-resistant cells. Among the nine induced genes, PLCB1 (phospholipase C β1; also known as phosphatidylinositol-specific phospholipase C), which is involved in AKT activation [ 28 ], was downregulated in re-sensitized cells. We also examined the tumor suppressors that could be involved in the regulation of AKT activation and LT resistance. A total of 24 tumor-suppressor protein-coding genes were changed more than two-fold by LT or LT + PCI ( Supplemental Table S3 ). We expected that the tumor suppressors involved in AKT inhibition were likely downregulated in LT-resistant cells but upregulated in LT re-sensitized cells. Among the 24 tumor suppressors, DESC1 (differentially expressed in squamous cell carcinoma 1), which is involved in the inhibition of AKT [ 29 ], was upregulated in LT re-sensitized cells. Therefore, we decided to further examine the roles of PLCB1 and DESC1 in AKT activation and resistance and re-sensitization to MEK1/2 inhibition. We confirmed that LT and U0126 greatly enhanced the PLCB1 mRNA expression, which was significantly inhibited by PCI through quantitative (q)PCR analysis ( Figure 4 A, left panel). Consistent with the mRNA levels, the Western blot analysis also showed that U0126 alone induced PLCB1 expression, which was inhibited by PCI ( Figure 4 B, left panel). Furthermore, HDAC8-targeting siRNAs also recapitulated the effects of PCI, inhibiting and enhancing the expression of PLCB1 in U0126-exposed cells ( Figure 4 C, left panel). Similarly, LT and U0126, together with PCI, greatly enhanced the DESC1 expression in the mRNA ( Figure 4 A, right panel) and protein ( Figure 4 B, right panel) levels. Of note, U0126 alone slightly induced DESC1 in the protein levels but not in the mRNA levels. The discrepancy could be due to the transient upregulation and/or short half-life of DESC1 mRNAs. Additionally, HDAC8-targeting siRNAs had similar effects as PCI in inducing DESC1 expression in U0126-treated cells ( Figure 4 C, right panel). Like HT-29 cells, B16-BL6 cells also induced the expression of PLCB1 in response to U0126, which was inhibited by PCI, and the expression of DESC1 in response to U0126 + PCI in both the mRNA ( Figure 4 D) and protein ( Figure 4 E) levels. These results suggest that HDAC8 is involved in positively and negatively regulating the expression of PLCB1 and DESC1, respectively, in LT- and U0126-resistant cells. 3.5. Inhibition of PLCB1 Prevents Resistance to LT and MEK1/2 Inhibition in HT-29 Cells To examine the role of PLCB1 in AKT activation and resistance to MEK1/2 inhibition, the effects of the PLCB1 inhibitor edelfosine [ 30 ] and PLCB1-targeting siRNAs were examined in LT- and/or U0126-resistant cells. Edelfosine at a noncytotoxic dose of 1.25 µM ( Figure 5 A) significantly inhibited cell proliferation in U0126-exposed cells ( Figure 5 B). Similarly, siRNA-targeting PLCB1 (si-PLCB1), which reduced the PLCB1 expression by 50% ( Figure 5 C), prevented cell proliferation in LT- and U0126-resistant HT-29 cells ( Figure 5 D,E). In line with these data, si-PLCB1 prevented AKT activation, which was induced by LT and U0126 ( Figure 5 F). These results suggest that PLCB1 is required for AKT activation and recovery from cell cycle arrest in LT- and U0126-exposed HT-29 cells. 3.6. DESC1 Prevents Resistance to LT and MEK1/2 Inhibition in HT-29 Cells To examine the role of DESC1 in resistance to LT and MEK1/2 inhibition, we first examined the effects of the broad-spectrum serine protease inhibitor AEBSF, which inhibits cell membrane-associated proteases, including DESC1 [ 31 ]. AEBSF was able to reverse the PCI effect on the resistance to U0126 ( Figure 6 A), suggesting that the increase of DESC1 expression was involved in preventing the resistance to MEK1/2 inhibition. To further confirm its role in resistance, we ectopically expressed DESC1 and examined the resistance to LT and U0126. As expected, the overexpression of DESC1 mimicked the effect of PCI and inhibited the resistance to LT and U0126 in HT-29 cells ( Figure 6 B). Additionally, the overexpression of DESC1 inhibited AKT activation in U0126-resistant cells ( Figure 6 C). Altogether, these results suggest that the upregulation of DESC1 by PCI in U0126-resistant cells inhibits AKT activation and re-sensitized cells to MEK1/2 inhibition. 4. Discussion The resistance of tumor cells to RAF/MEK inhibition is a transient and acquired adaptive response of the surviving cells due, in part, to the incomplete inactivation of ERK [ 2 , 3 ]. LT is a potential biological agent that effectively inhibits both MEK1/2-ERK and MEK3/6-p38 MAPK and, thus, is expected to render more pronounced effects on the cell cycle arrest and cell death [ 6 ]. The four cancer cell lines examined, harboring BRAF or NRAS mutations [ 6 , 20 , 21 , 22 , 23 ], were susceptible to LT ( Figure 1 A). Among the susceptible cells, MDA and SK-MEL human melanoma cells failed to develop a resistance to LT and, eventually, were all killed by LT ( Figure 1 B). Since MDA and SK-MEL cells develop a resistance to U0126 (data not shown) and BRAF inhibitors [ 6 , 18 ], but not LT ( Figure 1 B), it is possible that robust MEK1/2-ERK inhibition and/or the inhibition of both MEK1/2-ERK and MEK3/6-p38 signaling axes by LT prevented the development of resistance in these cells. Indeed, the incomplete inhibition of MEK1/2-ERK can lead to resistance due to residual or compensatory ERK activation [ 2 , 3 , 21 ]. These observations provided the theoretic grounds for combinatory therapies using both RAF and MEK inhibitors with positive outcomes. Unlike MDA and SK-MEL cells, murine melanoma B16-BL6 and human colorectal HT-29 cells still escaped from LT-induced MEK1/2-ERK inhibition ( Figure 1 B). In these cells, the MEK1/2 inhibitor U0126, but not the p38 inhibitor SB203580, mimicked LT cytotoxicity and the resistance profiles, suggesting that the inhibition of MEK1/2-ERK signaling is the main culprit of LT-induced cytotoxicity and activation of an alternative cell proliferation pathway(s). Among the various cell survival pathways, the PI3K-AKT signaling axis can induce cell proliferation in the absence of MEK1/2-ERK signaling. In previous studies, MEK1/2-ERK inhibition also induces activation of the PI3K-AKT signaling axis and leads to resistance [ 25 , 32 ]. We also showed that human macrophages develop a resistance to LT-induced cell cycle arrest through activating the PI3K-AKT signal pathway [ 26 ]. The role of PI3K/AKT signaling in cancer cell proliferation and drug resistance has been well-documented [ 33 ]. Similarly, there are various potential mechanisms that AKT protects the cells from cell cycle arrest. We and others showed that inhibition of the glycogen synthase kinase 3β (GSK3b) by AKT (mediated by the S9 phosphorylation of GSK3b) is a key downstream event that protects the cells from cell cycle arrest [ 26 ], enhances cell proliferation [ 34 ], and promotes the resistance to various stresses [ 35 ]. However, GSK3b is a multifaceted enzyme targeting numerous protein substrates involved in both tumor cell growth and suppression [ 36 ]. Therefore, the involvement of GSK3b in the resistance to MEK1/2 inhibition warrants further studies. Although, to date, the mechanism by which MEK1/2-ERK inhibition adaptively induces the PI3K-AKT signaling axis is not fully delineated, epigenetic reprogramming mediated by HDAC8 was shown to be involved in the resistance to MEK-ERK inhibitors [ 14 , 18 ]. In previous studies, we showed that HDAC8 suppresses the expression of the phosphatase-tensin homolog (PTEN; a negative regulator of PI3K) that enhances PI3K-AKT signaling in LT-resistant macrophages [ 14 ]. However, unlike in macrophages [ 14 ], LT and U0126 had no significant effects on PTEN expression in these cells (data not shown). In human melanoma, HDAC8 was also shown to induce a resistance to BRAF inhibition through targeting c-JUN [ 18 ]. In the study, HDAC8 directly deacetylates c-JUN at lysine 273, which enhances the transcriptional activation of receptor tyrosine kinases, such as EGFR, that induce a subsequent basal activation of ERK and AKT. Here, we found that the resistance to LT and U0126 also required HDAC8 in HT-29 and B16-BL6 cells ( Figure 1 , Figure 2 and Figure 3 ). Furthermore, we found that PLCB1 and DESC1 played key roles in the HDAC8-meidated resistance to MEK1/2 inhibition. PLCB1 cleaves phosphatidylinositol 4,5-biphosphate and produces inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These second messengers activate PKCs and intracellular Ca 2+ release in the cytoplasm [ 37 ]. PLCB1 also localizes in the nucleus, where it regulates transcription by releasing the second messengers and directly interacting with various nuclear proteins [ 38 ]. In various cells, the overexpression or activation of PLCB1 renders cell proliferation [ 39 , 40 ] and resistance to oxidative stresses [ 28 , 41 ] through activating PKC, ERK, and AKT and enhancing the expression of cyclin D3 and E. Here, we showed that PLCB1 was required for the resistance to LT and U0126 through, at least in part, by activating AKT in HT-29 cells ( Figure 5 ). Further studies are required to unravel how PLCB1 leads to AKT activation and whether it depends on PKCs and/or Ca 2+ release in the cytoplasm or nucleus. In addition, PLCB1 is activated by G-protein-coupled receptors (GPCRs), whose expression is one of the top-ranked protein classes associated with the resistance to MEK inhibitors in melanoma [ 42 ]. Therefore, it is possible that signaling from GPCRs could confer survival benefits for cells expressing high levels of PLCB1. The high expression of PLCB1 was also shown to be related to the development and poor prognosis of various cancers, including hepatocarcinoma [ 43 , 44 , 45 ], colorectal cancers [ 46 , 47 ], non-small cell lung carcinoma [ 45 ], breast cancer [ 48 ], and acute myeloid leukemia [ 49 ], suggesting its oncogenic role in different cancers. DESC1 is a member of the type II transmembrane serine protease (also known as transmembrane protease, serine 11E; TMPRSS11E) and downregulated in squamous cell carcinoma of the head and neck [ 50 ] and esophageal squamous cell carcinoma [ 51 ]. DESC1 was demonstrated to be a tumor suppressor that cleaves EGFR and inhibits AKT activation that sensitizes cell death in esophageal squamous cells carcinoma [ 29 , 52 ]. Here, we also found that the ectopic overexpression of DESC1 inhibited AKT activation ( Figure 6 C) and prevented the development of a resistance to LT and U0126 ( Figure 6 B). In contrast, the serine protease inhibitor AEBSF prevented the effect of PCI in U0126-treated cells ( Figure 6 A). These results suggest that HDAC8 inhibition also, at least in part, re-sensitized HT-29 cells to MEK1/2-ERK inhibition through inducing DESC1 expression. It is intriguing that HDAC8 renders a resistance by differently regulating the gene expression. Silencing the DESC1 expression by HDAC8 is anticipated, since HDAC8 deacetylates N-terminal tails of core histones and interacts with the corepressors [ 53 , 54 ]. Therefore, the inhibition of HDAC8 could lead to DESC1 transcription through targeting its cis-regulatory elements (promoter and enhancers). In addition, the DESC1 expression was shown to be regulated by the long non-coding RNA tumor-suppressor candidate 7 (TUSC7) that inactivates DESC1-targeting miR-224 [ 52 ], yet suggests an indirect regulation of DESC1 expression through noncoding RNAs. Further studies are needed to delineate the involvement of the cis-regulatory elements and/or TUSC7/miR-224 in regulating the DESC1 expression by HDAC8. Unlike DESCI, HDAC8 inhibition suppressed PLCB1 expression ( Figure 4 ). It is possible that HDAC8 induces PLCB1 through deacetylating/activating c-JUN, as in the BRAF inhibitor-resistant melanoma [ 18 ]. However, the involvement of c-JUN in PLCB1 expression has yet to be established. PLCB1 expression is also controlled by miRs, such as miR-3184 in hepatocellular carcinoma [ 44 ] and miR-423-5p in glioblastoma cells [ 55 ]. Since HDAC8 can suppress the expression of certain miRs [ 56 ], PLCB1 could be induced by HDAC8 through the negative regulation of miRs. Delineating the downstream mechanisms of HDAC8 will reveal more specific targets in controlling resistance and warrants further studies. In summary, MEK1/2 inhibitors or the biological agent LT, which can provide more potent and tumor-specific delivery, inhibit tumor cell growth by inhibiting the perpetually activated MEK1/2-ERK cell proliferation pathway ( Figure 7 , solid box). However, the inhibition of MEK1/2-ERK can lead to AKT activation through HDAC8 ( Figure 7 , dotted box). We found that HDAC8 induced the PLCB1 expression and subsequent AKT activation in low basal DESC1 expression/activity. The inhibition of HDAC8 prevented PLCB1 expression and, at the same time, increased DESC1 expression, both of which were involved in re-sensitizing cells to MEK1/2 inhibition. Therefore, targeting PLCB1 and DESC1 could be potential strategies for inhibiting the resistance to MEK1/2 inhibition in certain cancers with NRAS or BRAF mutations.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3304440/

Antibacterial Activities of Selected Cameroonian Plants and Their Synergistic Effects with Antibiotics against Bacteria Expressing MDR Phenotypes

The present work was designed to assess the antibacterial properties of the methanol extracts of some Cameroonian medicinal plants and the effect of their associations with currently used antibiotics on multidrug resistant (MDR) Gram-negative bacteria overexpressing active efflux pumps. The antibacterial activities of twelve methanol extracts of medicinal plants were evaluated using broth microdilution. The results of this test showed that three extracts Garcinia lucida with the minimal inhibitory concentrations (MIC) varying from 128 to 512 μ g/mL, Garcinia kola (MIC of 256 to 1024 μ g/mL), and Picralima nitida (MIC of 128 to 1024 μ g/mL) were active on all the twenty-nine studied bacteria including MDR phenotypes. The association of phenylalanine arginine β -naphthylamide (PA β N or efflux pumps inhibitor) to different extracts did not modify their activities. At the concentration of MIC/2 and MIC/5, the extracts of P. nitida and G. kola improved the antibacterial activities of some commonly used antibiotics suggesting their synergistic effects with the tested antibiotics. The results of this study suggest that the tested plant extracts and mostly those from P. nitida , G. lucida and G. kola could be used alone or in association with common antibiotics in the fight of bacterial infections involving MDR strains. 1. Introduction Bacterial infections are responsible for 90% of infections found in health care services. The emergence of MDR bacterial strains appears as the major cause of treatment failure [ 1 ]. Among the known mechanisms of resistances, active efflux via resistance-nodulation-cell division (RND) pumps is one of the most occurring system in Gram-negative bacterial strains [ 2 ]. Efflux pumps are transport proteins involved in the extrusion of toxic substrates (including virtually all classes of clinically relevant antibiotics). The present work was therefore designed to investigate the antibacterial potential against MDR bacteria expressing active efflux though RND pumps. Medicinal plants of Cameroon used in this study include the fruits of Citrus medica L. (Rutaceae), the bulbs of Allium sativum L. (Liliaceae) and Allium cepa L. (Liliaceae), the seeds of Carica papaya Linn (Caricaceae), Cola acuminata (P. Beauv.) Schott and Endl. (Sterculiaceae), Buchholzia coriacea Engl. (Capparidaceae), Garcinia kola Heckel (Guttifeare), and Garcinia lucida Vesque (Guttifeare), the seeds and fruits of Picralima nitida ; the potential of the extract from the above plant extracts to increase the activity of some antibiotics on MDR bacteria was also investigated as well as the role of bacterial efflux pumps in the resistance to the tested plant extracts. 2. Material and Methods 2.1. Plant Materials and Extraction The nine edible plants used in this work were purchased from Dschang local market, west region of Cameroon in January 2010. The collected vegetal material were the fruits of Citrus medica , the bulbs of Allium sativum and Allium cepa , the seeds of Carica papaya , Cola acuminata , Buchholzia coriacea , Garcinia kola , and Garcinia lucida , the seeds and fruits of Picralima nitida . The plants were identified by Mr. Tadjouteu Fulbert (Botanist) of the National Herbarium (Yaoundé, Cameroon) where voucher specimens were deposited under a reference number ( Table 1 ). The fresh or powdered air-dried sample (1 kg) from each plant was extracted with methanol (MeOH) for 48 h at room temperature. The extract was then concentrated under reduced pressure to give a residue that constituted the crude extract. They were then kept under 4°C until further use. 2.2. Preliminary Phytochemical Investigations The presence of major secondary metabolite classes, namely, alkaloids, flavonoids, phenols, saponins, tannins, anthocyanins, anthraquinones, sterol, and triterpenes was determined using common phytochemical methods as described by Harborne [ 3 ]. 2.3. Chemicals for Antimicrobial Assays Ciprofloxacin (CIP), chloramphenicol (CHL), streptomycin (STR), tetracycline (TET), norfloxacin (NFX), cloxacillin (CLX), ampicillin (AMP), erythromycin (ERY), kanamycin (KAN), and cefepim (CEF) (Sigma-Aldrich, St Quentin Fallavier, France) were used as reference antibiotics. p -Iodonitrotetrazolium chloride (INT) and phenylalanine arginine β -naphthylamide (PA β N) were used as microbial growth indicator and efflux pumps inhibitor (EPI), respectively. 2.4. Bacterial Strains and Culture Media The studied microorganisms include references (from the American Type Culture Collection) and clinical (Laboratory collection) strains of Escherichia coli, Enterobacter aerogenes, Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumonia, and Enterobacter cloacae ( Table 2 ). They were maintained on agar slant at 4°C and subcultured on a fresh appropriate agar plates 24 hrs prior to any antimicrobial test. Mueller Hinton Agar was used for the activation of bacteria. The Mueller Hinton Broth (MHB) was used for the MIC determinations. 2.5. Bacterial Susceptibility Determinations The respective MICs of samples on the studied bacteria were determined by using rapid INT colorimetric assay [ 4 ]. Briefly, the test samples were first dissolved in DMSO/MHB. The solution obtained was then added to MHB, and serially diluted twofold (in a 96-well microplate). One hundred microlitres (100 μ L) of inoculum (1.5 × 10 6 CFU/mL) prepared in MHB was then added. The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a shaker and incubated at 37°C for 18 hrs. The final concentration of DMSO was lower than 2.5% and does not affect the microbial growth. Wells containing MHB, 100 μ L of inoculums, and DMSO at a final concentration of 2.5% served as a negative control. Ciprofloxacin was used as reference antibiotic. The MICs of samples were detected after 18 hrs of incubation at 37°C, following addition (40 μ L) of 0.2 mg/mL INT and incubation at 37°C for 30 minutes [ 5 ]. Viable bacteria reduced the yellow dye to pink. MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of microbial growth. Samples were tested alone and then, in the presence of PA β N at 30 μ g/mL final concentration. Two of the best extracts, those from seeds of Garcinia kola and Picralima nitida fruits were also tested in association with antibiotics at MIC/2 and MIC/5. These concentrations were selected following a preliminary assay on one of the tested MDR bacteria, P. aeruginosa PA124 (see Supplemental Material S1 available online at doi:10.1155/2012/623723.). All assays were performed in triplicate and repeated thrice. Fractional inhibitory concentration (FIC) was calculated as the ratio of MIC Antibiotic  in  combination /MIC Antibiotic  alone and the interpretation made as follows: synergistic (FIC ≤ 0.5), indifferent (0.5 < FIC < 4), or antagonistic (FIC ≥ 4) [ 6 ]. (The FIC values are available in Supplemental Material S2). 2.1. Plant Materials and Extraction The nine edible plants used in this work were purchased from Dschang local market, west region of Cameroon in January 2010. The collected vegetal material were the fruits of Citrus medica , the bulbs of Allium sativum and Allium cepa , the seeds of Carica papaya , Cola acuminata , Buchholzia coriacea , Garcinia kola , and Garcinia lucida , the seeds and fruits of Picralima nitida . The plants were identified by Mr. Tadjouteu Fulbert (Botanist) of the National Herbarium (Yaoundé, Cameroon) where voucher specimens were deposited under a reference number ( Table 1 ). The fresh or powdered air-dried sample (1 kg) from each plant was extracted with methanol (MeOH) for 48 h at room temperature. The extract was then concentrated under reduced pressure to give a residue that constituted the crude extract. They were then kept under 4°C until further use. 2.2. Preliminary Phytochemical Investigations The presence of major secondary metabolite classes, namely, alkaloids, flavonoids, phenols, saponins, tannins, anthocyanins, anthraquinones, sterol, and triterpenes was determined using common phytochemical methods as described by Harborne [ 3 ]. 2.3. Chemicals for Antimicrobial Assays Ciprofloxacin (CIP), chloramphenicol (CHL), streptomycin (STR), tetracycline (TET), norfloxacin (NFX), cloxacillin (CLX), ampicillin (AMP), erythromycin (ERY), kanamycin (KAN), and cefepim (CEF) (Sigma-Aldrich, St Quentin Fallavier, France) were used as reference antibiotics. p -Iodonitrotetrazolium chloride (INT) and phenylalanine arginine β -naphthylamide (PA β N) were used as microbial growth indicator and efflux pumps inhibitor (EPI), respectively. 2.4. Bacterial Strains and Culture Media The studied microorganisms include references (from the American Type Culture Collection) and clinical (Laboratory collection) strains of Escherichia coli, Enterobacter aerogenes, Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumonia, and Enterobacter cloacae ( Table 2 ). They were maintained on agar slant at 4°C and subcultured on a fresh appropriate agar plates 24 hrs prior to any antimicrobial test. Mueller Hinton Agar was used for the activation of bacteria. The Mueller Hinton Broth (MHB) was used for the MIC determinations. 2.5. Bacterial Susceptibility Determinations The respective MICs of samples on the studied bacteria were determined by using rapid INT colorimetric assay [ 4 ]. Briefly, the test samples were first dissolved in DMSO/MHB. The solution obtained was then added to MHB, and serially diluted twofold (in a 96-well microplate). One hundred microlitres (100 μ L) of inoculum (1.5 × 10 6 CFU/mL) prepared in MHB was then added. The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a shaker and incubated at 37°C for 18 hrs. The final concentration of DMSO was lower than 2.5% and does not affect the microbial growth. Wells containing MHB, 100 μ L of inoculums, and DMSO at a final concentration of 2.5% served as a negative control. Ciprofloxacin was used as reference antibiotic. The MICs of samples were detected after 18 hrs of incubation at 37°C, following addition (40 μ L) of 0.2 mg/mL INT and incubation at 37°C for 30 minutes [ 5 ]. Viable bacteria reduced the yellow dye to pink. MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of microbial growth. Samples were tested alone and then, in the presence of PA β N at 30 μ g/mL final concentration. Two of the best extracts, those from seeds of Garcinia kola and Picralima nitida fruits were also tested in association with antibiotics at MIC/2 and MIC/5. These concentrations were selected following a preliminary assay on one of the tested MDR bacteria, P. aeruginosa PA124 (see Supplemental Material S1 available online at doi:10.1155/2012/623723.). All assays were performed in triplicate and repeated thrice. Fractional inhibitory concentration (FIC) was calculated as the ratio of MIC Antibiotic  in  combination /MIC Antibiotic  alone and the interpretation made as follows: synergistic (FIC ≤ 0.5), indifferent (0.5 < FIC < 4), or antagonistic (FIC ≥ 4) [ 6 ]. (The FIC values are available in Supplemental Material S2). 3. Results 3.1. Phytochemical Composition of the Plant Extracts The results of qualitative analysis showed that each plant contains various phytochemicals compounds such as alkaloids, anthocyanins, anthraquinons, flavonoids, phenols, saponins, tannins, and triterpenes as shown in Table 3 . 3.2. Antibacterial Activity of the Plant Extracts Extracts were tested for their antibacterial activities alone and in combination with PA β N on a panel of Gram-negative bacteria by the microdilution method. Results summarized in Table 4 showed that the most active extracts were those from Garcinia lucida (MIC ranged from 128 to 512 μ g/mL), Garcinia kola (MIC from 128 to 1024 μ g/mL), and the fruits of Picralima nitida (MIC from 256 to 1024 μ g/mL). The antibacterial activities of these plant species were recorded against all the 29 studied microorganisms. Other extracts exhibited weak activities against a limited number of strains studied. 3.3. Role of Efflux Pumps in Susceptibility of Gram-Negative Bacteria to the Tested Plants Extracts The various strains and MDR isolates were also tested for their susceptibility to the plants extracts, and reference antibiotic (ciprofloxacin) in the presence of PA β N, an EPI. Preliminary tests showed that PA β N did not have any antibacterial activity at 30 μ g/mL. The association of the PA β N with the extracts reduced the MIC values of some of the extracts on some tested bacteria ( Table 4 ). However, most of the studied extracts are not the substrates of the active efflux pumps. 3.4. Effects of the Association of Some Plants Extracts with Antibiotics The strain P. aeruginosa PA124 was used to find the appropriate subinhibitory concentration of the antibiotic-crude extract to be tested on other bacteria strains. The association of the extracts of P. nitida and G. kola reduced the MIC of ten antibiotics (CLX, AMP, ERY, KAN, CHL, TET, FEP, STR, CIP, and NOR) at MIC/2 and/or MIC/5 explaining the use of such concentrations. The associations of the extracts of P. nitida fruits and G. kola with antibiotics did not show any case of antagonism (FIC ≥ 4) meanwhile indifference was observed in some cases of the associations of the extracts with FEP, CLX, and AMP (see Tables 5 and 6 , Supplemental Material S2). Many cases of synergy were observed in most of the strains with the associations G. kola /ERY against CM64, P. nitida /NOR against KP63, and P. nitida /ERY against PA124. 3.1. Phytochemical Composition of the Plant Extracts The results of qualitative analysis showed that each plant contains various phytochemicals compounds such as alkaloids, anthocyanins, anthraquinons, flavonoids, phenols, saponins, tannins, and triterpenes as shown in Table 3 . 3.2. Antibacterial Activity of the Plant Extracts Extracts were tested for their antibacterial activities alone and in combination with PA β N on a panel of Gram-negative bacteria by the microdilution method. Results summarized in Table 4 showed that the most active extracts were those from Garcinia lucida (MIC ranged from 128 to 512 μ g/mL), Garcinia kola (MIC from 128 to 1024 μ g/mL), and the fruits of Picralima nitida (MIC from 256 to 1024 μ g/mL). The antibacterial activities of these plant species were recorded against all the 29 studied microorganisms. Other extracts exhibited weak activities against a limited number of strains studied. 3.3. Role of Efflux Pumps in Susceptibility of Gram-Negative Bacteria to the Tested Plants Extracts The various strains and MDR isolates were also tested for their susceptibility to the plants extracts, and reference antibiotic (ciprofloxacin) in the presence of PA β N, an EPI. Preliminary tests showed that PA β N did not have any antibacterial activity at 30 μ g/mL. The association of the PA β N with the extracts reduced the MIC values of some of the extracts on some tested bacteria ( Table 4 ). However, most of the studied extracts are not the substrates of the active efflux pumps. 3.4. Effects of the Association of Some Plants Extracts with Antibiotics The strain P. aeruginosa PA124 was used to find the appropriate subinhibitory concentration of the antibiotic-crude extract to be tested on other bacteria strains. The association of the extracts of P. nitida and G. kola reduced the MIC of ten antibiotics (CLX, AMP, ERY, KAN, CHL, TET, FEP, STR, CIP, and NOR) at MIC/2 and/or MIC/5 explaining the use of such concentrations. The associations of the extracts of P. nitida fruits and G. kola with antibiotics did not show any case of antagonism (FIC ≥ 4) meanwhile indifference was observed in some cases of the associations of the extracts with FEP, CLX, and AMP (see Tables 5 and 6 , Supplemental Material S2). Many cases of synergy were observed in most of the strains with the associations G. kola /ERY against CM64, P. nitida /NOR against KP63, and P. nitida /ERY against PA124. 4. Discussion 4.1. Antibacterial Activities and Chemicals Compositions of the Tested Extracts The phytochemical studies revealed the presence of at least two classes of secondary metabolites in each of the plant extracts. Several alkaloids, flavonoids, phenols, saponins, anthocyanins, anthraquinones, sterols, tannins, and triterpenes have been found active on pathogenic microorganisms [ 44 , 45 ]. Some of these compounds were found to be present in the plant species under this study, and they could contribute to the observed antimicrobial activities of some plant extracts. The results of the phytochemical test on G. kola are in accordance with those obtained by Onayade et al., [ 46 , 47 ]. Many compounds have been isolated from G. kola, such as kolaflavone and 2-hydroxybiflavone [ 48 – 50 ] but their antimicrobials activities have not been evaluated. However, Adegboye et al. [ 51 ] reported the activity of G. kola on some streptomycin-sensitive Gram-positive bacteria strain. The present study therefore provides additional information on the antibacterial potential of this plant on MDR bacteria. The previous phytochemical analyses on hexane extract from the seeds of G. lucida revealed several types of compounds [ 8 , 23 ]. These include terpenoids, anthocyanins, flavonoids, and saponins derivatives. This report therefore agrees well with the phytochemical data being reported herein. The results of the phytochemical analysis of the extract of fruits of P. nitida are similar to those obtained by Kouitcheu [ 52 ]. Several alkaloids previously isolated from this plant include akuammicine, akuammine, akuammidine, picraphylline, picraline, and pseudoakuammigine [ 32 , 53 ]. Their antibacterial activities have not yet been demonstrated but many alkaloids are known to be active on Gram-negative bacteria [ 33 ]. Differences were noted in the chemical composition of the seeds and fruits of P. nitida, evidently explaining the differences in the antibacterial activity of the two parts of this plant. In fact, the presence of tannins in the fruits may contributes to its better activity compared to the seeds as they were reported to inactivate the microbial adhesins, enzymes, transports proteins and cellular envelop [ 54 ]. Extracts from C. papaya , C. medica, B. coriacea , A. cepa , and C. acuminata showed weak activities against a limited number of strains. Nonetheless, the extracts from B. coriacea were rather reported to have good antibacterial activities. Their weak activities as observed in the present paper could therefore be due to the multidrug resistance of the studied bacteria. 4.2. Effects of the Association of Some Plants Extracts with Antibiotics Three of the most active plants extracts ( G. kola, G. lucida, and P. nitida ) were associated with antibiotics with the aim to evaluate the possible synergistic effects of their associations. A preliminary study using P. aeruginosa PA124, one of the ten MDR bacteria used in this paper, was carried out with ten antibiotics (CLX, AMP, ERY, KAN, CHL, TET, FEP, STR, CIP, and NOR) to select the appropriate sub-inhibitory concentrations of the extract to be used. The results (see Supplemental Material S1) allowed the selection of G. kola, G. lucida and their MIC/2 and MIC/5 as the sub-inhibitory concentrations. No antagonistic effect (FIC ≥ 4) was observed between extracts and antibiotics meanwhile indifference was observed in the case of CLX, FEP, AMP, which are β -lactams acting on the synthesis of the bacteria cell wall [ 55 ] (Tables 5 and 6 , Supplemental Material S2). Many studies demonstrated that efflux is the mechanism of resistance of bacteria for almost all antibiotic classes [ 56 ]. It is well demonstrated that the efflux pumps reduce the intracellular concentration of antibiotics and consequently their activities [ 57 ]. The MDR bacteria strains used in this paper are known for their ability to overexpress active efflux [ 58 ]. At MIC/2, synergistic effects were noted with the association of NOR, CHL, TET (on 100% the studied bacteria), ERY (on 80%), CIP (on 70%), and P. nitida extract meanwhile G. kola extract also increased the activity of NOR, TET (on 100%), ERY, and CIP (on 70%). Plant can be considered as an efflux pumps inhibitor if a synergistic effect with antibiotics is induced on more than 70% bacteria expressing active efflux pumps [ 6 ]. Therefore, the extracts from P. nitida and G. kola probably contain compounds that can acts as EPI. The results of the present paper corroborate with those of Iwu et al. [ 7 ] reporting the existence of synergy effects between G. kola extract and gatifloxacin ( G. kola /gatifloxacin in the proportions of 9/1, 8/2, 7/3, and 6/4) against Bacillus subtilis and the proportions of G. kola /gatifloxacin (at 9/1, 2/8, and 1/9) against Staphylococcus aureus. The overall results of the present work provide baseline information for the possible use of the studied plants and mostly G. Lucida, G. Kola, and P. Nitida extracts in the treatment of bacterial infections involving MDR phenotypes. In addition, the extracts of these plants could be used in association with common antibiotics to combat multidrug resistant pathogens. 4.1. Antibacterial Activities and Chemicals Compositions of the Tested Extracts The phytochemical studies revealed the presence of at least two classes of secondary metabolites in each of the plant extracts. Several alkaloids, flavonoids, phenols, saponins, anthocyanins, anthraquinones, sterols, tannins, and triterpenes have been found active on pathogenic microorganisms [ 44 , 45 ]. Some of these compounds were found to be present in the plant species under this study, and they could contribute to the observed antimicrobial activities of some plant extracts. The results of the phytochemical test on G. kola are in accordance with those obtained by Onayade et al., [ 46 , 47 ]. Many compounds have been isolated from G. kola, such as kolaflavone and 2-hydroxybiflavone [ 48 – 50 ] but their antimicrobials activities have not been evaluated. However, Adegboye et al. [ 51 ] reported the activity of G. kola on some streptomycin-sensitive Gram-positive bacteria strain. The present study therefore provides additional information on the antibacterial potential of this plant on MDR bacteria. The previous phytochemical analyses on hexane extract from the seeds of G. lucida revealed several types of compounds [ 8 , 23 ]. These include terpenoids, anthocyanins, flavonoids, and saponins derivatives. This report therefore agrees well with the phytochemical data being reported herein. The results of the phytochemical analysis of the extract of fruits of P. nitida are similar to those obtained by Kouitcheu [ 52 ]. Several alkaloids previously isolated from this plant include akuammicine, akuammine, akuammidine, picraphylline, picraline, and pseudoakuammigine [ 32 , 53 ]. Their antibacterial activities have not yet been demonstrated but many alkaloids are known to be active on Gram-negative bacteria [ 33 ]. Differences were noted in the chemical composition of the seeds and fruits of P. nitida, evidently explaining the differences in the antibacterial activity of the two parts of this plant. In fact, the presence of tannins in the fruits may contributes to its better activity compared to the seeds as they were reported to inactivate the microbial adhesins, enzymes, transports proteins and cellular envelop [ 54 ]. Extracts from C. papaya , C. medica, B. coriacea , A. cepa , and C. acuminata showed weak activities against a limited number of strains. Nonetheless, the extracts from B. coriacea were rather reported to have good antibacterial activities. Their weak activities as observed in the present paper could therefore be due to the multidrug resistance of the studied bacteria. 4.2. Effects of the Association of Some Plants Extracts with Antibiotics Three of the most active plants extracts ( G. kola, G. lucida, and P. nitida ) were associated with antibiotics with the aim to evaluate the possible synergistic effects of their associations. A preliminary study using P. aeruginosa PA124, one of the ten MDR bacteria used in this paper, was carried out with ten antibiotics (CLX, AMP, ERY, KAN, CHL, TET, FEP, STR, CIP, and NOR) to select the appropriate sub-inhibitory concentrations of the extract to be used. The results (see Supplemental Material S1) allowed the selection of G. kola, G. lucida and their MIC/2 and MIC/5 as the sub-inhibitory concentrations. No antagonistic effect (FIC ≥ 4) was observed between extracts and antibiotics meanwhile indifference was observed in the case of CLX, FEP, AMP, which are β -lactams acting on the synthesis of the bacteria cell wall [ 55 ] (Tables 5 and 6 , Supplemental Material S2). Many studies demonstrated that efflux is the mechanism of resistance of bacteria for almost all antibiotic classes [ 56 ]. It is well demonstrated that the efflux pumps reduce the intracellular concentration of antibiotics and consequently their activities [ 57 ]. The MDR bacteria strains used in this paper are known for their ability to overexpress active efflux [ 58 ]. At MIC/2, synergistic effects were noted with the association of NOR, CHL, TET (on 100% the studied bacteria), ERY (on 80%), CIP (on 70%), and P. nitida extract meanwhile G. kola extract also increased the activity of NOR, TET (on 100%), ERY, and CIP (on 70%). Plant can be considered as an efflux pumps inhibitor if a synergistic effect with antibiotics is induced on more than 70% bacteria expressing active efflux pumps [ 6 ]. Therefore, the extracts from P. nitida and G. kola probably contain compounds that can acts as EPI. The results of the present paper corroborate with those of Iwu et al. [ 7 ] reporting the existence of synergy effects between G. kola extract and gatifloxacin ( G. kola /gatifloxacin in the proportions of 9/1, 8/2, 7/3, and 6/4) against Bacillus subtilis and the proportions of G. kola /gatifloxacin (at 9/1, 2/8, and 1/9) against Staphylococcus aureus. The overall results of the present work provide baseline information for the possible use of the studied plants and mostly G. Lucida, G. Kola, and P. Nitida extracts in the treatment of bacterial infections involving MDR phenotypes. In addition, the extracts of these plants could be used in association with common antibiotics to combat multidrug resistant pathogens.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145597/

Rainfall trends and variation in the Maasai Mara ecosystem and their implications for animal population and biodiversity dynamics

Rainfall exerts a controlling influence on the availability and quality of vegetation and surface water for herbivores in African terrestrial ecosystems. We analyse temporal trends and variation in rainfall in the Maasai Mara ecosystem of East Africa and infer their implications for animal population and biodiversity dynamics. The data originated from 15 rain gauges in the Mara region (1965–2015) and one station in Narok Town (1913–2015), in Kenya's Narok County. This is the first comprehensive and most detailed analysis of changes in rainfall in the region of its kind. Our results do not support the current predictions of the International Panel of Climate Change (IPCC) of very likely increases of rainfall over parts of Eastern Africa. The dry season rainfall component increased during 1935–2015 but annual rainfall decreased during 1962–2015 in Narok Town. Monthly rainfall was more stable and higher in the Mara than in Narok Town, likely because the Mara lies closer to the high-precipitation areas along the shores of Lake Victoria. Predominantly deterministic and persistent inter-annual cycles and extremely stable seasonal rainfall oscillations characterize rainfall in the Mara and Narok regions. The frequency of severe droughts increased and floods intensified in the Mara but droughts became less frequent and less severe in Narok Town. The timings of extreme droughts and floods coincided with significant periodicity in rainfall oscillations, implicating strong influences of global atmospheric and oceanic circulation patterns on regional rainfall variability. These changing rainfall patterns have implications for animal population dynamics. The increase in dry season rainfall during 1935–2015 possibly counterbalanced the impacts of resource scarcity generated by the declining annual rainfall during 1965–2015 in Narok Town. However, the increasing rainfall extremes in the Mara can be expected to create conditions conducive to outbreaks of infectious animal diseases and reduced vegetation quality for herbivores, particularly when droughts and floods persist over multiple years. The more extreme wet season rainfall may also alter herbivore space use, including migration patterns. Introduction A better understanding of rainfall dynamics is indispensable for developing biodiversity conservation measures likely to be effective under climate change [ 1 ]. Such understanding requires carefully verified observational data to ensure accuracy and reliability. This is especially pertinent for Africa where high-quality observational rainfall datasets with sufficiently high spatial and temporal resolutions are rare [ 2 ] and noteworthy discrepancies often exist between digital datasets and original weather records [ 3 ] even for the same weather stations [ 4 ]. Here, we use carefully verified station rainfall data for the Maasai Mara ecosystem to answer the following questions. (1) Are there temporal trends in the monthly, annual and seasonal rainfall components? (2) Are there shifts in rainfall seasonality? (3) What are the dominant cycle periods of oscillations in the rainfall components and are the periods changing? (4) Are severe droughts and floods becoming more frequent and severe and do they persist over multiple years? (5) How might the changing rainfall patterns affect animal population and biodiversity dynamics based on known responses of animal abundance, reproduction, survival, disease susceptibility and migration to rainfall? Rainfall is the principal driver of the population dynamics of savanna herbivores [ 5 , 6 ] because it controls plant biomass production [ 7 , 8 ] and plant nutrient concentration [ 9 ], which affect herbivore birth [ 6 ] and survival [ 10 ] rates, susceptibility to predation [ 11 ] and, ultimately, biomass [ 12 , 13 ]. Not surprisingly, oscillatory dynamics in ungulate population size [ 5 ] and ungulate fecundity [ 14 ] are coupled with inter-annual and seasonal rainfall oscillations in African savannas, respectively. Droughts can cause substantial herbivore mortality and often regulate population size [ 15 ]. For example, 75% of wildebeest ( Connochaetes taurinus mearnsi (Burchell)) deaths in Serengeti were caused by undernutrition and rainfall was the most important factor determining food supply [ 16 ]. Concentrations of herbivores around water points during droughts [ 17 ] can elevate vegetation damage [ 18 ], and result in increased competition and predation [ 19 ]. Droughts and floods also facilitate infestations by parasites [ 20 ] and diseases such as the canine distemper virus outbreak among Serengeti lions following a severe drought in 1993 [ 21 ]. Excessive rainfall can adversely affect small herbivores that require high-quality forage through diluting plant nutrient concentration [ 22 ]. High rainfall also promotes fires because it increases fuel loads in grasslands [ 23 ]. But low rainfall can lead to more destructive and extensive fires in savanna woodlands and forests [ 24 ]. Rainfall distribution and seasonality principally drives animal migration [ 25 ] and dispersal [ 26 ] in savannas. Consequently, animals may alter both their migratory [ 25 ] and short-term movements rapidly in response to localised rainfall patterns. During low rainfall years, animals are forced to travel longer distances between water and foraging grounds, making their offspring more vulnerable to predation [ 27 ]. Temperatures have risen in recent decades in most parts of the eastern African region [ 2 , 28 – 30 ] but the contemporaneous changes in rainfall seem subtle and largely unpredictable [ 2 ]. Rainfall increased in parts of East Africa during 1951–2001 [ 31 ] and during 1979–2010 [ 2 ]. Thus, rainfall trends around Lake Victoria were predominantly positive over the 20 th century [ 32 ]. Likewise, General Circulation Models project increasing rainfall [ 33 – 36 ], more intense wet seasons and less severe droughts for most of East Africa [ 37 ]. Climate models also project strengthening of the El Niño Southern Oscillation (ENSO) and more frequent occurrences of the positive phase of the Indian Ocean Dipole [ 38 , 39 ] as temperatures rise, although evidence for the strengthening of the ENSO phenomenon remains controversial [ 40 , 41 ]. Such conditions facilitate moisture export from the Indian Ocean towards East Africa by weakening westerly winds [ 42 , 43 ] and lead to more intense wet seasons and floods. The above patterns are in contrast to the findings of other studies of temporal trends in East African rainfall, such as decreasing annual [ 30 , 33 ], wet season [ 44 – 46 ] and dry season rainfall [ 36 ] in recent decades. Concurrently, droughts became more severe during 1970–2006 in Eastern and Southern Africa [ 44 ]. La Niña events, which often follow extreme El Niño events [ 47 ], typically lead to severe drying in East Africa [ 48 ]. Rainfall also declines during negative phases of the Indian Ocean Dipole [ 49 ]. For example, the 2005–2006 East African drought was associated with both a strong negative Indian Ocean Dipole and La Niña-like conditions [ 50 , 51 ]. Contrasting rainfall trends have also been documented for the Mara-Serengeti ecosystem. Ritchie et al. [ 52 ] reported a decrease in the total annual and wet season rainfall during 1960–2001 but an increase in the dry season rainfall in the Serengeti during 1913–2001. In contrast, Ogutu et al. [ 53 ] reported a decline in the dry season rainfall in the Maasai Mara Reserve (Mara Reserve) during 1975–2003. However, dry season rainfall in Narok, a Kenyan town located 75 km north-east of the Mara Reserve, increased during 1940–2004 after a protracted drought during 1930–1939 [ 53 ]. These contrasting findings demonstrate considerable uncertainty inherent in trends and variation in the past and anticipated future rainfall scenarios [ 35 ]. Climate warming can change rainfall seasonality and cycle periods by modulating ocean-atmosphere circulations. Climate warming alters ocean temperatures, cloud and ice cover, leading to shifts in the movement of the Inter-Tropical Convergence Zone [ 54 ]. This belt of rising and convecting air masses driven by solar radiation is the causal agent for rainfall seasonality as it moves southwards from East Africa during the transition between the dry and the wet season (July-January) and northwards during the transition between the wet and the dry season (January-July) [ 55 ]. The dominant East African rainfall cycle periods ranging between about 2 to 12.5 years appear very variable in space and time [ 56 – 58 ]. Besides the ENSO phenomenon and the Indian Ocean Dipole, oscillations in Atlantic ocean temperatures can influence East African rainfall considerably [ 59 ] through its teleconnections (an influence occurring over large distances, typically thousands of kilometres) via the Indian Ocean [ 60 ] and the West African monsoon [ 61 ]. The highly variable local topography of the African Rift Valley may also contribute to temporal and spatial variability in rainfall [ 62 ]. The aim of this study was to quantify trends and variation in rainfall in the Maasai Mara ecosystem in East Africa as a background for understanding their past and possible future implications for animal population and biodiversity dynamics. Materials and methods Study area The Maasai Mara ecosystem is situated along the international border between Kenya and Tanzania in equatorial East Africa (34.7° to 35.4° E, 1.2° to 1.7° S, Fig 1 ). The elevation in the Mara Reserve ranges from about 1,450 m to about 2,100 m above sea level ( Fig 1 ). The Mara Reserve was established in 1961 and the adjoining Serengeti National Park in 1951 to protect the rainfall-driven migration of the numerous wildebeest, zebra ( Equus quagga burchellii ) and Thomson's gazelle ( Gazella thomsonii ) [ 63 ]. Multiple buffer zones with various degrees of protection and land use types now surround the protected areas [ 9 ] ( Fig 1 ). The Maasai Mara ecosystem of Kenya consists mostly of grasslands with the cover of shrubs and thorny bushes increasing towards its northern and eastern extremes [ 64 ]. The dominant vegetation type in the Serengeti is savanna with a mixture of grasses, shrubs and trees [ 63 ]. Riverine forests fringe various streams and drainage lines in the area [ 63 ] ( Fig 1 ) but much of this forest has been lost. 10.1371/journal.pone.0202814.g001 Fig 1 The Mara-Serengeti ecosystem (orange borders) straddling the international border (black line) between Tanzania and Kenya. The entire Mara-Serengeti ecosystem supports an extremely diverse and abundant community of herbivores [ 65 ] and carnivores [ 66 ]. Populations of many wildlife species are declining, concurrent with changes in climate, growing pressures from cultivation, livestock grazing and other anthropocentric influences that are driven by accelerating human population growth [ 30 , 63 , 67 ]. The major land use type in the Maasai Mara ecosystem is pastoralism [ 30 ]. Higher rainfall areas in the buffer zone more distant from the Mara Reserve are now largely converted into agriculture [ 64 ]. Settlements, extensive agriculture and sedentary livestock holdings characterize the western side of the Serengeti National Park today [ 63 ]. The climatic year in the Mara starts in November and ends in October of the following year. The seasonal rainfall distribution is strongly bimodal, with the wet season spanning November-June and the dry season spanning July-October. The wet season consists of the short (November-December) and the long (January-June) rains. January-February trends to be dry and hence is sometimes called the short dry season in contrast to the long dry season from July-October [ 68 ]. The annual rainfall in the Mara reserve follows a spatial continuum from about 650 mm in the southeast to about 1300 mm in the northwest [ 68 ]. The dry season rainfall in particular is higher and more stable close to Lake Victoria ([ 55 ], Fig 1 ), the largest lake in Africa. Climate models suggest that the difference in air temperature between the land and the lake water creates a local convergence zone [ 69 ] that interferes with large-scale atmospheric and oceanic circulation patterns [ 70 ] and brings rain to the north-western extensions of the Mara-Serengeti ecosystem [ 55 ]. Consequently, rainfall decreases away from Lake Victoria, reaching about 750 mm in Narok Town in Kenya and 350–450 mm on the south-eastern Serengeti plains in Tanzania [ 63 ]. During the dry season, the seasonal watercourses dry out and the remaining isolated pools, ponds, springs and the Mara River, the only permanent river in the Mara-Serengeti ecosystem, serve as the only sources of water for wildlife and livestock across the area [ 71 ]. As water levels drop, the water quality of these remaining sources becomes very poor, in part because wildlife concentrate near water as the dry season progresses [ 71 ]. Forage quality and quantity progressively decline with time after the wet season [ 72 ]. Consequently, wildebeest migrate back from the south-eastern Serengeti Plains to the Mara in Kenya where the dry season rainfall is higher [ 55 ]. Poor rainfall can force wildebeest to leave these nutrient-rich grass plains in Serengeti earlier than usual but increased rainfall can cause them to return earlier [ 25 ]. Direct correlations between rainfall and ENSO appear weak in the Mara-Serengeti ecosystem [ 53 , 73 ] but major El Niño events can cause substantial mortality of herbivores [ 74 ]. Similarly, La Niña events [ 50 , 51 ] are sometimes followed by marked reductions in herbivore biomass [ 75 ]. The dominant rainfall cycles in the Mara-Serengeti ecosystem have periods of about 5 to 10 years but cycle lengths can vary widely [ 76 ]. In addition to rainfall variability, water flow in the Mara River has been declining as a consequence of upstream deforestation of the Mau forest and excessive water abstraction for irrigation in Kenya [ 77 ]. Data sources and processing We obtained total monthly rainfall data for 15 gauges in the Mara spanning 1965–2014 ( S1 Data ) and for one gauge for Narok Town spanning 1913–2015 ( S2 Data ) from the sources listed in Table 1 . Thirteen of the 15 gauges in the Mara were operated by the World Wide Fund for Nature (WWF) and Friends of Conservation (FOC) as part of the Maasai Mara Ecological Monitoring Programme from 1989 to 2003. The records for 5 of the 15 gauges were taken daily and then summed to obtain monthly totals. Rainfall for the other gauges was measured at monthly intervals. 10.1371/journal.pone.0202814.t001 Table 1 The sources of rainfall records for 15 rain gauges in the Mara and a rain gauge in Narok Town in Kenya. Station Coordinates a Elevation (m) Period (year-month) Frequency % Missing monthly % Missing daily Eastings Northings Start End Narok Town b 819064 9878264 1,869 1913–04 2015–12 Daily 2) and damping factors ( ρ ) of the stochastic cycle components ( φ t ) with a time-varying amplitude and phase given by [ φ t φ t * ] = ρ [ c o s λ s i n λ − s i n λ c o s λ ] [ φ t − 1 φ t − 1 * ] + [ Ï t Ï t * ] , Ï t , Ï t * ∼ i . i . d . ( 0 , σ Ï 2 ) , (6) where 0 2) and damping factors ( ρ ) of the stochastic cycle components ( φ t ) with a time-varying amplitude and phase given by [ φ t φ t * ] = ρ [ c o s λ s i n λ − s i n λ c o s λ ] [ φ t − 1 φ t − 1 * ] + [ Ï t Ï t * ] , Ï t , Ï t * ∼ i . i . d . ( 0 , σ Ï 2 ) , (6) where 0 2) and damping factors ( ρ ) of the stochastic cycle components ( φ t ) with a time-varying amplitude and phase given by [ φ t φ t * ] = ρ [ c o s λ s i n λ − s i n λ c o s λ ] [ φ t − 1 φ t − 1 * ] + [ Ï t Ï t * ] , Ï t , Ï t * ∼ i . i . d . ( 0 , σ Ï 2 ) , (6) where 0  |t| ΔAICc c Effects Estimate Std. Error t Value Pr. > |t| Annual rainfall 0.05 0.0 Intercept 744 47 15.9  |t| ΔAICc c Effects Estimate Std. Error t Value Pr. > |t| Annual rainfall 0.05 0.0 Intercept 744 47 15.9 <0.0001 4.2 Intercept 425 28 15.1 <0.0001 Year 440 251 1.6 0.083 0.10 0.0 Intercept 752 34 22.3 <0.0001 8.7 Intercept 454 22 21.0 <0.0001 Year 526 243 2.2 0.0326 0.50 0.0 Intercept 1008 31 32.6 <0.0001 0.0 Intercept 698 30 22.3 <0.0001 0.90 10.8 Intercept 1162 40 29.0 <0.0001 0.0 Intercept 1071 61 17.6 <0.0001 Year 452 239 1.9 0.0648 0.95 4.4 Intercept 1251 61 20.5 <0.0001 0.0 Intercept 1115 55 20.3 <0.0001 Year 249 323 0.8 0.4448 Wet season rainfall 0.05 0.0 Intercept 546 29 19.0 <0.0001 4.8 Intercept 324 20 16.2 <0.0001 Year 296 170 1.7 0.0856 0.10 0.0 Intercept 741 32 17.6 <0.0001 0.0 Intercept 362 19 19.2 <0.0001 0.50 0.0 Intercept 742 39 19.1 <0.0001 0.0 Intercept 578 27 21.1 <0.0001 0.90 10.5 Intercept 941 32 29.9 <0.0001 0.0 Intercept 952 65 14.7 <0.0001 Year 306 222 1.4 0.1742 0.95 17.9 Intercept 991 41 24.1 <0.0001 0.0 Intercept 1021 43 23.7 <0.0001 Year 556 268 2.1 0.0413 Dry season rainfall 0.05 5.9 Intercept 110 12 9.4 <0.0001 0.0 Intercept 54 6 8.9 <0.0001 Year 141 86 1.6 0.108 0.10 0.0 Intercept 127 11 11.2 <0.0001 0.0 Intercept 60 4 14.2 <0.0001 0.50 0.0 Intercept 191 10 18.9 <0.0001 3.3 Intercept 109 6 17.9 <0.0001 Year 115 70 1.6 0.1048 0.90 0.0 Intercept 322 32 10.1 <0.0001 0.0 Intercept 193 14 13.9 <0.0001 0.95 0.0 Intercept 366 24 15.2 <0.0001 0.0 Intercept 221 27 8.1 <0.0001 a Rainfall recordings were derived from 15 gauges (Eq 3 in S1 Text ) during 1966–2014 (dry season: 1965–2014). b Rainfall was recorded during 1914–2015 (dry season: 1913–2015). The monthly rainfall records were summed to yield the annual and seasonal rainfall components. c ΔAICc are the deviations in AICc values of each model from that for the null (intercept-only) model for each quantile. Significant trends in rainfall quantiles are marked in bold-faced font. Extreme wet season floods (0.95 quantile) increased significantly in the Mara during 1966–2014 ( Table 5 , Fig 6C ). The increase in severe annual floods (0.90 quantile) was close to significance in the Mara ( Table 5 , Fig 6A ). The increase in the extreme (0.95 quantile) annual rainfall component ( Fig 6A ) and increase in the 0.90 quantile of the wet season rainfall component ( Fig 6C ) in the Mara were not significant ( Table 5 ). The median (0.50 quantile) dry season rainfall component increased substantially though non-significantly from about 90 mm in 1913 to about 125 mm in 2015 in Narok Town ( Table 5 , Fig 6F ). Otherwise, we found no evidence for significant trend over time in the median annual or seasonal rainfall components for the Mara or Narok Town ( Table 5 , Fig 6 ). Discussion We analysed temporal trends and variation in rainfall in the Maasai Mara ecosystem in Kenya as a background for understanding animal population and biodiversity dynamics in African terrestrial ecosystems. In contrast to IPCC's predictions [ 2 ] and projections of the General Circulation Models [ 33 – 35 ], which have difficulties capturing small-scale orographic rainfall variation [ 96 ], we found only minor empirical support for increases in rainfall over two areas in Eastern Africa. Our results show that only the dry season rainfall component for Narok Town increased during 1940–2015 based on UCM models. Likewise, the dry season rainfall increased during 1913–2001 in the Serengeti [ 52 ]. But our finding of decreasing annual rainfall during 1962–2015 in Narok Town reinforces results of earlier studies [ 36 , 44 – 46 ] that attributed decreasing East African rainfall to increasing temperatures of the northern hemisphere [ 36 ] and of the central Indian or west Pacific Oceans [ 44 – 46 ]. Similarly, Ritchie et al. [ 52 ] documented evidence of decreasing annual and wet season rainfall during 1960–2001 in the Serengeti. Rainfall also declined during 1960–2014 in 14 Kenyan counties but in another 6 Kenyan counties it declined initially and then switched to an upward trend according to Ogutu et al. [ 30 ], demonstrating spatial distinctions in rainfall trends. There was, however, no systematic change in the annual and seasonal rainfall components during 1965–2014 in the Mara according to our UCM analysis. The apparent discrepancies in the contrasting findings may partly reflect the strong spatial variation evident in East-African rainfall [ 55 , 97 ] and calls for considerable caution in applying results of climate modelling at large spatial scales to particular localities. Our result demonstrating virtually constant rainfall seasonality in the Mara or Narok Town suggests that regular movements of the Inter-Tropical Convergence Zone provide an extremely stable modulation of the rainfall seasons. The higher and less variable rainfall in the Mara than in Narok Town, in particular the higher and less variable dry season rainfall, is likely due to the closer proximity of the Mara to the high-precipitation areas near the eastern shores of Lake Victoria [ 55 ] and the influence of the Lake Victoria System. The modulation of Mara's rainfall by this system and the resulting higher dry season rainfall likely sustains the high abundance and diversity of wildlife and livestock there. The seasonal variation in rainfall both in the Mara and Narok Town agree well with the monthly discharge patterns for the Mara River, with peaks in December and April-May [ 98 ], implying that rainfall governs much of the Mara River discharge. The compensatory pattern we detected for the amounts of the wet and dry season rainfall, where periods with below-average dry season rainfall frequently received above-average wet season rainfall and vice versa, may possibly emerge from the interactions between the local Lake Victoria circulation and hemispheric level climate drivers. This compensatory dampening of rainfall seasonality has likely generated the stability and resilience of the Mara and allowed the high abundance and diversity of wildlife it supports. The positive Indian Ocean Dipole Mode and the El Niño–Southern Oscillation can increase East African rainfall primarily during the wet season by weakening westerly winds and enabling moisture transport from the Indian Ocean towards the land [ 42 , 43 , 99 ]. But weakened westerly winds may, in turn, reduce the dry season rainfall that typically originates from Lake Victoria and precipitates east of the lake in the Mara [ 55 ]. Correspondingly, La Niña-like conditions and negative Indian Ocean Dipole phases may enhance westerly winds from Lake Victoria that lead to above-average dry season rainfall but concurrently block moisture export from the Indian Ocean towards East Africa resulting in less wet season rainfall. The predominantly persistent rainfall cycle periods we estimated of between 2.1 and 3 years for the Mara and 2.3 to 5.2 years for Narok Town agree with the dominant cycle periods reported for East Africa ranging from about 2 to 11 years [ 56 – 58 , 76 ]. They are also within the range of the periods characteristic of the Indian Ocean Dipole (about 2 years), the El Niño-Southern Oscillation (3–6 years; [ 100 ]) and the quasi-biennial oscillations in the lower equatorial stratospheric zonal winds [ 101 ]. By means of wavelet analysis we were able to establish that periodicity in the monthly rainfall oscillations is not constant but varies over time and space in the Mara-Serengeti ecosystem. The cycle periods varied from just 0.75 to 1.5 years for several distinct episodes during 1970–1984 for the Mara to periods ranging from 4.5 to 8 years during 1951–1971 for Narok Town. Likewise, the amplitude and frequency of the Indian Ocean Dipole and the El Niño-Southern Oscillation can be highly variable [ 100 , 102 ]. In general, the wavelet analysis showed that extreme droughts and floods coincided with the times when the regional rainfall oscillations had statistically significant cycle periods. These results strongly suggest a possible association between oscillations in hemispheric atmospheric and oceanic circulations and regional rainfall oscillations in the Mara-Serengeti ecosystem that are similarly evident in East African rainfall [ 103 – 105 ]. For example, the first half of the wet season of 2006 was associated with both a strong negative Indian Ocean Dipole and La Niña-like conditions [ 50 , 51 ] with consequent severe drying in East Africa [ 48 , 75 , 82 ]. The flood of 1962 coincided with strong easterly winds [ 106 ] and an extreme dipole reversal of the Indian Ocean sea surface temperatures [ 42 ]. But there was no simultaneous El Niño effect during that time [ 42 ]. Similarly, the flood of 1998 was associated with the second strongest El Niño event on instrumental record during 1950–2016 [ 50 ], and coincided with a strong and positive Indian Ocean Dipole [ 42 , 50 , 51 ]. These circulations may also explain the simultaneous occurrence of overlapping rainfall cycles that were apparent both in UCM models and wavelet analysis. But the extreme dry season droughts of 1969 and 1993, and the extreme annual and wet season drought of 1982 in the Mara were not associated with any significant periodicity in rainfall oscillation. There was a moderate El Niño event during 1968–1969 and 1993, and the longest recorded El Niño episode during 1990–1995 [ 50 ]. The interactions between the El Niño events and Lake Victoria circulation patterns that probably underlie the compensatory pattern of the wet and dry season rainfall could also have contributed to the dry season droughts of 1969 and 1993. Our results indicating increasing frequency and severity of floods in the Mara during 1965–2014 and reduction in drought frequency and severity in Narok Town during 1913–2015 agree with the projections of General Circulation Models for most of East Africa of more intense wet seasons [ 107 ] and less severe droughts [ 74 ]. Precipitation extremes are expected as temperatures rise [ 107 ]. Such patterns may emerge from the projected strengthening of the El Niño Southern Oscillation [ 108 , 109 ] and more frequent occurrences of the positive phases of the Indian Ocean Dipole [ 38 , 39 ]. In contrast to Narok, severe droughts are apparently becoming more frequent in the Mara. Although some of the inconsistencies in our results might have arisen from using only one gauge in Narok Town versus 15 in the Mara, they show that the trends in extreme rainfall events can vary both locally and over time. Simulation models indicate that, although droughts may increase with increasing temperatures on a global scale, the changes expected for East Africa appear uncertain and comparatively small [ 47 ]. One possible cause of the recent severe droughts in the Mara could be due to the increasing frequency of extreme La Niña events that often follow extreme El Niño events [ 47 ]. These hemispheric circulation patterns may also underlie the multiannual persistence of severe droughts and floods that we detected in the annual and seasonal rainfall components in the Mara and in Narok Town. Implications for animal population and biodiversity dynamics The decreasing annual rainfall in Narok Town may have negative long-term effects on the reproductive performance of herbivores, including livestock, in that area through limitation of food and surface water availability [ 5 , 6 , 10 , 110 ]. Wildlife and livestock populations are also likely to suffer more from water and food scarcity in the Mara despite the stable rainfall levels during 1965–2014 due to the rising temperatures and increasing human impacts [ 30 ]. In addition, the combination of decreasing rainfall, rising temperatures and hence increased rates of evapo-transpiration can make wildfires more destructive and severe [ 24 ]. Such conditions can have potentially adverse effects on animal populations and biodiversity [ 30 ]. By contrast, the increase in dry season rainfall in Narok Town during 1940–2015 can improve the survival prospects of ungulates when resources are most limiting [ 10 , 16 ]. The predominantly deterministic and persistent primary rainfall cycles in the Mara likely have important implications for the dynamics and management of animal populations and their vegetation resources [ 5 , 14 ]. The changing periodicity in the rainfall oscillations and the increasing amplitude in the wet season rainfall oscillations in the Mara may compound impacts of the recurrent seasonal and cyclic variations in water and forage availability that animals already have to cope with by adaptively adjusting their reproduction, foraging and migration patterns. The increase in excessive wet season rainfall in the Mara can displace wildlife due to flooding [ 29 , 111 ], reduce recruitment [ 112 ] and forage quality due to excessive plant growth and dilution of plant nutrients and cause population declines [ 28 , 74 ]. The increasing severity of floods creates favourable conditions for the transmission of several diseases, including anthrax [ 113 ], Rift Valley Fever [ 114 ] and African horse sickness [ 115 ] and promotes infestation with parasites [ 20 ]. In the Serengeti, abnormally high wet season rainfall may enable wildebeest to use the nutrient-rich southern short grass plains for extended periods towards the dry season [ 25 ]. As a consequence, the excessive rainfall may reduce wildebeest's occupancy of their dry season range in the Mara in Kenya [ 25 ]. Rainfall-mediated migrations and local concentrations of large herbivores can have cascading and difficult-to-predict effects on the ecosystem by modifying nutrient cycling through grazing, urine and dung deposition [ 116 ] and through prey availability for predators and scavengers [ 117 , 118 ]. If the pattern of increasing drought frequency continues in the Mara it will likely have adverse and immediate impacts on both wildlife and livestock populations in the ecosystem [ 16 , 29 , 75 , 80 , 82 , 119 ], although some uncertainty remains owing to the effect of the opposing trend in drought frequency in Narok Town. Droughts also tend to be associated with outbreaks of infectious diseases among large herbivores [ 120 , 121 ], such as anthrax that spreads when herbivores graze short grass close to the ground. In particular, multi-year droughts and floods can have much stronger adverse impacts on herbivores than single-year droughts [ 15 , 75 ]. The changing drought and flood frequencies can also affect biodiversity in the region through their impact on the Mara River flow levels. Implications for animal population and biodiversity dynamics The decreasing annual rainfall in Narok Town may have negative long-term effects on the reproductive performance of herbivores, including livestock, in that area through limitation of food and surface water availability [ 5 , 6 , 10 , 110 ]. Wildlife and livestock populations are also likely to suffer more from water and food scarcity in the Mara despite the stable rainfall levels during 1965–2014 due to the rising temperatures and increasing human impacts [ 30 ]. In addition, the combination of decreasing rainfall, rising temperatures and hence increased rates of evapo-transpiration can make wildfires more destructive and severe [ 24 ]. Such conditions can have potentially adverse effects on animal populations and biodiversity [ 30 ]. By contrast, the increase in dry season rainfall in Narok Town during 1940–2015 can improve the survival prospects of ungulates when resources are most limiting [ 10 , 16 ]. The predominantly deterministic and persistent primary rainfall cycles in the Mara likely have important implications for the dynamics and management of animal populations and their vegetation resources [ 5 , 14 ]. The changing periodicity in the rainfall oscillations and the increasing amplitude in the wet season rainfall oscillations in the Mara may compound impacts of the recurrent seasonal and cyclic variations in water and forage availability that animals already have to cope with by adaptively adjusting their reproduction, foraging and migration patterns. The increase in excessive wet season rainfall in the Mara can displace wildlife due to flooding [ 29 , 111 ], reduce recruitment [ 112 ] and forage quality due to excessive plant growth and dilution of plant nutrients and cause population declines [ 28 , 74 ]. The increasing severity of floods creates favourable conditions for the transmission of several diseases, including anthrax [ 113 ], Rift Valley Fever [ 114 ] and African horse sickness [ 115 ] and promotes infestation with parasites [ 20 ]. In the Serengeti, abnormally high wet season rainfall may enable wildebeest to use the nutrient-rich southern short grass plains for extended periods towards the dry season [ 25 ]. As a consequence, the excessive rainfall may reduce wildebeest's occupancy of their dry season range in the Mara in Kenya [ 25 ]. Rainfall-mediated migrations and local concentrations of large herbivores can have cascading and difficult-to-predict effects on the ecosystem by modifying nutrient cycling through grazing, urine and dung deposition [ 116 ] and through prey availability for predators and scavengers [ 117 , 118 ]. If the pattern of increasing drought frequency continues in the Mara it will likely have adverse and immediate impacts on both wildlife and livestock populations in the ecosystem [ 16 , 29 , 75 , 80 , 82 , 119 ], although some uncertainty remains owing to the effect of the opposing trend in drought frequency in Narok Town. Droughts also tend to be associated with outbreaks of infectious diseases among large herbivores [ 120 , 121 ], such as anthrax that spreads when herbivores graze short grass close to the ground. In particular, multi-year droughts and floods can have much stronger adverse impacts on herbivores than single-year droughts [ 15 , 75 ]. The changing drought and flood frequencies can also affect biodiversity in the region through their impact on the Mara River flow levels. Conclusions For important and unique ecosystems such as the Maasai Mara in East Africa it is crucial to keep reliable historical and comprehensive contemporary climate records to provide a sound scientific basis for informing decisions on the nature and consequences of climate change to large herbivores and their predators, biodiversity and human livelihoods. Our analysis of verified station data for the Mara region and Narok Town in Kenya indicates that local trends and variation in rainfall can differ substantially from the patterns predicted for regional or continental scales. In particular, we found only minor support for IPCC's large-scale predictions of very likely increases in East African rainfall. Although the rainfall cycle periods in our study area correspond to the dominant cycle periods evident in East African rainfall, we found strong spatial and temporal variation in rainfall periodicity in the Maasai Mara ecosystem. While droughts are apparently becoming more frequent in the Mara, we detected the opposite pattern for Narok Town located only 75 km away. Similarly, wet season floods became more severe in the Mara but not in Narok Town. Significant changes in rainfall amounts and periodicity can profoundly affect animal population and biodiversity dynamics by lowering the availability and increasing the variability of food and water resources, increasing the risk of outbreaks of infectious diseases and altering ungulate migration and dispersal patterns. Supporting information S1 Data Mara monthly rainfall 1965–2015. Rainfall data comprising recordings from 15 stations in the Mara region of Kenya. KMD, Kenya Meteorological Department; MMEMP, Maasai Mara Ecological Monitoring Programme; NA, not available; Rainfall (mm), records verified for analysis; Source of verification; Rain_imp, rainfall records in mm including imputed values used in analyses. (XLSX) Click here for additional data file. S2 Data Narok monthly rainfall 1913–2015. Rainfall data comprising recordings from the gauge in Narok Town operated by the Kenya Meteorological Department. (XLSX) Click here for additional data file. S1 Text Rainfall standardisation methods. (DOCX) Click here for additional data file. S2 Text Modelling seasonal oscillations in rainfall. (DOCX) Click here for additional data file. S3 Text Using wavelet analysis to detect changing periodicity in monthly rainfall oscillations. (DOCX) Click here for additional data file. S4 Text Threshold selection for estimating return levels of droughts and floods. (DOCX) Click here for additional data file. S5 Text Estimation of the runs and intervals estimator to detect multiannual persistence of droughts and floods. (DOCX) Click here for additional data file. S6 Text Quantile regression to analyse trends in the severity of droughts and floods. (DOCX) Click here for additional data file. S1 File R code used to analyse the time series of rainfall derived from 15 gauges in the Mara (1966–2014) and a gauge in Narok Town (1913–2015). The code is provided for wavelet analysis for the total monthly rainfall, and extreme value analysis, extremal indices, runs tests and quantile regression for the annual and seasonal rainfall components. The standardisation methods for the Mara data are included. (TXT) Click here for additional data file. S2 File SAS code used to fit UCM models for the time series of rainfall derived from 15 gauges in the Mara (1966–2014) and a gauge in Narok Town (1913–2015). The analysis pertains to the time series of the total monthly rainfall, and to the annual and seasonal rainfall components. (TXT) Click here for additional data file. S1 Fig Standardisation methods for the time series of annual rainfall derived from 15 gauges in the Mara (1966–2014). Four different standardisation methods were applied: arithmetic mean (black line), mean-weighted average (orange line), gauge-adjusted mean (blue line) and Best Linear Unbiased Predictions (BLUPS) from a generalized linear mixed model with Tweedie error distribution and a log link function (red line). (TIFF) Click here for additional data file. S2 Fig Smoothed level component based on the structural time series analysis of monthly rainfall. (A) Rainfall recordings in the Mara were derived from 15 gauges (Eq 3 in S1 Text ) recording in the Mara during 1965–2014 and in (B) Narok Town in Kenya during 1913–2015. Significant components are marked in bold-faced font. (TIF) Click here for additional data file. S3 Fig Cumulative plot of decadal averages of the total monthly rainfall. (A) Rainfall in the Mara during 1965–2014. (B) Rainfall in Narok Town in Kenya during 1913–2015. (TIF) Click here for additional data file. S4 Fig The standardized (divided by the mean) moving averages of the wet season (red lines) and dry season (blue lines) rainfall components. The vertical needles are the standardized deviates and the solid curves are the 3-year (Mara), 5-year (wet season of Narok Town) and 2-year (dry season of Narok Town) moving averages. (A) Rainfall recordings in the Mara were derived from 15 gauges (Eq 3 in S1 Text ) during 1966–2014 (dry season: 1965–2014). (B) Rainfall in Narok Town in Kenya was recorded during 1914–2015 (dry season: 1913–2015). The wet season and dry season rainfall components were summed from the monthly rainfall records. (TIF) Click here for additional data file. S5 Fig Smoothed primary cycles for standardized rainfall based on the structural time series analysis. (A, C, D) Rainfall recordings in the Mara were derived from 15 gauges (Eq 3 in S1 Text ) during 1966–2014 (dry season: 1965–2014). (B, D, F) Rainfall in Narok Town in Kenya was recorded during 1914–2015 (dry season: 1913–2015). The (A, B) annual, (C, D) wet season and (E, F) dry season rainfall components were summed from the monthly rainfall records. (TIF) Click here for additional data file. S6 Fig Smoothed secondary cycles for standardized rainfall based on the structural time series analysis. (A, C) Rainfall recordings in the Mara were derived from 15 gauges (Eq 3 in S1 Text ) during 1966–2014 (dry season: 1965–2014). (B, D) Rainfall in Narok Town in Kenya was recorded during 1914–2015 (dry season: 1913–2015). The (A, B) annual and (C, D) wet season rainfall components were summed from the monthly rainfall records. (TIF) Click here for additional data file. S7 Fig Return periods and the corresponding return levels (blue lines) for extreme rainfall. (A, B, E, F, I, J) Rainfall recordings in the Mara were derived from 15 gauges (Eq 3 in S1 Text ) during 1966–2014 (dry season: 1965–2014). (C, D, G, H, K, L) Rainfall in Narok Town in Kenya was recorded during 1914–2015 (dry season: 1913–2015). The (A, B, C, D) annual, (E, F, G, H) wet season and (I, J, K, L) dry season rainfall components were summed from the monthly rainfall records. Blue polygons are the 95% normal approximate confidence bands. (TIFF) Click here for additional data file. S1 Table Significance analysis of components (based on the final state) of monthly rainfall. (XLSX) Click here for additional data file. S2 Table The estimated variances of the disturbance terms and the damping factor of the autoregressive component for the monthly rainfall series. (XLSX) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3043149/

Evaluation of Cryptococcus neoformans galactoxylomannan-protein conjugate as vaccine candidate against murine cryptococcosis

Galactoxylomannan (GalXM) is a complex polysaccharide produced by the human pathogenic fungus Cryptococcus neoformans that mediates profound immunological derangements in murine models. GalXM is essentially non-immunogenic and produces immune paralysis in mice. Previous studies have attempted to enhance immunogenicity by conjugating GalXM to a protein carrier, but only transient antibody responses were elicited. Here we report the generation of two GalXM conjugates with bovine serum albumin (BSA) and protective antigen (PA) of Bacillus anthracis , respectively, using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as the cyanylating reagent. Both conjugates induced potent and sustained antibody responses as detected by both cross antigen-based and CovaLink direct ELISAs. We confirmed the specificity of the response to GalXM by inhibition ELISA and immunofluorescence. The isotype composition analysis revealed that IgG and IgM were abundant in the immune sera against GalXM, consistent with the induction of a T cell-dependent response. IgG1 was the predominant IgG subclass against GalXM, while immunization with Quil A as adjuvant elicited a significantly higher production of IgG2a than with Freund's adjuvant. Immune sera were not opsonic for C. neoformans and there was no survival difference between immune and non-immune mice challenged with C. neoformans . These results demonstrated the effectiveness of the GalXM-protein conjugate to induce robust immune responses although no evidence was obtained that such responses contributed to host defense.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047682/

New Directions in Nicotine Vaccine Design and Use

Clinical trials of nicotine vaccines suggest that they can enhance smoking cessation rates but do not reliably produce the consistently high serum antibody concentrations required. A wide array of next-generation strategies are being evaluated to enhance vaccine efficacy or provide antibody through other mechanisms. Protein conjugate vaccines may be improved by modifications of hapten or linker design or by optimizing hapten density. Conjugating hapten to viruslike particles or disrupted virus may allow exploitation of naturally occurring viral features associated with high immunogenicity. Conjugates that utilize different linker positions on nicotine can function as independent immunogens, so that using them in combination generates higher antibody concentrations than can be produced by a single immunogen. Nanoparticle vaccines, consisting of hapten, T cell help peptides, and adjuvants attached to a liposome or synthetic scaffold, are in the early stages of development. Nanoparticle vaccines offer the possibility of obtaining precise and consistent control of vaccine component stoichiometry and spacing and immunogen size and shape. Passive transfer of nicotine-specific monoclonal antibodies offers a greater control of antibody dose, the ability to give very high doses, and an immediate onset of action but is expensive and has a shorter duration of action than vaccines. Viral vector-mediated transfer of genes for antibody production can elicit high levels of antibody expression in animals and may present an alternative to vaccination or passive immunization if the long-term safety of this approach is confirmed. Next-generation immunotherapies are likely to be substantially more effective than first-generation vaccines.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697458/

Evaluation of Immunogenicity and Efficacy of Anthrax Vaccine Adsorbed for Postexposure Prophylaxis

Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis -infected animals compared to antimicrobial treatment alone.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356051/

The Arg/N-Degron Pathway—A Potential Running Back in Fine-Tuning the Inflammatory Response?

Recognition of danger signals by a cell initiates a powerful cascade of events generally leading to inflammation. Inflammatory caspases and several other proteases become activated and subsequently cleave their target proinflammatory mediators. The irreversible nature of this process implies that the newly generated proinflammatory fragments need to be sequestered, inhibited, or degraded in order to cancel the proinflammatory program or prevent chronic inflammation. The Arg/N-degron pathway is a ubiquitin-dependent proteolytic pathway that specifically degrades protein fragments bearing N-degrons, or destabilizing residues, which are recognized by the E3 ligases of the pathway. Here, we report that the Arg/N-degron pathway selectively degrades a number of proinflammatory fragments, including some activated inflammatory caspases, contributing in tuning inflammatory processes. Partial ablation of the Arg/N-degron pathway greatly increases IL-1β secretion, indicating the importance of this ubiquitous pathway in the initiation and resolution of inflammation. Thus, we propose a model wherein the Arg/N-degron pathway participates in the control of inflammation in two ways: in the generation of inflammatory signals by the degradation of inhibitory anti-inflammatory domains and as an "off switch" for inflammatory responses through the selective degradation of proinflammatory fragments. 1. Introduction Recognition of a danger signal triggers a coordinated cascade of events, initiating the inflammatory response. In cells of the innate immune system such as neutrophils, granulocytes, or macrophages, danger signals lead to activation of inflammatory caspases (caspases-1, -4, -5, -11, and -12) [ 1 , 2 ] and subsequent proteolysis of a multitude of proinflammatory targets, including IL-1β and various activators of inflammation [ 3 , 4 ]. In the adaptive immune system, cytotoxic T lymphocytes (CTL) and natural killer (NK) cells influence proinflammatory cytokine secretion through the action of granzymes [ 5 , 6 ]. In a more general manner, inflammation can also be initiated through activation of the NF-κB pathway; triggering the transcription of numerous proinflammatory genes; and inducing the production of cytokines, chemokines, and additional proinflammatory mediators [ 7 ]. Proinflammatory fragments generated by inflammatory caspases are irreversibly modified, and only sequestration or degradation of the fragments will lead to the resolution of the inflammatory response. Ubiquitylation is known to regulate many inflammatory pathways such as those mediated by Toll-like receptors, NOD-like receptors, and cytokine receptors, all of which can initiate the NF-κB pathway, which is itself being tightly controlled by adding or removing ubiquitin chains [ 8 , 9 ]. For instance, the processing of NF-κB precursors, activation of the IκB kinase (IKK) complex, and degradation of the NF-κB inhibitor IκB all require ubiquitylation [ 10 , 11 ]. More specifically, when cleaved by calpains during sepsis or other inflammatory settings [ 12 ], the NF-κB inhibitor IκBα can be degraded by the Arg/N-degron pathway, due to the presence of the destabilizing residue Glu at the newly exposed N-terminus [ 13 ]. The negative regulation of inflammation is generally provided by deubiquitinating enzymes, which remove the ubiquitin-activating signal [ 8 , 14 ]. However, recent evidence suggests that the resolution of inflammation could also be mediated by E3 ligases and protein degradation through Lys48-linked ubiquitin chains [ 15 , 16 , 17 ]. An example is the control of cytokine signaling provided by the E3 ligases Suppressor of Cytokine (SOC) signaling proteins, which are themselves regulated by ubiquitin-mediated degradation [ 18 ]. The N-degron pathway is a proteolytic pathway that relates the in vivo half-life of a protein to the identity of its N-terminal residue [ 19 ]. N-degrons comprise a destabilizing residue, which can be recognized by an E3 ubiquitin ligase, as well as an internal lysine that acts as the polyubiquitylation site. N-degrons, generally formed after the cleavage of proteins by exo- or endopeptidases [ 13 , 20 , 21 , 22 ], are recognized by specific E3 ubiquitin ligases (N-recognins) that target the proteins for degradation through the proteasome or autophagy [ 23 , 24 ]. In mammals, these E3 ligases are UBR1, UBR2, UBR4, and UBR5 [ 25 ]. The eukaryote N-degron pathway consists of five branches [ 19 ], including the Arg/N-degron pathway, which targets specific nonacetylated N-terminal residues ( Figure 1 ) [ 26 ]. The primary destabilizing N-terminal residues are directly recognized by E3 ligases, whereas N-terminal Asp, Glu, Asn, Gln, and Cys amino acids function as destabilizing residues through their preliminary modifications, such as N-terminal arginylation by the ATE1 arginyl-transferase (R-transferase) ( Figure 1 ) [ 26 , 27 ]. Through the selective degradation of proteins, the mammalian Arg/N-degron pathway mediates multiple biological functions, including the regulation of apoptosis, repression of neurodegeneration, the elimination of aberrant proteins during stress, as well as the regulation of vascular development and cell motility ([ 19 ] and references therein). The regulation of apoptosis by the Arg/N-degron pathway involves the selective degradation of caspase-cleaved proapoptotic fragments that contain N-degrons [ 21 ]. In a similar manner to that of apoptosis, the onset of inflammation results in cleavage by activated proteases of multiple proteins, which could also be potential Arg/N-degron substrates. Recently, a role for the Arg/N-degron pathway in the development of the immune response against anthrax lethal factor (LF) was discovered. Upon infection, the LF protease cleaves the mouse inflammasome NLRP1B and generates a destabilized neo-N terminus that is recognized by the UBR2 E3 ligase of the Arg/N-degron pathway. Degradation of the N-terminal fragment frees the CARD domain of the NLRP1B, which can then recruit caspase-1 and initiate pyroptosis [ 28 , 29 ]. These studies were the first to indicate a role for N-terminal degradation in the onset of an inflammatory response. Here, we propose a broader mechanism where the Arg/N-degron pathway can participate in the control of inflammation by degrading proinflammatory fragments, thus protecting cells from the misfiring of inflammatory caspases and contributing in the resolution of the inflammatory state. Thus, we suggest that the Arg/N-degron pathway actively participates in the regulation of inflammation in a dual manner: through the selective degradation of inhibitory anti-inflammatory domains and a number of proinflammatory fragments, including some activated inflammatory caspases, contributing in tuning inflammatory processes. 2. Materials and Methods 2.1. Bioinformatic Analysis of Caspase Substrates Human inflammation caspase cleavage sites were collected from the MEROPS database [ 30 ]. The search for orthologous sites was produced using pBLAST [ 31 ] with an e-value cutoff of 1 × 10 −16 . The database subset was obtained from a nonredundant database restricted by the taxon Vertebrates (taxon id: 7742). Input sequences for pBLAST, including human octamer (P4-P4') +/−26 surrounding amino acids, 60 amino acids in total, were retrieved from the Uniprot database [ 32 ]. Subsequent data analysis was performed using an in-house package R language [ 33 ]. 2.2. Cell Culture, Transfections, and Stimulations The mouse J774A.1 macrophage cell line (ATCC ® Manassas, VA, USA, TIB-67 TM ) was grown in DMEM supplemented with 10% FBS (Gibco, Waltham, MA, USA, #161400) and 2-mM L-glutamine (Gibco, #250300) without antibiotics. Cells were split when they reached 80% confluency. siRNAs were transfected using Lipofectamine RNAiMAX (Invitrogen, Waltham, MA, USA, #13778), according to the manufacturer's instructions, at a total of 5 or 10 nM (1.25 or 2.5 nM for each UBR-ubiquitin ligase siRNA). Cells were stimulated with LPS (O11:B4, Millipore Sigma St. Louis, MO, USA, #L4391) at 1, 10, or 50 ng/mL for 6 h or 24 h, with ATP (5 mM) added during the last hour or with staurosporine at 500 nM for 24 h (Millipore Sigma, St. Louis, MO, USA, #S6942) in DMEM with 2% FBS. 2.3. Plasmids, cDNAs, and Primers DH5α Escherichia coli strain (Invitrogen, Waltham, MA, USA, #EC0111) was used for the cloning and production of plasmids. Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA, #M0530) was used for PCR. Sequences of all constructed plasmids were verified by Sanger sequencing. The plasmid pKP496 was constructed by the ligation of annealed primers 1447 and 1448 into SacII/XbaI-cut pcDNA3 f DHFR Ub R48 Xpr [ 34 ]. The resulting plasmid, which encoded the f DHFR-Ub K48R -MCS (SacII-EcoRI-XhoI-ClaI-EcoRV)-flag fusion, was used to construct the plasmids used in this study for the ubiquitin reference technique [ 35 ] ( Table S2 and descriptions). All plasmids generated in this study are available from the lead contact upon request. 2.4. In Vitro Transcription–Translation–Degradation Assay The TNT T7-Coupled Transcription/Translation System, (Promega, Madison, WI, USA, #L1170) was used to carry out transcription–translation–degradation assays. Reaction samples were prepared according to the manufacturer's instructions. Newly formed proteins in reticulocyte extract were pulse-labeled with l-[ 35 S]methionine (0.55 mCi/mL, 1′000 Ci/mmol; MP Biomedicals, Solon, OH, USA) for 5 min in a total volume of 30 μL. The labeling was quenched by the addition of cycloheximide and unlabeled methionine to the final concentrations of 0.1 mg/mL and 5 mM, respectively. Unless stated otherwise, the reactions were carried out at 30 °C and terminated by the addition of an equal volume of TDS (tris-dodecylsulfate) buffer (1% SDS, 5-mM DTT, 50-mM Tris·HCl, pH 7.4, also containing a complete protease inhibitor mixture; Roche, Indianapolis, IN, USA, 5892791001) followed by heating at 95 °C for 10 min. The resulting samples were diluted with 10 volumes of TNN (tris-nonidet-NaCl) buffer (0.5% nonidet P-40, 0.25-M NaCl, 5-mM EDTA, 50-mM Tris·HCl, pH 7.4, also containing the complete protease-inhibitor mixture; Roche, Indianapolis, IN, USA), and the amounts of 35 S were measured by precipitating an aliquot with 10% trichloroacetic acid, followed by counting in a liquid scintillation counter (Beckman Coulter, Brea, CA, USA, LS6000). For immunoprecipitation, samples were adjusted to contain equal amounts of total 35 S and were added to 10-μL beads with an immobilized antibody, anti-FLAG M2 (F1804; Sigma, St. Louis, MO, USA). The samples were incubated with rocking at 4 °C for 4 h, followed by four washes with TNN buffer, resuspension in a 20-μL SDS sample buffer, and heating at 95 °C for 10 min, followed by SDS 4–15% PAGE and autoradiography. Quantification of autoradiograms was carried out using PhosphorImager and ImageQuant 5.0 software (Molecular Dynamics, Chatsworth, CA, USA). 2.5. siRNA Description and Selection siRNAs targeting mouse UBR1, UBR2, UBR4, or UBR5 (NCBI Genbank accession codes NM_009461.2, NM_001177374.1, NM_001160319.1, and NM_001081359.3) were designed with the lowest off-target potential, including miRNA-like activity and decreased capacity to activate innate immunity. Screening and selection of the most efficient and potent siRNA ( Table S4 ) are described in [ 36 ]. The control siRNA targets the Firefly Luciferase gene. 2.6. cDNA Synthesis and qPCR Total RNA was purified by disrupting cells in Trizol (Invitrogen, Waltham, MA, #155960) using the MP FastPrep-24 instrument and Lysing Matrix D (MP Biomedicals, Solon, OH, USA, SKU 116913050-CF), followed by precipitation with isopropanol according to the manufacturer's instructions. cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA, #43688). Levels of mRNA were assessed by quantitative PCR using SYBR Green (Thermo Fisher, Waltham, MA, USA, 4364346) in the QuantStudio 5 thermocycler (Applied Biosystems Waltham, MA, USA). The mRNA levels were normalized to the level of the housekeeping gene (GAPDH) and to the average value of the control group. Specific primers used in qPCR are listed in Table S3 . 2.7. Cell Extracts and Western Blot Cells were lysed in RIPA buffer containing the complete protease inhibitor cocktail (Roche) using the MP FastPrep-24 instrument and Lysing Matrix D (MP Biomedicals, Solon, OH, USA). The extracts were centrifuged at 12,000× g for 10 min at 4 °C. Media samples were concentrated ~6× using ultracentrifugation filtration units with a MWCO of 3kDa (Amicon, St. Louis, MO, USA, UFC8003). Total protein concentrations in the lysates and supernatants were determined using the BCA assay (Pierce, Waltham, MA, USA, #2322). Samples were diluted in LDS sample buffer (ThermoFisher, Waltham, MA, USA, #NP0008) supplemented with 25-mM DTT and heated at 95 °C for 10 min, except for the detection of UBR4, where samples were heated at 56 °C for 30 min. Protein analysis was performed in SDS 5–12% PAGE, with 10–100 μg of total protein loaded per lane. PAGE-fractionated proteins were transferred onto the nitrocellulose membrane and analyzed by Western blot using the following antibodies: anti-UBR1 (Abcam, Cambridge, MA, USA, #156436), anti-UBR2 (Abcam #191505), anti-UBR4 (Abcam #86738), anti-EDD (Santa Cruz, Dallas, TX, USA, #515494), anti-GAPDH (Santa Cruz #sc32233), anti- IL-1β (Cell Signaling, Danvers, MA, USA, #12507), anticleaved-caspase-1 (P20) (Cell Signaling #4199), and anti-caspase-3 (Cell Signaling #14220). Immunoblots were visualized using the SuperSignal West Femto reagent (Pierce, Waltham, MA, USA, #34095) according to the manufacturer's instructions in the Fusion Solo S Imager (Vilber Lourmat, Marne-la-Vallée, France). 2.8. Caspase-1 Activity Assay Caspase-1 activity was measured using the bioluminescent Caspase-Glo 1 Inflammasome Assay (Promega, Madison, WI, USA) in cell supernatants from J774A.1 cells stimulated with 100-ng/mL LPS or 500-nM staurosporine for 24 h following the manufacturer's instructions, using Ac-YVAD-CHO as a specific caspase-1 inhibitor. 2.9. Cytokine Bead Assay (CBA) Cytokine levels were measured by the cytokine bead assay (BD Bioscience, Franklin Lakes, NJ, USA, Flex Set #560232) according to the manufacturer's instructions. Briefly, cell culture media was concentrated 5-6x using ultracentrifugation filtration units with a MWCO of 3 kDa (Amicon, UFC8003) following the manufacturer's instructions. Total protein concentrations in the supernatants were determined using the BCA assay, and 160 µg of protein was assessed in each sample. Data was acquired using a BD LSRFortessa TM , and analysis of flow cytometry data was performed using FlowJo software, version 10.4.1 (BD Bioscience). 2.10. Statistical Analysis Prism 7 (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses. A one-way ANOVA was used for statistical analysis unless otherwise indicated. A p < 0.01 was considered significant, unless otherwise indicated. 2.1. Bioinformatic Analysis of Caspase Substrates Human inflammation caspase cleavage sites were collected from the MEROPS database [ 30 ]. The search for orthologous sites was produced using pBLAST [ 31 ] with an e-value cutoff of 1 × 10 −16 . The database subset was obtained from a nonredundant database restricted by the taxon Vertebrates (taxon id: 7742). Input sequences for pBLAST, including human octamer (P4-P4') +/−26 surrounding amino acids, 60 amino acids in total, were retrieved from the Uniprot database [ 32 ]. Subsequent data analysis was performed using an in-house package R language [ 33 ]. 2.2. Cell Culture, Transfections, and Stimulations The mouse J774A.1 macrophage cell line (ATCC ® Manassas, VA, USA, TIB-67 TM ) was grown in DMEM supplemented with 10% FBS (Gibco, Waltham, MA, USA, #161400) and 2-mM L-glutamine (Gibco, #250300) without antibiotics. Cells were split when they reached 80% confluency. siRNAs were transfected using Lipofectamine RNAiMAX (Invitrogen, Waltham, MA, USA, #13778), according to the manufacturer's instructions, at a total of 5 or 10 nM (1.25 or 2.5 nM for each UBR-ubiquitin ligase siRNA). Cells were stimulated with LPS (O11:B4, Millipore Sigma St. Louis, MO, USA, #L4391) at 1, 10, or 50 ng/mL for 6 h or 24 h, with ATP (5 mM) added during the last hour or with staurosporine at 500 nM for 24 h (Millipore Sigma, St. Louis, MO, USA, #S6942) in DMEM with 2% FBS. 2.3. Plasmids, cDNAs, and Primers DH5α Escherichia coli strain (Invitrogen, Waltham, MA, USA, #EC0111) was used for the cloning and production of plasmids. Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA, #M0530) was used for PCR. Sequences of all constructed plasmids were verified by Sanger sequencing. The plasmid pKP496 was constructed by the ligation of annealed primers 1447 and 1448 into SacII/XbaI-cut pcDNA3 f DHFR Ub R48 Xpr [ 34 ]. The resulting plasmid, which encoded the f DHFR-Ub K48R -MCS (SacII-EcoRI-XhoI-ClaI-EcoRV)-flag fusion, was used to construct the plasmids used in this study for the ubiquitin reference technique [ 35 ] ( Table S2 and descriptions). All plasmids generated in this study are available from the lead contact upon request. 2.4. In Vitro Transcription–Translation–Degradation Assay The TNT T7-Coupled Transcription/Translation System, (Promega, Madison, WI, USA, #L1170) was used to carry out transcription–translation–degradation assays. Reaction samples were prepared according to the manufacturer's instructions. Newly formed proteins in reticulocyte extract were pulse-labeled with l-[ 35 S]methionine (0.55 mCi/mL, 1′000 Ci/mmol; MP Biomedicals, Solon, OH, USA) for 5 min in a total volume of 30 μL. The labeling was quenched by the addition of cycloheximide and unlabeled methionine to the final concentrations of 0.1 mg/mL and 5 mM, respectively. Unless stated otherwise, the reactions were carried out at 30 °C and terminated by the addition of an equal volume of TDS (tris-dodecylsulfate) buffer (1% SDS, 5-mM DTT, 50-mM Tris·HCl, pH 7.4, also containing a complete protease inhibitor mixture; Roche, Indianapolis, IN, USA, 5892791001) followed by heating at 95 °C for 10 min. The resulting samples were diluted with 10 volumes of TNN (tris-nonidet-NaCl) buffer (0.5% nonidet P-40, 0.25-M NaCl, 5-mM EDTA, 50-mM Tris·HCl, pH 7.4, also containing the complete protease-inhibitor mixture; Roche, Indianapolis, IN, USA), and the amounts of 35 S were measured by precipitating an aliquot with 10% trichloroacetic acid, followed by counting in a liquid scintillation counter (Beckman Coulter, Brea, CA, USA, LS6000). For immunoprecipitation, samples were adjusted to contain equal amounts of total 35 S and were added to 10-μL beads with an immobilized antibody, anti-FLAG M2 (F1804; Sigma, St. Louis, MO, USA). The samples were incubated with rocking at 4 °C for 4 h, followed by four washes with TNN buffer, resuspension in a 20-μL SDS sample buffer, and heating at 95 °C for 10 min, followed by SDS 4–15% PAGE and autoradiography. Quantification of autoradiograms was carried out using PhosphorImager and ImageQuant 5.0 software (Molecular Dynamics, Chatsworth, CA, USA). 2.5. siRNA Description and Selection siRNAs targeting mouse UBR1, UBR2, UBR4, or UBR5 (NCBI Genbank accession codes NM_009461.2, NM_001177374.1, NM_001160319.1, and NM_001081359.3) were designed with the lowest off-target potential, including miRNA-like activity and decreased capacity to activate innate immunity. Screening and selection of the most efficient and potent siRNA ( Table S4 ) are described in [ 36 ]. The control siRNA targets the Firefly Luciferase gene. 2.6. cDNA Synthesis and qPCR Total RNA was purified by disrupting cells in Trizol (Invitrogen, Waltham, MA, #155960) using the MP FastPrep-24 instrument and Lysing Matrix D (MP Biomedicals, Solon, OH, USA, SKU 116913050-CF), followed by precipitation with isopropanol according to the manufacturer's instructions. cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA, #43688). Levels of mRNA were assessed by quantitative PCR using SYBR Green (Thermo Fisher, Waltham, MA, USA, 4364346) in the QuantStudio 5 thermocycler (Applied Biosystems Waltham, MA, USA). The mRNA levels were normalized to the level of the housekeeping gene (GAPDH) and to the average value of the control group. Specific primers used in qPCR are listed in Table S3 . 2.7. Cell Extracts and Western Blot Cells were lysed in RIPA buffer containing the complete protease inhibitor cocktail (Roche) using the MP FastPrep-24 instrument and Lysing Matrix D (MP Biomedicals, Solon, OH, USA). The extracts were centrifuged at 12,000× g for 10 min at 4 °C. Media samples were concentrated ~6× using ultracentrifugation filtration units with a MWCO of 3kDa (Amicon, St. Louis, MO, USA, UFC8003). Total protein concentrations in the lysates and supernatants were determined using the BCA assay (Pierce, Waltham, MA, USA, #2322). Samples were diluted in LDS sample buffer (ThermoFisher, Waltham, MA, USA, #NP0008) supplemented with 25-mM DTT and heated at 95 °C for 10 min, except for the detection of UBR4, where samples were heated at 56 °C for 30 min. Protein analysis was performed in SDS 5–12% PAGE, with 10–100 μg of total protein loaded per lane. PAGE-fractionated proteins were transferred onto the nitrocellulose membrane and analyzed by Western blot using the following antibodies: anti-UBR1 (Abcam, Cambridge, MA, USA, #156436), anti-UBR2 (Abcam #191505), anti-UBR4 (Abcam #86738), anti-EDD (Santa Cruz, Dallas, TX, USA, #515494), anti-GAPDH (Santa Cruz #sc32233), anti- IL-1β (Cell Signaling, Danvers, MA, USA, #12507), anticleaved-caspase-1 (P20) (Cell Signaling #4199), and anti-caspase-3 (Cell Signaling #14220). Immunoblots were visualized using the SuperSignal West Femto reagent (Pierce, Waltham, MA, USA, #34095) according to the manufacturer's instructions in the Fusion Solo S Imager (Vilber Lourmat, Marne-la-Vallée, France). 2.8. Caspase-1 Activity Assay Caspase-1 activity was measured using the bioluminescent Caspase-Glo 1 Inflammasome Assay (Promega, Madison, WI, USA) in cell supernatants from J774A.1 cells stimulated with 100-ng/mL LPS or 500-nM staurosporine for 24 h following the manufacturer's instructions, using Ac-YVAD-CHO as a specific caspase-1 inhibitor. 2.9. Cytokine Bead Assay (CBA) Cytokine levels were measured by the cytokine bead assay (BD Bioscience, Franklin Lakes, NJ, USA, Flex Set #560232) according to the manufacturer's instructions. Briefly, cell culture media was concentrated 5-6x using ultracentrifugation filtration units with a MWCO of 3 kDa (Amicon, UFC8003) following the manufacturer's instructions. Total protein concentrations in the supernatants were determined using the BCA assay, and 160 µg of protein was assessed in each sample. Data was acquired using a BD LSRFortessa TM , and analysis of flow cytometry data was performed using FlowJo software, version 10.4.1 (BD Bioscience). 2.10. Statistical Analysis Prism 7 (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses. A one-way ANOVA was used for statistical analysis unless otherwise indicated. A p < 0.01 was considered significant, unless otherwise indicated. 3. Results 3.1. Evolutionary Conserved Proinflammatory Fragments Contain Destabilizing Residues at Their N-Terminus The initiation and progression of inflammation involves the activation of specific proteases that drive and amplify inflammatory signaling by cleaving their protein targets. We reasoned that some protein fragments resulting from processing by activated inflammatory caspases could be potential Arg/N-degron substrates. A search of online databases for human and orthologous proteins containing caspase-1 cleavage sites revealed more than 120 known substrates, 21% of which bear a destabilizing residue according to the Arg/N-degron pathway at their P1′ position (the first residue after the cleavage site) ( Table S1 ). From the shorter list of experimentally confirmed caspase-1 substrates with inflammatory functions, we identified nine fragments with possible N-degrons: Asn 120 -CASP1, Gln 81 -CASP4, Gln 138 -CASP5, Cys 149 Rab39a, Tyr 37 -IL-18, Tyr 49 -CCL3, Glu 245 and Leu 249 -Ataxin-3, Cys 50 -hnRNPA2 (heterogeneous nuclear ribonucleoproteins A2/B1), and Leu 680 -Matrin-3 (the fragments are summarized in Table S1 ). Interestingly, all of the destabilizing N-terminal residues studied are conserved through evolution, with few exceptions ( Figure 2 and Figure S1 ). These proinflammatory fragments are further described in the SI Results. The inclusion of a destabilizing residue after the self-cleavage site in caspase-1, -4, and -5 is conserved in most mammals in which this cleavage motif is present ( Figure 2 ). However, in rodents, even if caspase-1 and caspase-11 contain self-cleavage sites, the P1′ residue is not recognized by the Arg/N-degron pathway, indicating a possible evolutionary transition towards a degradable inflammatory caspase fragment in higher mammals. The degradation of activated caspase-1 and -11 in rodents is most likely driven by other mechanisms. In all other caspase-generated proinflammatory fragments examined in this study, the destabilizing residue at the N-terminus is well-conserved throughout vertebrates ( Figure 2 and Figure S1 ), with few exceptions. Tellingly, however, all of the changes in the P1′ residues remain destabilizing in the Arg/N-degron pathway. Knowing that more than 80% of the mapped caspase-cleavage sites in cellular proteins contain small residues such as Gly, Ser, Thr, and Ala at their P1′ positions [ 37 ], residues that are not recognized by the Arg/N-degron pathway, the change for a destabilizing residue in these proinflammatory fragments in higher mammals is significant and could indicate a fitness-increasing property that was later maintained by selection during evolution. Proinflammatory fragments containing destabilizing residues at their N-terminus are also generated by other endopeptidases such as DPP1 and proteinase-3. These fragments include Ile 29 -GRZA, Ile 27 -GRZM, Ile 26 -GRZK, Glu 6 -IL-36β, and Tyr 16 -IL36γ ( Figure 2 and Figure S1 ). The N-terminal destabilizing residues present in granzymes A, M, K, and IL36 are conserved in all organisms examined, illustrating the evolutionary pressure to keep the degradation signal and demonstrating the significance of the N-degron pathway in the regulation of these fragments. A detailed description of the proinflammatory fragments with possible N-degrons listed there is available in the Supplementary Materials . 3.2. Proinflammatory Fragments Generated by Proteases are Targeted for Degradation by the Arg/N-Degron Pathway To determine whether proinflammatory fragments such as caspase-1, -4, and -5 are degraded by the Arg/N-degron pathway, we used the Ubiquitin reference technique (URT) [ 21 , 38 ] where the test protein is fused to a reference f DHFR-Ub R48 , a FLAG-tagged derivative of the mouse dihydrofolate reductase ( Figure 3 a). Cotranslational cleavage of the fusion protein by deubiquitylases produces both the test protein and the reference polypeptide at an initial equimolar ratio. Relative degradation rates of the test protein can be quantified by normalization to the level of the f DHFR stable reference at the same time point. We first examined inflammatory caspases for degradation by the Arg/N-degron pathway. Self-cleavage of caspase-1 after the CARD domain is necessary to generate a more stable active form of the proteolytic enzyme and even serves to terminate inflammasome activity [ 39 ]. As for human caspases -4 and -5, activity can result of the nonprocessed or the cleaved forms of the enzymes [ 40 , 41 ], while murine caspase-11 autoproteolysis is essential for inflammasome activation [ 42 ]. To demonstrate that, after self-cleavage, the remaining C-terminal protein fragments are targets of the Arg/N-degron pathway, specific URT fusions tagged with the flag epitope at the C-terminus were labeled with 35 S-Met/Cys, followed by a chase, immunoprecipitation with a monoclonal anti-FLAG antibody, SDS/PAGE separation, and quantification by autoradiography ( Figure 3 b–g). Indeed, the P20-P10 fragments Asn 120 -CASP1, Gln 81 -CASP4, and Gln 138 -CASP5 were degraded quickly in the reticulocyte extract, whereas the otherwise identical fragments bearing the N-terminal Val residue were either stable or nearly stable. These results indicate that the three human inflammatory caspases contain Arg/N-degrons and are targeted for degradation by this pathway. Since caspases are multi-subunit proteins that function as dimers, degradation of one subunit is enough to stop the catalytic activity of the enzyme [ 43 ]. Therefore, degradation of the inflammatory caspases by the Arg/N-degron pathway could be an important mechanism to stop the propagation of the inflammation program. Next, we examined whether proinflammatory fragments generated by inflammatory caspase proteolysis or other proteases contained N-degrons. We confirmed experimentally that the Cys 149 -Rab39a fragment generated after caspase-1-mediated cleavage was indeed a substrate of the Arg/N-degron pathway by using the Ub-reference technique described above ( Figure 3 h,i). Although cysteine is a tertiary destabilizing residue ( Figure 1 ), it is rapidly oxidized, arginylated, and degraded by the N-degron pathway [ 27 , 44 ]. The oxidation of cysteine requires nitric oxide, which is always present in mammalian cells but increased during inflammation, accelerating the covalent modification of N-terminal Cys [ 45 ]. Finally, we examined the degradation of activated granzymes A and M, which are known to increase the production and release of inflammatory cytokines. Processing of the granzyme activation peptide by the protease DPP1 exposes an Ile, which can be recognized by the N-degron pathway. Indeed, both granzyme A and granzyme N were short-lived compared to identical fragments bearing a Val residue at the N-terminus ( Figure 3 j–m). However, changing the Ile to a Val did not completely stabilize the activated granzymes, suggesting multiple pathways for the degradation of these enzymes. In sum, we examined 6 of 14 proinflammatory fragments with potential N-degrons ( Figure 3 ) and found that all of them are degraded by the Arg/N-degron pathway. For all fragments except granzymes, the Arg/N-degron pathway is the main mechanism of degradation, at least in reticulocyte extracts. 3.3. Partial Downregulation of the Arg/N-Degron Pathway Leads to an Enhanced Inflammatory Response Since inflammatory caspases and some of their substrates are targets of the Arg/N-degron pathway, even a partial ablation of this pathway should stabilize proinflammatory fragments in the cell and enhance the inflammatory response. Rab39a is a necessary binding partner to caspase-1 for the cleavage and secretion of IL-1β [ 46 , 47 ]. As a result, the most straightforward consequence of the stabilization of caspase-1 or Rab39a would be the increased secretion of IL-1β. We used an RNAi approach to downregulate all four UBR-ubiquitin ligases of the Arg/N-degron pathway in the J774A.1 mouse macrophage cell line. The siRNA used were chemically modified and designed to avoid recognition by TLRs and initiation of an innate immune response [ 48 ]. Forty to sixty percent downregulation of mRNA and 50–90% downregulation of the proteins was achieved in J774A.1 cells after 72 h of exposure to siRNA ( Figure 4 a,b). In a previous work [ 36 ], we demonstrated robust UBR1 protein downregulation using the same siRNA. However, because of low levels of UBR1 expression in mouse macrophages, mRNA levels were relied on to confirm the downregulation in this study. To evaluate the levels of IL-1β production in macrophages with downregulated UBR-ubiquitin ligases, we stimulated J774A.1 cells with 1, 10, or 50 ng/mL of LPS for 6 h. Western blot and cytokine bead assay analysis revealed a significant increase in the secretion of IL-1β in the media of cells with a downregulated Arg/N-degron pathway ( Figure 4 d,e). The assays recognize both pro- and cleaved IL-1β, but the Western blot assay clearly demonstrated the dramatic increase of the cleaved portion of IL-1β in UBR knockdown compared to the control, especially at the lower LPS concentration. Predictably, the amounts of cleaved caspase-1 were not changed in UBR KD macrophages, since the mouse-cleaved caspase-1 is not a substrate of the Arg/N-degron pathway ( Figure 4 c). Importantly, there was no secretion of IL-1β without LPS stimulation, indicating that the downregulation of UBR-ubiquitin ligases did not induce inflammation in itself. This also indicates that the downregulation of the Arg/N-degron pathway only sensitizes cells to inflammation and requires a proinflammatory signal such as LPS to induce a cytokine response. Together with the URT assays, these results demonstrate that the Arg/N-degron pathway is capable of regulating the level of IL-1β secretion through its ability to degrade proinflammatory fragments. 3.4. N-Recognins are not Degraded During the Inflammatory Response Apoptotic caspases can cleave N-recognins of the Arg/N-degron pathway during late-apoptosis, once the proapoptotic signaling exceeds the antiapoptotic activity of the Arg/N-degron pathway [ 21 ]. Unlike apoptosis, cells can recover from a robust proinflammatory signal and would require a functional Arg/N-degron pathway in order to degrade proinflammatory proteins. Therefore, if the N-degron pathway is instrumental in the resolution of the inflammatory response, then the E3 ligases of the pathway should not get degraded after the activation of inflammatory caspases. To test this hypothesis, we used LPS to generate an inflammatory response and staurosporine to cause apoptosis in a murine macrophage cell line. We examined protein levels of N-recognins of the Arg/N-degron pathway after stimulation with LPS and found that these levels were comparable to controls, while they are significantly decreased in cells treated with staurosporine ( Figure 5 a). The activation of caspase-1 and caspase-3 was confirmed in the experimental setting ( Figure 5 b,c). These results indicate that, contrarily to apoptotic conditions, UBR1, UBR2, UBR4, and UBR5 were not cleaved or degraded during inflammation. 3.1. Evolutionary Conserved Proinflammatory Fragments Contain Destabilizing Residues at Their N-Terminus The initiation and progression of inflammation involves the activation of specific proteases that drive and amplify inflammatory signaling by cleaving their protein targets. We reasoned that some protein fragments resulting from processing by activated inflammatory caspases could be potential Arg/N-degron substrates. A search of online databases for human and orthologous proteins containing caspase-1 cleavage sites revealed more than 120 known substrates, 21% of which bear a destabilizing residue according to the Arg/N-degron pathway at their P1′ position (the first residue after the cleavage site) ( Table S1 ). From the shorter list of experimentally confirmed caspase-1 substrates with inflammatory functions, we identified nine fragments with possible N-degrons: Asn 120 -CASP1, Gln 81 -CASP4, Gln 138 -CASP5, Cys 149 Rab39a, Tyr 37 -IL-18, Tyr 49 -CCL3, Glu 245 and Leu 249 -Ataxin-3, Cys 50 -hnRNPA2 (heterogeneous nuclear ribonucleoproteins A2/B1), and Leu 680 -Matrin-3 (the fragments are summarized in Table S1 ). Interestingly, all of the destabilizing N-terminal residues studied are conserved through evolution, with few exceptions ( Figure 2 and Figure S1 ). These proinflammatory fragments are further described in the SI Results. The inclusion of a destabilizing residue after the self-cleavage site in caspase-1, -4, and -5 is conserved in most mammals in which this cleavage motif is present ( Figure 2 ). However, in rodents, even if caspase-1 and caspase-11 contain self-cleavage sites, the P1′ residue is not recognized by the Arg/N-degron pathway, indicating a possible evolutionary transition towards a degradable inflammatory caspase fragment in higher mammals. The degradation of activated caspase-1 and -11 in rodents is most likely driven by other mechanisms. In all other caspase-generated proinflammatory fragments examined in this study, the destabilizing residue at the N-terminus is well-conserved throughout vertebrates ( Figure 2 and Figure S1 ), with few exceptions. Tellingly, however, all of the changes in the P1′ residues remain destabilizing in the Arg/N-degron pathway. Knowing that more than 80% of the mapped caspase-cleavage sites in cellular proteins contain small residues such as Gly, Ser, Thr, and Ala at their P1′ positions [ 37 ], residues that are not recognized by the Arg/N-degron pathway, the change for a destabilizing residue in these proinflammatory fragments in higher mammals is significant and could indicate a fitness-increasing property that was later maintained by selection during evolution. Proinflammatory fragments containing destabilizing residues at their N-terminus are also generated by other endopeptidases such as DPP1 and proteinase-3. These fragments include Ile 29 -GRZA, Ile 27 -GRZM, Ile 26 -GRZK, Glu 6 -IL-36β, and Tyr 16 -IL36γ ( Figure 2 and Figure S1 ). The N-terminal destabilizing residues present in granzymes A, M, K, and IL36 are conserved in all organisms examined, illustrating the evolutionary pressure to keep the degradation signal and demonstrating the significance of the N-degron pathway in the regulation of these fragments. A detailed description of the proinflammatory fragments with possible N-degrons listed there is available in the Supplementary Materials . 3.2. Proinflammatory Fragments Generated by Proteases are Targeted for Degradation by the Arg/N-Degron Pathway To determine whether proinflammatory fragments such as caspase-1, -4, and -5 are degraded by the Arg/N-degron pathway, we used the Ubiquitin reference technique (URT) [ 21 , 38 ] where the test protein is fused to a reference f DHFR-Ub R48 , a FLAG-tagged derivative of the mouse dihydrofolate reductase ( Figure 3 a). Cotranslational cleavage of the fusion protein by deubiquitylases produces both the test protein and the reference polypeptide at an initial equimolar ratio. Relative degradation rates of the test protein can be quantified by normalization to the level of the f DHFR stable reference at the same time point. We first examined inflammatory caspases for degradation by the Arg/N-degron pathway. Self-cleavage of caspase-1 after the CARD domain is necessary to generate a more stable active form of the proteolytic enzyme and even serves to terminate inflammasome activity [ 39 ]. As for human caspases -4 and -5, activity can result of the nonprocessed or the cleaved forms of the enzymes [ 40 , 41 ], while murine caspase-11 autoproteolysis is essential for inflammasome activation [ 42 ]. To demonstrate that, after self-cleavage, the remaining C-terminal protein fragments are targets of the Arg/N-degron pathway, specific URT fusions tagged with the flag epitope at the C-terminus were labeled with 35 S-Met/Cys, followed by a chase, immunoprecipitation with a monoclonal anti-FLAG antibody, SDS/PAGE separation, and quantification by autoradiography ( Figure 3 b–g). Indeed, the P20-P10 fragments Asn 120 -CASP1, Gln 81 -CASP4, and Gln 138 -CASP5 were degraded quickly in the reticulocyte extract, whereas the otherwise identical fragments bearing the N-terminal Val residue were either stable or nearly stable. These results indicate that the three human inflammatory caspases contain Arg/N-degrons and are targeted for degradation by this pathway. Since caspases are multi-subunit proteins that function as dimers, degradation of one subunit is enough to stop the catalytic activity of the enzyme [ 43 ]. Therefore, degradation of the inflammatory caspases by the Arg/N-degron pathway could be an important mechanism to stop the propagation of the inflammation program. Next, we examined whether proinflammatory fragments generated by inflammatory caspase proteolysis or other proteases contained N-degrons. We confirmed experimentally that the Cys 149 -Rab39a fragment generated after caspase-1-mediated cleavage was indeed a substrate of the Arg/N-degron pathway by using the Ub-reference technique described above ( Figure 3 h,i). Although cysteine is a tertiary destabilizing residue ( Figure 1 ), it is rapidly oxidized, arginylated, and degraded by the N-degron pathway [ 27 , 44 ]. The oxidation of cysteine requires nitric oxide, which is always present in mammalian cells but increased during inflammation, accelerating the covalent modification of N-terminal Cys [ 45 ]. Finally, we examined the degradation of activated granzymes A and M, which are known to increase the production and release of inflammatory cytokines. Processing of the granzyme activation peptide by the protease DPP1 exposes an Ile, which can be recognized by the N-degron pathway. Indeed, both granzyme A and granzyme N were short-lived compared to identical fragments bearing a Val residue at the N-terminus ( Figure 3 j–m). However, changing the Ile to a Val did not completely stabilize the activated granzymes, suggesting multiple pathways for the degradation of these enzymes. In sum, we examined 6 of 14 proinflammatory fragments with potential N-degrons ( Figure 3 ) and found that all of them are degraded by the Arg/N-degron pathway. For all fragments except granzymes, the Arg/N-degron pathway is the main mechanism of degradation, at least in reticulocyte extracts. 3.3. Partial Downregulation of the Arg/N-Degron Pathway Leads to an Enhanced Inflammatory Response Since inflammatory caspases and some of their substrates are targets of the Arg/N-degron pathway, even a partial ablation of this pathway should stabilize proinflammatory fragments in the cell and enhance the inflammatory response. Rab39a is a necessary binding partner to caspase-1 for the cleavage and secretion of IL-1β [ 46 , 47 ]. As a result, the most straightforward consequence of the stabilization of caspase-1 or Rab39a would be the increased secretion of IL-1β. We used an RNAi approach to downregulate all four UBR-ubiquitin ligases of the Arg/N-degron pathway in the J774A.1 mouse macrophage cell line. The siRNA used were chemically modified and designed to avoid recognition by TLRs and initiation of an innate immune response [ 48 ]. Forty to sixty percent downregulation of mRNA and 50–90% downregulation of the proteins was achieved in J774A.1 cells after 72 h of exposure to siRNA ( Figure 4 a,b). In a previous work [ 36 ], we demonstrated robust UBR1 protein downregulation using the same siRNA. However, because of low levels of UBR1 expression in mouse macrophages, mRNA levels were relied on to confirm the downregulation in this study. To evaluate the levels of IL-1β production in macrophages with downregulated UBR-ubiquitin ligases, we stimulated J774A.1 cells with 1, 10, or 50 ng/mL of LPS for 6 h. Western blot and cytokine bead assay analysis revealed a significant increase in the secretion of IL-1β in the media of cells with a downregulated Arg/N-degron pathway ( Figure 4 d,e). The assays recognize both pro- and cleaved IL-1β, but the Western blot assay clearly demonstrated the dramatic increase of the cleaved portion of IL-1β in UBR knockdown compared to the control, especially at the lower LPS concentration. Predictably, the amounts of cleaved caspase-1 were not changed in UBR KD macrophages, since the mouse-cleaved caspase-1 is not a substrate of the Arg/N-degron pathway ( Figure 4 c). Importantly, there was no secretion of IL-1β without LPS stimulation, indicating that the downregulation of UBR-ubiquitin ligases did not induce inflammation in itself. This also indicates that the downregulation of the Arg/N-degron pathway only sensitizes cells to inflammation and requires a proinflammatory signal such as LPS to induce a cytokine response. Together with the URT assays, these results demonstrate that the Arg/N-degron pathway is capable of regulating the level of IL-1β secretion through its ability to degrade proinflammatory fragments. 3.4. N-Recognins are not Degraded During the Inflammatory Response Apoptotic caspases can cleave N-recognins of the Arg/N-degron pathway during late-apoptosis, once the proapoptotic signaling exceeds the antiapoptotic activity of the Arg/N-degron pathway [ 21 ]. Unlike apoptosis, cells can recover from a robust proinflammatory signal and would require a functional Arg/N-degron pathway in order to degrade proinflammatory proteins. Therefore, if the N-degron pathway is instrumental in the resolution of the inflammatory response, then the E3 ligases of the pathway should not get degraded after the activation of inflammatory caspases. To test this hypothesis, we used LPS to generate an inflammatory response and staurosporine to cause apoptosis in a murine macrophage cell line. We examined protein levels of N-recognins of the Arg/N-degron pathway after stimulation with LPS and found that these levels were comparable to controls, while they are significantly decreased in cells treated with staurosporine ( Figure 5 a). The activation of caspase-1 and caspase-3 was confirmed in the experimental setting ( Figure 5 b,c). These results indicate that, contrarily to apoptotic conditions, UBR1, UBR2, UBR4, and UBR5 were not cleaved or degraded during inflammation. 4. Discussion Proteolytic processing is a widespread mechanism used by cells to initiate and amplify signaling. One explicit example is the cleavage of multiple proteins by caspases once the apoptotic or inflammatory programs are initiated [ 49 , 50 ]. The irreversible nature of this process implies that activated fragments either need to be sequestered, inhibited, or degraded to turn off the signal. Previous studies demonstrated that the Arg/N-degron pathway contributes to the elimination of proapoptotic signals generated by caspases and calpains at the onset of programmed cell death [ 13 , 21 , 51 , 52 , 53 ]. These proapoptotic fragments are recognized by their newly generated N-terminus following cleavage by proteases. Similar activities occur after danger signals are perceived by cells and inflammatory endopeptidases are activated [ 54 , 55 ]. These newly generated proinflammatory molecules should be inactivated in order to cancel the inflammatory cascade and allow cells to recover from the inflammation. In the present study, we identified in silico fourteen proinflammatory protein fragments with destabilizing N-terminal amino acids, six of which (Asn 120 -CASP1, Gln 81 -CASP4, Gln 137 -CASP5, Cys 149 -Rab39a, Ile 29 -GRZA, and Ile 27 -GRZM) are experimentally shown to contain N-degrons and be substrates of the Arg/N-degron pathway ( Figure 3 ). Our results illustrate that one of the strategies used by cells to eliminate proteolytically activated fragments generated during inflammation is degradation via their built-in degron, which is exposed upon activation or proteolytic cleavage. Proof of the importance of this mechanism is through the strong evolutionary conservation of the destabilizing nature of N-terminal residues at the P1′ position of the proinflammatory fragments. A constraint of this kind could only be expected if a short in vivo half-life of a proinflammatory fragment was a fitness-increasing property. Additionally, as inflammation and apoptosis most probably originate from a common ancestor molecular pathway, where the evasion of pathogens occurred by "cell suicide" [ 56 , 57 , 58 ], it would be natural to conserve a common regulatory mechanism as well. However, unlike programmed cell death, cells can recover from the inflammatory process, which could explain why inflammatory caspase-1 does not cleave the E3 ligases of the Arg/N-degron pathway, leaving them intact and able to target proinflammatory fragments for degradation throughout all the stages of inflammation. By destroying proinflammatory activating signals, the Arg/N-degron pathway participates in the resolution of inflammation. One significant ramification from the suggested function of the Arg/N-degron pathway would be exaggerated inflammatory responses in cells with a malfunctioning Arg/N-degron pathway. Indeed, we found that, when the E3 ligases of this pathway are downregulated, the consequence upon inflammatory stimuli such as LPS is a much greater secretion of IL-1β, even at low concentrations of the endotoxin. In accordance with our findings, partial ablation of the Arg/N-degron pathway in humans and animal models also leads to increased inflammation or inflammation-prone phenotypes. Patients with the Johanson-Blizzard syndrome, who have mutations in the UBR1 gene leading to a loss-of-function of this protein, exhibit increased inflammatory infiltrates in the pancreas, leading to acinar cell destruction and pancreatic insufficiency [ 59 , 60 ]. Ubr1 −/− mice are also more sensitive to induced pancreatitis, as shown by increased elastase activity and an elevated systemic inflammatory response upon cerulean dosing [ 61 , 62 ]. In addition, postnatal deletion of the arginyltransferase Ate1 in mice causes brain edema [ 63 ], a general indicator of inflammation. Ate1 is essential for the recognition of destabilizing N-terminal residues requiring arginylation ( Figure 1 ), which are present in proinflammatory fragments Cys 149 -Rab39a, Glu 6 -IL36b, and Glu 245 -Ataxin 3, to name a few. Finally, partial inhibition of the Arg/N-degron pathway by the downregulation of UBR1, UBR2, UBR4, and UBR5 in the mouse liver causes the infiltration of neutrophils and eosinophils, known effectors of inflammation [ 36 ], and elevates serum IL-1β levels upon LPS stimulation (D. Leboeuf, T. Zatsepin, and K. Piatkov, unpublished data). In sum, these phenotypes indicate that ablation of the Arg/N-degron pathway not only impairs the resolution of inflammation but also increases susceptibility to a stronger inflammatory response, even in the presence of a weak stimulus. In light of the results presented in this study as well as recently published data, we propose a model wherein the Arg/N-degron pathway participates in the control of inflammation in two ways: (a) in the generation of inflammatory signals by the degradation of inhibitory anti-inflammatory domains and (b) in the resolution of inflammatory states through the selective degradation of inflammatory mediators or proinflammatory fragments ( Figure 6 ). This dual control mechanism of the immune response by the Arg/N-degron pathway is also known in plants. For instance, the knockout of key components of the Arg/N-degron pathway in Arabidopsis thaliana prevents mounting an immune response to a variety of pathogens, which suggests that this pathway is involved in the degradation of proteins involved in the initiation of the immune response [ 64 ]. Conversely, the Arg/N-degron pathway is involved in the degradation of protein fragments cleaved by the Pseudomonas syringae protease effector AvrRpt2, and the removal of these fragments could help in the resolution of the immune response in the plant [ 65 ]. Additional links between the Arg/N-degron pathway and inflammation can be made through its roles in the protein quality control mechanism [ 66 ], a cellular process present in all cells from yeast to mammals and, when impaired, is responsible for the generation of inflammation in diseases such as Alzheimer's and Parkinson's [ 67 , 68 , 69 ]. Moreover, due to the ubiquitous expression of the N-degron pathway, inflammation control mechanisms provided by this pathway are present in any cell type, acting as an on/off "switch" for the initiation of an inflammatory response and/or as a "pro-resolving" mediator by means of the degradation of proinflammatory fragments, depending on the cell type and on the source of the stimuli. 5. Conclusions This study provides new evidence that the Arg/N-degron pathway participates in the regulation of inflammation through the targeted degradation of proinflammatory fragments. While inflammation is critical to the response to danger signals and the healing process, it also contributes to the pathophysiology of many noninfectious diseases. Understanding the mechanisms behind the resolution of inflammation is key to the better management of this reaction in many disorders. Our findings propose that the Arg/N-degron pathway has dual functions in the control of inflammatory processes. Ablation of the Arg/N-degron pathway not only impairs the resolution of inflammation but also increases susceptibility to a stronger inflammatory response, even in the presence of a weak stimulus. This feature can be helpful for pharmacological intervention in inflammation, both in the context of cancer treatment and the resolution of chronic inflammation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10990187/

Development of a latex agglutination test based on VH antibody fragment for detection of Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is an important porcine pathogen that causes diseases in both swine and human. For rapid SS2 identification, a novel latex agglutination test (LAT) based on heavy-chain variable domain antibody (VH) was developed. Firstly, the soluble 47B3 VH antibody fragment from a phage display library, in which cysteine residues were engineered at the C-terminus, was expressed in Escherichia coli . The purified protein was then gently reduced to form monomeric soluble 47B3 VH subsequently used to coat with latex beads by means of site-specific conjugation. The resulting VH-coated beads gave a good agglutination reaction with SS2. The LAT was able to distinguish S . suis serotype 2 from serotype 1/2, which shares some common sugar residues, and showed no cross-reaction with other serotypes of S . suis or other related bacteria. The detection sensitivity was found to be as high as 1.85x10 6 cells. The LAT was stable at 4°C for at least six months without loss of activity. To the best of our knowledge, this is the first LAT based on a VH antibody fragment that can be considered as an alternative for conventional antibody-based LAT where VHs are the most favored recombinant antibody. Introduction During the past few years, the number of reported SS2 infections in humans has increased significantly. Most cases originate in Southeast Asia, where pig-rearing and eating uncooked pork is common [ 1 – 3 ]. Increased awareness and improved diagnostics have contributed to decrease the death rate from SS2 infections. To date, S . suis can be divided into 29 serotypes based on genetic analysis [ 4 , 5 ]. Some previously recognized serotypes; serotypes 20, 22, 26, 32, 33, and 34 were classified as other Streptococcus species [ 6 – 10 ]. Typically, diagnosis of SS2 infections and serotyping can be achieved by direct culture and biochemical tests followed by multiplex PCR [ 1 , 11 ], but these processes take several days to complete. Sometimes the biochemical tests show a false-positive with other Streptococcus species or it is often misidentified, resulting in the infection going undiagnosed [ 12 ]. Meanwhile, serological typing with CPS-specific antibodies also showed cross-reaction between serotypes 2, 1/2, as they share some common sugar residues [ 13 ]. Thus, loop-mediated isothermal amplification (LAMP) assay, mismatch amplification mutation assays (MAMA-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and other molecular based techniques have been developed to differentiate between S . suis serotype 2 from 1/2 based on a single nucleotide polymorphism (SNP) in the cpsK gene; however, these methods required high-cost, specific tools and an examination specialist for the examination [ 14 – 18 ]. The latex agglutination test (LAT) is an alternative way to diagnose and serotype SS2 infection. It is easy, rapid, and can be used to test directly from a colony after culturing or for direct antigen detection in specimens. The method does not require specific laboratory tools for its operation, which makes it suitable for use in general hospital laboratories or health stations that encounter a high risk of SS2 infected patients and limited resources. In recent years, heavy-chain variable domains (VH), also called single domain antibodies, have become one of the more attractive recombinant antibody fragments [ 19 , 20 ]. The utility of VH for a broad range of applications, ranging from therapy as cancer targeting and diagnosis as bacterial contamination detection, has been demonstrated [ 21 – 23 ]. The VH antibody fragment can be derived from the heavy-chain variable domains of antibodies from camels, cartilaginous fishes , or human [ 24 , 25 ]. Compared to full-length antibodies, the VH is made of one polypeptide and so they can recognize more epitopes, particularly some "hidden" or cryptic epitopes, and they can be easily produced in bacteria or yeast [ 26 – 28 ]. Recently, we successfully isolated the phage clone 47B3 VH from a human VH phage library, selected as that with the highest specific binding activity to SS2 with no cross-reactivity with SS1/2, a serotype that shares a common antigenic epitope with serotype 2 [ 29 ]. It is likely that 47B3 VH can bind to cryptic epitopes that can differentiate among these subtypes. Conjugation with latex beads have endowed versatile functions to antibodies, resulting in antibody conjugates that are capable of being used for the LAT. To avoid interference with the antibody target binding, site specific conjugation methods have been developed with the aim of directing the conjugation to a specific location on the antibody [ 30 ]. In this paper, we describe the development of a LAT using 47B3 VH, which is directed against the capsule polysaccharide antigen of SS2. Phage clone 47B3 VH was converted to soluble 47B3 VH. The specific unpaired C-terminal cysteine on VH was used to site-specifically conjugate the VH to maleimide linker latex beads. The specificity of the derived LAT, based upon the agglutination reactivity of the VH-coated latex particles, was evaluated with different concentrations of SS2 cells. Materials and methods Construction and expression of soluble 47B3 VH The phage clone 47B3 VH, which showed a specific binding to the capsule polysaccharide of SS2, was selected to express as a soluble VH antibody. The coding sequence described in a previous report was synthetically prepared by Invitrogen company (Genscript, USA) using the codon preference of E . coli and the amber stop codon was replaced with glutamine [ 20 ]. The sequences, including a 5' restriction site for Nco I, 6 x His tag, unpaired cysteine at the C-terminus, and a 3' restriction site for Not I were added in the sequence, respectively. The Nco I- Not I fragment containing the coding sequence was excised from the derived 47B3-pUC57 plasmid and ligated into the pET28b+ vector (Genscript, USA) that had likewise been digested with Nco I and Not I enzymes. The resulting recombinant 47B3 VH plasmid was transformed into competent E . coli SHuffle ® T7 cells (New England Biolabs, Massachusetts, USA). The recombinant 47B3 VH plasmid-transformed E . coli SHuffle was grown in fresh Luria-Bertani broth supplemented with 50 μg/mL kanamycin and cultured at 30°C to an optical density at 600 nm of 0.6–0.8. Next, the expression of the recombinant protein was induced by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and culturing for 20 h at 30°C. The cells were harvested by centrifugation at 6,000 x g for 15 min and stored at -80°C until used for protein extraction. For protein extraction, the pellet was resuspended in lysis buffer [150 mM NaCl, 1% (w/v) Triton x-100, 50 mM Tris-HCl, and 20 mM imidazole] and incubated on ice for 15 min before being lysed by sonication (10 s pulse cycle at 35% amplitude) on ice. The crude lysate was centrifuged at 6,000 x g for 15 min and the pellet and supernatant were separately harvested. The clear crude lysate (supernatant) was purified using nickel-nitrilotriacetic acid (Ni-NTAA) agarose column chromatography (ACC; Cytiva, Uppsala, Sweden). The lysate was loaded onto a pre-equilibrated Ni-NTAA column using binding buffer that contained 20 mM imidazole. After that, the column was washed in washing buffer containing 40 mM imidazole and then the bound soluble 47B3 VH was eluted from the column using eluting buffer containing 400 mM imidazole. The eluted fractions were screened for protein by 15% (w/v) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under a reducing condition and western blot, was detected by mouse anti-His-tag AP conjugate and BCIP/NBT AP substrate (Surmodics IVD, Inc., Eden Prairie, USA). Characterization of soluble 47B3 VH To determine the cross-reactivity of soluble 47B3 VH, ATCC reference strain of S . suis serotypes 2, 1/2, 1, 5, 6, 14, 16, and 24, plus three serotype 2 human clinical isolates were used [ 22 ]. Furthermore, other bacteria that can be found in the bloodstream of sepsis patients, such as Streptococcus pyogenes ( S . pyogenes ), Staphylococcus aureus (S . aureus) , Escherichia coli (E . coli) , Pseudomonas aeruginosa (P . aeruginosa) , and Enterobacter aerogenes (E . aerogenes) ATCC reference strain were also used to test for cross-reactivity by ELISA. Bacterial cells (1.5 x 10 7 cells/well) were coated at 4°C overnight. The plate was washed with phosphate buffered saline pH 7.4 (PBS) and non-specific binding was blocked with PBS containing 2% (w/v) powdered milk (MPBS) for 1 h at 37°C. After washing with PBS, 50 μL of soluble 47B3 VH in two-fold dilutions from 20 μg/mL to 0.625 μg/mL diluted in MPBS were added. The plate was further incubated at 37°C for 1 h, then washed with PBS containing 0.01% (v/v) Tween-20 (0.01% PBST). The cell-bound VH were sequentially incubated with anti-his tag antibody (1:2,000; Cell Signaling Technology, Massachusetts, USA) in 1% MPBS and horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (1:2,000; GE Healthcare, UK) at 37°C for 1 h. The plate was subsequently washed five times with 0.01% PBST. The HRP activity was determined using the TMB-substrate (Surmodics IVD, Inc., Eden Prairie, USA) and monitoring the color change at 450 nm (A 450 ) using a CALIOstar Microplate reader (BMG LABTECH, Ortenberg, Germany). Preparation of 47B3 VH for site-specific conjugation To obtain the monomeric 47B3 VH-SH for site specific conjugation, the soluble 47B3 VH was first reduced with different molar ratios of VH: dithiothreitol (DTT) at 1:40, 1:60, 1:80, and 1:100 and incubated at 37 ˚C for 1 h. After the respective incubation time, the reduced antibody was mixed with loading dyes and evaluated for the presence of the dimer and monomer forms by non-reducing 15% (w/v) SDS-PAGE and visualized by coomassie brilliant blue g-250. After the reduction process, the excess DTT was removed from the reduced antibody using a vivaspin 500 3-kDa cutoff centricon filter (Cytiva, Uppsala, Sweden). The binding ability of the reduced soluble 47B3 VH with SS2 was tested by ELISA. The overnight culture of SS2 (1.5x 10 7 cells/well was coated in ELISA wells. The plate was washed five times with PBS. Non-specific binding was blocked with MPBS for 1 h at 37°C. After washing five times with PBS, 5 μg/mL of non-reduced or reduced 47B3 VH (at VH: DTT mole ratios of 1:40 and 1:60) were added and the plate was incubated at 37°C for 1 h and then washed five times with 0.05% PBST. The cell-bound Abs were detected using a 1:2000 dilution of sheep anti-mouse IgG-HRP conjugate (GE Healthcare, UK) in MPBS. Unbound antibodies were removed by washing with 0.05% PBST. The HRP activity was detected as described in the cross-reactivity test. Preparation of VH-coated beads Firstly, to create the maleimide-linked latex beads, 25 μL of 5% (w/v) 0.8-μm amino polystyrene latex beads (Bangs Laboratories, Inc., Indiana, USA) were washed three times with PBS (pH 7.2). The latex bead suspension was then mixed with (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) linker (Thermo Fisher Scientific, Massachusetts, USA) at a bead: Sulfo-SMCC ratio of 1:6 and rotated on a rotary shaker for 30 mins at room temperature (RT). After incubation, the beads were washed three times with PBS to remove excess sulfo-SMCC and then resuspended in 25 μL of PBS. Next, 25 μg of reduced soluble 47B3 VH was combined with the freshly made maleimide-linked latex bead preparation and rotated on a rotary shaker for 1 h at RT to allow conjugation. After conjugation, the 47B3 VH-coated beads were washed five times with PBS to remove the excess VH and then resuspended in PBS containing 0.1% (w/v) bovine serum albumen and 0.1% (w/v) sodium azide to give a 0.8% (w/v) suspension of VH-coated bead particles. The LAT The LATs were conducted on a glass slide with black background. On each slide, 25 μL of 47B3 VH-coated bead suspension and 25 μL of SS2 suspension (1 colony in 150 μL PBS) were combined thoroughly, and then the glass slide was manually rocked from side to side for up to 5 min to provoke the agglutination reaction. Non-coated beads were likewise mixed with SS2 suspensions as a negative control. In addition, S . suis serotypes 1/2, 1, 5, 6, 14, 16, and 24 as well as other bacteria that can be found in the bloodstream of sepsis patients, such as S . pyogenes , S . aureus , E . coli , P aeruginosa , and E . aerogenes were also used as above to test for cross-reactivity. Moreover, several dilutions of an overnight-grown culture of SS2 were applied as above in the LAT to ascertain the sensitivity of the test. The agglutination results were evaluated by visual inspection macroscopically with the naked eye and scored as follows: (i) +++, a strong and clear agglutination appeared within 1 min; (ii) ++, visible agglutinated clumps appeared after a delay of 1–3 min; (iii) +, the latex suspensions were converted to visible white clumps within 5 min; and (iv) -, no agglutination was observed during 5 min. For stability evaluation, the 47B3 VH-coated beads were kept at 4°C and agglutination tests were performed against SS2 and E . coli , as the negative control, every week for 6 months. Statistical analysis Data are expressed as the mean ± one standard deviation (SD). Statistical analysis was performed using the SPSS version 22.0 software (SPSS Inc., Chicago, IL, USA). Construction and expression of soluble 47B3 VH The phage clone 47B3 VH, which showed a specific binding to the capsule polysaccharide of SS2, was selected to express as a soluble VH antibody. The coding sequence described in a previous report was synthetically prepared by Invitrogen company (Genscript, USA) using the codon preference of E . coli and the amber stop codon was replaced with glutamine [ 20 ]. The sequences, including a 5' restriction site for Nco I, 6 x His tag, unpaired cysteine at the C-terminus, and a 3' restriction site for Not I were added in the sequence, respectively. The Nco I- Not I fragment containing the coding sequence was excised from the derived 47B3-pUC57 plasmid and ligated into the pET28b+ vector (Genscript, USA) that had likewise been digested with Nco I and Not I enzymes. The resulting recombinant 47B3 VH plasmid was transformed into competent E . coli SHuffle ® T7 cells (New England Biolabs, Massachusetts, USA). The recombinant 47B3 VH plasmid-transformed E . coli SHuffle was grown in fresh Luria-Bertani broth supplemented with 50 μg/mL kanamycin and cultured at 30°C to an optical density at 600 nm of 0.6–0.8. Next, the expression of the recombinant protein was induced by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and culturing for 20 h at 30°C. The cells were harvested by centrifugation at 6,000 x g for 15 min and stored at -80°C until used for protein extraction. For protein extraction, the pellet was resuspended in lysis buffer [150 mM NaCl, 1% (w/v) Triton x-100, 50 mM Tris-HCl, and 20 mM imidazole] and incubated on ice for 15 min before being lysed by sonication (10 s pulse cycle at 35% amplitude) on ice. The crude lysate was centrifuged at 6,000 x g for 15 min and the pellet and supernatant were separately harvested. The clear crude lysate (supernatant) was purified using nickel-nitrilotriacetic acid (Ni-NTAA) agarose column chromatography (ACC; Cytiva, Uppsala, Sweden). The lysate was loaded onto a pre-equilibrated Ni-NTAA column using binding buffer that contained 20 mM imidazole. After that, the column was washed in washing buffer containing 40 mM imidazole and then the bound soluble 47B3 VH was eluted from the column using eluting buffer containing 400 mM imidazole. The eluted fractions were screened for protein by 15% (w/v) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under a reducing condition and western blot, was detected by mouse anti-His-tag AP conjugate and BCIP/NBT AP substrate (Surmodics IVD, Inc., Eden Prairie, USA). Characterization of soluble 47B3 VH To determine the cross-reactivity of soluble 47B3 VH, ATCC reference strain of S . suis serotypes 2, 1/2, 1, 5, 6, 14, 16, and 24, plus three serotype 2 human clinical isolates were used [ 22 ]. Furthermore, other bacteria that can be found in the bloodstream of sepsis patients, such as Streptococcus pyogenes ( S . pyogenes ), Staphylococcus aureus (S . aureus) , Escherichia coli (E . coli) , Pseudomonas aeruginosa (P . aeruginosa) , and Enterobacter aerogenes (E . aerogenes) ATCC reference strain were also used to test for cross-reactivity by ELISA. Bacterial cells (1.5 x 10 7 cells/well) were coated at 4°C overnight. The plate was washed with phosphate buffered saline pH 7.4 (PBS) and non-specific binding was blocked with PBS containing 2% (w/v) powdered milk (MPBS) for 1 h at 37°C. After washing with PBS, 50 μL of soluble 47B3 VH in two-fold dilutions from 20 μg/mL to 0.625 μg/mL diluted in MPBS were added. The plate was further incubated at 37°C for 1 h, then washed with PBS containing 0.01% (v/v) Tween-20 (0.01% PBST). The cell-bound VH were sequentially incubated with anti-his tag antibody (1:2,000; Cell Signaling Technology, Massachusetts, USA) in 1% MPBS and horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (1:2,000; GE Healthcare, UK) at 37°C for 1 h. The plate was subsequently washed five times with 0.01% PBST. The HRP activity was determined using the TMB-substrate (Surmodics IVD, Inc., Eden Prairie, USA) and monitoring the color change at 450 nm (A 450 ) using a CALIOstar Microplate reader (BMG LABTECH, Ortenberg, Germany). Preparation of 47B3 VH for site-specific conjugation To obtain the monomeric 47B3 VH-SH for site specific conjugation, the soluble 47B3 VH was first reduced with different molar ratios of VH: dithiothreitol (DTT) at 1:40, 1:60, 1:80, and 1:100 and incubated at 37 ˚C for 1 h. After the respective incubation time, the reduced antibody was mixed with loading dyes and evaluated for the presence of the dimer and monomer forms by non-reducing 15% (w/v) SDS-PAGE and visualized by coomassie brilliant blue g-250. After the reduction process, the excess DTT was removed from the reduced antibody using a vivaspin 500 3-kDa cutoff centricon filter (Cytiva, Uppsala, Sweden). The binding ability of the reduced soluble 47B3 VH with SS2 was tested by ELISA. The overnight culture of SS2 (1.5x 10 7 cells/well was coated in ELISA wells. The plate was washed five times with PBS. Non-specific binding was blocked with MPBS for 1 h at 37°C. After washing five times with PBS, 5 μg/mL of non-reduced or reduced 47B3 VH (at VH: DTT mole ratios of 1:40 and 1:60) were added and the plate was incubated at 37°C for 1 h and then washed five times with 0.05% PBST. The cell-bound Abs were detected using a 1:2000 dilution of sheep anti-mouse IgG-HRP conjugate (GE Healthcare, UK) in MPBS. Unbound antibodies were removed by washing with 0.05% PBST. The HRP activity was detected as described in the cross-reactivity test. Preparation of VH-coated beads Firstly, to create the maleimide-linked latex beads, 25 μL of 5% (w/v) 0.8-μm amino polystyrene latex beads (Bangs Laboratories, Inc., Indiana, USA) were washed three times with PBS (pH 7.2). The latex bead suspension was then mixed with (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) linker (Thermo Fisher Scientific, Massachusetts, USA) at a bead: Sulfo-SMCC ratio of 1:6 and rotated on a rotary shaker for 30 mins at room temperature (RT). After incubation, the beads were washed three times with PBS to remove excess sulfo-SMCC and then resuspended in 25 μL of PBS. Next, 25 μg of reduced soluble 47B3 VH was combined with the freshly made maleimide-linked latex bead preparation and rotated on a rotary shaker for 1 h at RT to allow conjugation. After conjugation, the 47B3 VH-coated beads were washed five times with PBS to remove the excess VH and then resuspended in PBS containing 0.1% (w/v) bovine serum albumen and 0.1% (w/v) sodium azide to give a 0.8% (w/v) suspension of VH-coated bead particles. The LAT The LATs were conducted on a glass slide with black background. On each slide, 25 μL of 47B3 VH-coated bead suspension and 25 μL of SS2 suspension (1 colony in 150 μL PBS) were combined thoroughly, and then the glass slide was manually rocked from side to side for up to 5 min to provoke the agglutination reaction. Non-coated beads were likewise mixed with SS2 suspensions as a negative control. In addition, S . suis serotypes 1/2, 1, 5, 6, 14, 16, and 24 as well as other bacteria that can be found in the bloodstream of sepsis patients, such as S . pyogenes , S . aureus , E . coli , P aeruginosa , and E . aerogenes were also used as above to test for cross-reactivity. Moreover, several dilutions of an overnight-grown culture of SS2 were applied as above in the LAT to ascertain the sensitivity of the test. The agglutination results were evaluated by visual inspection macroscopically with the naked eye and scored as follows: (i) +++, a strong and clear agglutination appeared within 1 min; (ii) ++, visible agglutinated clumps appeared after a delay of 1–3 min; (iii) +, the latex suspensions were converted to visible white clumps within 5 min; and (iv) -, no agglutination was observed during 5 min. For stability evaluation, the 47B3 VH-coated beads were kept at 4°C and agglutination tests were performed against SS2 and E . coli , as the negative control, every week for 6 months. Statistical analysis Data are expressed as the mean ± one standard deviation (SD). Statistical analysis was performed using the SPSS version 22.0 software (SPSS Inc., Chicago, IL, USA). Results Construction and expression of soluble 47B3 VH A single transformed E . coli colony containing the recombinant 47B3 VH plasmid was selected for expression of the soluble VH in subsequent experiments. To optimize the condition for inducing protein expression, recombinant 47B3 VH in the E . coli SHuffle ® T7 was induced by different concentrations of IPTG (0.1, 0.5, and 1 mM) at 30°C for 20 h, lysed by sonication, and centrifuged. The expression of soluble 47B3 VH in the cell pellet and supernatant was analyzed by SDS-PAGE. The recombinant protein with a size of approximately 14 kDa was successfully expressed and was found at the highest expression level in the supernatant fraction following induction with 0.5 mM IPTG. Given the recombinant soluble 47B3 VH had a 6 x his tag sequence, it was further purified from the induced supernatant using Ni-NTAA-ACC. The eluted fractions were screened by SDS-PAGE analysis ( Fig 1 ). The eluted fractions containing the soluble 47B3 VH were pooled and the total protein concentration determined using a Bradford protein assay, revealing a net purified protein yield of approximately 1.69 mg/L of culture. 10.1371/journal.pone.0299691.g001 Fig 1 Representative SDS-PAGE analysis showing the purification of the recombinant soluble 47B3 VH protein. M: Marker; S: soluble 47B3 VH in the supernatant before purification; 1–5: elution fractions 1 to 5 of soluble 47B3 VH after purification. Characterization of soluble 47B3 VH The cross-reactivity profiles of the soluble 47B3 VH were determined against other bacteria that could cause a false-positive when testing samples suspected to be SS2. The tested bacteria were divided in two groups. In addition to SS2 in each group, the first group was comprised of S . suis serotypes 1/2, 1, 5, 6, 14, 16, and 24, which have been occasionally reported of human cases. The second group was comprised of some of the bacteria that can be found in the bloodstream in sepsis patients, such as S . pyogenes , S . aureus , E . coli , P . aeruginosa , and E . aerogenes , plus SS2 for direct comparison. In the ELISA results, soluble 47B3 VH had no cross-reactivity with any of these other tested S . suis serotypes (group 1) or bacteria (group 2) but was specific for SS2 ( Fig 2 ). 10.1371/journal.pone.0299691.g002 Fig 2 The specificity and cross-reactivity of soluble 47B3 VH against tested bacteria. (A) S . suis serotypes 2, 1/2, 1, 14, 5, 6, 16, and 24, and (B) S . pyogenes , S . aureus , E . coli , P . aeruginosa , and E . aerogenes , as tested by ELISA. Bars represent the mean of three replicate wells and error bars indicate the SD of the mean (n = 3). Moreover, the soluble 47B3 VH had a high binding activity with all three tested SS2 human clinical isolates in a specific and dose-dependent manner ( Fig 3 ). 10.1371/journal.pone.0299691.g003 Fig 3 The specificity of soluble 47B3 VH against SS2 and three serotype 2 human clinical isolates, as tested by ELISA. Bars represent the mean of three replicate wells and error bars indicate the standard deviation of the mean (n = 3). Preparation of 47B3 VH for site-specific conjugation Soluble 47B3 VH was reduced with different molar ratios of VH: DTT (1:40, 1:60, 1:80, and 1:100) and then visualized for monomer or dimers using non-reducing SDS-PAGE ( Fig 4 ). Following reduction, there was a clear decrease in the intensity of the dimer band (at around 28 kDa) and an increase in the intensity of the monomer band (at around 14 kDa) at all VH: DTT ratios. Thus, VH: DTT molar ratios between 1:40 to 1:100 were appropriate to reduce the 47B3 to give 98% in the monomer form determined in non-reducing SDS-PAGE. Since VH: DTT molar ratios above 1:60 did not show any reduced dimeric and increased monomeric VH, compared to that at a 1:60 ratio, but could risk reducing intradomain disulfide bonds and compromise the antigen binding capacity, VH: DTT molar ratios of 1:40 and 1:60 were chosen to reduce the 47B3 VH and test for antibody activity after reduction. 10.1371/journal.pone.0299691.g004 Fig 4 Representative non-reducing SDS-PAGE analysis showing the dimer and monomer forms of 47B3 VH after reduction with different VH: DTT molar ratios. Lane 1: protein MW marker; Lane 2: non-reduced soluble VH fraction; Lanes 3–6: soluble 47B3 VH fraction reduced with VH: DTT molar ratios of 1:40, 1:60, 1:80, and 1:100, respectively. Whether the soluble 47B3 VH still maintained its binding ability to SS2 after DTT reduction was evaluated by ELISA. The binding activity of the reduced 47B3 VH at a VH: DTT molar ratio of 1:40 was not significantly reduced, whereas it was at a 1:60 ratio ( Fig 5 ). Therefore, we chose a VH: DTT molar ratio of 1:40 as optimal for the preparation of the monomeric 47B3 VH-SH for site directed conjugation. 10.1371/journal.pone.0299691.g005 Fig 5 The SS2 binding activity of soluble 47B3 VH after being reduced at various VH: DTT molar ratios, as tested by ELISA. Data are shown as the mean ± 1SD (n = 3). *P < 0.05 compared to the non-reduced 47B3 VH. The LAT Following the immobilization of the 47B3 VH on the latex beads, the functionality of the 47B3 VH-coated beads was verified by combining equal volumes (25 μL) of the test bacterial suspension with the 47B3 VH-coated beads on a glass slide. The relative strength of the agglutination was graded from +++ to + and -, depending on the strength of agglutination, as described in methods. The agglutination test showed that the newly developed LAT had a satisfactory agglutination result with SS2 with the strength of agglutination of +++. There was no cross reaction with SS1/2, which is important as these two serotypes cannot be differentiated by multiplex PCR. Rather, it gave a strong (+++) and negative (-) reaction to serotype 2 and 1/2, respectively ( Fig 6 ). The non-coated beads gave a completely negative agglutination reaction with SS2. 10.1371/journal.pone.0299691.g006 Fig 6 Representative images of the LAT using 47B3 VH-coated beads mixed with tested bacteria. (A) SS2 cells and (B) SS1/2 cells; and (C) non-coated beads mixed with SS2 cells (negative control). In addition, a strong positive agglutination reaction (+++) was seen with all three of the tested SS2 human clinical isolates, while no agglutination (-) was seen for all other tested S . suis serotypes, which were those that occasionally cause infections in human, and all the other tested bacteria that can be found in the bloodstream of sepsis patients, were also negative ( Fig 7 ). 10.1371/journal.pone.0299691.g007 Fig 7 Representative images of the LAT using 47B3 VH-coated beads mixed with suspensions of different bacterial serotypes or species to test for cross-reactivity. Comparison of the sensitivity of the 47B3 VH-coated beads against different concentrations of SS2 cells revealed a positive reaction against as few as 1.85 x 10 6 cells ( Fig 8 ). 10.1371/journal.pone.0299691.g008 Fig 8 Representative images showing the agglutination reactivity of 47B3 VH-coated beads against different cell densities of SS2, with that for E . coli cells as a negative control. The stability of the 47B3 VH-coated beads was determined by testing them after storage at 4°C with SS2 suspensions. They showed the agglutination (+++) after storage at 4°C for at least 6 months without loss of activity. Construction and expression of soluble 47B3 VH A single transformed E . coli colony containing the recombinant 47B3 VH plasmid was selected for expression of the soluble VH in subsequent experiments. To optimize the condition for inducing protein expression, recombinant 47B3 VH in the E . coli SHuffle ® T7 was induced by different concentrations of IPTG (0.1, 0.5, and 1 mM) at 30°C for 20 h, lysed by sonication, and centrifuged. The expression of soluble 47B3 VH in the cell pellet and supernatant was analyzed by SDS-PAGE. The recombinant protein with a size of approximately 14 kDa was successfully expressed and was found at the highest expression level in the supernatant fraction following induction with 0.5 mM IPTG. Given the recombinant soluble 47B3 VH had a 6 x his tag sequence, it was further purified from the induced supernatant using Ni-NTAA-ACC. The eluted fractions were screened by SDS-PAGE analysis ( Fig 1 ). The eluted fractions containing the soluble 47B3 VH were pooled and the total protein concentration determined using a Bradford protein assay, revealing a net purified protein yield of approximately 1.69 mg/L of culture. 10.1371/journal.pone.0299691.g001 Fig 1 Representative SDS-PAGE analysis showing the purification of the recombinant soluble 47B3 VH protein. M: Marker; S: soluble 47B3 VH in the supernatant before purification; 1–5: elution fractions 1 to 5 of soluble 47B3 VH after purification. Characterization of soluble 47B3 VH The cross-reactivity profiles of the soluble 47B3 VH were determined against other bacteria that could cause a false-positive when testing samples suspected to be SS2. The tested bacteria were divided in two groups. In addition to SS2 in each group, the first group was comprised of S . suis serotypes 1/2, 1, 5, 6, 14, 16, and 24, which have been occasionally reported of human cases. The second group was comprised of some of the bacteria that can be found in the bloodstream in sepsis patients, such as S . pyogenes , S . aureus , E . coli , P . aeruginosa , and E . aerogenes , plus SS2 for direct comparison. In the ELISA results, soluble 47B3 VH had no cross-reactivity with any of these other tested S . suis serotypes (group 1) or bacteria (group 2) but was specific for SS2 ( Fig 2 ). 10.1371/journal.pone.0299691.g002 Fig 2 The specificity and cross-reactivity of soluble 47B3 VH against tested bacteria. (A) S . suis serotypes 2, 1/2, 1, 14, 5, 6, 16, and 24, and (B) S . pyogenes , S . aureus , E . coli , P . aeruginosa , and E . aerogenes , as tested by ELISA. Bars represent the mean of three replicate wells and error bars indicate the SD of the mean (n = 3). Moreover, the soluble 47B3 VH had a high binding activity with all three tested SS2 human clinical isolates in a specific and dose-dependent manner ( Fig 3 ). 10.1371/journal.pone.0299691.g003 Fig 3 The specificity of soluble 47B3 VH against SS2 and three serotype 2 human clinical isolates, as tested by ELISA. Bars represent the mean of three replicate wells and error bars indicate the standard deviation of the mean (n = 3). Preparation of 47B3 VH for site-specific conjugation Soluble 47B3 VH was reduced with different molar ratios of VH: DTT (1:40, 1:60, 1:80, and 1:100) and then visualized for monomer or dimers using non-reducing SDS-PAGE ( Fig 4 ). Following reduction, there was a clear decrease in the intensity of the dimer band (at around 28 kDa) and an increase in the intensity of the monomer band (at around 14 kDa) at all VH: DTT ratios. Thus, VH: DTT molar ratios between 1:40 to 1:100 were appropriate to reduce the 47B3 to give 98% in the monomer form determined in non-reducing SDS-PAGE. Since VH: DTT molar ratios above 1:60 did not show any reduced dimeric and increased monomeric VH, compared to that at a 1:60 ratio, but could risk reducing intradomain disulfide bonds and compromise the antigen binding capacity, VH: DTT molar ratios of 1:40 and 1:60 were chosen to reduce the 47B3 VH and test for antibody activity after reduction. 10.1371/journal.pone.0299691.g004 Fig 4 Representative non-reducing SDS-PAGE analysis showing the dimer and monomer forms of 47B3 VH after reduction with different VH: DTT molar ratios. Lane 1: protein MW marker; Lane 2: non-reduced soluble VH fraction; Lanes 3–6: soluble 47B3 VH fraction reduced with VH: DTT molar ratios of 1:40, 1:60, 1:80, and 1:100, respectively. Whether the soluble 47B3 VH still maintained its binding ability to SS2 after DTT reduction was evaluated by ELISA. The binding activity of the reduced 47B3 VH at a VH: DTT molar ratio of 1:40 was not significantly reduced, whereas it was at a 1:60 ratio ( Fig 5 ). Therefore, we chose a VH: DTT molar ratio of 1:40 as optimal for the preparation of the monomeric 47B3 VH-SH for site directed conjugation. 10.1371/journal.pone.0299691.g005 Fig 5 The SS2 binding activity of soluble 47B3 VH after being reduced at various VH: DTT molar ratios, as tested by ELISA. Data are shown as the mean ± 1SD (n = 3). *P < 0.05 compared to the non-reduced 47B3 VH. The LAT Following the immobilization of the 47B3 VH on the latex beads, the functionality of the 47B3 VH-coated beads was verified by combining equal volumes (25 μL) of the test bacterial suspension with the 47B3 VH-coated beads on a glass slide. The relative strength of the agglutination was graded from +++ to + and -, depending on the strength of agglutination, as described in methods. The agglutination test showed that the newly developed LAT had a satisfactory agglutination result with SS2 with the strength of agglutination of +++. There was no cross reaction with SS1/2, which is important as these two serotypes cannot be differentiated by multiplex PCR. Rather, it gave a strong (+++) and negative (-) reaction to serotype 2 and 1/2, respectively ( Fig 6 ). The non-coated beads gave a completely negative agglutination reaction with SS2. 10.1371/journal.pone.0299691.g006 Fig 6 Representative images of the LAT using 47B3 VH-coated beads mixed with tested bacteria. (A) SS2 cells and (B) SS1/2 cells; and (C) non-coated beads mixed with SS2 cells (negative control). In addition, a strong positive agglutination reaction (+++) was seen with all three of the tested SS2 human clinical isolates, while no agglutination (-) was seen for all other tested S . suis serotypes, which were those that occasionally cause infections in human, and all the other tested bacteria that can be found in the bloodstream of sepsis patients, were also negative ( Fig 7 ). 10.1371/journal.pone.0299691.g007 Fig 7 Representative images of the LAT using 47B3 VH-coated beads mixed with suspensions of different bacterial serotypes or species to test for cross-reactivity. Comparison of the sensitivity of the 47B3 VH-coated beads against different concentrations of SS2 cells revealed a positive reaction against as few as 1.85 x 10 6 cells ( Fig 8 ). 10.1371/journal.pone.0299691.g008 Fig 8 Representative images showing the agglutination reactivity of 47B3 VH-coated beads against different cell densities of SS2, with that for E . coli cells as a negative control. The stability of the 47B3 VH-coated beads was determined by testing them after storage at 4°C with SS2 suspensions. They showed the agglutination (+++) after storage at 4°C for at least 6 months without loss of activity. Discussion Many alternative methods have been developed for the rapid detection of SS2, including immunological and molecular based detection assays [ 31 – 34 ]. The advantages of the LAT over other newly developed methods are that no specific tools are required for the examination, it is simple, inexpensive, and rapid to perform. To develop the test, the conjugation of selected antibodies to latex beads can be performed in several ways, such as adsorption on plain beads or randomly immobilized by cross-linking with functionalized beads [ 35 – 37 ]. However, the selected antibody will be docked onto the bead in a random distribution resulting in a loss in the binding activity [ 30 ]. The addition of a free cysteine at the C terminus of VH provides a free thiol (—SH) group, which serves for the site-specific conjugation to maleimide-functionalized particles, forming a stable thioether bond [ 38 ]. This strategy was intentionally employed in our study to conjugate the 47B3 VH to the latex beads. Since the VH has one pair of disulfide bonds that play a significant role in the protein folding and binding ability, the 47B3 VH was then selected to be expressed as a soluble protein in E . coli SHuffle, an engineered strain that can promote disulfide bond formation in its oxidizing cytoplasmic part [ 39 , 40 ]. The soluble 47B3 VH with an unpaired cysteine at the C-terminus was expressed in the E . coli cytoplasm in both monomeric and dimeric forms (the latter having a disulfide bridge at the C terminal cysteine), as seen in the non-reducing SDS-PAGE analysis. After expression and purification, the soluble 47B3VH still retained its bioactivity against SS2. The most serious problem in conjugation via a free cysteine is that reducing the dimeric VH to obtain the homo-monomeric VH can negate its binding ability due to the reduction of the intradomain disulfide bonds. To overcome this, we firstly optimized the VH: DTT molar ratio to obtain VH monomers without interfering with the antigen binding ability. The results indicated that a 1:40 VH: DTT molar ratio was appropriate for the reduction of the 47B3 VH dimer, which was assumed to give approximately one free–SH residue per VH molecule. The 47B3 VH-SH was subsequently conjugated to the maleimide groups on the latex beads in a reaction mixture at pH 6.5–7.5 to spontaneously produce the 47B3 VH-coated beads. The maleimide linker beads were generated in-house by linking Sulfo-SMCC with NH 2 -polystyrene latex beads of 0.8 μm diameter, since previous reports on LATs demonstrated that the agglutination activity was highest with 0.8-μm diameter latex beads [ 41 ]. Once conjugation was achieved, the functionality of the 47B3 VH-coated beads was then verified by combining an equal volume of SS2 cell suspension with 47B3 VH-coated beads on a glass side with a black background. The 47B3 VH-coated beads were able to trigger a strong agglutination reaction with SS2 cells, both the ATCC stain and three clinical isolates. Thus, the optimized VH-bead conjugation described here did not markedly compromise the antigen binding ability of the 47B3 VH antibody. Additionally, the 47B3 VH-coated beads gave a positive reaction with a SS2 cell suspension of only 1.85 x 10 6 cells, which was slightly less sensitive than that reported before for a LAT based on scFv antibody fragment that gave a positive reaction with 0.23 x 10 6 cells [ 35 ]. The outstanding feature of our LAT that is different from other immunological and molecular based assays is that there is no cross-reactivity with S . suis serotype 1/2, a serotype that shares a common antigenic epitope with serotype 2 [ 31 , 34 , 42 ]. This is because of the advantage of the VH format that can bind to cryptic epitopes that can differentiate among these subtypes. Another report on the LAT using polyclonal rabbit anti-CPS antibody for detecting SS2 showed cross-reactivity to S . pyogenes and has not been reported to differentiate between serotypes 2 and ½ [ 43 ]. The 47B3 VH-coated beads were stable for at least six months at 4°C, which is broadly similar to that for other LATs based on conventional antibodies that have a stability of 4–12 months at 4°C [ 44 – 47 ]. The LAT described herein could be easily implemented for the identification of suspected S. suis colonies grown on an agar plate in regions where SS2 is highly prevalent. In the further study, the sensitivity and specificity of LAT assay with all S . suis serotypes and related bacteria will be tested before it can be proposed as a reliable diagnostic tool. Supporting information S1 Raw images Raw image of gel data shown in Figs 1 and 4 . (PDF) S1 Table Raw data of Fig 5 . (PDF)

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8399609/

A Suggested Diagnostic Approach for Sporadic Anthrax in Cattle to Protect Public Health

The repeated occurrence of anthrax in grazing animals should be a reminder of a widespread presence of Bacillus anthracis spores in the environment. Its rapid diagnosis is critical to protect public health. Here, we report a case of anthrax in cattle that was investigated using conventional and molecular methods. In 2015, six cows suddenly died within three days and the number of dead animals increased to a total of 12 within two weeks. At necropsy, anthrax was suspected. Therefore, spleen tissue samples were collected (from 6/12 animals) and laboratory tests (microscopy, cultivation, and real-time PCR) performed. The results of tissue staining for microscopy and cultivation were in congruence, while B. anthracis real-time PCR outperformed both. Spleen tissues from all six animals were real-time PCR-positive, while B. anthracis was successfully cultivated and detected by microscopy from the spleen of only three animals. Additionally, the ear tissue from another (1/12) cow tested positive by real-time PCR, supporting the suitability of ear clippings for molecular confirmation of B. anthracis . Genotyping of the isolates using multiple-locus variable-number tandem repeat analysis (MLVA) revealed a common source of infection as all three typed isolates had an indistinguishable MLVA genotype, which has not been observed previously in Europe. The results indicate that molecular testing should be selected as the first-line tool for confirming anthrax outbreaks in animals to ensure timely protection of public health. 1. Introduction Anthrax, an ancient zoonosis caused by Bacillus anthracis , is distributed globally and is enzootic in many regions of the world, especially in Asia, sub-Saharan Africa, and Central and South America [ 1 ]. The true worldwide incidence of anthrax is not known; however, epizootics occur each year, resulting in the death of hundreds to thousands of animals and transmission of the disease to humans. It is estimated that between 2000 and 20,000 human anthrax cases occur worldwide yearly [ 2 , 3 ]. Anthrax is not a major human or animal health issue in developed countries; in Europe, it is a rare disease with only a few cases reported annually. Between 2007 and 2019, 97 confirmed human anthrax cases were reported in Europe, ranging from one to 32 cases per year [ 4 ]. Animal anthrax cases were reported in Italy, Croatia, and Romania in 2020 and 2021 [ 5 ]. In Slovenia, the last human anthrax case dates back to 1983 (Maja Sočan, personal communication). Since then, fewer than ten animal cases have been confirmed in Slovenia, with the last case described here in 2015. Rapid diagnosis of anthrax is necessary to prevent the spread of the bacteria. Several conventional and molecular microbiological methods for the detection of B. anthracis are available [ 6 ], but diagnostic algorithms vary worldwide from necropsy to various laboratory methods. Selecting the most appropriate methods is challenging, especially in countries where sporadic anthrax cases occur only every few decades. Here, we describe the most recent animal anthrax cases in Slovenia and compare the performance of methods available in our laboratory. 2. Materials and Methods In August 2015, 6 cows from farm A were found dead in a marshland pasture in the central part of Slovenia. As a part of the national disease surveillance activities, 2 carcasses were sent to the National Veterinary Institute (NVI) laboratory for necropsy. In the following 2 weeks, another 6 cows from 4 farms (A–D) died in a nearby pasture; 4 carcasses, each from a different farm, were transported separately to the NVI laboratory. Therefore, samples from 6/12 dead cows (1/A, 2/A, 4/B, 5/A, 6/C and 7/D in Table 1 ) were examined for the presence of B. anthracis using laboratory tests, namely, microscopy of the stained spleen tissue, cultivation, and real-time PCR. Additionally, a clipped piece of an ear from another (1/12) cow was collected and subjected to real-time PCR (3/A in Table 1 ). Spleen tissue smears were stained with methylene blue (Becton Dickinson, Franklin Lakes, NJ, USA) and examined under a light microscope for the presence of encapsulated B. anthracis cells. Samples were inoculated onto 5% sheep blood agar plates (Columbia Blood Agar Base, Oxoid by Thermo Fischer Scientific, Hampshire, UK) and incubated at 37 °C for 24 h. Suspect and ambiguous colonies (based on morphology) were tested using B. anthracis specific real-time PCR assay as described below for the tissue samples. DNA from bacterial colonies was extracted using a rapid lysis method (boiling of cell suspensions for 15 min, followed by centrifugation for 2 min at 14,000× g and filtration [0.45 μm membrane filter] of the supernatant). DNA from tissue samples (spleen, ear clipping) was extracted using a commercial kit (DNA Isolation from Complex Samples, Institute of Metagenomics and Microbial Technologies, Ljubljana, Slovenia), following the manufacturer's instructions. The protocol included bead-beating (45 s at 6400 rpm) for 3 times using a tissue homogenizer (MagNA Lyser Instrument, Roche, Basel, Switzerland), combined with enzymatic and heat-induced lysis between mechanical shearing. A previously described and validated real-time PCR assay targeting the capC and pag genes of fully pathogenic B. anthracis was employed for DNA amplification (both for tissue samples and suspect colonies) [ 7 ], which was interpreted as an indication of the presence of B. anthracis because the result was supported by pathological and epidemiological data. Briefly, the reaction mix for each gene contained 10 µL of 2× master mix (TaqMan Fast Universal PCR Master Mix, Applied Biosystems by Thermo Fisher Scientific, Foster City, CA, USA), 1 µL of primer-probe mix with final concentrations of 900 nM for each primer and 250 nM for the probe, 2 µL of template DNA, and PCR-grade water to the final volume of 20 µL. PCR amplification (20 s at 95 °C, followed by 40 cycles of 3 s at 95 °C and 30 s at 60 °C) and amplicon detection were performed in a thermocycler 7500 Fast Real-Time PCR System (Applied Biosystems by Thermo Fisher Scientific). Three obtained B. anthracis isolates (designated 1/A, 5/A, and 6/C in Table 1 ) were subjected to multiple-locus variable-number tandem repeat analysis (MLVA). Subsequently, an additional B. anthracis isolate (271/15) obtained 3 months later from a diseased cow, located approx. 50 km from the initial anthrax cases, was also MLVA-genotyped. MLVA was performed on 8 loci ( vrrA , vrrB 1 , vrrB 2 , vrrC 1 , vrrC 2 , CG 3 , pXO1, pXO2) as described by Keim et al. [ 8 ]. For MLVA, DNA from bacterial colonies was isolated using the commercially available QIAcube DNA Mini Kit and the QIAcube system (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The reaction mix for each MLVA locus contained 10 µL of 2× master mix (HotStarTaq Master Mix, Qiagen), 0.5 μM of each primer pair specific for the target locus, 2 μL of template DNA, and PCR-grade water to the final volume of 20 µL. PCR amplification was adopted from Keim et al. [ 8 ] and performed in a thermocycler ProFlex PCR System (Applied Biosystems by Thermo Fisher Scientific). Amplicons were analyzed by QIAxcel capillary electrophoresis (Qiagen) using the QIAxcel DNA High Resolution Kit, QX Alignment Marker 15–5 kb, QX Size Marker 100 bp–2.5 kb, and OM1700 separation method. From MLVA results, amplicon lengths were converted into the number of individual repeats according to Keim et al. [ 8 ]. The number of individual repeats was further confirmed by Sanger sequencing of PCR amplicons (Macrogen, Amsterdam, The Netherlands). The results were presented as 8-digit numerical codes. The obtained codes were analyzed by applying the categorical coefficient to construct a minimum spanning tree using BioNumerics version 8.0 (bioMérieux, Applied Maths NV, Sint-Martens-Latem, Belgium). Data were compared with results from different countries deposited in the public B. anthracis v4_1 database (available at https://microbesgenotyping.i2bc.paris-saclay.fr/databases/view/9 (accessed on 18 June 2021)). 3. Results Necropsy of the first two bloated cows examined (1/A and 2/A in Table 1 ) revealed rapid decomposition and bleeding from the nose and inner corners of the eyes. The blood was dark and unclotted, the spleen was severely congested and enlarged, and hemorrhagic content in the abomasum and small intestine was noted. The lungs were moderately edematous and the diaphragm was swollen. To avoid further contamination of the necropsy room and for biosafety reasons, samples from the other four suspect animals were harvested directly through an incision in the upper left corner of the abdominal wall. In the spleen samples from 6/12 animals subjected to laboratory tests, the presence of B. anthracis was confirmed by at least one method: real-time PCR was positive in all six samples, in contrast to microscopy and cultivation, where only three samples (1/A, 5/A, and 6/C) were positive ( Table 1 ). Staining results (microscopy) were in accordance with cultivation. Suspect bacterial colonies were obtained also from the spleen sample of animal 4/B, but B. anthracis was not confirmed by real-time PCR. Because cultivation yielded inconclusive results, additional samples from the small intestine and blood of animal 4/B were examined; cultivation of B. anthracis from the small intestine failed, but ambiguous results were again noticed for the blood sample (real-time PCR identification of colonies was negative for B. anthracis ). In addition, real-time PCR was performed for the blood sample after DNA extraction, and it was positive for B. anthracis , but only pag was detected. The threshold cycle (Ct) value for the pag gene was 34.91, indicating a low B. anthracis load in the blood sample. In general, the Ct values for the pag gene were lower than for the capC gene ( Table 1 ), which could be the reason for the observed negative capC result. To inspect the suitability of a non-invasive sampling method for molecular confirmation of anthrax, a clipped piece of an ear from 1/12 dead cows (3/A) was collected and subjected to real-time PCR, which was positive for B. anthracis ( Table 1 , see Note 2). The Ct values obtained were the second lowest compared to the other six positive (spleen) samples, indicating a high B. anthracis load in the ear clipping. All three isolates (1/A, 5/A, and 6/C) belonged to the same genotype, while the isolate 271/15 differed from the other typed isolates ( Table 2 ). All Slovenian isolates had a unique MLVA genotype in comparison to the other European B. anthracis strains ( Figure 1 ). 4. Discussion Anthrax is a rare disease in Slovenia and Europe, but a prompt response to outbreaks in animals is crucial to minimize the risk of zoonotic transmission. One of the prerequisites for timely outbreak investigation is rapid and effective disease detection and confirmation, based on expert personnel of various specializations, from field veterinarians to pathologists and microbiologists. In the outbreak described here, the disease was confirmed in the laboratory within one day, and measures to prevent the spread of anthrax were immediately implemented on farms and pastures, including disinfection, animal movement bans, and vaccination. The State Centre for Disease Control defined a new anthrax district, an area where vaccination of all susceptible animals, grazing or receiving feed from the district, is mandatory for the next 50 years. All persons who came in contact with the dead and suspect animals were instructed to seek medical attention and were given antibiotic prophylaxis. The results of the outbreak investigation presented here indicate that molecular detection of B. anthracis in spleen tissue by real-time PCR should be considered as the method of choice for rapid confirmation of anthrax, as only 3/6 cows were found positive by staining and cultivation, but 6/6 by real-time PCR. In addition, the positive real-time PCR result for the ear sample indicates the suitability of ear clippings for rapid real-time PCR confirmation of B. anthracis without necropsy. In comparison to conventional PCR, real-time PCR is more sensitive [ 9 ]. Similarly to our case report, the superiority of PCR compared to cultivation and microscopy for the detection of B. anthracis in blood smears has also been reported [ 10 ]. In the latter study, PCR yielded positive results even from blood smears with degraded capsules that led to false-negative staining results. In conclusion, classical bacteriological methods appear as useful to complement the molecular methods to obtain B. anthracis isolates for subsequent genotyping purposes, such as MLVA or whole-genome sequencing (WGS). In our case, MLVA confirmed that the observed anthrax cases in cows in August 2015 were part of an outbreak, as all three typed isolates showed the same genotype, which was unique among other B. anthracis strains from Europe. The proposed diagnostic algorithm for anthrax outbreaks should therefore include real-time PCR as a first-line tool for the confirmation of B. anthracis , followed by genotyping of the obtained isolates to delineate the outbreak. In the present study, sheep blood agar plates were used for cultivation of B. anthracis from spleen tissue after necropsy, although PLET (polymyxin–lysozyme–EDTA–thallous acetate) agar is recommended when anthrax is suspected [ 11 ]; it is a suitable selective medium for contaminated samples but is not routinely used in our laboratory due to the small number of anthrax cases. Not using the PLET agar plates could represent a disadvantage in the efficient cultivation of B. anthracis in countries where anthrax occurs sporadically. However, in our case, contamination did not significantly hinder the isolation of B. anthracis , as Proteus sp. swarmed across the agar plate in only one sample. Although several chromogenic and selective agars are known for the detection of B. anthracis [ 12 , 13 ], maintaining their (including PLET agars) supply is difficult and expensive in laboratories that cover only a few anthrax cases every few years. Another issue for laboratories faced with the diagnosis of sporadic anthrax is the use of M'Fadyean alternative commercial methylene blue stains, which often give mixed results and lead to diagnostic failures [ 14 ]. In countries with regular anthrax cases, outbreaks may occur in very remote or challenging environments, and the samples collected may not be suitable for cultivation when they reach the laboratory [ 15 ]. Therefore, in countries with both endemic and sporadic anthrax, real-time PCR should be a preferred method for detecting B. anthracis in tissue samples. We collected spleen tissue at necropsy, but blood samples also proved suitable [ 10 ]. However, in our study, spleen samples were clearly superior to blood and intestinal samples collected postmortem . On the other hand, the non-invasively collected ear clipping sample performed well with real-time PCR, like the skin samples showing high sensitivity and specificity in a previous study [ 15 ]. Because anthrax is a fatal disease with a high mortality rate in humans and animals if not diagnosed and treated in time, rapid and reliable diagnosis is of utmost importance.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC155543/

Global plagues and the Global Fund: Challenges in the fight against HIV, TB and malaria

Background Although a grossly disproportionate burden of disease from HIV/AIDS, TB and malaria remains in the Global South, these infectious diseases have finally risen to the top of the international agenda in recent years. Ideal strategies for combating these diseases must balance the advantages and disadvantages of 'vertical' disease control programs and 'horizontal' capacity-building approaches. Discussion The Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM) represents an important step forward in the struggle against these pathogens. While its goals are laudable, significant barriers persist. Most significant is the pitiful lack of funds committed by world governments, particularly those of the very G8 countries whose discussions gave rise to the Fund. A drastic scaling up of resources is the first clear requirement for the GFATM to live up to the international community's lofty intentions. A directly related issue is that of maintaining a strong commitment to the treatment of the three diseases along with traditional prevention approaches, with the ensuing debates over providing affordable access to medications in the face of the pharmaceutical industry's vigorous protection of patent rights. Summary At this early point in the Fund's history, it remains to be seen how these issues will be resolved at the programming level. Nevertheless, it is clear that significant structural changes are required in such domains as global spending priorities, debt relief, trade policy, and corporate responsibility. HIV/AIDS, tuberculosis and malaria are global problems borne of gross socioeconomic inequality, and their solutions require correspondingly geopolitical solutions. Background Although a grossly disproportionate burden of disease from HIV/AIDS, TB and malaria remains in the Global South, these infectious diseases have finally risen to the top of the international agenda in recent years. Ideal strategies for combating these diseases must balance the advantages and disadvantages of 'vertical' disease control programs and 'horizontal' capacity-building approaches. Discussion The Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM) represents an important step forward in the struggle against these pathogens. While its goals are laudable, significant barriers persist. Most significant is the pitiful lack of funds committed by world governments, particularly those of the very G8 countries whose discussions gave rise to the Fund. A drastic scaling up of resources is the first clear requirement for the GFATM to live up to the international community's lofty intentions. A directly related issue is that of maintaining a strong commitment to the treatment of the three diseases along with traditional prevention approaches, with the ensuing debates over providing affordable access to medications in the face of the pharmaceutical industry's vigorous protection of patent rights. Summary At this early point in the Fund's history, it remains to be seen how these issues will be resolved at the programming level. Nevertheless, it is clear that significant structural changes are required in such domains as global spending priorities, debt relief, trade policy, and corporate responsibility. HIV/AIDS, tuberculosis and malaria are global problems borne of gross socioeconomic inequality, and their solutions require correspondingly geopolitical solutions. Background World Health Organization (WHO) statistics estimate that over 5.6 million people are killed by HIV/AIDS, tuberculosis and malaria every year, with virtually all of these deaths occurring in the developing world [ 1 ]. This virus, bacterium and parasite are the top infectious disease killers in the world today. While the morbidity and mortality stemming from the latter two agents have devastated populations for centuries, the HIV/AIDS pandemic has helped stimulate a recent surge of high-level interest in infectious disease control launching all three diseases to the forefront of public attention over just a few short years. Out of the growing momentum over these diseases has emerged a potentially golden opportunity, in the form of the new Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM). Its creation demonstrates a significant step forward, though important policy issues remain to be worked out. What specific strategies will allow us to successfully generate and maintain the global public good of AIDS, tuberculosis and malaria control? This paper will discuss the GFATM and important priorities for the control of these diseases. An overview of the policy context is presented first, discussing the reasons for the recent surge of interest in these diseases and outlining two general ways of conceptualizing disease control: 'vertical', disease-specific control programs, and 'horizontal', broader-based approaches. A brief analysis of the GFATM's initial round of grants follows, examining its balance between vertical and horizontal approaches, as well as between treatment and prevention. Finally, the paper describes the major obstacles to the GFATM's success – namely, that of funding – and identifies specific political and economic strategies for the international community regarding the control of these diseases in the future. Why All the Interest? It has long been recognized that infectious diseases constitute a significant global burden of disease. In 2001, HIV/AIDS, tuberculosis and malaria together accounted for 11.4% of all disability-adjusted life-years (DALYs) globally and 31.5% in Africa [ 1 ]. Based on these numbers alone, the desire to combat these three diseases has been considerable. But a variety of factors have increased this interest still further. Not least among them is a shifting conceptualization of infectious diseases which reclassifies them as a threat to international security. The historical record shows how uncontrolled infectious diseases have been critical in the rise and fall of human societies, from the annihilation of Native Americans to the fall of the Byzantine Empire [ 2 ]. Present-day ecological changes such as the rise of mega-cities, the ease of international transport, and the destruction of the natural environment continue to increase the infectious disease burden. Coupled with increasing social inequalities, these global shifts may overwhelm states' capacities for governance and economic growth, and exacerbate the threat of intra- or inter-state conflict [ 3 , 4 ]. These dangers prompted the United Nations Security Council to convene an unprecedented session on the threat to Sub-Saharan Africa of HIV/AIDS in January 2000, and prompted the Clinton administration in the United States to appoint a National Science Council on [the security threat posed by] Emerging and Re-Emerging Infectious Diseases. Clinton himself publicly declared AIDS an international security threat at a World AIDS Day commemoration in December 2000 [ 5 ]. Another motivation for high-level engagement around AIDS, tuberculosis and malaria relates to domestic disease control concerns. For instance, in most developed countries, tuberculosis is predominantly a disease of the foreign-born, albeit with a notable disease burden among urban disenfranchised and other poor communities. In such settings, it makes far more sense both economically and in terms of public health to 'turn off the tap' of disease burden by controlling tuberculosis at a global level than to 'mop' the global tide of infectious disease through the screening and management of individual patients [ 6 ]. There is increasing recognition of the need to broaden horizons beyond our borders and to acknowledge that global forces and international policy shape the disease patterns of our domestic populations. Finally, a moral-rational model of 'global public goods' has recently been advanced, which urges us to reconcieve the benefits of infectious disease control in the developing world as a common good with benefits for all humanity. Global public goods are 'non-excludable' inputs to the public domain which by definition are available for all to enjoy. 'Enlightened self-interest' is one way of understanding of how one's own well-being is intrinsically tied to that of one's neighbours, but for many, the gross health inequalities between the industrialized and developing world have become intolerable on purely ethical and moral grounds [ 7 ]. Why All the Interest? It has long been recognized that infectious diseases constitute a significant global burden of disease. In 2001, HIV/AIDS, tuberculosis and malaria together accounted for 11.4% of all disability-adjusted life-years (DALYs) globally and 31.5% in Africa [ 1 ]. Based on these numbers alone, the desire to combat these three diseases has been considerable. But a variety of factors have increased this interest still further. Not least among them is a shifting conceptualization of infectious diseases which reclassifies them as a threat to international security. The historical record shows how uncontrolled infectious diseases have been critical in the rise and fall of human societies, from the annihilation of Native Americans to the fall of the Byzantine Empire [ 2 ]. Present-day ecological changes such as the rise of mega-cities, the ease of international transport, and the destruction of the natural environment continue to increase the infectious disease burden. Coupled with increasing social inequalities, these global shifts may overwhelm states' capacities for governance and economic growth, and exacerbate the threat of intra- or inter-state conflict [ 3 , 4 ]. These dangers prompted the United Nations Security Council to convene an unprecedented session on the threat to Sub-Saharan Africa of HIV/AIDS in January 2000, and prompted the Clinton administration in the United States to appoint a National Science Council on [the security threat posed by] Emerging and Re-Emerging Infectious Diseases. Clinton himself publicly declared AIDS an international security threat at a World AIDS Day commemoration in December 2000 [ 5 ]. Another motivation for high-level engagement around AIDS, tuberculosis and malaria relates to domestic disease control concerns. For instance, in most developed countries, tuberculosis is predominantly a disease of the foreign-born, albeit with a notable disease burden among urban disenfranchised and other poor communities. In such settings, it makes far more sense both economically and in terms of public health to 'turn off the tap' of disease burden by controlling tuberculosis at a global level than to 'mop' the global tide of infectious disease through the screening and management of individual patients [ 6 ]. There is increasing recognition of the need to broaden horizons beyond our borders and to acknowledge that global forces and international policy shape the disease patterns of our domestic populations. Finally, a moral-rational model of 'global public goods' has recently been advanced, which urges us to reconcieve the benefits of infectious disease control in the developing world as a common good with benefits for all humanity. Global public goods are 'non-excludable' inputs to the public domain which by definition are available for all to enjoy. 'Enlightened self-interest' is one way of understanding of how one's own well-being is intrinsically tied to that of one's neighbours, but for many, the gross health inequalities between the industrialized and developing world have become intolerable on purely ethical and moral grounds [ 7 ]. Discussion The Policy Context: Vertical Disease Control Programs vs. Horizontal Approaches Traditionally, there are two general approaches to disease control: 'vertical' disease-specific programs which are independent of the rest of the health care system, and 'horizontal', broader-based approaches to improving health. Vertical control programs are exemplified by the standardized public health approach to TB. In the wake of skyrocketing TB rates around the world and the WHO's ensuing declaration of TB as a "global public health emergency" in 1993, international authorities have rallied around a public health package involving directly-observed therapy, short-course (DOTS) as an absolute requirement for control of the disease [ 8 , 9 ]. DOTS has achieved good results in numerous settings around the world [ 10 , 11 ]. A similar vertical approach to disease management has been recently proposed for HIV/AIDS as well, calling for centralized, national programs for the delivery of antiretrovirals in the developing world, either alongside or directly integrated with existing TB programs [ 12 ]. The strength of such vertical mechanisms is in the attention paid to all aspects of disease control, spanning the continuum from prevention to treatment and follow-up, and from government commitment to standardized clinical care. Indeed, the text of the recent United Nations General Assembly Special Session (UNGASS) Declaration of Commitment on HIV/AIDS addresses a wide array of HIV-related imperatives ranging from clinical prevention and treatment to human rights, from socioeconomic impact to research and development [ 13 ]. The fierce commitment of such programs' proponents offers inspiration and hope to those who refuse to 'write off' the health challenges of the developing world as unsustainably expensive or not 'cost effective'. Vertical programs are not without their critics, however. Often, they are seen as focussing too exclusively on one health problem while unjustly ignoring others. In investing solely in one disease, they may fail to build local capacities to foster broader health benefits. In particular, DOTS vertical programming has received a wealth of criticism, though generally for reasons specific to TB-DOTS programs. For instance, observers of operational research on DOTS have commented that there is a lack of standardization regarding the exact program inputs in studies reporting on DOTS program successes [ 14 ]. Foucauldian critiques consider the program's fixation on 'supervised swallowing' to be dehumanizing and authoritarian [ 15 ]. Of great practical consideration, there is widespread concern that DOTS may simply not be attainable in resource-poor settings. In fact, two randomized controlled trials comparing DOTS with self-supervision, family supervision, or both, failed to demonstrate the superiority of direct observation by a health worker, suggesting that cheaper and more convenient forms of treatment supervision than DOTS merit consideration. The first trial, conducted in South Africa, reported greater treatment success among self-supervised patients (60%) than DOTS patients (54%) [ 15 ]. The second, conducted in Pakistan, revealed similar results for all forms of direct observation: 64% cure rates for DOTS, 55% for family supervision, and 62% for self-supervision [ 16 ]. Suggestions that downplay the supervisory role of health care professional have thus emerged, arguing that the counselling and personal support capacities of treatment observers are more important factors in the program's success [ 17 , 18 ]. More complex analyses of tuberculosis control point to the insidious emergence of multi-drug resistant tuberculosis (MDRTB) as both evidence and predictor of DOTS' shortcomings. MDRTB refers to strains of the bacterium which are resistant to at least isoniazid and rifampin, the two drugs at the foundation of standard DOTS therapy (though strains may be resistant to other drugs as well). Mathematical modeling [ 19 ], medical anthropological studies [ 20 ], and epidemiological surveys in at least 6 countries [ 21 ] demonstrate how the formulaic application of DOTS in the era of multidrug resistance can fuel a growing epidemic of MDRTB. The failure of vertical programming, it is argued, lies in the inadequacy of underlying health care systems and, more fundamentally, in the virulence of a globalizing world system which marginalizes the poor [ 22 , 23 ]. Despite its virtues, broader approaches are clearly needed which address the complex ways in which poverty and inadequate access breed resistance. An alternative framework for addressing these systemic forces is a 'horizontal' approach to public health, in which emphasis is placed on basic needs and essential health infrastructure. Indeed, it is widely pointed out that throughout the world, 95% of people living with HIV/AIDS lack access to basic health care services [ 24 ]. Since 1978, one popular way of conceiving this approach is through the promotion of Primary Health Care, the focus of an oft-cited WHO International Conference in Alma-Ata, USSR, in that year [ 25 ]. The Declaration of Alma-Ata advocates a multifactorial approach involving health education, an adequate food supply, nutrition, safe water and sanitation, maternal and child health, immunization, disease control and prevention, and the provision of essential drugs. More recently, the international development community has promoted another horizontal approach to known as a 'Sector Investment Program' or, when applied to the health domain, a 'Sector-Wide Approach' (SWAp). This technique, first proposed by the World Bank in 1995 and pioneered in several countries in Sub-Saharan Africa and South Asia, represents a novel approach to 'development' and 'aid' which builds on the well-described failures of project-oriented funding and structural adjustment program approaches [ 26 - 28 ]. SWAps involve a series of crucial steps that differentiate them from previous models. First, agreement is reached in advance among local government, donors, and locally active non-governmental organizations (NGOs) on a clear set of priorities within a given sector (e.g., the health sector). Donor monies are then pooled into a transparently-monitored, locally-controlled fund, which is used to finance sector activities in order of pre-specified priority. A SWAp thereby does away with the fragmentation and inefficiency of project-by-project, donor-by-donor approaches, in favour of a coherent, mutually-agreed upon policy framework which is under local, rather than donor, control. Poverty reduction and the rational prioritization of development initiatives are key components of the strategy, making it a potentially effective mechanism for addressing horizontal public health goals. Present experience with SWAps has been limited to only a few African and Asian countries, and tensions remain over the changes demanded of funding agencies (e.g., financing of recurrent rather than one-time expenditures), the balance between government and NGO priorities, and other key areas [ 28 ]. Nevertheless, results have been encouraging thus far, and their potential to broadly address the health problems of the global South remains significant. But horizontal approaches, too, have their limitations. Since the Alma-Ata commitment to 'Health for All by the year 2000' was announced, for instance, HIV/AIDS has emerged and flourished, while the continuing death tolls of malaria and tuberculosis rates seem to demand specific attention. There is also widespread concern that vertical, disease-specific programs – notwithstanding their own inadequacies – will not fit into SWAp frameworks at all. Indeed, a recent report from Zambia describes how SWAp policy reform in that country led to the collapse of its previously effective tuberculosis program [ 29 ]. The challenge ahead for the control of HIV, tuberculosis and malaria will be in achieving an appropriate balance between targeted interventions for these major killers and broader programs targeting the underlying inadequacies which predispose people to poor health. As argued recently in the Bulletin of the World Health Organization , The two approaches are not in opposition to each other and a false choice between vertical and horizontal approaches should not threaten international cooperation for disease control, nor should disease control be promoted at the cost of health sector development or of focusing on non-communicable diseases . . . To accomplish targeted health-policy outcomes, the international community should therefore encourage organizations such as WHO to complement horizontal, health sector programmes with vertical multisector, multilevel initiatives [ 7 ]. Disease-specific interventions could, for instance, be used as a springboard for investments into the health care systems which buttress them. In the realm of HIV control, for instance, targeted efforts to decrease mother-to-child HIV transmission via AZT/nevirapine prophylaxis should be conceived not as stand-alone interventions, but rather as integral parts of a maternal and child health care package [ 30 ]. Such an integrated approach would ideally include disease-specific interventions such as antiretroviral therapy for the mother, voluntary counseling testing for her sexual/blood contacts, as well as psychosocial support. At the same time, it would strengthen health infrastructure through pre- and postnatal care, STD counseling and screening, nutritional supplementation and family planning. In fact, experience with integrating HIV- and tuberculosis-specific interventions into broader community health programs has already been reported from the developing world. Through its 'HIV Equity Initiative' in rural Haiti, one group has employed community health workers, social and economic support for families, and simplified clinical treatment algorithms to integrate vertical HIV prophylaxis and highly-active antiretroviral therapy (HAART) into an existing community clinic [ 31 ]. The same group has also advocated a newer vertical approach to MDRTB known as DOTS – Plus, a standard DOTS protocol enhanced by individually-tailored pharmacotherapy [ 32 ]. While critics may contend that this is too costly and technically demanding for a resource-poor setting, its advocates contend that the treatment of MDRTB is the only rational and morally acceptable medical and public health response to this phenomenon. They and others have demonstrated its feasibility in rural Haiti, the slums of Peru and urban Turkey [ 20 , 33 , 34 ], and have successfully integrated the program into a community-based health care setting which addresses patients' broader health needs. Further, to bolster this aggressive approach, encouraging progress has been made in negotiating price reductions for second line anti-tuberculous drugs through a WHO-convened Working Group and its 'Green Light Committee' [ 35 ]. Clearly, vertical disease control programs must be complemented by broader, horizontal approaches to health care and health infrastructure if AIDS, tuberculosis and malaria are to be controlled. By extension, policy makers must consider the broader socioeconomic inputs to health if outcomes are to be improved – an approach which may require higher-level political commitments and systemic, structural change. The Global Fund: A Golden Opportunity? The GFATM is an international financing initiative representing the new-found determination of the international community to address the health impact of the three diseases. The Fund was born out of discussions at the Okinawa G8 Summit in July 2000, and was made concrete by UN Secretary General Kofi Annan's call to action in April 2001. Bolstered by the subsequent United Nations General Assembly Special Session on HIV/AIDS (UNGASS) in June 2001, and by the G8 Summit in Genoa, July 2001, the Fund has become operational in a remarkably short period of time. After a rigorous selection process, the Fund announced its first round of grants in April 2002 [ 36 ], through which $616 million will be dispersed over two years [ 37 ]. Two issues are of particular interest in this granting process. The first relates to how the Fund tackled the question of balancing vertical, disease-specific 'product support' against horizontal health systems support. Though its name suggests a distinctly vertical approach, the Fund's stated intentions are to "address the three diseases in ways that will contribute to strengthening health systems". It aims to support proposals which "build on, complement and coordinate with existing . . . national policies, priorities and partnerships, including Poverty Reduction Strategies and sector-wide approaches" [ 36 ]. On paper, its list of approved projects from the April 2002 round of grants appears to have favoured disease-specific programming, including comprehensive 'prevention and control' strategies, the social marketing of disease prevention, DOTS expansion programs, and a few cases of product support (eg. bednets in Tanzania, antiretrovirals in Nigeria) [ 36 ]. One strategy the Fund has employed to ensure a broad consensus on an individual country's programming is to require that every application be done through a partnership that includes representatives from governments, civil society, and people affected by the diseases. The goal of these Country Coordinating Mechanisms (CCMs) is to improve coordination of their activities and to avoid duplication [ 36 ]. But the extent to which the GFATM lives true to its goal of building broad-based health systems thus remains to be seen. The Global Alliance for Vaccines and Immunizations, another global body active in the arena of international health, appears to provide a model for balancing these approaches: The alliance's executive director is reported to have said that an optimum balance might be 60% of funds for new vaccines and 40% for strengthening immunization services. To encourage recipients to meet the targets set, additional funds for strengthening health systems will be released only once the countries have reached higher levels of immunization coverage [ 38 ]. But ironically, "failure to meet targets could indicate the need for greater support to weak health systems rather than withholding of funds" [ 38 ]. Investing in horizontal programs for developing health systems would not only provide much-needed basic services for individuals suffering from these and other diseases at present, but would also lay essential groundwork for the future delivery of targeted interventions such as medications and immunizations, once they become available. The way ahead lies in fostering innovative solutions that integrate vertical, disease-specific programming for AIDS, tuberculosis and malaria with much-needed health systems support. The second issue of interest in the Fund's granting process was how the Fund balanced treatment programs with prevention programs in allocating its monies. It is now widely recognized that antiretroviral treatment is a cornerstone of HIV/AIDS control that must not play second fiddle to prevention, and that treatment for HIV plays an important role in controlling tuberculosis and malaria infection as well [ 39 ]. In its official call for funding applications in January, the GFATM articulated a commitment to the "prevention, treatment, care and support of the infected and directly affected" [ 36 ], and in his July 2002 speech to the XIV International AIDS Conference in Barcelona, executive director Richard Feachem reiterated this dedication. He stated that the first round of grants "will double the current number of people receiving Highly Active Anti-Retroviral Therapy (HAART) in the developing world and in Africa HAART recipients will increase six fold as a result of these commitments" [ 40 ]. But as Feachem himself acknowledges, these achievements are "nothing like enough". The inadequacy may largely be due to the continual reluctance of industrialized countries to finance treatment programs – particularly for expensive antiretroviral (ARV) therapy. Indeed, officials from Malawi and other countries were allegedly encouraged by donor countries to remove a treatment component from their GFATM proposal [ 37 ]. Yet at around the same time, the WHO made the groundbreaking move of adding ARVs to its Essential Medicines List [ 41 ]. The debate over funding medicines encompasses a deeper issue, about balancing the potentially limitless need for costly pharmaceuticals with the financial interests of the pharmaceutical companies that manufacture them. Macroeconomic analysis by the United Kingdom's Performance and Innovation Unit (UKPIU) [ 24 ] asserts that in order to establish any reasonable hope for the widespread availability of medications, vaccines and other health products for these diseases in the future, the Fund should provide a secure market for affordably-priced goods. Further, it should signal this commitment through advance-purchase commitments. The document argues that it is only through a willingness to cover the costs of manufacture, as well as the financial risks of research, that the global community can hope to drive research and development into essential medicines and vaccines. But wherein lies the balance of power in a system which lays corporate bottom lines at the foundation of a global effort to combat the diseases of poverty? Half the members of the commission that wrote the UKPIU report are from the pharmaceutical industry. By no means does this invalidate their findings – indeed, the report applies sound economic theory in reaching rational, pragmatic conclusions. But already, the public sector provides most of the market for pharmaceutical products, through public health insurance schemes. Furthermore, a review of the chemotherapeutic agents developed by the pharmaceutical industry over the past 25 years reveals how despite increased protection in the form of extended patent durations, the industry has not shown a concomitant increase in innovation [ 42 ]. The lack of new medicines for neglected diseases of the world's poor populations is palpable: of 1393 new chemical entities marketed over this time period, only 16 were for tropical diseases and tuberculosis [ 42 ]. Taken together, these arguments form the basis for calls for innovative new mechanisms for improving the development of and access to therapies for major infectious diseases. Contrasting the views of industry are the recommendations of another global constituency with vested interests in the GFATM's design – namely, the health NGO sector. Deeply concerned about the Fund becoming a mere 'pharmaceutical industry subsidy' [ 43 ], NGOs have made passionate pleas that priority be given to finding the most affordable, effective treatment available when GFATM monies are used to purchase medications. As observed by Director of Médecins Sans Frontières' Access to Essential Medicines Campaign Bernard Pecoul, an explicit statement of this commitment is conspicuously missing from the Fund's official documentation [ 44 ]. Practically speaking, the Fund must ensure that expensive, brand-name antiretroviral drugs are not blindly purchased where legal mechanisms could allow the purchase of up to three times the quantity of an equally efficacious generic version of the same medications. In the excitement over ensuring that large-scale efforts to treat the three diseases maintain adequate respect for intellectual property rights, potential beneficiaries must be explicitly reminded that obtaining generic and branded medicines through alternative mechanisms such as compulsory licensing and parallel import arrangements are entirely consistent with the Trade-Related Aspects of Intellectual Property Rights agreement (TRIPS) [ 45 ]. The legality of these measures was explicitly agreed to at the WTO's 2001 meeting in Doha, Qatar. Paragraph four of the declaration reads: "We agree that the TRIPS Agreement does not and should not prevent Members from taking measures to protect public health. Accordingly, while reiterating our commitments to the TRIPS Agreement, we affirm that the Agreement can and should be interpreted and implemented in a manner supportive of WTO Members' right to protect public health and, in particular, to promote access to medicines for all" [ 46 ]. Practical legal experience such as that garnered through Brazil's success with using the mechanism of compulsory licensing to reduce the price of both generic and brand ARVs down should be translated elsewhere, so that potential applicants might gain access to the most affordable quality medicines. Of relevance to this discussion of pharmaceutical and NGO interests, another set of policy tensions in the GFATM's history relates to its governance. The very composition of the Fund's Executive Board was under contentious debate in preliminary sectoral consultations by the Fund's Transitional Working Group. Since its inception, the Fund has billed itself as a "public-private partnership", yet consultation with the NGO sector initially recommended that no representatives of the pharmaceutical industry be members of the Board. Meanwhile, the private sector itself asked for more than the proposed two allotted positions on the Fund's 15-person Board, requesting in the interim an additional ex-officio observer seat. For the present, both constituencies retain two Board positions, which will hopefully preserve a balance between private and public perspectives. The Challenge of Funding the Fund The Global Fund represents an important opportunity for visionary leadership and meaningful action towards reducing the horrific tolls of HIV/AIDS, TB and malaria. The challenges which lie ahead for the GFATM lie in fostering and funding innovative projects which integrate vertical approaches with horizontal approaches, and balance preventive programs with treatment. But even beyond these programming dilemmas, how easily will it reach its lofty goals? A quick survey of the GFATM's progress to date reveals its first major barrier, in the form of grossly inadequate funds. If the international community is truly as committed to stamping out these three diseases as it would have the world believe, it must drastically scale up its financial commitments. At this writing, the fund totals little more than $2.1 billion [ 36 ] – a relatively paltry sum when compared to a recent report that put the minimum price tag for global HIV control at $7.5 billion annually for that disease alone [ 47 ]. Commendable shows of leadership have been made by Kofi Annan himself, who initiated the fund by personally donating $100,000 of prize money from his Philadelphia Liberty Medal, and by the governments of Sub-Saharan Africa, which set target commitments of 15% of their annual national budgets to be devoted to health sector improvements for HIV/AIDS at an April 2001 summit in Abuja, Nigeria. But what of the leadership from the G8 countries, out of whose own summits the very idea of the Global Fund first arose? To date, the G8 have collectively committed about $1.6 billion of the $2.1 billion total [ 36 ]. The Unites States has pledged by far the greatest proportion of this amount, at $500 million. But these seemingly impressive dollar figures fall far sufficient of the money needed. At least $US 1.3 billion each year is required to support basic commodities for prevention and treatment for malaria among vulnerable groups [ 48 ]. Thus far, less that $US 23 million has been awarded by the Fund for Malaria. African countries, which represent 90% of the global malaria burden, gets only $US 12.7 million [ 48 ]. In the wake of post-September 11 anthrax scares, for instance, the 2003 US Homeland Security Budget has proposed $5.9 billion to defend against bioterrorism [ 49 ]. Similarly, the seemingly astronomical price tag of $7–10 billion for HIV/AIDS control is dwarfed by the still more astronomical annual expenditures on military and defense budgets the world over. Global military spending totaled $1 trillion in 1990 alone, and industrialized countries spend 5.3% of GNP on military expenditures each year [ 23 ]. In contrast, these same countries spend less than 0.3% of GNP on overseas development assistance (ODA) each year – far short of their mutually-agreed upon target of 0.7–1.0% of GNP [ 23 ]. A glimmer of hope shone over the weeks leading up to the recent June 2002 G8 Summit in Kananaskis, Canada, where a 'New Partnership for African Development' (NEPAD) was placed high on the agenda. Drafted by African leaders themselves, the proposal's great innovation was that aid spending on the continent would be more reliably spent, since NEPAD required them to pass an African peer review process. But despite the enthusiasm about this attention to Africa, the industrialized countries still failed to make AIDS, tuberculosis and malaria a priority in Kananaskis. UN Envoy on AIDS in Africa Stephen Lewis made this point abundantly clear in his speech to the Alternative Summit that ran parallel to the official G8 proceedings [ 50 ]. He notes that while the NEPAD document sets admirable goals (an annual growth rate of 7% for fifteen years, halving poverty by 2015, a two-thirds reduction in infant mortality, a 25% reduction in maternal mortality, and education for all children), none of them are realistically attainable unless the HIV/AIDS pandemic receives the attention it deserves. Yet, NEPAD pays little attention to the disease in its proposals. The global community must rethink its approach to 'development', with HIV/AIDS and the other major infectious diseases at the core of its analysis. This is precisely the argument of the much-heralded recent WHO Report of the Commission on Macroeconomics and Health. In it, Jeffrey Sachs asserts that The burden of disease in some low-income regions, especially sub-Saharan Africa, stands as a stark barrier to economic growth and therefore must be addressed frontally and centrally in any comprehensive development strategy. The AIDS pandemic represents a unique challenge of unprecedented urgency and intensity. This single epidemic can undermine Africa's development over the next generation, and may cause tens of millions of deaths in India, China, and other developing countries unless addressed by greatly increased efforts [ 51 ]. The importance, then, of combatting AIDS, tuberculosis and malaria has been made clear in the global arena. If global health is truly understood as a 'global public good', the necessary finances must be mobilized by whatever means necessary. One proposal offers compelling reasons to open up national health budgets to fund health development in the international arena [ 7 ]. By this argument, money budgeted for investment in the health of one country's population is just as appropriately spent on global health problems as domestic ones, since the health of the world's populations are so closely intertwined. Regardless of the accounting logistics, it must ultimately be realized that the funds required for the control of AIDS, tuberculosis and malaria do exist, and must be made available through a careful re-examination of funding priorities. The Way Ahead The GFATM holds considerable promise for harnessing true international commitment to addressing the three diseases. But even if the Global Fund attains its massive targets of $7–10 billion US per year, does it truly have the capacity to mend the damage from diseases so mired in centuries of growing global inequality? HIV/AIDS, tuberculosis and malaria are diseases that demand consideration of populations' underlying predisposition to disease in the forms of socioeconomic inequality and abject poverty. While the motivations of the international community for addressing the diseases include pragmatic concerns of international security, economic prosperity and domestic health status, they must ultimately include the ethical responsibility to redress gross inequalities. Adequate attention to the systemic forces underlying these infections thus necessitates correspondingly systemic solutions. As discussed already, not least among these is the need to mobilize far larger sums of money to invest in world health and in redressing social inequalities. For decades, much of Africa has been left to stagnate in a perpetual "poverty trap" [ 52 ], in which the state is simply too poor to provide adequate basic living conditions for the population. Infectious diseases are both a cause and a consequence of this lack of health care, education and infrastructure. Direct financial transfers and investment in basic needs in such countries are the only viable solutions to the continent's ongoing health and economic crises. Hand in hand with this financial commitment is the need to relieve those developing countries with unreasonable debt burdens of these outlandish costs. Debt repayment schedules paralyze national budgets and lock them into paying back unsustainable sums of money to high-income countries and financial institutions every year. On average, debtor countries pay one and a half times as much in servicing debt as they do on health care [ 53 ]. Debt relief is imperative if the Global South's bankrupt governments are ever to address their populations' basic health needs. Economic theory asserts that for a creditor nation, "the outright cancellation of debt becomes . . . a necessary part of its foreign policy" if it hopes to promote economic growth as well as its own strategic interests in bankrupt states [ 47 , 52 ]. Similarly, industrialized countries must reform trade policies that create absurd financial barriers to the integration of poor countries' economies into the global marketplace. Import tariffs on many African goods destined for the United States, for examples, can reach levels of up to 33% – up to 15 times the average US tariff rate of 2% [ 54 ]. Structural barriers such as these clearly impair the capacity of poor countries to attain anything close to equal footing with rich ones in their attempt to bring economic prosperity to their people. Finally, in our increasingly globalized economic system, the 'right' of transnational corporations to global patent protection for medicines that are almost exclusively sold to the minority in the rich West must become more intimately tied to their international responsibilities, in the form of technology transfer, local capacity building, and investments in basic infrastructure. Nowhere is this more true than for the pharmaceutical industry, where the public sector must play an active role in obliging its private sector partners to invest in research and development for neglected diseases, commit to equitable pricing schemes and participate in technology transfer. It is only by linking rights and responsibilities that we can hope to achieve improved health for all the world's inhabitants. The Policy Context: Vertical Disease Control Programs vs. Horizontal Approaches Traditionally, there are two general approaches to disease control: 'vertical' disease-specific programs which are independent of the rest of the health care system, and 'horizontal', broader-based approaches to improving health. Vertical control programs are exemplified by the standardized public health approach to TB. In the wake of skyrocketing TB rates around the world and the WHO's ensuing declaration of TB as a "global public health emergency" in 1993, international authorities have rallied around a public health package involving directly-observed therapy, short-course (DOTS) as an absolute requirement for control of the disease [ 8 , 9 ]. DOTS has achieved good results in numerous settings around the world [ 10 , 11 ]. A similar vertical approach to disease management has been recently proposed for HIV/AIDS as well, calling for centralized, national programs for the delivery of antiretrovirals in the developing world, either alongside or directly integrated with existing TB programs [ 12 ]. The strength of such vertical mechanisms is in the attention paid to all aspects of disease control, spanning the continuum from prevention to treatment and follow-up, and from government commitment to standardized clinical care. Indeed, the text of the recent United Nations General Assembly Special Session (UNGASS) Declaration of Commitment on HIV/AIDS addresses a wide array of HIV-related imperatives ranging from clinical prevention and treatment to human rights, from socioeconomic impact to research and development [ 13 ]. The fierce commitment of such programs' proponents offers inspiration and hope to those who refuse to 'write off' the health challenges of the developing world as unsustainably expensive or not 'cost effective'. Vertical programs are not without their critics, however. Often, they are seen as focussing too exclusively on one health problem while unjustly ignoring others. In investing solely in one disease, they may fail to build local capacities to foster broader health benefits. In particular, DOTS vertical programming has received a wealth of criticism, though generally for reasons specific to TB-DOTS programs. For instance, observers of operational research on DOTS have commented that there is a lack of standardization regarding the exact program inputs in studies reporting on DOTS program successes [ 14 ]. Foucauldian critiques consider the program's fixation on 'supervised swallowing' to be dehumanizing and authoritarian [ 15 ]. Of great practical consideration, there is widespread concern that DOTS may simply not be attainable in resource-poor settings. In fact, two randomized controlled trials comparing DOTS with self-supervision, family supervision, or both, failed to demonstrate the superiority of direct observation by a health worker, suggesting that cheaper and more convenient forms of treatment supervision than DOTS merit consideration. The first trial, conducted in South Africa, reported greater treatment success among self-supervised patients (60%) than DOTS patients (54%) [ 15 ]. The second, conducted in Pakistan, revealed similar results for all forms of direct observation: 64% cure rates for DOTS, 55% for family supervision, and 62% for self-supervision [ 16 ]. Suggestions that downplay the supervisory role of health care professional have thus emerged, arguing that the counselling and personal support capacities of treatment observers are more important factors in the program's success [ 17 , 18 ]. More complex analyses of tuberculosis control point to the insidious emergence of multi-drug resistant tuberculosis (MDRTB) as both evidence and predictor of DOTS' shortcomings. MDRTB refers to strains of the bacterium which are resistant to at least isoniazid and rifampin, the two drugs at the foundation of standard DOTS therapy (though strains may be resistant to other drugs as well). Mathematical modeling [ 19 ], medical anthropological studies [ 20 ], and epidemiological surveys in at least 6 countries [ 21 ] demonstrate how the formulaic application of DOTS in the era of multidrug resistance can fuel a growing epidemic of MDRTB. The failure of vertical programming, it is argued, lies in the inadequacy of underlying health care systems and, more fundamentally, in the virulence of a globalizing world system which marginalizes the poor [ 22 , 23 ]. Despite its virtues, broader approaches are clearly needed which address the complex ways in which poverty and inadequate access breed resistance. An alternative framework for addressing these systemic forces is a 'horizontal' approach to public health, in which emphasis is placed on basic needs and essential health infrastructure. Indeed, it is widely pointed out that throughout the world, 95% of people living with HIV/AIDS lack access to basic health care services [ 24 ]. Since 1978, one popular way of conceiving this approach is through the promotion of Primary Health Care, the focus of an oft-cited WHO International Conference in Alma-Ata, USSR, in that year [ 25 ]. The Declaration of Alma-Ata advocates a multifactorial approach involving health education, an adequate food supply, nutrition, safe water and sanitation, maternal and child health, immunization, disease control and prevention, and the provision of essential drugs. More recently, the international development community has promoted another horizontal approach to known as a 'Sector Investment Program' or, when applied to the health domain, a 'Sector-Wide Approach' (SWAp). This technique, first proposed by the World Bank in 1995 and pioneered in several countries in Sub-Saharan Africa and South Asia, represents a novel approach to 'development' and 'aid' which builds on the well-described failures of project-oriented funding and structural adjustment program approaches [ 26 - 28 ]. SWAps involve a series of crucial steps that differentiate them from previous models. First, agreement is reached in advance among local government, donors, and locally active non-governmental organizations (NGOs) on a clear set of priorities within a given sector (e.g., the health sector). Donor monies are then pooled into a transparently-monitored, locally-controlled fund, which is used to finance sector activities in order of pre-specified priority. A SWAp thereby does away with the fragmentation and inefficiency of project-by-project, donor-by-donor approaches, in favour of a coherent, mutually-agreed upon policy framework which is under local, rather than donor, control. Poverty reduction and the rational prioritization of development initiatives are key components of the strategy, making it a potentially effective mechanism for addressing horizontal public health goals. Present experience with SWAps has been limited to only a few African and Asian countries, and tensions remain over the changes demanded of funding agencies (e.g., financing of recurrent rather than one-time expenditures), the balance between government and NGO priorities, and other key areas [ 28 ]. Nevertheless, results have been encouraging thus far, and their potential to broadly address the health problems of the global South remains significant. But horizontal approaches, too, have their limitations. Since the Alma-Ata commitment to 'Health for All by the year 2000' was announced, for instance, HIV/AIDS has emerged and flourished, while the continuing death tolls of malaria and tuberculosis rates seem to demand specific attention. There is also widespread concern that vertical, disease-specific programs – notwithstanding their own inadequacies – will not fit into SWAp frameworks at all. Indeed, a recent report from Zambia describes how SWAp policy reform in that country led to the collapse of its previously effective tuberculosis program [ 29 ]. The challenge ahead for the control of HIV, tuberculosis and malaria will be in achieving an appropriate balance between targeted interventions for these major killers and broader programs targeting the underlying inadequacies which predispose people to poor health. As argued recently in the Bulletin of the World Health Organization , The two approaches are not in opposition to each other and a false choice between vertical and horizontal approaches should not threaten international cooperation for disease control, nor should disease control be promoted at the cost of health sector development or of focusing on non-communicable diseases . . . To accomplish targeted health-policy outcomes, the international community should therefore encourage organizations such as WHO to complement horizontal, health sector programmes with vertical multisector, multilevel initiatives [ 7 ]. Disease-specific interventions could, for instance, be used as a springboard for investments into the health care systems which buttress them. In the realm of HIV control, for instance, targeted efforts to decrease mother-to-child HIV transmission via AZT/nevirapine prophylaxis should be conceived not as stand-alone interventions, but rather as integral parts of a maternal and child health care package [ 30 ]. Such an integrated approach would ideally include disease-specific interventions such as antiretroviral therapy for the mother, voluntary counseling testing for her sexual/blood contacts, as well as psychosocial support. At the same time, it would strengthen health infrastructure through pre- and postnatal care, STD counseling and screening, nutritional supplementation and family planning. In fact, experience with integrating HIV- and tuberculosis-specific interventions into broader community health programs has already been reported from the developing world. Through its 'HIV Equity Initiative' in rural Haiti, one group has employed community health workers, social and economic support for families, and simplified clinical treatment algorithms to integrate vertical HIV prophylaxis and highly-active antiretroviral therapy (HAART) into an existing community clinic [ 31 ]. The same group has also advocated a newer vertical approach to MDRTB known as DOTS – Plus, a standard DOTS protocol enhanced by individually-tailored pharmacotherapy [ 32 ]. While critics may contend that this is too costly and technically demanding for a resource-poor setting, its advocates contend that the treatment of MDRTB is the only rational and morally acceptable medical and public health response to this phenomenon. They and others have demonstrated its feasibility in rural Haiti, the slums of Peru and urban Turkey [ 20 , 33 , 34 ], and have successfully integrated the program into a community-based health care setting which addresses patients' broader health needs. Further, to bolster this aggressive approach, encouraging progress has been made in negotiating price reductions for second line anti-tuberculous drugs through a WHO-convened Working Group and its 'Green Light Committee' [ 35 ]. Clearly, vertical disease control programs must be complemented by broader, horizontal approaches to health care and health infrastructure if AIDS, tuberculosis and malaria are to be controlled. By extension, policy makers must consider the broader socioeconomic inputs to health if outcomes are to be improved – an approach which may require higher-level political commitments and systemic, structural change. The Global Fund: A Golden Opportunity? The GFATM is an international financing initiative representing the new-found determination of the international community to address the health impact of the three diseases. The Fund was born out of discussions at the Okinawa G8 Summit in July 2000, and was made concrete by UN Secretary General Kofi Annan's call to action in April 2001. Bolstered by the subsequent United Nations General Assembly Special Session on HIV/AIDS (UNGASS) in June 2001, and by the G8 Summit in Genoa, July 2001, the Fund has become operational in a remarkably short period of time. After a rigorous selection process, the Fund announced its first round of grants in April 2002 [ 36 ], through which $616 million will be dispersed over two years [ 37 ]. Two issues are of particular interest in this granting process. The first relates to how the Fund tackled the question of balancing vertical, disease-specific 'product support' against horizontal health systems support. Though its name suggests a distinctly vertical approach, the Fund's stated intentions are to "address the three diseases in ways that will contribute to strengthening health systems". It aims to support proposals which "build on, complement and coordinate with existing . . . national policies, priorities and partnerships, including Poverty Reduction Strategies and sector-wide approaches" [ 36 ]. On paper, its list of approved projects from the April 2002 round of grants appears to have favoured disease-specific programming, including comprehensive 'prevention and control' strategies, the social marketing of disease prevention, DOTS expansion programs, and a few cases of product support (eg. bednets in Tanzania, antiretrovirals in Nigeria) [ 36 ]. One strategy the Fund has employed to ensure a broad consensus on an individual country's programming is to require that every application be done through a partnership that includes representatives from governments, civil society, and people affected by the diseases. The goal of these Country Coordinating Mechanisms (CCMs) is to improve coordination of their activities and to avoid duplication [ 36 ]. But the extent to which the GFATM lives true to its goal of building broad-based health systems thus remains to be seen. The Global Alliance for Vaccines and Immunizations, another global body active in the arena of international health, appears to provide a model for balancing these approaches: The alliance's executive director is reported to have said that an optimum balance might be 60% of funds for new vaccines and 40% for strengthening immunization services. To encourage recipients to meet the targets set, additional funds for strengthening health systems will be released only once the countries have reached higher levels of immunization coverage [ 38 ]. But ironically, "failure to meet targets could indicate the need for greater support to weak health systems rather than withholding of funds" [ 38 ]. Investing in horizontal programs for developing health systems would not only provide much-needed basic services for individuals suffering from these and other diseases at present, but would also lay essential groundwork for the future delivery of targeted interventions such as medications and immunizations, once they become available. The way ahead lies in fostering innovative solutions that integrate vertical, disease-specific programming for AIDS, tuberculosis and malaria with much-needed health systems support. The second issue of interest in the Fund's granting process was how the Fund balanced treatment programs with prevention programs in allocating its monies. It is now widely recognized that antiretroviral treatment is a cornerstone of HIV/AIDS control that must not play second fiddle to prevention, and that treatment for HIV plays an important role in controlling tuberculosis and malaria infection as well [ 39 ]. In its official call for funding applications in January, the GFATM articulated a commitment to the "prevention, treatment, care and support of the infected and directly affected" [ 36 ], and in his July 2002 speech to the XIV International AIDS Conference in Barcelona, executive director Richard Feachem reiterated this dedication. He stated that the first round of grants "will double the current number of people receiving Highly Active Anti-Retroviral Therapy (HAART) in the developing world and in Africa HAART recipients will increase six fold as a result of these commitments" [ 40 ]. But as Feachem himself acknowledges, these achievements are "nothing like enough". The inadequacy may largely be due to the continual reluctance of industrialized countries to finance treatment programs – particularly for expensive antiretroviral (ARV) therapy. Indeed, officials from Malawi and other countries were allegedly encouraged by donor countries to remove a treatment component from their GFATM proposal [ 37 ]. Yet at around the same time, the WHO made the groundbreaking move of adding ARVs to its Essential Medicines List [ 41 ]. The debate over funding medicines encompasses a deeper issue, about balancing the potentially limitless need for costly pharmaceuticals with the financial interests of the pharmaceutical companies that manufacture them. Macroeconomic analysis by the United Kingdom's Performance and Innovation Unit (UKPIU) [ 24 ] asserts that in order to establish any reasonable hope for the widespread availability of medications, vaccines and other health products for these diseases in the future, the Fund should provide a secure market for affordably-priced goods. Further, it should signal this commitment through advance-purchase commitments. The document argues that it is only through a willingness to cover the costs of manufacture, as well as the financial risks of research, that the global community can hope to drive research and development into essential medicines and vaccines. But wherein lies the balance of power in a system which lays corporate bottom lines at the foundation of a global effort to combat the diseases of poverty? Half the members of the commission that wrote the UKPIU report are from the pharmaceutical industry. By no means does this invalidate their findings – indeed, the report applies sound economic theory in reaching rational, pragmatic conclusions. But already, the public sector provides most of the market for pharmaceutical products, through public health insurance schemes. Furthermore, a review of the chemotherapeutic agents developed by the pharmaceutical industry over the past 25 years reveals how despite increased protection in the form of extended patent durations, the industry has not shown a concomitant increase in innovation [ 42 ]. The lack of new medicines for neglected diseases of the world's poor populations is palpable: of 1393 new chemical entities marketed over this time period, only 16 were for tropical diseases and tuberculosis [ 42 ]. Taken together, these arguments form the basis for calls for innovative new mechanisms for improving the development of and access to therapies for major infectious diseases. Contrasting the views of industry are the recommendations of another global constituency with vested interests in the GFATM's design – namely, the health NGO sector. Deeply concerned about the Fund becoming a mere 'pharmaceutical industry subsidy' [ 43 ], NGOs have made passionate pleas that priority be given to finding the most affordable, effective treatment available when GFATM monies are used to purchase medications. As observed by Director of Médecins Sans Frontières' Access to Essential Medicines Campaign Bernard Pecoul, an explicit statement of this commitment is conspicuously missing from the Fund's official documentation [ 44 ]. Practically speaking, the Fund must ensure that expensive, brand-name antiretroviral drugs are not blindly purchased where legal mechanisms could allow the purchase of up to three times the quantity of an equally efficacious generic version of the same medications. In the excitement over ensuring that large-scale efforts to treat the three diseases maintain adequate respect for intellectual property rights, potential beneficiaries must be explicitly reminded that obtaining generic and branded medicines through alternative mechanisms such as compulsory licensing and parallel import arrangements are entirely consistent with the Trade-Related Aspects of Intellectual Property Rights agreement (TRIPS) [ 45 ]. The legality of these measures was explicitly agreed to at the WTO's 2001 meeting in Doha, Qatar. Paragraph four of the declaration reads: "We agree that the TRIPS Agreement does not and should not prevent Members from taking measures to protect public health. Accordingly, while reiterating our commitments to the TRIPS Agreement, we affirm that the Agreement can and should be interpreted and implemented in a manner supportive of WTO Members' right to protect public health and, in particular, to promote access to medicines for all" [ 46 ]. Practical legal experience such as that garnered through Brazil's success with using the mechanism of compulsory licensing to reduce the price of both generic and brand ARVs down should be translated elsewhere, so that potential applicants might gain access to the most affordable quality medicines. Of relevance to this discussion of pharmaceutical and NGO interests, another set of policy tensions in the GFATM's history relates to its governance. The very composition of the Fund's Executive Board was under contentious debate in preliminary sectoral consultations by the Fund's Transitional Working Group. Since its inception, the Fund has billed itself as a "public-private partnership", yet consultation with the NGO sector initially recommended that no representatives of the pharmaceutical industry be members of the Board. Meanwhile, the private sector itself asked for more than the proposed two allotted positions on the Fund's 15-person Board, requesting in the interim an additional ex-officio observer seat. For the present, both constituencies retain two Board positions, which will hopefully preserve a balance between private and public perspectives. The Challenge of Funding the Fund The Global Fund represents an important opportunity for visionary leadership and meaningful action towards reducing the horrific tolls of HIV/AIDS, TB and malaria. The challenges which lie ahead for the GFATM lie in fostering and funding innovative projects which integrate vertical approaches with horizontal approaches, and balance preventive programs with treatment. But even beyond these programming dilemmas, how easily will it reach its lofty goals? A quick survey of the GFATM's progress to date reveals its first major barrier, in the form of grossly inadequate funds. If the international community is truly as committed to stamping out these three diseases as it would have the world believe, it must drastically scale up its financial commitments. At this writing, the fund totals little more than $2.1 billion [ 36 ] – a relatively paltry sum when compared to a recent report that put the minimum price tag for global HIV control at $7.5 billion annually for that disease alone [ 47 ]. Commendable shows of leadership have been made by Kofi Annan himself, who initiated the fund by personally donating $100,000 of prize money from his Philadelphia Liberty Medal, and by the governments of Sub-Saharan Africa, which set target commitments of 15% of their annual national budgets to be devoted to health sector improvements for HIV/AIDS at an April 2001 summit in Abuja, Nigeria. But what of the leadership from the G8 countries, out of whose own summits the very idea of the Global Fund first arose? To date, the G8 have collectively committed about $1.6 billion of the $2.1 billion total [ 36 ]. The Unites States has pledged by far the greatest proportion of this amount, at $500 million. But these seemingly impressive dollar figures fall far sufficient of the money needed. At least $US 1.3 billion each year is required to support basic commodities for prevention and treatment for malaria among vulnerable groups [ 48 ]. Thus far, less that $US 23 million has been awarded by the Fund for Malaria. African countries, which represent 90% of the global malaria burden, gets only $US 12.7 million [ 48 ]. In the wake of post-September 11 anthrax scares, for instance, the 2003 US Homeland Security Budget has proposed $5.9 billion to defend against bioterrorism [ 49 ]. Similarly, the seemingly astronomical price tag of $7–10 billion for HIV/AIDS control is dwarfed by the still more astronomical annual expenditures on military and defense budgets the world over. Global military spending totaled $1 trillion in 1990 alone, and industrialized countries spend 5.3% of GNP on military expenditures each year [ 23 ]. In contrast, these same countries spend less than 0.3% of GNP on overseas development assistance (ODA) each year – far short of their mutually-agreed upon target of 0.7–1.0% of GNP [ 23 ]. A glimmer of hope shone over the weeks leading up to the recent June 2002 G8 Summit in Kananaskis, Canada, where a 'New Partnership for African Development' (NEPAD) was placed high on the agenda. Drafted by African leaders themselves, the proposal's great innovation was that aid spending on the continent would be more reliably spent, since NEPAD required them to pass an African peer review process. But despite the enthusiasm about this attention to Africa, the industrialized countries still failed to make AIDS, tuberculosis and malaria a priority in Kananaskis. UN Envoy on AIDS in Africa Stephen Lewis made this point abundantly clear in his speech to the Alternative Summit that ran parallel to the official G8 proceedings [ 50 ]. He notes that while the NEPAD document sets admirable goals (an annual growth rate of 7% for fifteen years, halving poverty by 2015, a two-thirds reduction in infant mortality, a 25% reduction in maternal mortality, and education for all children), none of them are realistically attainable unless the HIV/AIDS pandemic receives the attention it deserves. Yet, NEPAD pays little attention to the disease in its proposals. The global community must rethink its approach to 'development', with HIV/AIDS and the other major infectious diseases at the core of its analysis. This is precisely the argument of the much-heralded recent WHO Report of the Commission on Macroeconomics and Health. In it, Jeffrey Sachs asserts that The burden of disease in some low-income regions, especially sub-Saharan Africa, stands as a stark barrier to economic growth and therefore must be addressed frontally and centrally in any comprehensive development strategy. The AIDS pandemic represents a unique challenge of unprecedented urgency and intensity. This single epidemic can undermine Africa's development over the next generation, and may cause tens of millions of deaths in India, China, and other developing countries unless addressed by greatly increased efforts [ 51 ]. The importance, then, of combatting AIDS, tuberculosis and malaria has been made clear in the global arena. If global health is truly understood as a 'global public good', the necessary finances must be mobilized by whatever means necessary. One proposal offers compelling reasons to open up national health budgets to fund health development in the international arena [ 7 ]. By this argument, money budgeted for investment in the health of one country's population is just as appropriately spent on global health problems as domestic ones, since the health of the world's populations are so closely intertwined. Regardless of the accounting logistics, it must ultimately be realized that the funds required for the control of AIDS, tuberculosis and malaria do exist, and must be made available through a careful re-examination of funding priorities. The Way Ahead The GFATM holds considerable promise for harnessing true international commitment to addressing the three diseases. But even if the Global Fund attains its massive targets of $7–10 billion US per year, does it truly have the capacity to mend the damage from diseases so mired in centuries of growing global inequality? HIV/AIDS, tuberculosis and malaria are diseases that demand consideration of populations' underlying predisposition to disease in the forms of socioeconomic inequality and abject poverty. While the motivations of the international community for addressing the diseases include pragmatic concerns of international security, economic prosperity and domestic health status, they must ultimately include the ethical responsibility to redress gross inequalities. Adequate attention to the systemic forces underlying these infections thus necessitates correspondingly systemic solutions. As discussed already, not least among these is the need to mobilize far larger sums of money to invest in world health and in redressing social inequalities. For decades, much of Africa has been left to stagnate in a perpetual "poverty trap" [ 52 ], in which the state is simply too poor to provide adequate basic living conditions for the population. Infectious diseases are both a cause and a consequence of this lack of health care, education and infrastructure. Direct financial transfers and investment in basic needs in such countries are the only viable solutions to the continent's ongoing health and economic crises. Hand in hand with this financial commitment is the need to relieve those developing countries with unreasonable debt burdens of these outlandish costs. Debt repayment schedules paralyze national budgets and lock them into paying back unsustainable sums of money to high-income countries and financial institutions every year. On average, debtor countries pay one and a half times as much in servicing debt as they do on health care [ 53 ]. Debt relief is imperative if the Global South's bankrupt governments are ever to address their populations' basic health needs. Economic theory asserts that for a creditor nation, "the outright cancellation of debt becomes . . . a necessary part of its foreign policy" if it hopes to promote economic growth as well as its own strategic interests in bankrupt states [ 47 , 52 ]. Similarly, industrialized countries must reform trade policies that create absurd financial barriers to the integration of poor countries' economies into the global marketplace. Import tariffs on many African goods destined for the United States, for examples, can reach levels of up to 33% – up to 15 times the average US tariff rate of 2% [ 54 ]. Structural barriers such as these clearly impair the capacity of poor countries to attain anything close to equal footing with rich ones in their attempt to bring economic prosperity to their people. Finally, in our increasingly globalized economic system, the 'right' of transnational corporations to global patent protection for medicines that are almost exclusively sold to the minority in the rich West must become more intimately tied to their international responsibilities, in the form of technology transfer, local capacity building, and investments in basic infrastructure. Nowhere is this more true than for the pharmaceutical industry, where the public sector must play an active role in obliging its private sector partners to invest in research and development for neglected diseases, commit to equitable pricing schemes and participate in technology transfer. It is only by linking rights and responsibilities that we can hope to achieve improved health for all the world's inhabitants. Summary Research abounds which links these glaring inequalities and potential solutions to epidemic infectious diseases. The UNGASS Declaration of Commitment on HIV/AIDS already represents one forum in which the global call for such measures as ODA increases and debt relief has been clearly spelled out [ 13 ], and the Commission on Macroeconomics and Health has provided another powerful voice for increased international commitment. The challenge ahead lies in directly transforming research data and our moral obligation into concrete international policy. HIV/AIDS, TB and malaria are diseases mired in longstanding fundamental inequalities. Structural changes in policy such as a realignment of spending priorities, debt relief, equitable trade policies and a commitment to global corporate responsibility must complement other economic aid mechanisms for improving public health in the future. Achieving an appropriate balance between vertical and horizontal programming and between prevention and treatment strategies are issues to be resolved by the GFATM and other institutions at the programming level. At a more fundamental level, the G8 countries that spearheaded the GFATM initiative must show political and financial leadership in putting these cold, sobering realities at the forefront of the international agenda if their intentions to curb the toll of these three diseases are to be effectively realized. Competing interests None Authors' contributions DT wrote the initial and final drafts of the manuscript. RU and NF provided valuable support and instructive comments on all drafts. All authors read and approved the final manuscript. Opinions expressed are those of the authors and may not represent the official views of the employing organization. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgments Dr Upshur is supported by a New Investigator Award from the Canadian Institutes of Health Research and a Research Scholar Award from the Department of Family and Community Medicine, University Of Toronto. The authors are very grateful to Shari Gruman for her expert assistance in the preparation of the manuscript.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923779/

Iron Acquisition in Bacillus cereus : The Roles of IlsA and Bacillibactin in Exogenous Ferritin Iron Mobilization

In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus , a surface protein named IlsA ( I ron-regulated l eucine rich s urface protein type A ) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis. Introduction Iron is an essential nutrient for most forms of life. Owing to the high variability of the Fe 3+ /Fe 2+ redox potential, this transition metal fulfills a large number of biological processes including respiration and DNA synthesis. However, because of its low solubility and propensity to generate highly reactive hydroxyl radicals, iron is a double-edged element and its homeostasis must be finely tuned [1] . Given that most microorganisms require micromolar iron concentrations for growth and multiplication [2] , the ability to obtain iron is thus an important adaptation factor requiring intricately sophisticated iron uptake systems [3] . Upon infection, the host sets up a form of nutritional immunity aimed at depriving the invader of nutritional iron through iron redistribution in the organism and scavenging of certain microbial siderophores [4] , [5] . The importance of this strategy is evidenced by the effects of iron homeostasis disorders on both innate and acquired immune responses [2] , [6] . In this battle for iron, and to circumvent host-iron withholding, pathogenic bacteria are able to acquire iron via siderophore-based systems or by surface and membrane anchored proteins which interfere with host iron-containing proteins such as transferrins, hemoproteins or ferritins [7] . Most of these iron acquisition systems are under the control of the global regulator Fur (Ferric uptake regulator) which also regulates the expression of multiple virulence factors [8] . Over the past 10 years, our understanding of iron import into bacteria has been greatly improved [9] , [10] . The most impressive advances concerned heme acquisition in Gram-positive bacteria. One major discovery has been made with the characterization of the Isd ( I ron-regulated s urface d eterminant) system in Staphylococcus aureus [11] . Heme or hemoglobin interaction with this system relies on cell wall–anchored proteins that act as hemoprotein receptors by means of their NEAT ( NEA r iron T ransporter) domains. Since then, a growing number of studies have emphasized the role of NEAT domains in heme binding in several Gram-positive bacteria including S. aureus , Streptococcus pyogenes , Bacillus anthracis (for review, see [12] ) or Bacillus cereus (Abi Khalil et al. , unpublished data). Although most iron is bound to hemoglobin in vertebrates, ferritin can represent another important source of iron for microbes [13] , [14] , [15] , [16] , [17] , [18] , [19] . Ferritin is a well-studied ubiquitous protein found in both prokaryotes and eukaryotes. In animals, it is composed of 24 subunits that self-assemble through non-covalent interactions into a hollow spherical shell. Due to the presence of two types of subunits, H (heavy) and L (light), multiple ferritin isoforms exist whereby up to 4500 iron atoms can be mineralized inside the nanocage [20] . Whereas the main role of ferritin is to store iron under a safe and bioavailable form, other biological functions have been proposed [21] . This extremely stable protein represents a concentrated source of iron and thus could be a perfect target for microbes. However, the molecular basis of ferritin utilization by pathogens remains poorly documented. Recent studies have shown that the intracellular bacterium Neisseria meningitidis can indirectly utilize ferritin by inducing an iron starvation state within epithelial cells leading to ferritin degradation and release of free iron [17] . Other in vitro studies showed that ferritin utilization relies on proteolytic degradation in the cystic fibrosis-associated pathogen Burkholderia cenocepacia [19] and on a reductase activity in Listeria monocytogenes [22] . To our knowledge, only two molecular determinants directly involved in iron acquisition from ferritin have been identified: (i) Als3, a Candida albicans invasin-like protein [13] and (ii) IlsA, a Bacillus cereus surface protein [14] . It has been suggested that both proteins are ferritin receptors but direct in vitro binding to ferritin has only been reported in B. cereus [14] . B. cereus is a Gram-positive, spore-forming bacterium. This opportunistic human pathogen is frequently associated with food-borne infections due to the production of diarrheal and emetic toxins. Rare non-gastrointestinal infections such as meningitis, pneumonia, endophthalmitis or gas gangrene-like cutaneous infections have also been observed [23] . As a member of the B. cereus sensu lato group, B. cereus is closely related to other species of this group such as B. anthracis , the etiological agent of anthrax in mammals and the entomopathogen, Bacillus thuringiensis [24] . The ability of B. cereus and B. thuringiensis to colonize the host (mammal or insect) is linked to the presence of multiple adaptation and virulence factors, one of which is the capacity to acquire iron [25] . Several host iron sources can be used by B. cereus , including hemoproteins and ferritin [14] , [26] , [27] , [28] , [29] , [30] , [31] . In the past few years, various systems involved in iron acquisition have been discovered and some of them are required for full virulence in animal models ( Table S1 ). The adaptation of B. cereus to iron paucity in host tissues is also illustrated by the Fur-regulation of the cytotoxin HlyII [32] . Among the iron uptake systems characterized in B. cereus , a surface protein, IlsA (NCBI gene number Bc1331), is involved in both ferritin and heme/hemoglobin acquisition [14] , [33] . This protein is composed of a unique combination of three conserved domains: an N-Terminal NEAT domain followed by 13 LRRs ( L eucine R ich R epeat) and three C-Terminal SLH ( S - L ayer H omology) domains and has been shown to interact with heme and hemoglobin via the NEAT domain [14] (Abi Khalil et al. , unpublished data, a revised manuscript has been re-submitted to Metallomics). Affinity tests revealed that IlsA binds to ferritin although the details of this interaction have not been described [14] . As a Fur-regulated gene, ilsA is specifically expressed in the insect hemocoel and under iron-depleted conditions [14] , [33] . Moreover, the virulence and the survival of the ΔilsA mutant are reduced in the lepidopteran insect model Galleria mellonella suggesting IlsA involvement in optimal colonization of a susceptible host [14] . These results prompted us to investigate the interaction between IlsA and ferritin and examine the possible involvement of two siderophores produced by B. cereus , bacillibactin and petrobactin, as partners of IlsA in ferritin utilization. Here, we demonstrate that IlsA acts as ferritin receptor on the surface of B. cereus . Isothermal titration calorimetry data indicate a binding stoichiometry of 24 IlsA per ferritin molecule (i.e. one IlsA per ferritin subunit). In vitro iron release kinetics showed significant increase of iron mobilization from ferritin in the presence of IlsA. In addition, our in vivo results show that bacillibactin is essential for iron acquisition from ferritin and for full virulence of B. cereus in an insect model, suggesting that IlsA and bacillibactin may work synergistically to effectively mobilize iron from host ferritin. Results IlsA is required for ferritin binding in vivo Earlier studies showed that under iron-restricted conditions IlsA was located on the surface of B. cereus , and ELISA assays and Surface Plasmon Resonance measurements indicated a possible interaction between IlsA and ferritin in vitro [14] . To demonstrate the involvement of IlsA in the binding of ferritin to bacterial cells in vivo , ferritin localization was followed by immunofluorescence using the polyclonal antibody anti-HoSF ( Ho rse S pleen F erritin) ( Figure 1 ). When the wild-type strain was grown in ferritin-enriched LB medium, a condition under which ilsA is not expressed [14] , [33] , ferritin was not immuno-detected on the bacterial cell surface. In sharp contrast, in iron-depleted medium supplemented with HoSF, ferritin aggregation was observed on the surface along the chains of bacterial cells. Moreover, ferritin recruitment was abolished in the ΔilsA mutant whereas complementation of this mutation with a wild-type copy of ilsA restored ferritin aggregation on the bacterial surface ( Figure 1 ). Because B. cereus has its own bacterial ferritins, the antibodies were tested against the bacteria and no staining was observed in living cells; fluorescence was only detected in permeable dead cells ( Figure S1 ). Collectively, our data are in agreement with the expression profile and the localization of IlsA during iron starvation [14] , [33] and indicate that IlsA acts as a ferritin receptor in vivo (bacterial culture) too. 10.1371/journal.ppat.1003935.g001 Figure 1 IlsA is essential for ferritin binding on B. cereus cell surface. B. cereus wild type (WT; A–F), ΔilsA mutant (G–I) and the complemented ΔilsAΩilsA (J–L) strains were grown in LB+0,3 µM HoSF ( Ho rse S pleen F erritin) medium (only the wild type; A–C) and in iron-depleted LB (Dip) +0,3 µM HoSF medium (D–L). Bacterial cells were washed before fluorescence microscopy analysis using HoSF Alexa Fluor 594 labelled polyclonal antibody (B,E,H,K) or DAPI to stain DNA. The merged images (C,F,I,L) show DAPI in blue and ferritin in red. Experiments were performed three times. In iron rich LB medium, IlsA is not produced [14] and no ferritin is detected on the bacterial surface in these conditions (A–C). Ferritin aggregates only on the surface in iron-depleted medium supplemented with ferritin (D–F). Absence of llsA in the Δ ilsA mutant compromises ferritin binding on the bacterial surface (G–I) whereas ilsA complementation restores ferritin aggregation (J–L). IlsA interacts with each ferritin subunit To further investigate the interaction of IlsA with the ferritin shell, the binding between the two purified proteins was followed in vitro by isothermal titration calorimetry (ITC). This highly sensitive thermodynamic technique measures heat variations during molecular interactions. In a single experiment, a complete thermodynamic profile of the interaction is obtained with the concomitant determination of the binding constant ( K ), the binding stoichiometry ( n ), the enthalpy (Δ H °), the entropy (Δ S °) and the free energy (Δ G °) changes of binding. The ITC technique has been proven very successful in obtaining accurate thermodynamic data for a number of molecular interactions involving ferritin [34] , [35] or IlsA (Abi Khalil et al. , unpublished data). Each ITC experiment was repeated two to four times to ensure accuracy and reproducibility. Figure 2 shows the injection heats for IlsA binding to recombinant human H-chain ferritin (HuHF) at pH 7.0 and 25°C (A) and the integrated heats (μJ) for each injection against the molar ratio of IlsA to HuHF after subtraction of the control heats (B). The other ferritins tested (recombinant human homopolymer L-chain, recombinant human heteropolymer ferritin composed of ∼20H-chains and 4L-chains and recombinant mouse homopolymer H-chain) showed similar ITC isotherms ( Figure S2 ). All of the experimental thermodynamic parameters obtained from curve fittings of the integrated heats to a model of one set of independent binding sites are compiled in Table 1 . As the concentration of IlsA increases following successive injections into the ITC reaction cell containing ferritin, saturation is reached and subsequently less heat is absorbed on further addition of IlsA. The positive upward peaks seen in Figure 2A correspond to an exothermic reaction with a binding stoichiometry of ∼24 IlsA per ferritin shell and an apparent dissociation constant (K d ) of ∼540 nM. The binding of one IlsA molecule to one ferritin subunit did not alter binding of additional IlsA to the remaining subunits suggesting similar affinities and direct interactions between IlsA molecules and the 24-mer protein. The negative enthalpy change (∼−4 to −10 kJ/mol) and the large and positive entropy of binding (∼85 to 110 J/(mol.K)) observed with all IlsA-ferritin interactions indicate that the interaction is both enthalpically and entropically driven. The most likely contributions to the large positive ΔS 0 values are probably due to changes in the hydration of the two proteins upon binding leading to an overall increase of the disorder of the system. To determine whether IlsA NEAT or LRR domains are involved in ferritin binding, dot blot experiments were performed separately on either domain following their individual expression and purification. No significant binding was observed between HoSF and the NEAT domain and HoSF was found to bind weakly to the LRR domains while a strong binding was apparent with full-length IlsA ( Figure S3 ). These results suggest that the presence of both the NEAT and LRR domains may be crucial for optimal binding of ferritin to IlsA. However, we cannot exclude the possibility that the observed weak binding of HosF with the purified domains is a consequence of incorrect domain folding. 10.1371/journal.ppat.1003935.g002 Figure 2 Calorimetric titration of recombinant human H-chain ferritin with IlsA. ( A ): ITC ( I sothermal T itration C alorimetry) raw data. ( B ): Plot of the integrated heat versus the number of injections of IlsA. Conditions: 1 µM holoHuHF (recombinant Hu man H -chain F erritin) titrated with 3 µl injections of 229 µM IlsA solution in 50 mM Tris/HCl buffer, 150 mM NaCl, 1 mM EDTA and 1 mM DTT, pH = 7.0 and 25°C. ITC binding experiments were repeated at least two times with similar results and thermodynamic data are listed in Table 1 . 10.1371/journal.ppat.1003935.t001 Table 1 Best fit parameters for ITC measurements of IlsA binding to ferritins a . Protein 1 Protein 2 n K (M −1 ) Δ H 0 (kJ/mol) Δ G 0 (kJ/mol) b Δ S 0 (J/(mol.K)) c Holo-IlsA HuHF 25.21±2.62 (1.86±0.99) ×10 6 −4.11±0.11 −35.78±1.32 106.24±4.44 Holo-IlsA HuLF 23.88±0.1 (8.36±0.24) ×10 5 −8.71±3.15 −33.80±0.07 84.16±10.56 Holo-IlsA HuH/LF 23.79±0.89 (1.08±0.21) ×10 6 −8.72±2.47 −34.44±0.48 86.26±8.44 Holo-IlsA MoHF 24.94±1.73 (2.07±0.66) ×10 6 −3.35±0.32 −36.05±0.79 109.68±2.85 a The reported thermodynamic quantities are apparent values and include the contributions to the overall equilibrium from ferritin and buffer species in different states of protonation. Standard errors from replicate determinations are indicated. b Calculated from Δ G 0 = − RT ln K . c Calculated from Δ S 0 = (Δ H °−Δ G °)/ T . HuHF, recombinant human H-chain ferritin; HuLF, recombinant human L-chain ferritin; HuH/LF, recombinant heteropolymer ferritin of 21H-chains and 4L-chains; MoHF, recombinant mouse H-chain ferritin. All experiments were repeated two to four times. IlsA enhances iron release from ferritin To examine whether IlsA plays any role in iron mobilization from ferritin, in vitro spectrophotometric kinetic experiments using HuHF in the presence of IlsA were performed under aerobic non-reducing conditions. Because bacillibacftin is not available commercially and is very hard to purify, we used the bacterial siderophore DFO ( D e f er o xamine B) as a reporter molecule (i.e. an Fe(III)-chelator) to follow the kinetics of iron release from ferritin. Figure 3 shows that DFO alone (in the absence of IlsA) was able to release only a small amount of iron from HuHF loaded with ∼500 Fe/protein at a very slow rate (less than 5% after 90 minutes), a result in accord with earlier data obtained with other ferritins [36] , [37] , [38] . However, in the presence of llsA and DFO, a faster rate and a significant amount of iron was released from the protein (∼25% of total iron present within the protein) during the same time period ( Figure 3 ) suggesting a role of IlsA in enhancing iron mobilization from ferritin. It is conceivable that IlsA might alter the ferritin structural integrity rendering the iron core more accessible to iron chelators such as microbial siderophores. 10.1371/journal.ppat.1003935.g003 Figure 3 Role of IlsA in iron mobilization from ferritin. Demineralization of recombinant holoHuHF (recombinant Hu man H -chain F erritin, 1 µM) containing 500 Fe/shell was followed by the absorption of the Fe(III)-DFO ( D e f er o xamine B) complex at 425 nm in the presence (black line) or absence (dotted line) of IlsA (5 µM). The experiment was repeated in triplicate using different protein preparations. Curves are averages of three independent runs and error bars are SEM from mean values. Production of siderophores in B. cereus In iron-depleted medium, B. cereus and B. anthracis synthesize bacillibactin and petrobactin, two catechol-based siderophores that are differently regulated and have different affinity for iron [39] , [40] . The organization of the biosynthetic gene clusters for both siderophores is highly conserved in the two species. Petrobactin and bacillibactin productions rely on the asbABCDEF and entA-dhbBCF clusters, respectively. Mutant strains were obtained from deletions of the asbABCDEF cluster and entA gene in the wild-type strain ( Figure 4 ). To evaluate the relative contribution of the two siderophores in catechol production, the total siderophore production in wild-type and isogenic mutant strains Δasb and ΔentA and double mutant ΔentAΔasb were compared using the Arnow assay [41] ( Figure 5 ). The expression of the asbA and entA genes was activated in the wild type strain grown under iron-depleted conditions (data not shown) and catechol production was detected in the wild type. The production of catechols was almost four times lower in the bacillibactin ΔentA mutant and the wild-type production was restored following complementation of ΔentA mutant with entA gene. In contrast, catechol production was not impaired in Δasb strain, suggesting a possible overproduction of bacillibactin to compensate for the absence of petrobactin. The strongest reduction in catechol production was observed in the ΔentAΔasb double mutant ( Figure 5 ). These data indicate that bacillibactin represents the primary siderophore produced by B. cereus in low iron environment. 10.1371/journal.ppat.1003935.g004 Figure 4 Construction of B. cereus siderophore mutants. Genetic organization of petrobactin ( A ) and bacillibactin ( B ) biosynthetic gene clusters in B. cereus strain ATCC 14579 is represented. The deleted genes (in black) and the orientation of the antibiotic cassettes (tetracycline, tet R and kanamycin, km R ) are depicted. Deletions were created by integrative recombination in the loci using upstream and downstream region (∼1 kb each) amplified with primer pairs (+) and (x) for the asb locus or (*) and (°) for entA gene. In addition, the promoter region (359 bp) of entA was cloned between the km R cassette and dhbC ( P in gray box) to ensure transcription of downstream genes. For ΔentA complementation, (□) represent the primers used to amplify the fragment cloned in pHT304 plasmid. 10.1371/journal.ppat.1003935.g005 Figure 5 Production of catechol siderophores in Δasb and ΔentA mutants. Culture supernatants were collected for each strain from overnight (20 h) cultures in low iron conditions. Measurement of catechol productions was achieved using the Arnow assay. Data were normalized to the OD 600 of cultures and percentages of wild-type (WT) catechol level are shown. Error bars represent SEM from mean values of three independent experiments. * Significant difference compared to wild type ( P <0.001). ** Significant difference compared to ΔentA strain ( P <0.05). Bacillibactin is essential for iron acquisition from ferritin The ability of siderophores to remove iron from ferritin in vitro has been documented [42] . To investigate the ability of B. cereus siderophores to extract iron from ferritin in vivo , the growth of the wild type and mutant strains in different media was followed. No difference in growth was noticed in LB ( Figure 6A ). In iron-depleted medium, bacterial growth was strongly reduced for all strains (OD max after 16 h∼0.1), the ΔentA and ΔentAΔasb mutants being the most affected strains ( Figure 6B ). Supplementation with ferritin as sole iron source restored the growth of the wild type and the Δasb strains whereas the mutants disrupted in bacillibactin production were still unable to grow under these conditions ( Figure 6C ). The importance of entA (and therefore of bacillibactin) in iron acquisition from ferritin was further confirmed with the ΔentΩentA complemented strain since its ability to grow with ferritin was fully rescued ( Figure 6C ). Zawadzka et al. showed that the B. cereus FeuA transporter could bind both bacillibactin and the exogenous E. coli siderophore enterobactin [43] . Our data showed that the growth defect with ferritin due to the entA mutation was recovered when E. coli enterobactin was added to the culture medium ( Figure 6D ) suggesting that the FeuA transporter might be involved in the import process of bacillibactin too. The results of our study emphasize an important role of bacillibactin in iron acquisition from ferritin in B. cereus in contrast to previous work with B. anthracis pointing out a major role of the siderophore petrobactin in bacterial growth under iron starvation [44] , [45] , [46] , [47] . 10.1371/journal.ppat.1003935.g006 Figure 6 Iron acquisition from ferritin relies on bacillibactin production. Growth kinetics of B. cereus wild type (WT; black square), Δ asb petrobactin mutant (blue triangle), Δ entA bacillibactin mutant (red circle), complemented ΔentAΩentA strains (purple diamond) and double Δ entA Δ asb mutant (grey cross). The strains were grown at 37°C in LB medium ( A ) and in LB medium treated with 2,2′-dipyridyl without addition of iron sources ( B ) or supplemented with 0.3 µM ferritin only ( C ) or with 0.3 µM ferritin and 5 µM enterobactin ( D ). Bacterial growth was monitored during 16 hours by measuring the optical density (OD) at 600 nm every hour. Curves are averages of three independent experiments and error bars are SEM from mean values. Bacillibactin is important for bacterial virulence in an insect In addition to iron storage, ferritin serves as iron transporter in insects. High amounts of ferritin are found in the insect hemocoel (mg/L quantity) compared to the low level of vertebrate plasma ferritin (µg/L quantity) [4] , [48] . Thus, insect ferritins represent an easily accessible iron-concentrated source for extracellular pathogens such as B. cereus and have been purified from the tissues and the hemolymph of the greater wax moth Galleria mellonella [49] , [50] , a very useful model to study bacterial pathogenesis [51] . While earlier studies have reported on the importance of IlsA in growth and survival of B. cereus in G. mellonella [14] , [33] , our current data suggest that bacillibactin acts in unison with IlsA in ferritin utilization. To investigate whether B. cereus siderophores are also involved in bacterial pathogenicity, virulence of the siderophore mutants was assayed in G. mellonella by direct injection into the hemocoel of various doses of mid-log phase bacteria (1×10 3 to 3×10 4 ). The number of living larvae was recorded for three days ( Figure 7 ) and the 50% lethal doses (LD 50 ) 24 hours after infection were evaluated by Probit analysis ( Table 2 ). ΔentA and ΔentAΔasb mutants were significantly less virulent than the wild-type strain ( Table S2 ) with a 5.6-fold and 6.7-fold decrease, respectively. At the highest dose, only the double mutant was affected. The virulence of the ΔentA strain complemented with wild-type entA ( ΔentAΩentA ) was partially restored and no difference was observed between the Δasb mutant and the wild-type strain. While most of the larvae died 24 hours after the injection of the wild type or the Δasb mutant, survival with the ΔentA mutant continued to decrease after the first 24 hours ( Figure 7 ). Since petrobactin is the first siderophore produced upon bacterial outgrowth from spores in B. anthracis [47] , the role of petrobactin in B. cereus virulence following inoculation with spores was then investigated. The infection tests with wild type and mutant spores yielded the same results as the previous assays with vegetative cells (data not shown). These results confirm that, in an insect model, bacillibactin plays a more important role than petrobactin in B. cereus virulence. Our data indicate that the strains impaired in bacillibactin production are still able to kill their host but slower than the wild type suggesting that bacillibactin is an important adaptation factor that allows B. cereus to disseminate into the low iron environment encountered in the insect hemocoel. 10.1371/journal.ppat.1003935.g007 Figure 7 Effects of siderophore deficiency on B. cereus virulence in G. mellonella are dose- and time-dependent. Wild type and mutant strains were injected separately into the hemocoel. For each strain, twenty last-instar larvae were infected with 3×10 4 ( A ), 1×10 4 ( B ) or 3×10 3 ( C ) of mid-log phase bacteria. The survival rate (% of alive/total number of infected larvae) was monitored for 72 hours after infection with the wild type (black square), Δ asb (blue diamond), Δ entA (red triangle), ΔentAΩentA (purple triangle), Δ entA Δ asb (grey circle) strains or PBS (green cross). Results are mean values of three to seven independent experiments and error bars indicate SEM from mean values. Based on these data, LD 50 were estimated and are reported in Table 2 . ( D ) Illustrates white alive and dead black larvae. 10.1371/journal.ppat.1003935.t002 Table 2 Virulence of siderophore mutants in G. mellonella. Strain LD 50 a LD 50 Confidence Limits Wild type BcATCC14579 1.8×10 3 3.1×10 2 –3.3×10 3 Δasb 3.4×10 3 1.2×10 3 –5.6×10 3 ΔentA 1.0×10 4 8.8×10 3 –1.2×10 4 ΔentAΩentA 6.4×10 3 4.7×10 3 –8.2×10 3 ΔentAΔasb 1.2×10 4 8.5×10 3 –1.6×10 4 a The 50% lethal doses, in number of colony forming units (cfu), were evaluated by Probit survival. analysis ( p <0.05). IlsA is required for ferritin binding in vivo Earlier studies showed that under iron-restricted conditions IlsA was located on the surface of B. cereus , and ELISA assays and Surface Plasmon Resonance measurements indicated a possible interaction between IlsA and ferritin in vitro [14] . To demonstrate the involvement of IlsA in the binding of ferritin to bacterial cells in vivo , ferritin localization was followed by immunofluorescence using the polyclonal antibody anti-HoSF ( Ho rse S pleen F erritin) ( Figure 1 ). When the wild-type strain was grown in ferritin-enriched LB medium, a condition under which ilsA is not expressed [14] , [33] , ferritin was not immuno-detected on the bacterial cell surface. In sharp contrast, in iron-depleted medium supplemented with HoSF, ferritin aggregation was observed on the surface along the chains of bacterial cells. Moreover, ferritin recruitment was abolished in the ΔilsA mutant whereas complementation of this mutation with a wild-type copy of ilsA restored ferritin aggregation on the bacterial surface ( Figure 1 ). Because B. cereus has its own bacterial ferritins, the antibodies were tested against the bacteria and no staining was observed in living cells; fluorescence was only detected in permeable dead cells ( Figure S1 ). Collectively, our data are in agreement with the expression profile and the localization of IlsA during iron starvation [14] , [33] and indicate that IlsA acts as a ferritin receptor in vivo (bacterial culture) too. 10.1371/journal.ppat.1003935.g001 Figure 1 IlsA is essential for ferritin binding on B. cereus cell surface. B. cereus wild type (WT; A–F), ΔilsA mutant (G–I) and the complemented ΔilsAΩilsA (J–L) strains were grown in LB+0,3 µM HoSF ( Ho rse S pleen F erritin) medium (only the wild type; A–C) and in iron-depleted LB (Dip) +0,3 µM HoSF medium (D–L). Bacterial cells were washed before fluorescence microscopy analysis using HoSF Alexa Fluor 594 labelled polyclonal antibody (B,E,H,K) or DAPI to stain DNA. The merged images (C,F,I,L) show DAPI in blue and ferritin in red. Experiments were performed three times. In iron rich LB medium, IlsA is not produced [14] and no ferritin is detected on the bacterial surface in these conditions (A–C). Ferritin aggregates only on the surface in iron-depleted medium supplemented with ferritin (D–F). Absence of llsA in the Δ ilsA mutant compromises ferritin binding on the bacterial surface (G–I) whereas ilsA complementation restores ferritin aggregation (J–L). IlsA interacts with each ferritin subunit To further investigate the interaction of IlsA with the ferritin shell, the binding between the two purified proteins was followed in vitro by isothermal titration calorimetry (ITC). This highly sensitive thermodynamic technique measures heat variations during molecular interactions. In a single experiment, a complete thermodynamic profile of the interaction is obtained with the concomitant determination of the binding constant ( K ), the binding stoichiometry ( n ), the enthalpy (Δ H °), the entropy (Δ S °) and the free energy (Δ G °) changes of binding. The ITC technique has been proven very successful in obtaining accurate thermodynamic data for a number of molecular interactions involving ferritin [34] , [35] or IlsA (Abi Khalil et al. , unpublished data). Each ITC experiment was repeated two to four times to ensure accuracy and reproducibility. Figure 2 shows the injection heats for IlsA binding to recombinant human H-chain ferritin (HuHF) at pH 7.0 and 25°C (A) and the integrated heats (μJ) for each injection against the molar ratio of IlsA to HuHF after subtraction of the control heats (B). The other ferritins tested (recombinant human homopolymer L-chain, recombinant human heteropolymer ferritin composed of ∼20H-chains and 4L-chains and recombinant mouse homopolymer H-chain) showed similar ITC isotherms ( Figure S2 ). All of the experimental thermodynamic parameters obtained from curve fittings of the integrated heats to a model of one set of independent binding sites are compiled in Table 1 . As the concentration of IlsA increases following successive injections into the ITC reaction cell containing ferritin, saturation is reached and subsequently less heat is absorbed on further addition of IlsA. The positive upward peaks seen in Figure 2A correspond to an exothermic reaction with a binding stoichiometry of ∼24 IlsA per ferritin shell and an apparent dissociation constant (K d ) of ∼540 nM. The binding of one IlsA molecule to one ferritin subunit did not alter binding of additional IlsA to the remaining subunits suggesting similar affinities and direct interactions between IlsA molecules and the 24-mer protein. The negative enthalpy change (∼−4 to −10 kJ/mol) and the large and positive entropy of binding (∼85 to 110 J/(mol.K)) observed with all IlsA-ferritin interactions indicate that the interaction is both enthalpically and entropically driven. The most likely contributions to the large positive ΔS 0 values are probably due to changes in the hydration of the two proteins upon binding leading to an overall increase of the disorder of the system. To determine whether IlsA NEAT or LRR domains are involved in ferritin binding, dot blot experiments were performed separately on either domain following their individual expression and purification. No significant binding was observed between HoSF and the NEAT domain and HoSF was found to bind weakly to the LRR domains while a strong binding was apparent with full-length IlsA ( Figure S3 ). These results suggest that the presence of both the NEAT and LRR domains may be crucial for optimal binding of ferritin to IlsA. However, we cannot exclude the possibility that the observed weak binding of HosF with the purified domains is a consequence of incorrect domain folding. 10.1371/journal.ppat.1003935.g002 Figure 2 Calorimetric titration of recombinant human H-chain ferritin with IlsA. ( A ): ITC ( I sothermal T itration C alorimetry) raw data. ( B ): Plot of the integrated heat versus the number of injections of IlsA. Conditions: 1 µM holoHuHF (recombinant Hu man H -chain F erritin) titrated with 3 µl injections of 229 µM IlsA solution in 50 mM Tris/HCl buffer, 150 mM NaCl, 1 mM EDTA and 1 mM DTT, pH = 7.0 and 25°C. ITC binding experiments were repeated at least two times with similar results and thermodynamic data are listed in Table 1 . 10.1371/journal.ppat.1003935.t001 Table 1 Best fit parameters for ITC measurements of IlsA binding to ferritins a . Protein 1 Protein 2 n K (M −1 ) Δ H 0 (kJ/mol) Δ G 0 (kJ/mol) b Δ S 0 (J/(mol.K)) c Holo-IlsA HuHF 25.21±2.62 (1.86±0.99) ×10 6 −4.11±0.11 −35.78±1.32 106.24±4.44 Holo-IlsA HuLF 23.88±0.1 (8.36±0.24) ×10 5 −8.71±3.15 −33.80±0.07 84.16±10.56 Holo-IlsA HuH/LF 23.79±0.89 (1.08±0.21) ×10 6 −8.72±2.47 −34.44±0.48 86.26±8.44 Holo-IlsA MoHF 24.94±1.73 (2.07±0.66) ×10 6 −3.35±0.32 −36.05±0.79 109.68±2.85 a The reported thermodynamic quantities are apparent values and include the contributions to the overall equilibrium from ferritin and buffer species in different states of protonation. Standard errors from replicate determinations are indicated. b Calculated from Δ G 0 = − RT ln K . c Calculated from Δ S 0 = (Δ H °−Δ G °)/ T . HuHF, recombinant human H-chain ferritin; HuLF, recombinant human L-chain ferritin; HuH/LF, recombinant heteropolymer ferritin of 21H-chains and 4L-chains; MoHF, recombinant mouse H-chain ferritin. All experiments were repeated two to four times. IlsA enhances iron release from ferritin To examine whether IlsA plays any role in iron mobilization from ferritin, in vitro spectrophotometric kinetic experiments using HuHF in the presence of IlsA were performed under aerobic non-reducing conditions. Because bacillibacftin is not available commercially and is very hard to purify, we used the bacterial siderophore DFO ( D e f er o xamine B) as a reporter molecule (i.e. an Fe(III)-chelator) to follow the kinetics of iron release from ferritin. Figure 3 shows that DFO alone (in the absence of IlsA) was able to release only a small amount of iron from HuHF loaded with ∼500 Fe/protein at a very slow rate (less than 5% after 90 minutes), a result in accord with earlier data obtained with other ferritins [36] , [37] , [38] . However, in the presence of llsA and DFO, a faster rate and a significant amount of iron was released from the protein (∼25% of total iron present within the protein) during the same time period ( Figure 3 ) suggesting a role of IlsA in enhancing iron mobilization from ferritin. It is conceivable that IlsA might alter the ferritin structural integrity rendering the iron core more accessible to iron chelators such as microbial siderophores. 10.1371/journal.ppat.1003935.g003 Figure 3 Role of IlsA in iron mobilization from ferritin. Demineralization of recombinant holoHuHF (recombinant Hu man H -chain F erritin, 1 µM) containing 500 Fe/shell was followed by the absorption of the Fe(III)-DFO ( D e f er o xamine B) complex at 425 nm in the presence (black line) or absence (dotted line) of IlsA (5 µM). The experiment was repeated in triplicate using different protein preparations. Curves are averages of three independent runs and error bars are SEM from mean values. IlsA enhances iron release from ferritin To examine whether IlsA plays any role in iron mobilization from ferritin, in vitro spectrophotometric kinetic experiments using HuHF in the presence of IlsA were performed under aerobic non-reducing conditions. Because bacillibacftin is not available commercially and is very hard to purify, we used the bacterial siderophore DFO ( D e f er o xamine B) as a reporter molecule (i.e. an Fe(III)-chelator) to follow the kinetics of iron release from ferritin. Figure 3 shows that DFO alone (in the absence of IlsA) was able to release only a small amount of iron from HuHF loaded with ∼500 Fe/protein at a very slow rate (less than 5% after 90 minutes), a result in accord with earlier data obtained with other ferritins [36] , [37] , [38] . However, in the presence of llsA and DFO, a faster rate and a significant amount of iron was released from the protein (∼25% of total iron present within the protein) during the same time period ( Figure 3 ) suggesting a role of IlsA in enhancing iron mobilization from ferritin. It is conceivable that IlsA might alter the ferritin structural integrity rendering the iron core more accessible to iron chelators such as microbial siderophores. 10.1371/journal.ppat.1003935.g003 Figure 3 Role of IlsA in iron mobilization from ferritin. Demineralization of recombinant holoHuHF (recombinant Hu man H -chain F erritin, 1 µM) containing 500 Fe/shell was followed by the absorption of the Fe(III)-DFO ( D e f er o xamine B) complex at 425 nm in the presence (black line) or absence (dotted line) of IlsA (5 µM). The experiment was repeated in triplicate using different protein preparations. Curves are averages of three independent runs and error bars are SEM from mean values. Production of siderophores in B. cereus In iron-depleted medium, B. cereus and B. anthracis synthesize bacillibactin and petrobactin, two catechol-based siderophores that are differently regulated and have different affinity for iron [39] , [40] . The organization of the biosynthetic gene clusters for both siderophores is highly conserved in the two species. Petrobactin and bacillibactin productions rely on the asbABCDEF and entA-dhbBCF clusters, respectively. Mutant strains were obtained from deletions of the asbABCDEF cluster and entA gene in the wild-type strain ( Figure 4 ). To evaluate the relative contribution of the two siderophores in catechol production, the total siderophore production in wild-type and isogenic mutant strains Δasb and ΔentA and double mutant ΔentAΔasb were compared using the Arnow assay [41] ( Figure 5 ). The expression of the asbA and entA genes was activated in the wild type strain grown under iron-depleted conditions (data not shown) and catechol production was detected in the wild type. The production of catechols was almost four times lower in the bacillibactin ΔentA mutant and the wild-type production was restored following complementation of ΔentA mutant with entA gene. In contrast, catechol production was not impaired in Δasb strain, suggesting a possible overproduction of bacillibactin to compensate for the absence of petrobactin. The strongest reduction in catechol production was observed in the ΔentAΔasb double mutant ( Figure 5 ). These data indicate that bacillibactin represents the primary siderophore produced by B. cereus in low iron environment. 10.1371/journal.ppat.1003935.g004 Figure 4 Construction of B. cereus siderophore mutants. Genetic organization of petrobactin ( A ) and bacillibactin ( B ) biosynthetic gene clusters in B. cereus strain ATCC 14579 is represented. The deleted genes (in black) and the orientation of the antibiotic cassettes (tetracycline, tet R and kanamycin, km R ) are depicted. Deletions were created by integrative recombination in the loci using upstream and downstream region (∼1 kb each) amplified with primer pairs (+) and (x) for the asb locus or (*) and (°) for entA gene. In addition, the promoter region (359 bp) of entA was cloned between the km R cassette and dhbC ( P in gray box) to ensure transcription of downstream genes. For ΔentA complementation, (□) represent the primers used to amplify the fragment cloned in pHT304 plasmid. 10.1371/journal.ppat.1003935.g005 Figure 5 Production of catechol siderophores in Δasb and ΔentA mutants. Culture supernatants were collected for each strain from overnight (20 h) cultures in low iron conditions. Measurement of catechol productions was achieved using the Arnow assay. Data were normalized to the OD 600 of cultures and percentages of wild-type (WT) catechol level are shown. Error bars represent SEM from mean values of three independent experiments. * Significant difference compared to wild type ( P <0.001). ** Significant difference compared to ΔentA strain ( P <0.05). Bacillibactin is essential for iron acquisition from ferritin The ability of siderophores to remove iron from ferritin in vitro has been documented [42] . To investigate the ability of B. cereus siderophores to extract iron from ferritin in vivo , the growth of the wild type and mutant strains in different media was followed. No difference in growth was noticed in LB ( Figure 6A ). In iron-depleted medium, bacterial growth was strongly reduced for all strains (OD max after 16 h∼0.1), the ΔentA and ΔentAΔasb mutants being the most affected strains ( Figure 6B ). Supplementation with ferritin as sole iron source restored the growth of the wild type and the Δasb strains whereas the mutants disrupted in bacillibactin production were still unable to grow under these conditions ( Figure 6C ). The importance of entA (and therefore of bacillibactin) in iron acquisition from ferritin was further confirmed with the ΔentΩentA complemented strain since its ability to grow with ferritin was fully rescued ( Figure 6C ). Zawadzka et al. showed that the B. cereus FeuA transporter could bind both bacillibactin and the exogenous E. coli siderophore enterobactin [43] . Our data showed that the growth defect with ferritin due to the entA mutation was recovered when E. coli enterobactin was added to the culture medium ( Figure 6D ) suggesting that the FeuA transporter might be involved in the import process of bacillibactin too. The results of our study emphasize an important role of bacillibactin in iron acquisition from ferritin in B. cereus in contrast to previous work with B. anthracis pointing out a major role of the siderophore petrobactin in bacterial growth under iron starvation [44] , [45] , [46] , [47] . 10.1371/journal.ppat.1003935.g006 Figure 6 Iron acquisition from ferritin relies on bacillibactin production. Growth kinetics of B. cereus wild type (WT; black square), Δ asb petrobactin mutant (blue triangle), Δ entA bacillibactin mutant (red circle), complemented ΔentAΩentA strains (purple diamond) and double Δ entA Δ asb mutant (grey cross). The strains were grown at 37°C in LB medium ( A ) and in LB medium treated with 2,2′-dipyridyl without addition of iron sources ( B ) or supplemented with 0.3 µM ferritin only ( C ) or with 0.3 µM ferritin and 5 µM enterobactin ( D ). Bacterial growth was monitored during 16 hours by measuring the optical density (OD) at 600 nm every hour. Curves are averages of three independent experiments and error bars are SEM from mean values. Bacillibactin is important for bacterial virulence in an insect In addition to iron storage, ferritin serves as iron transporter in insects. High amounts of ferritin are found in the insect hemocoel (mg/L quantity) compared to the low level of vertebrate plasma ferritin (µg/L quantity) [4] , [48] . Thus, insect ferritins represent an easily accessible iron-concentrated source for extracellular pathogens such as B. cereus and have been purified from the tissues and the hemolymph of the greater wax moth Galleria mellonella [49] , [50] , a very useful model to study bacterial pathogenesis [51] . While earlier studies have reported on the importance of IlsA in growth and survival of B. cereus in G. mellonella [14] , [33] , our current data suggest that bacillibactin acts in unison with IlsA in ferritin utilization. To investigate whether B. cereus siderophores are also involved in bacterial pathogenicity, virulence of the siderophore mutants was assayed in G. mellonella by direct injection into the hemocoel of various doses of mid-log phase bacteria (1×10 3 to 3×10 4 ). The number of living larvae was recorded for three days ( Figure 7 ) and the 50% lethal doses (LD 50 ) 24 hours after infection were evaluated by Probit analysis ( Table 2 ). ΔentA and ΔentAΔasb mutants were significantly less virulent than the wild-type strain ( Table S2 ) with a 5.6-fold and 6.7-fold decrease, respectively. At the highest dose, only the double mutant was affected. The virulence of the ΔentA strain complemented with wild-type entA ( ΔentAΩentA ) was partially restored and no difference was observed between the Δasb mutant and the wild-type strain. While most of the larvae died 24 hours after the injection of the wild type or the Δasb mutant, survival with the ΔentA mutant continued to decrease after the first 24 hours ( Figure 7 ). Since petrobactin is the first siderophore produced upon bacterial outgrowth from spores in B. anthracis [47] , the role of petrobactin in B. cereus virulence following inoculation with spores was then investigated. The infection tests with wild type and mutant spores yielded the same results as the previous assays with vegetative cells (data not shown). These results confirm that, in an insect model, bacillibactin plays a more important role than petrobactin in B. cereus virulence. Our data indicate that the strains impaired in bacillibactin production are still able to kill their host but slower than the wild type suggesting that bacillibactin is an important adaptation factor that allows B. cereus to disseminate into the low iron environment encountered in the insect hemocoel. 10.1371/journal.ppat.1003935.g007 Figure 7 Effects of siderophore deficiency on B. cereus virulence in G. mellonella are dose- and time-dependent. Wild type and mutant strains were injected separately into the hemocoel. For each strain, twenty last-instar larvae were infected with 3×10 4 ( A ), 1×10 4 ( B ) or 3×10 3 ( C ) of mid-log phase bacteria. The survival rate (% of alive/total number of infected larvae) was monitored for 72 hours after infection with the wild type (black square), Δ asb (blue diamond), Δ entA (red triangle), ΔentAΩentA (purple triangle), Δ entA Δ asb (grey circle) strains or PBS (green cross). Results are mean values of three to seven independent experiments and error bars indicate SEM from mean values. Based on these data, LD 50 were estimated and are reported in Table 2 . ( D ) Illustrates white alive and dead black larvae. 10.1371/journal.ppat.1003935.t002 Table 2 Virulence of siderophore mutants in G. mellonella. Strain LD 50 a LD 50 Confidence Limits Wild type BcATCC14579 1.8×10 3 3.1×10 2 –3.3×10 3 Δasb 3.4×10 3 1.2×10 3 –5.6×10 3 ΔentA 1.0×10 4 8.8×10 3 –1.2×10 4 ΔentAΩentA 6.4×10 3 4.7×10 3 –8.2×10 3 ΔentAΔasb 1.2×10 4 8.5×10 3 –1.6×10 4 a The 50% lethal doses, in number of colony forming units (cfu), were evaluated by Probit survival. analysis ( p <0.05). Discussion For pathogens, the ability to cope with the low iron environment encountered in the host is essential for tissue colonization. Thus, the production of efficient iron acquisition systems represents key factors. Because ferritin is the major iron storage protein found in living organisms, pathogens have developed efficient mechanisms to use this iron source and gain rapid access to sufficient quantities of iron. However, studies of the microbial determinants involved in host ferritin iron theft remain scarce. The present study showed that the bacterial surface protein IlsA interacts directly with the ferritin shell perhaps altering its structural integrity and leading to an amplification of iron release from the nanocage. In an earlier work [14] , an in vitro interaction between recombinant IlsA and horse spleen ferritin was reported. Here, a more detailed in vivo characterization of this interaction demonstrates that IlsA is required for ferritin recognition and recruitment at the bacterial surface. The observed binding stoichiometry of 24 IlsA per ferritin molecule argues in favor of a direct interaction of one IlsA molecule per ferritin subunit, irrespective of the ferritin source or type ( Table 1 ) suggesting a role for IlsA as ferritin receptor. The similarities between the thermodynamic parameters reported in Table 1 using different ferritin types (i.e. homopolymers composed of 24 H-subunits or 24 L-subunits and heteropolymers composed of 21 H- and 4 L-subunits) suggest that either ferritin subunit binds IlsA equally well. This novel finding advances our understanding of iron acquisition by pathogens since to our knowledge no host-ferritin receptor has been identified thus far in microorganisms. The only existing clue was described in the pathogenic fungus C. albicans , where Als3, which has no structural homology with IlsA, was required for ferritin binding to hypha. However, the authors did not shown any direct interaction between Als3 and ferritin [13] . To further understand how IlsA interacts with ferritin and which domain(s) is involved in the binding, we searched for ferritin-binding receptors that share some degree of homology with IlsA. In mammals, several receptors associated with serum ferritin internalization have been described. Tim-2, a T cell immunoglobulin-domain and a mucin-domain protein expressed in various mouse tissues and TfR-1, the human transferrin receptor-1 are both able to recognize H-chain ferritin [52] , [53] whereas Scara5, a class A scavenger receptor binds L-chain ferritin and is used to deliver iron to mouse kidney cells [54] . However, no homology or conserved domains exist or is evident between IlsA and these ferritin receptors. IlsA has an original structure consisting of LRR and NEAT domains. LRR motifs are found in a broad range of proteins and are frequently involved in protein-protein interactions [55] . The NEAT domains are known to interact with heme and hemoproteins. However, the NEAT protein IsdA from S. aureus was shown to bind several non-heme host proteins [56] . A recent investigation from our laboratory showed that B. cereus IlsA NEAT domain alone exhibited affinity for heme binding (Abi Khalil et al. , unpublished data). Here, we tested individually the LRR and NEAT domains of B cereus IlsA for their ability to bind ferritin. Although a weak interaction was observed with the LRR domains alone but not with the NEAT domain, only the full length IlsA protein was able to effectively bind ferritin. However, we cannot exclude structural modifications in the LRR domains during purification, which would explain the weak binding affinity observed with these domains. Therefore, further in-depth structural and mutagenesis studies are needed to pinpoint the exact location of the binding site between IlsA and ferritin. Among the NEAT proteins, only a few of them also carry LRR domains and have been found exclusively in the Firmicutes phylum. Besides IlsA, two other members of this composite NEAT protein family have been described in S. pyogenes (Shr) and in B. anthracis (Hal). Similarly to IlsA, both NEAT proteins are involved in heme acquisition and bacterial virulence but no role in ferritin iron acquisition has been reported yet [57] , [58] , [59] , [60] . Interestingly, B. anthracis possesses an ORF encoding a protein termed BslL ( Ba1346 ) [61] that is nearly identical to the last three fourths of IlsA with LRR domains followed by three SLH domains. BslK (Ba1093), another B. anthracis protein, shares some similarities with both IlsA NEAT and SLH domains [61] . BslK binds and directionally transfers heme to the Isd system [62] . However, the involvement of these proteins in ferritin iron acquisition has not been studied. Further experiments are needed to determine whether the ability of IlsA to bind ferritin is a universally conserved feature of the composite NEAT-LRR proteins found in other pathogenic bacteria. IlsA-ferritin interaction is also shown to enhance the rate of iron release from ferritin. This result constitutes a major finding since no direct effect of a microbial protein on iron mobilization from host ferritin has ever been reported. As the supramolecular structure of the ferritin shell is extremely robust, it is possible that IlsA induces small conformational modifications upon binding leading to local destabilization of the ferritin subunits. This observation is in part supported by the large positive ΔS 0 values obtained by ITC reflecting an increase in the system's disorder. Several models for iron mobilization from the protein nanocage have been proposed but the exact in vivo mechanism of iron release remains poorly understood [63] . It has been suggested that iron ions exit through the eight gated-pores located at the 3-fold symmetry axis of ferritin [64] . Mutations of specific residues near the ferritin entry pores lead to an increase in iron release and are associated with localized unfolding without changes in the overall protein assembly [65] . Moreover, the 3-fold channels can be altered by chaotropic agents [66] and specific ferritin binding peptides [67] . By interfering with the protein intramolecular forces, these small molecules can significantly alter the rate of iron mobilization from ferritin, underlying the importance of pore flexibility in the transfer of iron in or out of the nanocage cavity. In addition to several ferritin receptors described above, a number of studies have reported the existence of other proteins that bind ferritin (for review, see [21] ). In the absence of a clear mechanism of iron release from ferritin, it has been suggested that ferritin-binding proteins may cause opening or closing of the 3-fold channels and regulate iron storage or release. Hence, it is tempting to speculate that IlsA might act as a chaperone protein that dock around the 3-fold channels causing pore opening and rapid iron mobilization from ferritin. In contrast, the slow iron release rate observed with DFO alone (less than 5% of the total iron present in ferritin) is probably a consequence of the chelator size, which is too large to pass through the narrow ferritin pores. Direct chelation at the surface of the iron core has been reported with small bidentate Fe (III)-chelator [68] , [69] and is unlikely to be relevant in our case. However, our proposed model of iron release involving IlsA contrasts with previous models for microbial iron acquisition from host ferritin. For instance, B. cenocepacia and N. meningitidis adopt direct or indirect proteolytic degradation strategy, respectively [17] , [19] . Preliminary tests using protease inhibitors suggest that ferritin utilization in B. cereus does not rely on proteolysis in vivo (data not shown). Another strategy used by microbes is based on a reductase activity as proposed for L. monocytogenes and C. albicans [13] , [22] . However, DFO can remove iron from ferritin in a non-reductive process [38] and our in vitro experiments were carried out under non-reducing conditions. Thus, it is likely that the reductive pathway is not relevant in B. cereus although enhancement of iron mobilization through redox processes is not excluded by the present study. The effect of microbial siderophores on iron mobilization from ferritin has been emphasized thirty years ago [42] . Although two B. cereus siderophores are produced in iron-depleted media, our data suggest that only bacillibactin enables iron transfer from ferritin. Compared to earlier studies with the ΔilsA mutant [14] , the ΔentA strain deficient in bacillibactin production displayed a more pronounced growth defect on ferritin. This indicates that bacillibactin is essential for iron uptake from ferritin and that IlsA may facilitate this process, as evidenced in the in vitro iron release kinetics ( Figure 3 ). Furthermore, as the ferric iron core of ferritin is highly insoluble in aerobic conditions and neutral pH (K sp (Fe(OH) 3 ) = 10 −39 ) [63] , the major influence of bacillibactin could be ascribed to its stronger affinity for ferric ions (K f = 10 47.6 ) [70] compared to petrobactin (K f = 10 23 ) [71] . Bacillibactin is not only important for growth with ferritin but also for bacterial virulence in an insect model. These results contrast with the major role played by petrobactin in B. anthracis . In this closely related species, petrobactin is the primary siderophore synthesized under conditions of iron starvation [45] and is important for virulence in mice and survival in macrophages [44] . Petrobactin, but not bacillibactin, possesses a unique ability to evade the mammalian siderophore scavenger protein named siderocalin [72] . It has been proposed that petrobactin is probably a trait required for pathogenesis in mammals [39] . Considering that no siderocalin homolog has been described in insects to date and that ferritin is more abundant in the insect hemocoel than in the vertebrate blood, the relevance of bacillibactin in G. mellonella is meaningful. However, depending on the host infected, the type of tissues and the available iron source, variation in the relative importance of the B. cereus siderophores might be expected. Further studies in mammal models are needed to elucidate this possibility. In conclusion, a new model of iron mobilization from host ferritin in bacteria is proposed ( Figure 8 ). This working model involves the destabilization of the ferritin subunits by IlsA leading to an enhancement of iron release from the ferritin mineral core. The B. cereus siderophore bacillibactin then acquires the mobilized iron needed for bacterial growth. The results of our current study and additional work from our laboratory [14] (Abi Khalil et al. , in revision) provide new insights into iron uptake by pathogens and ascribe a multifaceted role for IlsA in iron acquisition from structurally different host iron sources. 10.1371/journal.ppat.1003935.g008 Figure 8 Schematic representation of iron uptake from ferritin in B. cereus . (1) In low iron environments, IlsA is produced and anchored to the surface. IlsA binds to each ferritin subunit, resulting in ferritin recognition and recruitment on the bacterial cell surface. (2) Following binding interaction, IlsA is proposed to alter ferritin pores openings or subunit-subunit interactions leading to protein destabilization or an increased accessibility to the ferritin iron core. (3) Because IlsA itself does not bind iron (data not shown), the iron released from ferritin by the action of IlsA is chelated by bacillibactin (and may involve other molecules) whereby the iron-siderophore complex is transported into the cell probably via the FeuA transporter leading to iron release into the cytosol. Materials and Methods Bacterial strains and growth conditions Bacillus cereus strain ATCC14579 (laboratory stock) was used throughout this study. The mutant B. cereus ATCC14579 ΔilsA was previously constructed by homologous recombination [33] and complemented with the pHT304Ω ilsA plasmid [14] . E. coli K12 strain TG1 was used as a host for cloning experiments. Dam − /Dcm − E. coli strain ET12567 (laboratory stock) was used to generate unmethylated DNA for electro-transformation in B. cereus . E. coli strains M15 (laboratory stock) and C600 ΔhemA [73] were used for protein overproduction. All the strains used in this study are listed in Table 3 . E. coli and B. cereus were cultured in LB (Luria-Bertani) broth, with vigorous shaking (175 rpm) at 37°C and E. coli C600 Δ hemA was cultured in BHI (Brain Heart Infusion, Difco) broth, without shaking. For electro-transformation, B. cereus was grown in BHI. E. coli and B. cereus strains were transformed by electroporation as previously described [74] , [75] . The following concentrations of antibiotic were used for bacterial selection: ampicillin 100 µg ml −1 and kanamycin 25 µg ml −1 for E. coli ; kanamycin 200 µg ml −1 , tetracycline 10 µg ml −1 and erythromycin 10 µg ml −1 for B. cereus . The iron chelator, 2,2′-dipyridyl and the horse spleen ferritin (HoSF) were purchased from Sigma-Aldrich. 2,2′-dipyridyl was used at final concentrations of 200, 450 and 600 µM and ferritin at 300 nM. 10.1371/journal.ppat.1003935.t003 Table 3 Strains and plasmids used in this work. Strain or plasmid Characteristics Reference Strain Bacillus cereus ATCC14579 Wild type Laboratory stock Bc ΔilsA ATCC14579 mutant; Δbc1331 ; Tet R [33] Bc ΔilsAΩilsA ΔilsA strain carrying pHT304Ω ilsA plasmid; Tet R , Erm R [14] Bc Δasb ATCC14579 mutant; Δbc1978–1983 ; Tet R This study Bc ΔentA ATCC14579 mutant; Δbc2302 ; Kan R This study Bc ΔentAΩentA ΔentA strain carrying pHT304Ω entA plasmid; Kan R , Erm R This study Bc ΔentAΔasb Δasb mutation into ΔentA strain; Kan R , Tet R This study Escherichia coli K12 strain TG1 Strain used as host for cloning experiments Laboratory stock Ec ET12567 Strain used for generation of unmethylated DNA Laboratory stock Ec C600 ΔhemA Heme-deficient mutant used for protein overproduction; Kan R [73] Ec C600 ΔhemA GST-IlsA Strain C600 ΔhemA carrying pGEX6P1- ilsA ; Kan R , Amp R This study Ec C600 ΔhemA GST-NEAT IlsA Strain C600 ΔhemA carrying pGEX6P1- NEAT IlsA , Kan R , Amp R This study Ec M15 Strain carrying pREP4, used for protein overproduction; Km R Laboratory stock Ec M15 GST-LRR IlsA Strain M15 carrying pREP4 and pGEX6P1- LRR IlsA ; Kan R , Amp R This study Plasmid pHT304 Shuttle vector used for complementation; Amp R , Erm R [80] pMAD Shuttle vector, thermosensitive origin of replication; Amp R , Erm R [78] pRN5101 Shuttle vector, thermosensitive origin of replication; Amp R , Erm R [83] pHTS1 Vector carrying the tetracycline resistance cassette ( tet ) [81] pDG783 Vector carrying the kanamycin resistance cassette ( aphA3 ) [82] pGEX6P1 Vector for inducible GST-tagged protein overproduction; Amp R GE Healthcare pMAD Ωasb :: tet pMAD with bc1978–1983 deletion fragment This study pRN5101 ΩentA :: kan pRN5101 with bc2302 deletion fragment This study pHT304 ΩentA pHT304 with wild-type entA fragment This study pGEX6P1- ilsA pGEX6P1 with the whole i lsA sequence (without signal peptide) This study pGEX6P1- NEAT IlsA pGEX6P1 with NEAT domain sequence of ilsA This study pGEX6P1- LRR IlsA pGEX6P1 with LRR domains sequence of ilsA This study Bc , B. cereus ; Ec , E. coli ; Tet, tetracycline; Erm, erythromycin; Kan, kanamycin; Amp, ampicillin. Immunofluorescence analysis B. cereus wild type, ΔilsA and ΔilsAΩilsA strains were grown overnight in LB medium supplemented with 200 µM 2,2′-dipyridyl. These cultures were used to inoculate several media (LB/LB+0.3 µM HoSF/LB+450 µM 2,2′-dipyridyl/LB+450 µM 2,2′-dipyridyl+0.3 µM HoSF) at 37°C and a final OD of 0.1. Mid-log phase bacteria were collected, washed twice in PBS buffer and used immediately. Bacterial cells (∼10 8 ) were fixed with 4% formaldehyde dissolved in PBS on poly-L-Lysine slides (Labomoderne). After 20 min, bacteria were washed with PBS, blocked with 1% BSA and incubated for one hour at room temperature with an anti-HoSF polyclonal antibody (Sigma-Aldrich) labeled with Alexa Fluor 594-conjugate (Invitrogen) at a dilution of 1∶60 in 1% BSA. Then, bacteria were washed with PBS, fixed a second time with 4% formaldehyde and bacterial DNA was counterstained with (4′,6-diamidino-2-phenylindole) DAPI diluted at 1∶300 in 1% BSA. Finally, slides were rinsed with water, cover-slips were sticked with the antifading Polyvinyl alcohol mounting medium with 1,4-diazabicyclo[2.2.2]octane (DABCO) from Fluka and dried at 37°C for 30 min. At least two experiments in duplicates were examined by phase contrast and epifluoresence microscopy using a Zeiss Observer Z1 microscope and the Axiovision imaging software. A representative picture of each strain was selected. Overproduction and purification of IlsA and its NEAT and LRR domains GST-IlsA, GST-NEAT IlsA and GST-LRR IlsA were purified as recombinant proteins from E. coli using the expression plasmids pGEX6P1- ilsA , pGEX6P1- NEAT IlsA and pGEX6P1- LRR IlsA . In order to purify recombinant IlsA protein and its NEAT and LRR domains, sequences corresponding to Ala29-Lys760, Thr24-Gly163 and Lys208-Asn492 respectively were amplified from B. cereus strain ATCC14579 with paired primers FIlsA/RIlsA, FNEAT/RNEAT and FLRR/RLRR respectively ( Table 4 ) and cloned into the plasmid pGEX6P1 holding the tag GST (GE Healthcare) after digestion of the PCR products with EcoRI/XhoI. Recombinant LRR IlsA were overexpressed in E. coli M15 as previously described in [14] , except that the bacterial culture was incubated for 3 h at 30°C and overnight at 15°C. IlsA and NEAT IlsA were produced in apo-form ((without heme bound to the NEAT domain) by using E. coli strain C600 ΔhemA impaired in heme biosynthesis [73] . The recombinant apo-proteins were expressed in BHI (Brain Heart Infusion, Difco) supplemented with appropriate antibiotics and the cultures were grown into bottles, in static phase, at 37°C to an OD 600 = 0.8–0.9. The same protocol used for LRR IlsA purification is then followed. The Bulk GST Purification Module (GE Healthcare) was used as recommended by the manufacturer. GST tag was removed by eluting the proteins with the PreScission Protease (GE Healthcare). The purified proteins were concentrated by ultrafiltration and stored at −20°C. For apo-IlsA and apo-NEAT IlsA , in order to reconstitute holo-proteins, hemin (Sigma-Aldrich) was added to protein preparations until saturation of 80%. 10.1371/journal.ppat.1003935.t004 Table 4 Primers used in this work. Name Sequence 5′-3′ Restriction site (underlined) FIlsA CG GAATTC GCATTAAAAGTTGAAGCAAATC EcoRI RIlsA CC CTCGAG TTATTTCTTTATTGCATTATAC XhoI FNEAT IlsA CCG GAATTC ACTCCAGCATTAGCGGCA EcoRI RNEAT IlsA CC CTCGAG ACCTACAGTTGGATCTTTAAT XhoI FLRR CCG GAATTC AAAGATTTAAATACACC EcoRI RLRR CC CTCGAG TCAATTTTGGACATTAATATAA XhoI FpetU G GAATTC GATAGTTGGAAAGCAACG EcoRI RpetU CG GGATCC ATACAAAGTAACGTTCTG BamHI FpetD AA CTGCAG AATGGTTTGGACATAATTC PstI RpetD GC GTCGAC CTTGAATCGCTCTACCG SalI FbacU CC AAGCTT GGTATTTACTTCGTATGTGTAG HindIII RbacU GC TCTAGA GCCTATGCCTTGTGCTGCA XbaI FbacD CC CTCGAG GCACAACCTTCAGAAGTTGC XhoI RbacD CG GGATCC CGCTTCACTATGAATAACTGAT BamHI FbacP AA CTGCAG AAGCATTGTAAATGAACGTATC PstI RbacP CC CTCGAG GGTTTCTCTATCCTTTCACATA XhoI FbacComp AA CTGCAG CATTGTAAATGAACGTATC PstI RbacComp GC TCTAGA TTAAACTCCTAACGTAGC XbaI Microcalorimetry Isothermal titration calorimetry (ITC) experiments were performed at 25°C on a low volume (185 µl) NanoITC (TA Instruments). Titrant and sample solutions were made from the same stock buffer solution (50 mM Tris- HCl pH 7, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT). IlsA and its purified domains were obtained as described above. Concerning the ferritin samples, recombinant mouse H-chain (MoHF), human H-chain (HuHF), human L-chain (HuLF) and human heteropolymer H/L (HuH/LF) were purified as previously described [76] , [77] . To test for the interaction between IlsA (or its NEAT and LRR domains) and ferritins, an automated sequence of 16 injections, each of 3 µl titrant (229 µM holo-IlsA) into the sample cell containing 1 µM ferritin, was performed at intervals of 5 min to allow complete equilibration, with the equivalence point coming at the area midpoint of the titration. The protein solution was stirred at 250 rpm to ensure rapid mixing of the titrant upon injection. The area under the resulting peak following each injection is proportional to the heat of interaction, which is normalized by the concentration of the added titrant and corrected for the dilution heat using the buffer solution alone to give the molar binding enthalpy ΔH°. The data were collected automatically and analyzed using NanoAnalyze fitting program (TA Instruments). The standard enthalpy change (ΔH°), the binding constant (K), and the stoichiometry of binding (n) are determined by a single ITC experiment. From these values, the standard Gibbs free energy change (ΔG°), and standard entropy change (ΔS°) are calculated using the following equations: ΔG° = −RTlnK and TΔS° = ΔH°−ΔG° where R is the universal gas constant (1.9872 cal mol −1 K −1 ) and T is the temperature in Kelvin degrees. The dissociation constant is expressed as K d = 1/K (in mol l −1 ). All experiments were repeated two to four times and control experiments (IlsA or ferritin alone in the buffer) did not show any significant heat changes. Iron release assays Apoferritin (HuHF) was loaded aerobically with 500 Fe atoms/nanocage. Typically, the FeSO 4 solution was prepared in pH 2 DI water and loaded into ferritin via ten additions of 50 Fe(II) per shell. The iron release experiments were conducted in 50 mM Tris-HCl pH 7 and 150 mM NaCl in presence of 1 µM ferritin, 1 mM deferoxamine B (DFO – Sigma-Aldrich) chelator and with or without purified IlsA at 5 µM. The kinetics of iron release were performed under aerobic conditions at 25°C and monitored by the increase in the characteristic MLCT absorption band of the Fe(III)-DFO complex (425 nm). The percent of iron release from ferritin was calculated using experimentally determined UV-Vis molar extinction coefficient of the Fe(III)-DFO complex at 425 nm (3500 M −1 cm −1 ). Experiments were repeated three times with different protein preparations. DNA manipulations and plasmid constructions Chromosomal DNA was extracted from B. cereus cells with the Puregene Yeast/Bact. Kit B (QIAgen). Plasmid DNA was extracted from E. coli and B. cereus using QIAprep spin columns (QIAgen). For B. cereus , 5 mg ml −1 of lysozyme was added and cells were incubated at 37°C for 1 h. Restriction enzymes and T4 DNA ligase were used according to the manufacturer's instructions (New England Biolabs). Oligonucleotide primers ( Table 4 ) were synthesized by Sigma-Proligo. PCRs were performed in an Applied Biosystem 2720 Thermak cycler (Applied Biosystem) with Phusion High-Fidelity or Taq DNA Polymerase (New England Biolabs). Amplified fragments were purified using the QIAquick PCR purification Kit (QIAgen). Digested DNA fragments were separated by electrophoresis on 0.8% agarose gels and extracted from gels using the QIAquick gel extraction Kit (QIAgen). Nucleotide sequences were determined by Beckman Coulter Genomics. The thermosensitive plasmids pMAD [78] and pRN5101 [79] were used for homologous recombination. The low-copy-number plasmid pHT304 [80] was used for complementation experiments with wild-type entA gene under its own promoter. The vector pGEX6P1 (GE Healthcare) was used to overproduce Glutathione S-transferase (GST)-tagged protein under the control of a tac promoter. All the plasmids used in this study are reported in Table 3 . Construction of the B. cereus mutant strains B. cereus Δ asb and ΔentA were constructed as follows. For asbABCDEF ( bc1978–1983 ) deletion, a 956 bp EcoRI/BamHI DNA fragment and a 985 bp PstI/SalI DNA fragment, corresponding to the chromosomal regions located immediately upstream and downstream from the asb locus, were generated by PCR, using B. cereus strain ATCC14579 chromosomal DNA as a template and oligonucleotide pairs FpetU–RpetU and FpetD–RpetD respectively ( Table 4 ). A Tet cassette, conferring resistance to tetracycline, was purified from pHTS1 [81] as a 1.6 kb PstI/BamHI fragment carrying the tet gene from B. cereus . The amplified DNA fragments and the Tet R cassette were digested with the appropriate enzymes and inserted between the EcoRI and SalI sites of the thermosensitive plasmid pMAD [78] by ligation using the T4 DNA ligase. For entA ( bc2302 ) deletion, a 996 bp HindIII/XbaI and a 957 bp XhoI/BamHI DNA regions upstream and downstream the entA gene, were respectively amplified by PCR, using chromosomal DNA of the ATCC14579 strain of B. cereus as template and FbacU/RbacU, FbacD/RbacD as primers ( Table 4 ). In addition, a 359 bp PstI/XhoI DNA fragment corresponding to the putative regulatory region of entA-dhbBCF was amplified using the same template and the primer pair FbacP/RbacP ( Table 4 ). A Kan R cassette containing aphA3 gene, conferring resistance to kanamycin, was purified from pDG783 [82] as a 1.5 kb PstI/XbaI. The amplified DNA fragments and the Kan R cassette were digested with the appropriate enzymes and inserted between the HindIII and BamHI sites of the thermosensitive plasmid pRN5101 [83] as illustrated in Figure 4B . The resulting plasmids pMADΩ asb :: tet and pRN5101Ω entA :: kan were produced in E. coli , and then used to transform B. cereus wild type strain by electroporation. Integrants resistant to tetracycline (for Δasb ) or kanamycin (for ΔentA ) and sensitive to erythromycin arose through a double cross-over event, in which the chromosomal wild-type copies of asbABCEDF and entA coding sequences were deleted and replaced by the Tet R and Kan R cassette respectively. The chromosomal allelic exchanges were checked by PCR, using appropriate primers and by sequencing the insertion sites. The genetic complementation of the ΔentA mutant was carried out as follows. A 1142 bp DNA fragment corresponding to the entA gene and its putative promoter was amplified by PCR using the B. cereus ATCC14579 genomic DNA as a template and FbacComp/RbacComp as primers ( Table 4 ). The PCR product was digested with PstI and XbaI restriction enzymes and inserted into the plasmid pHT304 [80] . The resulting plasmid (pHT304Ω entA ) was amplified in E. coli and then introduced into the ΔentA mutant strain of B. cereus by electroporation. Measurement of catechol production Extracellular levels of catechols were measured using the Arnow assay [41] . Bacteria were grown overnight (20 h) at 37°C in LB medium +200 µM 2,2′-dipyridyl. Then, samples of cultures were collected, centrifuged and filtered to remove bacteria. Samples were mixed sequentially with equal volumes of 0.5 N HCl, nitrite-molybdate reagent (10% sodium nitrite and 10% sodium molybdate), and 1 N NaOH. Positive reactions produce a red colour and absorbance was determined at 510 nm. Data were normalized to OD600 of the original culture and percentages of wild-type catechol level in culture supernatants are presented. Three independent replicates were statistically analyzed using the Student's T-test. Growth assays B. cereus strains were grown overnight at 37°C under low iron conditions by inoculating strains in LB medium supplemented with 200 µM 2,2′-dipyridyl. Overnight cultures were inoculated into a new LB medium +200 µM 2,2′-dipyridyl at a final OD of 0.01. Bacteria from mid-log phase culture were washed twice in LB medium containing 600 µM 2,2′-dipyridyl, then inoculated to a final optical density (OD) of about 0.005 into LB medium or LB+600 µM 2,2′-dipyridyl +0.3 µM HoSF supplemented or not with 5 µM Enterobactin (Sigma-Aldrich). Stock solution of ferritin was prechelated in 2 mM 2,2′-dipyridyl for two hours in order to eliminate the free iron. B. cereus cells were grown at 37°C in 96-wells microtiter plate under continuous shaking. The OD was measured at 600 nm every hour over 16 hours using a TECAN Infinite M200 Microplate Reader (TECAN Group). The assays were repeated at least three times. Virulence assays Bacterial strains were grown in LB medium and bacterial concentrations were monitored by optical density measurements and plating dilutions onto LB agar plates. B. cereus wild-type and mutant strains were injected separately into the hemocoel of G. mellonella . Insect eggs were incubated at 25°C and the larvae reared on beeswax and pollen (Naturalim). Last-instar larvae weighing about 200 mg were injected with 10 µl of mid-log phase bacteria (or spores) suspended in PBS, using the microinjector (Buckard Scientific) as previously described [84] . Various doses of bacteria (1×10 3 to 3×10 4 bacteria/larva) were used, and each experiment was repeated at least three times with 20 larvae. A control group of larvae was injected with PBS only and no effect was observed. The survival rate (% of alive/total number of infected larvae) was recorded during 72 hours after infection. Statistical analysis was performed using the Log-rank test. Based on the data obtained, LD 50 were estimated by Probit analysis with StatPlus software (AnalystSoft). Bacterial strains and growth conditions Bacillus cereus strain ATCC14579 (laboratory stock) was used throughout this study. The mutant B. cereus ATCC14579 ΔilsA was previously constructed by homologous recombination [33] and complemented with the pHT304Ω ilsA plasmid [14] . E. coli K12 strain TG1 was used as a host for cloning experiments. Dam − /Dcm − E. coli strain ET12567 (laboratory stock) was used to generate unmethylated DNA for electro-transformation in B. cereus . E. coli strains M15 (laboratory stock) and C600 ΔhemA [73] were used for protein overproduction. All the strains used in this study are listed in Table 3 . E. coli and B. cereus were cultured in LB (Luria-Bertani) broth, with vigorous shaking (175 rpm) at 37°C and E. coli C600 Δ hemA was cultured in BHI (Brain Heart Infusion, Difco) broth, without shaking. For electro-transformation, B. cereus was grown in BHI. E. coli and B. cereus strains were transformed by electroporation as previously described [74] , [75] . The following concentrations of antibiotic were used for bacterial selection: ampicillin 100 µg ml −1 and kanamycin 25 µg ml −1 for E. coli ; kanamycin 200 µg ml −1 , tetracycline 10 µg ml −1 and erythromycin 10 µg ml −1 for B. cereus . The iron chelator, 2,2′-dipyridyl and the horse spleen ferritin (HoSF) were purchased from Sigma-Aldrich. 2,2′-dipyridyl was used at final concentrations of 200, 450 and 600 µM and ferritin at 300 nM. 10.1371/journal.ppat.1003935.t003 Table 3 Strains and plasmids used in this work. Strain or plasmid Characteristics Reference Strain Bacillus cereus ATCC14579 Wild type Laboratory stock Bc ΔilsA ATCC14579 mutant; Δbc1331 ; Tet R [33] Bc ΔilsAΩilsA ΔilsA strain carrying pHT304Ω ilsA plasmid; Tet R , Erm R [14] Bc Δasb ATCC14579 mutant; Δbc1978–1983 ; Tet R This study Bc ΔentA ATCC14579 mutant; Δbc2302 ; Kan R This study Bc ΔentAΩentA ΔentA strain carrying pHT304Ω entA plasmid; Kan R , Erm R This study Bc ΔentAΔasb Δasb mutation into ΔentA strain; Kan R , Tet R This study Escherichia coli K12 strain TG1 Strain used as host for cloning experiments Laboratory stock Ec ET12567 Strain used for generation of unmethylated DNA Laboratory stock Ec C600 ΔhemA Heme-deficient mutant used for protein overproduction; Kan R [73] Ec C600 ΔhemA GST-IlsA Strain C600 ΔhemA carrying pGEX6P1- ilsA ; Kan R , Amp R This study Ec C600 ΔhemA GST-NEAT IlsA Strain C600 ΔhemA carrying pGEX6P1- NEAT IlsA , Kan R , Amp R This study Ec M15 Strain carrying pREP4, used for protein overproduction; Km R Laboratory stock Ec M15 GST-LRR IlsA Strain M15 carrying pREP4 and pGEX6P1- LRR IlsA ; Kan R , Amp R This study Plasmid pHT304 Shuttle vector used for complementation; Amp R , Erm R [80] pMAD Shuttle vector, thermosensitive origin of replication; Amp R , Erm R [78] pRN5101 Shuttle vector, thermosensitive origin of replication; Amp R , Erm R [83] pHTS1 Vector carrying the tetracycline resistance cassette ( tet ) [81] pDG783 Vector carrying the kanamycin resistance cassette ( aphA3 ) [82] pGEX6P1 Vector for inducible GST-tagged protein overproduction; Amp R GE Healthcare pMAD Ωasb :: tet pMAD with bc1978–1983 deletion fragment This study pRN5101 ΩentA :: kan pRN5101 with bc2302 deletion fragment This study pHT304 ΩentA pHT304 with wild-type entA fragment This study pGEX6P1- ilsA pGEX6P1 with the whole i lsA sequence (without signal peptide) This study pGEX6P1- NEAT IlsA pGEX6P1 with NEAT domain sequence of ilsA This study pGEX6P1- LRR IlsA pGEX6P1 with LRR domains sequence of ilsA This study Bc , B. cereus ; Ec , E. coli ; Tet, tetracycline; Erm, erythromycin; Kan, kanamycin; Amp, ampicillin. Immunofluorescence analysis B. cereus wild type, ΔilsA and ΔilsAΩilsA strains were grown overnight in LB medium supplemented with 200 µM 2,2′-dipyridyl. These cultures were used to inoculate several media (LB/LB+0.3 µM HoSF/LB+450 µM 2,2′-dipyridyl/LB+450 µM 2,2′-dipyridyl+0.3 µM HoSF) at 37°C and a final OD of 0.1. Mid-log phase bacteria were collected, washed twice in PBS buffer and used immediately. Bacterial cells (∼10 8 ) were fixed with 4% formaldehyde dissolved in PBS on poly-L-Lysine slides (Labomoderne). After 20 min, bacteria were washed with PBS, blocked with 1% BSA and incubated for one hour at room temperature with an anti-HoSF polyclonal antibody (Sigma-Aldrich) labeled with Alexa Fluor 594-conjugate (Invitrogen) at a dilution of 1∶60 in 1% BSA. Then, bacteria were washed with PBS, fixed a second time with 4% formaldehyde and bacterial DNA was counterstained with (4′,6-diamidino-2-phenylindole) DAPI diluted at 1∶300 in 1% BSA. Finally, slides were rinsed with water, cover-slips were sticked with the antifading Polyvinyl alcohol mounting medium with 1,4-diazabicyclo[2.2.2]octane (DABCO) from Fluka and dried at 37°C for 30 min. At least two experiments in duplicates were examined by phase contrast and epifluoresence microscopy using a Zeiss Observer Z1 microscope and the Axiovision imaging software. A representative picture of each strain was selected. Overproduction and purification of IlsA and its NEAT and LRR domains GST-IlsA, GST-NEAT IlsA and GST-LRR IlsA were purified as recombinant proteins from E. coli using the expression plasmids pGEX6P1- ilsA , pGEX6P1- NEAT IlsA and pGEX6P1- LRR IlsA . In order to purify recombinant IlsA protein and its NEAT and LRR domains, sequences corresponding to Ala29-Lys760, Thr24-Gly163 and Lys208-Asn492 respectively were amplified from B. cereus strain ATCC14579 with paired primers FIlsA/RIlsA, FNEAT/RNEAT and FLRR/RLRR respectively ( Table 4 ) and cloned into the plasmid pGEX6P1 holding the tag GST (GE Healthcare) after digestion of the PCR products with EcoRI/XhoI. Recombinant LRR IlsA were overexpressed in E. coli M15 as previously described in [14] , except that the bacterial culture was incubated for 3 h at 30°C and overnight at 15°C. IlsA and NEAT IlsA were produced in apo-form ((without heme bound to the NEAT domain) by using E. coli strain C600 ΔhemA impaired in heme biosynthesis [73] . The recombinant apo-proteins were expressed in BHI (Brain Heart Infusion, Difco) supplemented with appropriate antibiotics and the cultures were grown into bottles, in static phase, at 37°C to an OD 600 = 0.8–0.9. The same protocol used for LRR IlsA purification is then followed. The Bulk GST Purification Module (GE Healthcare) was used as recommended by the manufacturer. GST tag was removed by eluting the proteins with the PreScission Protease (GE Healthcare). The purified proteins were concentrated by ultrafiltration and stored at −20°C. For apo-IlsA and apo-NEAT IlsA , in order to reconstitute holo-proteins, hemin (Sigma-Aldrich) was added to protein preparations until saturation of 80%. 10.1371/journal.ppat.1003935.t004 Table 4 Primers used in this work. Name Sequence 5′-3′ Restriction site (underlined) FIlsA CG GAATTC GCATTAAAAGTTGAAGCAAATC EcoRI RIlsA CC CTCGAG TTATTTCTTTATTGCATTATAC XhoI FNEAT IlsA CCG GAATTC ACTCCAGCATTAGCGGCA EcoRI RNEAT IlsA CC CTCGAG ACCTACAGTTGGATCTTTAAT XhoI FLRR CCG GAATTC AAAGATTTAAATACACC EcoRI RLRR CC CTCGAG TCAATTTTGGACATTAATATAA XhoI FpetU G GAATTC GATAGTTGGAAAGCAACG EcoRI RpetU CG GGATCC ATACAAAGTAACGTTCTG BamHI FpetD AA CTGCAG AATGGTTTGGACATAATTC PstI RpetD GC GTCGAC CTTGAATCGCTCTACCG SalI FbacU CC AAGCTT GGTATTTACTTCGTATGTGTAG HindIII RbacU GC TCTAGA GCCTATGCCTTGTGCTGCA XbaI FbacD CC CTCGAG GCACAACCTTCAGAAGTTGC XhoI RbacD CG GGATCC CGCTTCACTATGAATAACTGAT BamHI FbacP AA CTGCAG AAGCATTGTAAATGAACGTATC PstI RbacP CC CTCGAG GGTTTCTCTATCCTTTCACATA XhoI FbacComp AA CTGCAG CATTGTAAATGAACGTATC PstI RbacComp GC TCTAGA TTAAACTCCTAACGTAGC XbaI Microcalorimetry Isothermal titration calorimetry (ITC) experiments were performed at 25°C on a low volume (185 µl) NanoITC (TA Instruments). Titrant and sample solutions were made from the same stock buffer solution (50 mM Tris- HCl pH 7, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT). IlsA and its purified domains were obtained as described above. Concerning the ferritin samples, recombinant mouse H-chain (MoHF), human H-chain (HuHF), human L-chain (HuLF) and human heteropolymer H/L (HuH/LF) were purified as previously described [76] , [77] . To test for the interaction between IlsA (or its NEAT and LRR domains) and ferritins, an automated sequence of 16 injections, each of 3 µl titrant (229 µM holo-IlsA) into the sample cell containing 1 µM ferritin, was performed at intervals of 5 min to allow complete equilibration, with the equivalence point coming at the area midpoint of the titration. The protein solution was stirred at 250 rpm to ensure rapid mixing of the titrant upon injection. The area under the resulting peak following each injection is proportional to the heat of interaction, which is normalized by the concentration of the added titrant and corrected for the dilution heat using the buffer solution alone to give the molar binding enthalpy ΔH°. The data were collected automatically and analyzed using NanoAnalyze fitting program (TA Instruments). The standard enthalpy change (ΔH°), the binding constant (K), and the stoichiometry of binding (n) are determined by a single ITC experiment. From these values, the standard Gibbs free energy change (ΔG°), and standard entropy change (ΔS°) are calculated using the following equations: ΔG° = −RTlnK and TΔS° = ΔH°−ΔG° where R is the universal gas constant (1.9872 cal mol −1 K −1 ) and T is the temperature in Kelvin degrees. The dissociation constant is expressed as K d = 1/K (in mol l −1 ). All experiments were repeated two to four times and control experiments (IlsA or ferritin alone in the buffer) did not show any significant heat changes. Iron release assays Apoferritin (HuHF) was loaded aerobically with 500 Fe atoms/nanocage. Typically, the FeSO 4 solution was prepared in pH 2 DI water and loaded into ferritin via ten additions of 50 Fe(II) per shell. The iron release experiments were conducted in 50 mM Tris-HCl pH 7 and 150 mM NaCl in presence of 1 µM ferritin, 1 mM deferoxamine B (DFO – Sigma-Aldrich) chelator and with or without purified IlsA at 5 µM. The kinetics of iron release were performed under aerobic conditions at 25°C and monitored by the increase in the characteristic MLCT absorption band of the Fe(III)-DFO complex (425 nm). The percent of iron release from ferritin was calculated using experimentally determined UV-Vis molar extinction coefficient of the Fe(III)-DFO complex at 425 nm (3500 M −1 cm −1 ). Experiments were repeated three times with different protein preparations. DNA manipulations and plasmid constructions Chromosomal DNA was extracted from B. cereus cells with the Puregene Yeast/Bact. Kit B (QIAgen). Plasmid DNA was extracted from E. coli and B. cereus using QIAprep spin columns (QIAgen). For B. cereus , 5 mg ml −1 of lysozyme was added and cells were incubated at 37°C for 1 h. Restriction enzymes and T4 DNA ligase were used according to the manufacturer's instructions (New England Biolabs). Oligonucleotide primers ( Table 4 ) were synthesized by Sigma-Proligo. PCRs were performed in an Applied Biosystem 2720 Thermak cycler (Applied Biosystem) with Phusion High-Fidelity or Taq DNA Polymerase (New England Biolabs). Amplified fragments were purified using the QIAquick PCR purification Kit (QIAgen). Digested DNA fragments were separated by electrophoresis on 0.8% agarose gels and extracted from gels using the QIAquick gel extraction Kit (QIAgen). Nucleotide sequences were determined by Beckman Coulter Genomics. The thermosensitive plasmids pMAD [78] and pRN5101 [79] were used for homologous recombination. The low-copy-number plasmid pHT304 [80] was used for complementation experiments with wild-type entA gene under its own promoter. The vector pGEX6P1 (GE Healthcare) was used to overproduce Glutathione S-transferase (GST)-tagged protein under the control of a tac promoter. All the plasmids used in this study are reported in Table 3 . Construction of the B. cereus mutant strains B. cereus Δ asb and ΔentA were constructed as follows. For asbABCDEF ( bc1978–1983 ) deletion, a 956 bp EcoRI/BamHI DNA fragment and a 985 bp PstI/SalI DNA fragment, corresponding to the chromosomal regions located immediately upstream and downstream from the asb locus, were generated by PCR, using B. cereus strain ATCC14579 chromosomal DNA as a template and oligonucleotide pairs FpetU–RpetU and FpetD–RpetD respectively ( Table 4 ). A Tet cassette, conferring resistance to tetracycline, was purified from pHTS1 [81] as a 1.6 kb PstI/BamHI fragment carrying the tet gene from B. cereus . The amplified DNA fragments and the Tet R cassette were digested with the appropriate enzymes and inserted between the EcoRI and SalI sites of the thermosensitive plasmid pMAD [78] by ligation using the T4 DNA ligase. For entA ( bc2302 ) deletion, a 996 bp HindIII/XbaI and a 957 bp XhoI/BamHI DNA regions upstream and downstream the entA gene, were respectively amplified by PCR, using chromosomal DNA of the ATCC14579 strain of B. cereus as template and FbacU/RbacU, FbacD/RbacD as primers ( Table 4 ). In addition, a 359 bp PstI/XhoI DNA fragment corresponding to the putative regulatory region of entA-dhbBCF was amplified using the same template and the primer pair FbacP/RbacP ( Table 4 ). A Kan R cassette containing aphA3 gene, conferring resistance to kanamycin, was purified from pDG783 [82] as a 1.5 kb PstI/XbaI. The amplified DNA fragments and the Kan R cassette were digested with the appropriate enzymes and inserted between the HindIII and BamHI sites of the thermosensitive plasmid pRN5101 [83] as illustrated in Figure 4B . The resulting plasmids pMADΩ asb :: tet and pRN5101Ω entA :: kan were produced in E. coli , and then used to transform B. cereus wild type strain by electroporation. Integrants resistant to tetracycline (for Δasb ) or kanamycin (for ΔentA ) and sensitive to erythromycin arose through a double cross-over event, in which the chromosomal wild-type copies of asbABCEDF and entA coding sequences were deleted and replaced by the Tet R and Kan R cassette respectively. The chromosomal allelic exchanges were checked by PCR, using appropriate primers and by sequencing the insertion sites. The genetic complementation of the ΔentA mutant was carried out as follows. A 1142 bp DNA fragment corresponding to the entA gene and its putative promoter was amplified by PCR using the B. cereus ATCC14579 genomic DNA as a template and FbacComp/RbacComp as primers ( Table 4 ). The PCR product was digested with PstI and XbaI restriction enzymes and inserted into the plasmid pHT304 [80] . The resulting plasmid (pHT304Ω entA ) was amplified in E. coli and then introduced into the ΔentA mutant strain of B. cereus by electroporation. Measurement of catechol production Extracellular levels of catechols were measured using the Arnow assay [41] . Bacteria were grown overnight (20 h) at 37°C in LB medium +200 µM 2,2′-dipyridyl. Then, samples of cultures were collected, centrifuged and filtered to remove bacteria. Samples were mixed sequentially with equal volumes of 0.5 N HCl, nitrite-molybdate reagent (10% sodium nitrite and 10% sodium molybdate), and 1 N NaOH. Positive reactions produce a red colour and absorbance was determined at 510 nm. Data were normalized to OD600 of the original culture and percentages of wild-type catechol level in culture supernatants are presented. Three independent replicates were statistically analyzed using the Student's T-test. Growth assays B. cereus strains were grown overnight at 37°C under low iron conditions by inoculating strains in LB medium supplemented with 200 µM 2,2′-dipyridyl. Overnight cultures were inoculated into a new LB medium +200 µM 2,2′-dipyridyl at a final OD of 0.01. Bacteria from mid-log phase culture were washed twice in LB medium containing 600 µM 2,2′-dipyridyl, then inoculated to a final optical density (OD) of about 0.005 into LB medium or LB+600 µM 2,2′-dipyridyl +0.3 µM HoSF supplemented or not with 5 µM Enterobactin (Sigma-Aldrich). Stock solution of ferritin was prechelated in 2 mM 2,2′-dipyridyl for two hours in order to eliminate the free iron. B. cereus cells were grown at 37°C in 96-wells microtiter plate under continuous shaking. The OD was measured at 600 nm every hour over 16 hours using a TECAN Infinite M200 Microplate Reader (TECAN Group). The assays were repeated at least three times. Virulence assays Bacterial strains were grown in LB medium and bacterial concentrations were monitored by optical density measurements and plating dilutions onto LB agar plates. B. cereus wild-type and mutant strains were injected separately into the hemocoel of G. mellonella . Insect eggs were incubated at 25°C and the larvae reared on beeswax and pollen (Naturalim). Last-instar larvae weighing about 200 mg were injected with 10 µl of mid-log phase bacteria (or spores) suspended in PBS, using the microinjector (Buckard Scientific) as previously described [84] . Various doses of bacteria (1×10 3 to 3×10 4 bacteria/larva) were used, and each experiment was repeated at least three times with 20 larvae. A control group of larvae was injected with PBS only and no effect was observed. The survival rate (% of alive/total number of infected larvae) was recorded during 72 hours after infection. Statistical analysis was performed using the Log-rank test. Based on the data obtained, LD 50 were estimated by Probit analysis with StatPlus software (AnalystSoft). Supporting Information Figure S1 Immunofluorescence control observations with anti-HoSF on B. cereus. B. cereus wild type (A–D) was grown in iron rich LB medium. B: HoSF Alexa Fluor 594 labelled polyclonal antibody. C: DAPI, D: merged images (anti-HoSF: red, DAPI: blue). B. cereus ferrtitin is revealed inside lysed (dead bacterial) cells only, compare with DAPI staining in panel C and also with Figure 1 . Experiments were performed three times. (TIF) Click here for additional data file. Figure S2 Calorimetric titration of various recombinant ferritins with IlsA. ( A, C, E ): ITC raw data. ( B, D, F ): Plot of the integrated heat versus the number of injections of IlsA. Conditions: 1 µM HuLF ( Hu man L -chain F erritin; A, B) or HuH/LF ( Hu man heteropolymer H/L F erritin; C, D) or MoHF ( Mo use H -chain F erritin; E, F) titrated with 3 µl injections of 229 µM IlsA solution in 50 mM Tris/HCl buffer, 150 mM NaCl, 1 mM EDTA and 1 mM DTT, pH = 7.0 and 25°C. ITC binding experiments were repeated at least two times with similar results and thermodynamic data are listed in Table 1 . (TIF) Click here for additional data file. Figure S3 Roles of the IlsA-NEAT and LRR domains in ferritin binding. Dot blot experiments were carried as follows: 12 pmol of IlsA and the NEAT and LRR domains of IlsA purified separately were spotted on PVDF membranes and then incubated for 1 hour with HoSF at 1 µg/ml. The signals were obtained with the HRP (horse radish peroxidase) ECL (enhanced chemiluminescent) system using an anti-HoSF polyclonal antibody at a dilution of 1∶1000 in TBS pH 7.4 buffer with 1% fat free milkpowder. (TIF) Click here for additional data file. Table S1 The table refers to B.cereus genes, which have been studied in relation to iron acquisition particularly with attention to genes analyzed in an insect environment. (DOC) Click here for additional data file. Table S2 Galleria mellonella larvae were infected by injection of several doses of B. cereus wildtype (WT) and various mutant strains of the EntA (bacillibactin) and Asb (petrobactin) siderophores. Controls were infected with PBS buffer only. For survival curves see Figure 7 . (DOC) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392503/

Biomedical Applications of Biomolecules Isolated from Methanotrophic Bacteria in Wastewater Treatment Systems

Wastewater treatment plants and other remediation facilities serve important roles, both in public health, but also as dynamic research platforms for acquiring useful resources and biomolecules for various applications. An example of this is methanotrophic bacteria within anaerobic digestion processes in wastewater treatment plants. These bacteria are an important microbial source of many products including ectoine, polyhydroxyalkanoates, and methanobactins, which are invaluable to the fields of biotechnology and biomedicine. Here we provide an overview of the methanotrophs' unique metabolism and the biochemical pathways involved in biomolecule formation. We also discuss the potential biomedical applications of these biomolecules through creation of beneficial biocompatible products including vaccines, prosthetics, electronic devices, drug carriers, and heart stents. We highlight the links between molecular biology, public health, and environmental science in the advancement of biomedical research and industrial applications using methanotrophic bacteria in wastewater treatment systems. 1. Introduction In recent years, a global movement has engaged targeting the development of alternative bio-based therapeutic products for biomedical applications in order to reduce or eliminate the adverse side effects associated with the use of non-biocompatible compounds by the human immune system [ 1 , 2 ]. A broad spectrum of naturally occurring compounds derived from animals, plants, or microbes has been tested for their employment in modern medicine [ 3 ]. In the early 2000s, twenty naturally derived therapeutic drugs were developed and brought to market [ 4 ], though this clearly was not the first instance of using biologically-derived molecules in medicine. The use of naturally occurring compounds has been documented in ancient civilizations such as Egypt and China, where they relied on plant extracts and honey for remediation and healing purposes [ 5 ], and of course Alexander Fleming's discovery of penicillin opened the door for the use of antibiotics to fight infection, but also highlighted the potential for microbially synthesized products in the pharmaceutical and medical industries [ 6 ]. The employment of bioactive composites for use in many industries has become a more robust and efficient process in many applications including: (i) the manufacturing of biopolymers, nanoparticles, pigments for the productions of drug capsules, optical fibers, electronic devices, and paint [ 7 , 8 , 9 ], (ii) the extraction of catalytic enzymes, organic acids, and surfactants to employ in drug, food, and soap industries [ 10 , 11 , 12 ], and (iii) the use of organic vitamins, lipids, and proteins, and microbial metabolites to generate synthetic hormones, nutritional supplements, targeted therapies, vaccines anti-cancer agents, anti-inflammatory drugs, and the overall medicinal industry [ 13 , 14 , 15 , 16 , 17 ]. Interestingly, wastewater treatment plants (WWTPs) comprise an important asset for its public health, environmental and economic contributions, in addition to their function as a remediation facility. WWTPs encompass an important revenue stream for their role in resource recovery [ 18 ]. More recently, WWTPs have been viewed as biorefinery facilities; they exploit the organic matter within wastewater as microbial substrates in order to sustainably generate electricity, remove contaminants, and recover resources [ 19 ]. WWTPs can be considered large biodiverse microbial populations that are distinct within each stage of the water treatment process. Each microbial population can also be characterized with the ability to produce a variety of value-added products, each of which is suitable for use in a variety of implementations in different sectors of biomedical industries [ 18 , 20 ]. WWTPs have a broad variety of different important bacterial genera such as purple sulfur bacteria, ammonia oxidizing bacteria, nitrate oxidizing bacteria, Pseudomonas , Mycobacterium , and Methylobacterium [ 21 ]. Each bacterial population represents important contributors for resource recovery for the production of biopolymers, catalytic enzymes, lipids, and proteins [ 19 ]. While bioproduct resource recovery from WWTPs can be more challenging compared to purely synthetic methods, it is a more sustainable option and is essential to overcome limitations in resource availability. For instance, more synthetic routes for production of high-value biomolecules require cost-intensive processes and bio-refineries for realistic applications. On the other hand, while there are many technical challenges in the processes of optimal bacterial cultivation, biomolecule extraction, and purification from resource pools such as WWTPs, many of these challenges are offset by the large and renewable feedstock, leading to lower input costs associated with bioproduct development. In this review we highlight the role of methane oxidizing bacteria, namely the methanotrophs found in anerobic digestion processes of WWTPs, as a multiple high-value bioproduct generating system with diverse potential biomedical applications. Furthermore, we provide an overview of the enzymatic pathways employed by methanotrophs to generate different metabolites and demonstrate the dynamic interactions of different types of biomolecules. 2. Methanotrophic Bacteria in Water Treatment Systems Methanotrophic bacteria have a unique metabolism that relies on a single carbon substrate, methane. However, its sophistication allows it to manufacture a mixture of value-added organic compounds. Methanotrophs utilize methane gas as an electron donor in order to produce sufficient energy required for cellular growth and biosynthesis of different metabolites [ 22 ]. While methanotrophs might not be a top producer for certain products, this is compensated by its ability to perform multiple roles simultaneously which begins with the oxidation of methane gas to produce methanol, an important biofuel [ 23 ]. Methane gas is the second most potent greenhouse gas after carbon dioxide; therefore, methanotrophic bacteria's ability to mitigate this harmful biogas is crucial in the global methane cycle. WWTPs are responsible for an estimated 4% of the global methane production; the gas is normally flared into the atmosphere contributing to global warming [ 24 ]. Whenever methanotrophs are present in WWTPs, they are associated with the availability of methane gas produced from the anaerobic digestion process, and with the right allocations the generated biogas can be exploited for resource recovery purposes [ 25 ]. Methanotrophs are Gram-negative bacteria and are a subgroup of a broader bacterial group known as methylotrophs [ 26 ]. They are distinct in their reliance on methane oxidization, unlike methylotrophs that have the potential to utilize different single carbon substrate such as methanol, halomethanes, and methylated amines [ 27 ]. Thus, aerobic methanotrophs metabolism relies on the role of oxygen to oxidize the methane substrate to ultimately generate carbon dioxide (CO 2 ) and water [ 28 ]. There are three types of aerobic methanotrophs that differ phylogenetically, types I, II, or X, each of which follow a distinct metabolic pathway. All three types share a common methane oxidation pathway to produce formaldehyde, after which, each group carries on in a different enzymatic pathway. Type I methanotrophs employ the ribulose monophosphate (RuMP), and type II methanotrophs utilize the serine pathway ( Figure 1 ) [ 29 ]. Type X methanotrophs exhibit similarities with type I methanotrophs in that they use the RuMP pathway; however, it differs from type I as they also have low concentrations of the serine pathway enzyme ribulose–bisphosphate carboxylase [ 30 ]. 2.1. Taxonomy and Phenotype Aerobic methanotrophs are taxonomically classified based on their phenotype, ability for spore formation, possession of specific membrane bound proteins, and their metabolic properties [ 24 ]. There are three main groups of methane oxidizing bacteria, types I, II, and X, each of which undertake a unique enzymatic pathway [ 31 ]. Type I and X methanotrophs belong to gamma-proteobacteria and reside in families Methylococcaceae and Methylothermaceae [ 32 ], whereas type II methanotrophs belong to alpha-proteobacteria from families Methylocystaceae and Beijerinckiaceae . Over time other groups of methanotrophs have emerged, including filamentous methane oxidizers with unusual methane monooxygenase, and extremely acidophilic bacteria of the phylum Verrucomicrobia . The classifications of methane-utilizing bacteria has of course evolved; however, the terms types I, II, and X are still commonly used when discussing the methanotrophs due to the distinctiveness of metabolic pathways for carbon fixation across phylogenetic groupings [ 26 , 30 ]. Morphologically, methanotrophs exhibit red coloring under Gram-staining and have various physical morphologies. For instance, type X methanotrophs are mainly found as paired cocci, while type II methanotrophs are crescent shaped rods and can occur in rosettes, and finally, type I methanotrophs can be found as either single cocci or rods [ 24 ]. 2.2. EcoPhysiology Aerobic methanotrophic bacteria inhabit oxic zones where oxygen is present as an electron acceptor and organic carbon, methane, is present for cellular biosynthesis, such as soils and freshwater, rice paddies, and WWTP sludge [ 33 ]. Most methanotrophs are mesophilic and prefer neutral pH; however, some thermophilic genera inhabit areas with high methane profusion and high temperatures such as volcanoes and soil paddies [ 34 , 35 ]. Moreover, a few psychrophilic species found in temperatures between 4 and 10 °C mainly belonging to type I methanotrophs have been reported in arctic regions [ 36 , 37 , 38 ]. Different types of methanotrophs follow distinct metabolic pathways after the oxidation of methane to formaldehyde and formate, which lead to the formation of industrially and biomedically valuable biomolecules and biopolymers as illustrated in Figure 2 . First, methane is converted to methanol by the action of methane monooxygenase (MMO), then methanol dehydrogenase (MDH) further oxidizes methanol into formaldehyde [ 39 ]. There are two types of MMO: (i) the membrane bound and copper reliant particulate (p)MMO and (ii) soluble (s)MMO. Nonetheless, the pmoA gene encoding for pMMO is considered a universal marker for methanotrophic bacteria and is expressed by nearly all types of methanotrophs. Conversely, sMMO is typically expressed by type II and some type X methanotrophs under conditions of copper scarcity, where it utilizes iron as an alternative [ 40 ]. Following the common methane oxidation pathway, type I methanotrophs undergo the RuMP cycle, which is responsible for formaldehyde assimilation and detoxification. In the RuMP cycle, formaldehyde is fixed with ribulose 5-phsosphate to form 3-hexulose-6-phophate, which is then converted to glyceraldehyde-3-phosphate to finally generate pyruvate. There is an interplay between the RuMP pathway and the pentose phosphate pathway (PPP), where the latter ensures the regeneration of ribulose-5-phsosphate, while the former produces fructose-6-phosphote that is further metabolized via the PPP [ 41 ]. Thereafter, the pentose phosphate shunt is responsible for the generation of NADPH and ribose, which is of course important for the formation of nucleotide based biological molecules. These two pathways are crucial precursors of amino acid, nucleotide, and lipid biosynthesis. Pyruvate is further converted into acetyl-CoA for incorporation into the tricarboxylic acid (TCA) cycle and the electron transport chain for energy generation [ 27 ]. In type II methanotrophs, formaldehyde produced from the common metabolic pathway is further oxidized to formate and then converted to 5,10-methylenetetrahydrofolate to form serine and yields phosphoglycerate in several stepwise reactions of the serine cycle. The serine pathway requires 3 ATP and 2 NADH for activation unlike the RuMP cycle which only requires a single ATP. The serine pathway is simultaneously synced with the ethyl malonyl Co-A (EMC) pathway which includes several CoA thioesters, starting with malyl-CoA in the TCA which is converted into acetyl-CoA and eventually ethyl malonyl-CoA is cleaved into glyoxylate and propionyl-CoA where the former is an intermediate for the formation of oxaloacetate and succinyl-CoA for the latter, which are important originators for creating high-end products [ 42 ]. Furthermore, an intermediary cycle for glyoxylate recycling, the glyoxylate regeneration cycle (GRC), overlaps with the TCA and EMC cycles in type II methanotrophs and is considered as an additional route for glyoxylate regeneration [ 40 , 43 ]. Thus, the serine, EMC, and GRC pathways lead to mapping the derivatization of the secondary metabolites in type II methanotrophs. In nutrient-deficient conditions, an additional pathway to store energy in the form of polyhydroxybutyrate (PHB) granules is engaged [ 44 ], and is discussed in the following section. 2.1. Taxonomy and Phenotype Aerobic methanotrophs are taxonomically classified based on their phenotype, ability for spore formation, possession of specific membrane bound proteins, and their metabolic properties [ 24 ]. There are three main groups of methane oxidizing bacteria, types I, II, and X, each of which undertake a unique enzymatic pathway [ 31 ]. Type I and X methanotrophs belong to gamma-proteobacteria and reside in families Methylococcaceae and Methylothermaceae [ 32 ], whereas type II methanotrophs belong to alpha-proteobacteria from families Methylocystaceae and Beijerinckiaceae . Over time other groups of methanotrophs have emerged, including filamentous methane oxidizers with unusual methane monooxygenase, and extremely acidophilic bacteria of the phylum Verrucomicrobia . The classifications of methane-utilizing bacteria has of course evolved; however, the terms types I, II, and X are still commonly used when discussing the methanotrophs due to the distinctiveness of metabolic pathways for carbon fixation across phylogenetic groupings [ 26 , 30 ]. Morphologically, methanotrophs exhibit red coloring under Gram-staining and have various physical morphologies. For instance, type X methanotrophs are mainly found as paired cocci, while type II methanotrophs are crescent shaped rods and can occur in rosettes, and finally, type I methanotrophs can be found as either single cocci or rods [ 24 ]. 2.2. EcoPhysiology Aerobic methanotrophic bacteria inhabit oxic zones where oxygen is present as an electron acceptor and organic carbon, methane, is present for cellular biosynthesis, such as soils and freshwater, rice paddies, and WWTP sludge [ 33 ]. Most methanotrophs are mesophilic and prefer neutral pH; however, some thermophilic genera inhabit areas with high methane profusion and high temperatures such as volcanoes and soil paddies [ 34 , 35 ]. Moreover, a few psychrophilic species found in temperatures between 4 and 10 °C mainly belonging to type I methanotrophs have been reported in arctic regions [ 36 , 37 , 38 ]. Different types of methanotrophs follow distinct metabolic pathways after the oxidation of methane to formaldehyde and formate, which lead to the formation of industrially and biomedically valuable biomolecules and biopolymers as illustrated in Figure 2 . First, methane is converted to methanol by the action of methane monooxygenase (MMO), then methanol dehydrogenase (MDH) further oxidizes methanol into formaldehyde [ 39 ]. There are two types of MMO: (i) the membrane bound and copper reliant particulate (p)MMO and (ii) soluble (s)MMO. Nonetheless, the pmoA gene encoding for pMMO is considered a universal marker for methanotrophic bacteria and is expressed by nearly all types of methanotrophs. Conversely, sMMO is typically expressed by type II and some type X methanotrophs under conditions of copper scarcity, where it utilizes iron as an alternative [ 40 ]. Following the common methane oxidation pathway, type I methanotrophs undergo the RuMP cycle, which is responsible for formaldehyde assimilation and detoxification. In the RuMP cycle, formaldehyde is fixed with ribulose 5-phsosphate to form 3-hexulose-6-phophate, which is then converted to glyceraldehyde-3-phosphate to finally generate pyruvate. There is an interplay between the RuMP pathway and the pentose phosphate pathway (PPP), where the latter ensures the regeneration of ribulose-5-phsosphate, while the former produces fructose-6-phosphote that is further metabolized via the PPP [ 41 ]. Thereafter, the pentose phosphate shunt is responsible for the generation of NADPH and ribose, which is of course important for the formation of nucleotide based biological molecules. These two pathways are crucial precursors of amino acid, nucleotide, and lipid biosynthesis. Pyruvate is further converted into acetyl-CoA for incorporation into the tricarboxylic acid (TCA) cycle and the electron transport chain for energy generation [ 27 ]. In type II methanotrophs, formaldehyde produced from the common metabolic pathway is further oxidized to formate and then converted to 5,10-methylenetetrahydrofolate to form serine and yields phosphoglycerate in several stepwise reactions of the serine cycle. The serine pathway requires 3 ATP and 2 NADH for activation unlike the RuMP cycle which only requires a single ATP. The serine pathway is simultaneously synced with the ethyl malonyl Co-A (EMC) pathway which includes several CoA thioesters, starting with malyl-CoA in the TCA which is converted into acetyl-CoA and eventually ethyl malonyl-CoA is cleaved into glyoxylate and propionyl-CoA where the former is an intermediate for the formation of oxaloacetate and succinyl-CoA for the latter, which are important originators for creating high-end products [ 42 ]. Furthermore, an intermediary cycle for glyoxylate recycling, the glyoxylate regeneration cycle (GRC), overlaps with the TCA and EMC cycles in type II methanotrophs and is considered as an additional route for glyoxylate regeneration [ 40 , 43 ]. Thus, the serine, EMC, and GRC pathways lead to mapping the derivatization of the secondary metabolites in type II methanotrophs. In nutrient-deficient conditions, an additional pathway to store energy in the form of polyhydroxybutyrate (PHB) granules is engaged [ 44 ], and is discussed in the following section. 3. Microbially Recovered Resources from Methanotrophic Bacteria and Their Biomedical Applications The unique metabolism of the methanotrophs enable them to both mitigate the greenhouse gas methane and remove harmful contaminants such as ammonia and nitrate found in water systems. Furthermore, they have the potential to produce valuable bioactive derivatives through methane uptake and phosphorylation pathways including single cell protein, biopolymer, S-layer, the copper binding protein methanobactin, methanol biogas, organic acids, ectoine, vitamin B 12 , and various enzyme catalysts. These products have significant value in diverse biomedical fields as they can be used as tools to aid in overcoming biomedical obstacles ( Figure 3 ). 3.1. Exopolysaccharides The extra-polymeric substance or exopolysaccharide (EPS) is a biocompatible, non-toxic, and decomposable high molecular weight carbohydrate-based polymer [ 45 ]. EPS is a primary component of biofilms in environments with low nutrient availability and/or high contamination to protect bacterial cells from environmental toxicity [ 46 ]. They are mainly formed by polysaccharide and protein integration, with low amounts of DNA and lipids being detected [ 46 ], and perform many roles as they ensure structural stability of the bacterial biofilm and act as a filter to allow the passage of certain nutrients while blocking the entrance of other molecules [ 47 ]. EPS production in methanotrophs has been found to be associated with high carbon-to-oxygen and -nitrogen ratios. Methane-rich environments such as soil interfaces are found to possess the thickest EPS biofilms [ 46 ]. The EPS is thought to play a role in carbon assimilation in different environments where nitrogen is depleted and type I methanotrophs are unable to fix nitrogen; EPS is produced through the RuMP pathway as a carbon reservoir [ 47 ]. On the other hand, type II methanotrophs have been known to occasionally produce the EPS polymer to catalyze nitrogen fixation by limiting oxygen penetration, which in turn causes oxygen depletion that triggers enzymatic activation [ 44 ]. Generally, in both type I and II methanotrophs, glycolysis is the main pathway for EPS synthesis as it metabolically overlaps with both the serine and RuMP pathways to provide nucleoside diphosphate saccharides that polymerize into EPS [ 48 ]. Microbial extracted EPS have better properties for industrial applications than its algal- and plant-based counterparts due to the replicability and sustainability of its production process and the higher quality polysaccharide polymer [ 47 ]. Additionally, the microbial EPS is of a stronger titer than the plant-based product. The safe biocompatible nature of EPS makes it an ideal candidate for many medicinal and pharmaceutical applications. An example of a microbially produced EPS is dextran, first used as a plasma expander to control bleeding in hypovolemic patients [ 49 ]. Moreover, bacterial alginates and other EPSs such as xanthan, pullulan, and bacterial cellulose are suitable in different applications, such drug encapsulation, dental casting material, treatment of acid reflux, and as scaffolds or wound dressings [ 50 ]. Moreover, hyaluronic acid produced by many bacterial species is used for many cosmetic purposes including skin regeneration, as well as in many operative procedures due to its ability to accelerate healing, and as a curative approach for arthritis [ 51 ]. Bacterial gellan is an EPS useful in the manufacturing of different medicinal drugs [ 52 ]. Similarly, EPS has seen application as additives to vaccine formulations and integration in devices for diagnostic imaging. Lastly, due to their physiological potency, EPS is often explored as a component for applications in immunoregulation and cancer treatments. 3.2. Polyhydroxyalkanoate Polyhydroxyalkanoates (PHAs) are optically active microbial synthesized polyesters of hydroxy-acids; repeating monomers accumulated intracellularly as hydrophobic inclusion bodies ranging from 0.2–0.5 μm in diameter within the cytoplasm. These polymers are stored as an energy reserve material and can reach as much as 90% of the cell dry weight (CDW) in some species such as Bacillus megaterium . PHAs are accumulated under limiting conditions of phosphorus, nitrogen, and oxygen as well as excess carbon [ 53 ]. PHAs are non-toxic, biocompatible, isotactic and insoluble in water. PHAs tend to have a high polymerization rate, crystallinity and high molecular mass, which is a comparable property with regular plastics such as polypropylene (PP) [ 54 ]. PHAs have been classified into more than 150 different types based on the large number of different hydroxy-alkanoic acid monomeric structures with different side chain lengths. Therefore, they are divided into two main categories; short chain length (SCL) with 3-5 carbon molecules of hydroxy-acids monomers such as poly 3-hydroxybutyrate (PHB) and polyhydroxyvalerate (PHV) or medium chain length (MCL) 6-16 carbon molecules such as polyhydroxyoctanoate (PHO) [ 11 ]. The precursors of these PHA categories encompass the type of bacteria and the carbon substrate in the culture media; each category is characterized with different chemical and physical properties as well as designated industrial applications. For instance, the MCL PHAs have lower crystallinity and are more flexible than PHB or PHV [ 14 , 55 ], while SCL PHB is considered the most well studied PHA. Bacterial PHB has 55–80% crystallinity and mechanical properties similar to that of PP. However, it is less likely to break and is remarkably more durable than PP [ 56 ]. PHA can be produced using both Gram-negative and Gram-positive bacteria. However, it is noteworthy that most of the PHA production research have been focusing on Gram-negative bacteria even though Gram-positive bacteria, especially Bacillus spp., have long been found to produce large amounts of PHB. Moreover, Gram-negative microorganisms have a lipid bilayer that is not biocompatible and stimulate defense reaction by the body; hence, Gram-positive bacteria are better suited for biomedical applications. PHB is synthesized by type II methanotrophs using the serine pathway in order to produce malyl-CoA, which is responsible for the initiation of the EMC cycle. PHB synthase is the terminal enzyme involved in the process of moderate final monomer formation. It is part of a group of enzymes encoded by the genes phaA , phaB, and phaC . An important group of PHA associated proteins, the so-called phasins, have been found to modulate the accumulation of the PHB polymer through regulation of the activation of phaA , phaB , and phaC expression. Methanotrophs have been found to produce high yields of PHB (up to 70% CDW) and have a vital role in the industrial accumulation of PHB [ 7 , 57 ]. PHA industrialization began in the 1990s where the mass production of this bioplastic was manufactured for multiple medicinal and non-medicinal purposes. While PHA has been offered as an answer to petroleum-based plastics, its high capital cost made it more suitable for biomedical and pharmaceutical applications [ 58 ]. Since PHA is an ecofriendly and biocompatible material, with an array of diverse physicochemical properties that can be modified to meet suitable strength and elasticity, it could be employed for a variety of applications ranging from hard to soft tissue engineering, implant manufacturing [ 58 ], tissue regeneration [ 7 ], vascular systems [ 59 ], heart valves [ 60 , 61 ], and bones and cartilage [ 16 ]. The integration of PHA polymers and copolymers within the human body help overcome some of the problems associated with using other materials including general fragility, danger of contamination, high chance of occlusions, or inducing an immunological response due to non-biocompatibility [ 60 ]. Hence, current applications of PHB and PHO have been employed as copolymers to create stents, bioactive tissue patches for heart and vascular systems, orthopedic pins, nerve guides, and repair devices, etc. [ 61 , 62 , 63 , 64 , 65 ]. Moreover, many important pharmaceutical applications include different form of drug carriers with the ability of controlled drug release [ 66 ]. 3.3. Surface Layers Surface layers (S-layer) are polymeric proteins that cover the outside of a microbial cell. They are of special importance as they can comprise ~15% of the total protein content of the cell [ 67 ]. These crystalline structures are characterized with an amorphous layout that mainly consists of hydrophobic molecules, acidic amino acids, and lysine [ 64 ]. This nanoscale lattice matrix is characterized with identical pore sizes, allowing for self-assembly in a variety of environments [ 65 ]. The negatively charged two-dimensional crystalline protein or glycoprotein lattice embodies the complex network of proteins involved in vital cellular functions such as synthesis, excretion, and the layout of membrane bound proteins [ 68 ]. While the specific role of S-layer has been controversial, some studies illustrate that the S-layer might be serving a different function for different bacterial groups, as part of an adaptation evolutionary mechanism in a diversity of eco-niches [ 68 ]. For example, the S-layer serves as a protective layer against high osmotic pressure where the porous membrane has the ability to counteract the external pressure in halophilic bacteria. Its ability to withstand high temperature and mechanical stress has been linked to maintenance of cell shape and structural integrity for archaeal groups [ 46 ], and the S-layer provides gram positive bacteria with a periplasmic space [ 69 ]. The S-layer guarantees the presence of binding sites for different exoenzymes, for instance providing linkage sites for thermostable proteases enabling tolerance to thermophilic environments, or the ability to incorporate enzymes like exo-amylase in Bacillus stearothermophilus [ 45 ]. The S-layer can also provide cellular adhesion capabilities [ 47 ], act as a selective semipermeable membrane to protect from lyase activity, and prevent parasite and toxin penetration, while at the same time modulating the passage of essential molecules and some important enzymes, and aid in retention of organic substrates and metal compounds bound to cellular membrane [ 65 ]. Lastly, in particular to methanotrophs, S-layer proteins have been linked with conveying copper ions to pMMO, which aids in maintenance of the equilibrium in regard of copper concentrations [ 49 ]. The prevalence of copper-binding proteins CorA and MopE, and diheme periplasmic cytochrome C peroxidase CorB/Mca was detected in Methylomicrobium album BG8, Methylotuvimicrobium alcaliphilum 20Z, and Methylococcus capsulatus (Bath) [ 70 ]. This protein combination corresponds to an iron chelating compound, methanobactin; methanotrophs that do not possess S-layers do not express CorA nor MopE polypeptide pairs [ 70 ]. The surface structure of aerobic methanotrophs is uniquely different since it permits multifunctionality which gives a communicative advantage between these microorganisms and their surroundings. However, it is important to unravel the genes responsible for the protein structure of S-layers in aerobic methanotrophs in order to understand the means of interaction between methanobactin and cell wall machineries that are able to make S-layers. Slightly over four hundred different species of prokarya have been identified as possessing genes responsible for expression of S-layer [ 71 ]. It is noteworthy that the S-layer has little to no structural similarities between different taxonomical groups [ 65 ]. Furthermore, short sequences of post-translational mRNA are usually responsible for transcription of a single protein that makes up the S-layer crystalline matrix. The promoter's sequences responsible for translation of S protein have dented long end 5′ untranslated region (UTR) which protects the protein from RNase dissimilative activity. S-proteins are characterized with a longer half-life, which is why it is stringently modulated to be synthesized in the stationary phase rather than the log phase that is achieved through regulatory repressive gene sequences to stop the translation of the S protein in the growth phase namely, splA in Thermus thermophilus . Hence, some species possess different genes responsible for each phase of cell synthesis [ 31 ]. In Gram-positive bacteria, S-layers are most commonly attached to the peptidoglycan layer through the SLH (S-layer homology) motif. On the other hand, in Gram-negative bacteria, S-layers are attached to the outer membrane lipid bilayer by ionically binding to the lipopolysaccharide or covalently joining N-terminal end to the S-layer and hydrophobic interactions as it can be joined with lipids via van der Waals forces. Accordingly, most methanotrophic bacteria such as Methylococcus , Methylothermus , and Methylomicrobium have an encapsulating S-layer with varying structure of the S-layer between methanotrophs [ 67 ]. Most methanotrophs S-layer form planar (p2, p4) symmetry, cup-shaped, or conical arrangements having hexagonal (p6) symmetry. S-layers are highly organized matrices packed with proteins and glycoproteins; they have been found to be useful for many applications in biotechnology and nanomedicine ( Figure 4 ) [ 64 ]. The S-layer differs within different bacterial species each with unique network of antigenic proteins [ 69 ]. Furthermore, S-layers possess good thermomechanical stability and the ability to capture different bioactive molecules [ 68 ]. In addition, S-layers are surrounded with a set of functional groups including protein biomarkers that have a role in preventing cellular damage by reducing oxidative stress, which are useful in different target therapy technologies [ 67 ]. Therefore, these crystalline formations show great promise in gene therapy and as drug nanocarriers. However, in some pathogenic microorganisms the S-layer can reflect virulence and potency. An example of this is Bacillus anthracis where the surface layer of this pathogen is responsible for causing anthrax. Therefore, S-layers are employed in vaccine development either as an attenuated pathogen, adjuvant, a hapten added to an immunization formula, or as vaccine carrier [ 72 ]. It has been found that S-layer containing vaccines to be a treatment for different hypersensitivity disorders such as type I allergy [ 73 ]. In addition, S-layers form highly stable ultrafiltration membranes that have uniform isoporous lattice with desirable mechanochemical stability that is formed by inter and intramolecular interactions which is useful for retaining important biological molecules and in the formation of lipid membranes [ 65 ]. S-layers have also been studied for creating immobilization matrices that can be used as a diagnostic tool to identify various diseases such as type I allergy [ 74 , 75 ]. The external layer contain a set of fusion proteins such as rSbpA, STII, and Cys, which are used in synthesizing gold nanoparticles [ 76 ]. Furthermore, some studies have demonstrated that S-layers belonging to different Lactobacillus strains have a role in protecting the human intestine as they form a protective coating that binds to the lining of the gut and creates a barrier in order to avert the attachment of many opportunistic pathogens like E. coli [ 77 ]. 3.4. Methanobactin Methanobactins (Mbns) are peptide chalkophores that bind copper, which is needed for cell synthesis [ 79 ]. They were originally characterized in the methanotrophic bacterial species Methylococcus capsulatus and were found to be required for the functioning of methane oxidizing pMMO [ 68 ], which is transported outside of the cell to trap and bind to Cu(I) ions [ 80 ]. This chelating effect also reduces Cu(II), which is toxic to the bacterial cell, and to Cu(I) in copper-deficient environments [ 81 , 82 ]. The Mbn-Cu(I) complex then enters the cell through active transport to convey copper for important metabolic functions in methanotrophic bacteria [ 83 ]. Mbns exhibit significant sequence variability between bacterial strains [ 84 ], and use a modified rRNA to regulate its translation [ 85 ]. Methanobactin acts as a chelating agent with the ability to bind to copper, mercury and gold particles [ 86 ]. This is especially important as the ability of reducing gold has successfully generated gold nanoparticles that have many biomedical applications. These applications include antimicrobial activity, incorporation in fuel cells, photo-thermal and dynamic therapy, cancer treatment, act as an epitope, and tether to immunogenic molecules that have the ability to attach to cancer biomarkers (antigens), and in the development of many medically advanced technologies such as plasmonic biosensors, for visualization, and bioimaging [ 80 ]. Mbns also show great potential in the treatment of copper-associated illnesses such as Wilson disease (WD), Alzheimer disease (AD), fatty liver disease, and BRAF-positive cancers by preventing copper buildup in the liver and consequently other body tissues, and thereby preventing permanent liver and neurological tissue damage [ 83 ]. 3.5. Antibacterial Proteins Methylocystis minimus and Methylobacter luteus have been found to produce a thermostable protein capable of killing pathogenic bacteria, which is currently under investigation for possible application as an antibiotic [ 13 ]. The bacteria encode genes that produce peptidase enzymes, which was found to function as bacteriocin [ 87 , 88 ]. 3.6. Single Cell Protein (SCP) Single cell protein (SCP) is a protein derived from microbial cells that feed on a range of organic carbon sources. Many microorganisms, including algae, blue-green fungi, and bacteria, which can make up to 80% CDW [ 18 ]. The current vernacular usage of SCP arose in 1966 following a substitution of an older term microbial proteins, which was used to describe dried microbial cells serving as an ingredient or a substitute for protein-rich foods [ 64 ]. In view of an insufficient world food supply, the use of biomass produced by industrially scaled reactors could in theory provide a resource for SCP recovery [ 89 ]. SCP is of great nutritional value because of its high protein, vitamins, and lipid and essential amino acid content [ 90 ]. Methanotrophs are a well-known source for SCP production where the proteinaceous substance within methanotrophic bacteria is estimated to be 60–65% [ 89 ]. SCP offers a source of vitamins, amino acids, minerals, crude fibers, etc., which are important for healthy eyes and skin [ 76 ]. It is used as a protein supplement for undernourished children as it is added to improve the nutritional value of many consumed products as well as for athletes as they consume to derive energy [ 91 ]. Furthermore, it is also used for animal nourishment including pigs, cattle, and poultry [ 92 ]. Currently, Spirulina tablets are prescribed as dietary supplement as it lowers blood sugar levels in diabetic patients due to the presence of gamma-linolenic acid and prevents the accumulation of cholesterol in human body [ 93 ]. 3.7. Ectoine Ectoine (C 6 H 10 N 2 O 2 ) and its derivative hydroxyectoine (C 6 H 10 N 2 O 3 ) are high value compatible solutes [ 94 ], and are secreted intracellularly by microbial cells in order to overcome an environment's hypersalinity and balance the osmotic pressure [ 64 ]. It is noteworthy that this osmoregulatory can also be excreted to the extracellular environment as a response to hyposalinity [ 95 ]. Furthermore, ectoine synthesis was found to be associated with many amino acids, lipids, and proteins, which ensure their structural stability [ 31 ]. Thus, this cyclic imino acid has many medical and biotechnological applications and considered an expensive ingredient that currently retails for around $1000/kg [ 96 ]. Interestingly, in halophilic environments, methanotrophic bacteria synthesize ectoine by the conversion of oxaloacetate to aspartate followed by activation of a cascade reaction catalyzed by diaminobutyric acid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA), and ectoine synthase (EctC) as shown in Figure 5 [ 97 ]. This process is regulated by the MarR-like transcriptional regulator EctR1 that is usually found in conjunction with ectABC operon [ 94 ]. An important function for ectoine and hydroxyectoine is stabilizing and protecting nucleic acids and proteins from mutations. It is a key contributor to the prevention and treatment of many illnesses that are linked to protein and nucleic acid conformation such as Alzheimer's disease (AD), Machado–Joseph disease (MJD), and spongiform encephalopathies that occur due to amyloid deformation [ 98 ]. Therefore, ectoine is important in mediating the hinderance of protein aggregation and chain elongation in these illnesses while reducing cytotoxicity [ 99 , 100 ]. Moreover, ectoine has an anti-inflammatory and hydrating nature which can prevent hostile immune responses [ 96 ]. This is achieved through reducing neutrophils activity and stabilizing epithelial cells through creating a barrier and obstructing inflammatory cell signaling initiation in the lungs, thus preventing diseases like oral mucositis (chemotherapy induced in cancer patients) and in the airways like Allergic Rhinitis (AR) and Rhinosinusitis (ARS). These hypersensitivity-related conditions are responsible for airway damage due to the increased oxidative stress caused by polluting nanoparticles in the air [ 101 ]. Topical application of ectoine solute as nasal or oral sprays have been shown to limit these conditions and to be effective as an allergy medication in order to prevent irritation [ 102 ]. Similarly, ectoine-based eye drops hydrating potential exhibit an analogous effect in controlling and treating eye inflammation which can be caused by many medically associated conditions causing eye dryness or irritation [ 103 ]. Ectoine is also employed and integrated in many skin care product formulations as it can protect skin from harmful UV-A radiation (due to its DNA stabilizing capability) while soothing skin irritations through a barrier to trap moisture and protect skin from damage [ 104 ]. Lastly, ectoine has an important role in the regeneration of body tissue and healing ulcers, treating conditions, such as vascular leak syndrome (VLS) and neurodermatitis [ 105 ]. 3.8. Carotenoids Carotenoids occur as a group of pigmented biomolecules that help in photosynthesis in a wide range of organisms such as plants, fungi, and bacteria. Carotenoids are divided into two classes: carotenes and xanthophylls. The former are biological precursors for the formation of vitamin A1 and retinol while the latter provide protection against excess absorbed light energy, therefore acting as a shield for organisms against photo-oxidative stress [ 106 ]. Carotenoids play an important role in biomedicine due to their antioxidant activity and oxygen reducing ability which endow a protective function to several organs such as the heart and pancreas. For instance, the presence of lycopene has been correlated with heart health and the prevention of cervical intraepithelial neoplasia [ 107 ] and myocardial infarction [ 108 ]. Furthermore, carotenoids has been found to regulate gene expression which in turn affects cell growth and overall immunity [ 109 ]. Cohort studies have demonstrated an association between high carotenoid intake and a reduced occurrence of breast, cervical, ovarian, colorectal cancers, cardiovascular, and eye diseases [ 110 ]. The Methylomonas genus of methanotrophic bacteria has been found to produce one form of xanthophylls, namely lycopene. Lycopene consists of a 40 carbon chain and is present in lipophilic mediums such as membranes and lipoproteins commonly found in type I methanotrophic bacteria [ 111 ]. This xanthophyll derivative is a well-known antioxidant that protects the cell against reactive oxygen species (ROS) and oxidative damage and is often associated with various chronic illnesses, including cancer and cardiovascular diseases. Likewise, studies have shown that a drop in lycopene levels in blood serum as one ages has been considered to have a role in the incidence of prostatic, cervical, and pancreatic cancers [ 110 ]. Furthermore, lycopene plays a pivotal role in protecting against coronary atherosclerosis and myocardial infraction by being a part of low-density lipoproteins (LDL) and promoting immunity against free radicals damage [ 112 ]. Due to the aforementioned functional role of carotenoids, they have been employed in the drug industry and cosmetics, since they are biological precursors for vitamin A production, an fundamental nutrient in biological functions comprising vision, reproduction, and immunity [ 113 ]. As well, carotenoids are used as additives in the form of lutein and astaxanthin which exhibit anti-inflammatory and neuroprotective effect [ 114 ]. The administration of a mixture of lutein, β-cryptoxanthin, lycopene, zeaxanthin, and fucoxanthin have been found to inhibit function of the p16 and p73 oncogenes [ 115 ]. Different types of carotenoids are used in treatment of several illnesses such as diabetes, Parkinson's, Alzheimer's ( Table 1 ). 3.1. Exopolysaccharides The extra-polymeric substance or exopolysaccharide (EPS) is a biocompatible, non-toxic, and decomposable high molecular weight carbohydrate-based polymer [ 45 ]. EPS is a primary component of biofilms in environments with low nutrient availability and/or high contamination to protect bacterial cells from environmental toxicity [ 46 ]. They are mainly formed by polysaccharide and protein integration, with low amounts of DNA and lipids being detected [ 46 ], and perform many roles as they ensure structural stability of the bacterial biofilm and act as a filter to allow the passage of certain nutrients while blocking the entrance of other molecules [ 47 ]. EPS production in methanotrophs has been found to be associated with high carbon-to-oxygen and -nitrogen ratios. Methane-rich environments such as soil interfaces are found to possess the thickest EPS biofilms [ 46 ]. The EPS is thought to play a role in carbon assimilation in different environments where nitrogen is depleted and type I methanotrophs are unable to fix nitrogen; EPS is produced through the RuMP pathway as a carbon reservoir [ 47 ]. On the other hand, type II methanotrophs have been known to occasionally produce the EPS polymer to catalyze nitrogen fixation by limiting oxygen penetration, which in turn causes oxygen depletion that triggers enzymatic activation [ 44 ]. Generally, in both type I and II methanotrophs, glycolysis is the main pathway for EPS synthesis as it metabolically overlaps with both the serine and RuMP pathways to provide nucleoside diphosphate saccharides that polymerize into EPS [ 48 ]. Microbial extracted EPS have better properties for industrial applications than its algal- and plant-based counterparts due to the replicability and sustainability of its production process and the higher quality polysaccharide polymer [ 47 ]. Additionally, the microbial EPS is of a stronger titer than the plant-based product. The safe biocompatible nature of EPS makes it an ideal candidate for many medicinal and pharmaceutical applications. An example of a microbially produced EPS is dextran, first used as a plasma expander to control bleeding in hypovolemic patients [ 49 ]. Moreover, bacterial alginates and other EPSs such as xanthan, pullulan, and bacterial cellulose are suitable in different applications, such drug encapsulation, dental casting material, treatment of acid reflux, and as scaffolds or wound dressings [ 50 ]. Moreover, hyaluronic acid produced by many bacterial species is used for many cosmetic purposes including skin regeneration, as well as in many operative procedures due to its ability to accelerate healing, and as a curative approach for arthritis [ 51 ]. Bacterial gellan is an EPS useful in the manufacturing of different medicinal drugs [ 52 ]. Similarly, EPS has seen application as additives to vaccine formulations and integration in devices for diagnostic imaging. Lastly, due to their physiological potency, EPS is often explored as a component for applications in immunoregulation and cancer treatments. 3.2. Polyhydroxyalkanoate Polyhydroxyalkanoates (PHAs) are optically active microbial synthesized polyesters of hydroxy-acids; repeating monomers accumulated intracellularly as hydrophobic inclusion bodies ranging from 0.2–0.5 μm in diameter within the cytoplasm. These polymers are stored as an energy reserve material and can reach as much as 90% of the cell dry weight (CDW) in some species such as Bacillus megaterium . PHAs are accumulated under limiting conditions of phosphorus, nitrogen, and oxygen as well as excess carbon [ 53 ]. PHAs are non-toxic, biocompatible, isotactic and insoluble in water. PHAs tend to have a high polymerization rate, crystallinity and high molecular mass, which is a comparable property with regular plastics such as polypropylene (PP) [ 54 ]. PHAs have been classified into more than 150 different types based on the large number of different hydroxy-alkanoic acid monomeric structures with different side chain lengths. Therefore, they are divided into two main categories; short chain length (SCL) with 3-5 carbon molecules of hydroxy-acids monomers such as poly 3-hydroxybutyrate (PHB) and polyhydroxyvalerate (PHV) or medium chain length (MCL) 6-16 carbon molecules such as polyhydroxyoctanoate (PHO) [ 11 ]. The precursors of these PHA categories encompass the type of bacteria and the carbon substrate in the culture media; each category is characterized with different chemical and physical properties as well as designated industrial applications. For instance, the MCL PHAs have lower crystallinity and are more flexible than PHB or PHV [ 14 , 55 ], while SCL PHB is considered the most well studied PHA. Bacterial PHB has 55–80% crystallinity and mechanical properties similar to that of PP. However, it is less likely to break and is remarkably more durable than PP [ 56 ]. PHA can be produced using both Gram-negative and Gram-positive bacteria. However, it is noteworthy that most of the PHA production research have been focusing on Gram-negative bacteria even though Gram-positive bacteria, especially Bacillus spp., have long been found to produce large amounts of PHB. Moreover, Gram-negative microorganisms have a lipid bilayer that is not biocompatible and stimulate defense reaction by the body; hence, Gram-positive bacteria are better suited for biomedical applications. PHB is synthesized by type II methanotrophs using the serine pathway in order to produce malyl-CoA, which is responsible for the initiation of the EMC cycle. PHB synthase is the terminal enzyme involved in the process of moderate final monomer formation. It is part of a group of enzymes encoded by the genes phaA , phaB, and phaC . An important group of PHA associated proteins, the so-called phasins, have been found to modulate the accumulation of the PHB polymer through regulation of the activation of phaA , phaB , and phaC expression. Methanotrophs have been found to produce high yields of PHB (up to 70% CDW) and have a vital role in the industrial accumulation of PHB [ 7 , 57 ]. PHA industrialization began in the 1990s where the mass production of this bioplastic was manufactured for multiple medicinal and non-medicinal purposes. While PHA has been offered as an answer to petroleum-based plastics, its high capital cost made it more suitable for biomedical and pharmaceutical applications [ 58 ]. Since PHA is an ecofriendly and biocompatible material, with an array of diverse physicochemical properties that can be modified to meet suitable strength and elasticity, it could be employed for a variety of applications ranging from hard to soft tissue engineering, implant manufacturing [ 58 ], tissue regeneration [ 7 ], vascular systems [ 59 ], heart valves [ 60 , 61 ], and bones and cartilage [ 16 ]. The integration of PHA polymers and copolymers within the human body help overcome some of the problems associated with using other materials including general fragility, danger of contamination, high chance of occlusions, or inducing an immunological response due to non-biocompatibility [ 60 ]. Hence, current applications of PHB and PHO have been employed as copolymers to create stents, bioactive tissue patches for heart and vascular systems, orthopedic pins, nerve guides, and repair devices, etc. [ 61 , 62 , 63 , 64 , 65 ]. Moreover, many important pharmaceutical applications include different form of drug carriers with the ability of controlled drug release [ 66 ]. 3.3. Surface Layers Surface layers (S-layer) are polymeric proteins that cover the outside of a microbial cell. They are of special importance as they can comprise ~15% of the total protein content of the cell [ 67 ]. These crystalline structures are characterized with an amorphous layout that mainly consists of hydrophobic molecules, acidic amino acids, and lysine [ 64 ]. This nanoscale lattice matrix is characterized with identical pore sizes, allowing for self-assembly in a variety of environments [ 65 ]. The negatively charged two-dimensional crystalline protein or glycoprotein lattice embodies the complex network of proteins involved in vital cellular functions such as synthesis, excretion, and the layout of membrane bound proteins [ 68 ]. While the specific role of S-layer has been controversial, some studies illustrate that the S-layer might be serving a different function for different bacterial groups, as part of an adaptation evolutionary mechanism in a diversity of eco-niches [ 68 ]. For example, the S-layer serves as a protective layer against high osmotic pressure where the porous membrane has the ability to counteract the external pressure in halophilic bacteria. Its ability to withstand high temperature and mechanical stress has been linked to maintenance of cell shape and structural integrity for archaeal groups [ 46 ], and the S-layer provides gram positive bacteria with a periplasmic space [ 69 ]. The S-layer guarantees the presence of binding sites for different exoenzymes, for instance providing linkage sites for thermostable proteases enabling tolerance to thermophilic environments, or the ability to incorporate enzymes like exo-amylase in Bacillus stearothermophilus [ 45 ]. The S-layer can also provide cellular adhesion capabilities [ 47 ], act as a selective semipermeable membrane to protect from lyase activity, and prevent parasite and toxin penetration, while at the same time modulating the passage of essential molecules and some important enzymes, and aid in retention of organic substrates and metal compounds bound to cellular membrane [ 65 ]. Lastly, in particular to methanotrophs, S-layer proteins have been linked with conveying copper ions to pMMO, which aids in maintenance of the equilibrium in regard of copper concentrations [ 49 ]. The prevalence of copper-binding proteins CorA and MopE, and diheme periplasmic cytochrome C peroxidase CorB/Mca was detected in Methylomicrobium album BG8, Methylotuvimicrobium alcaliphilum 20Z, and Methylococcus capsulatus (Bath) [ 70 ]. This protein combination corresponds to an iron chelating compound, methanobactin; methanotrophs that do not possess S-layers do not express CorA nor MopE polypeptide pairs [ 70 ]. The surface structure of aerobic methanotrophs is uniquely different since it permits multifunctionality which gives a communicative advantage between these microorganisms and their surroundings. However, it is important to unravel the genes responsible for the protein structure of S-layers in aerobic methanotrophs in order to understand the means of interaction between methanobactin and cell wall machineries that are able to make S-layers. Slightly over four hundred different species of prokarya have been identified as possessing genes responsible for expression of S-layer [ 71 ]. It is noteworthy that the S-layer has little to no structural similarities between different taxonomical groups [ 65 ]. Furthermore, short sequences of post-translational mRNA are usually responsible for transcription of a single protein that makes up the S-layer crystalline matrix. The promoter's sequences responsible for translation of S protein have dented long end 5′ untranslated region (UTR) which protects the protein from RNase dissimilative activity. S-proteins are characterized with a longer half-life, which is why it is stringently modulated to be synthesized in the stationary phase rather than the log phase that is achieved through regulatory repressive gene sequences to stop the translation of the S protein in the growth phase namely, splA in Thermus thermophilus . Hence, some species possess different genes responsible for each phase of cell synthesis [ 31 ]. In Gram-positive bacteria, S-layers are most commonly attached to the peptidoglycan layer through the SLH (S-layer homology) motif. On the other hand, in Gram-negative bacteria, S-layers are attached to the outer membrane lipid bilayer by ionically binding to the lipopolysaccharide or covalently joining N-terminal end to the S-layer and hydrophobic interactions as it can be joined with lipids via van der Waals forces. Accordingly, most methanotrophic bacteria such as Methylococcus , Methylothermus , and Methylomicrobium have an encapsulating S-layer with varying structure of the S-layer between methanotrophs [ 67 ]. Most methanotrophs S-layer form planar (p2, p4) symmetry, cup-shaped, or conical arrangements having hexagonal (p6) symmetry. S-layers are highly organized matrices packed with proteins and glycoproteins; they have been found to be useful for many applications in biotechnology and nanomedicine ( Figure 4 ) [ 64 ]. The S-layer differs within different bacterial species each with unique network of antigenic proteins [ 69 ]. Furthermore, S-layers possess good thermomechanical stability and the ability to capture different bioactive molecules [ 68 ]. In addition, S-layers are surrounded with a set of functional groups including protein biomarkers that have a role in preventing cellular damage by reducing oxidative stress, which are useful in different target therapy technologies [ 67 ]. Therefore, these crystalline formations show great promise in gene therapy and as drug nanocarriers. However, in some pathogenic microorganisms the S-layer can reflect virulence and potency. An example of this is Bacillus anthracis where the surface layer of this pathogen is responsible for causing anthrax. Therefore, S-layers are employed in vaccine development either as an attenuated pathogen, adjuvant, a hapten added to an immunization formula, or as vaccine carrier [ 72 ]. It has been found that S-layer containing vaccines to be a treatment for different hypersensitivity disorders such as type I allergy [ 73 ]. In addition, S-layers form highly stable ultrafiltration membranes that have uniform isoporous lattice with desirable mechanochemical stability that is formed by inter and intramolecular interactions which is useful for retaining important biological molecules and in the formation of lipid membranes [ 65 ]. S-layers have also been studied for creating immobilization matrices that can be used as a diagnostic tool to identify various diseases such as type I allergy [ 74 , 75 ]. The external layer contain a set of fusion proteins such as rSbpA, STII, and Cys, which are used in synthesizing gold nanoparticles [ 76 ]. Furthermore, some studies have demonstrated that S-layers belonging to different Lactobacillus strains have a role in protecting the human intestine as they form a protective coating that binds to the lining of the gut and creates a barrier in order to avert the attachment of many opportunistic pathogens like E. coli [ 77 ]. 3.4. Methanobactin Methanobactins (Mbns) are peptide chalkophores that bind copper, which is needed for cell synthesis [ 79 ]. They were originally characterized in the methanotrophic bacterial species Methylococcus capsulatus and were found to be required for the functioning of methane oxidizing pMMO [ 68 ], which is transported outside of the cell to trap and bind to Cu(I) ions [ 80 ]. This chelating effect also reduces Cu(II), which is toxic to the bacterial cell, and to Cu(I) in copper-deficient environments [ 81 , 82 ]. The Mbn-Cu(I) complex then enters the cell through active transport to convey copper for important metabolic functions in methanotrophic bacteria [ 83 ]. Mbns exhibit significant sequence variability between bacterial strains [ 84 ], and use a modified rRNA to regulate its translation [ 85 ]. Methanobactin acts as a chelating agent with the ability to bind to copper, mercury and gold particles [ 86 ]. This is especially important as the ability of reducing gold has successfully generated gold nanoparticles that have many biomedical applications. These applications include antimicrobial activity, incorporation in fuel cells, photo-thermal and dynamic therapy, cancer treatment, act as an epitope, and tether to immunogenic molecules that have the ability to attach to cancer biomarkers (antigens), and in the development of many medically advanced technologies such as plasmonic biosensors, for visualization, and bioimaging [ 80 ]. Mbns also show great potential in the treatment of copper-associated illnesses such as Wilson disease (WD), Alzheimer disease (AD), fatty liver disease, and BRAF-positive cancers by preventing copper buildup in the liver and consequently other body tissues, and thereby preventing permanent liver and neurological tissue damage [ 83 ]. 3.5. Antibacterial Proteins Methylocystis minimus and Methylobacter luteus have been found to produce a thermostable protein capable of killing pathogenic bacteria, which is currently under investigation for possible application as an antibiotic [ 13 ]. The bacteria encode genes that produce peptidase enzymes, which was found to function as bacteriocin [ 87 , 88 ]. 3.6. Single Cell Protein (SCP) Single cell protein (SCP) is a protein derived from microbial cells that feed on a range of organic carbon sources. Many microorganisms, including algae, blue-green fungi, and bacteria, which can make up to 80% CDW [ 18 ]. The current vernacular usage of SCP arose in 1966 following a substitution of an older term microbial proteins, which was used to describe dried microbial cells serving as an ingredient or a substitute for protein-rich foods [ 64 ]. In view of an insufficient world food supply, the use of biomass produced by industrially scaled reactors could in theory provide a resource for SCP recovery [ 89 ]. SCP is of great nutritional value because of its high protein, vitamins, and lipid and essential amino acid content [ 90 ]. Methanotrophs are a well-known source for SCP production where the proteinaceous substance within methanotrophic bacteria is estimated to be 60–65% [ 89 ]. SCP offers a source of vitamins, amino acids, minerals, crude fibers, etc., which are important for healthy eyes and skin [ 76 ]. It is used as a protein supplement for undernourished children as it is added to improve the nutritional value of many consumed products as well as for athletes as they consume to derive energy [ 91 ]. Furthermore, it is also used for animal nourishment including pigs, cattle, and poultry [ 92 ]. Currently, Spirulina tablets are prescribed as dietary supplement as it lowers blood sugar levels in diabetic patients due to the presence of gamma-linolenic acid and prevents the accumulation of cholesterol in human body [ 93 ]. 3.7. Ectoine Ectoine (C 6 H 10 N 2 O 2 ) and its derivative hydroxyectoine (C 6 H 10 N 2 O 3 ) are high value compatible solutes [ 94 ], and are secreted intracellularly by microbial cells in order to overcome an environment's hypersalinity and balance the osmotic pressure [ 64 ]. It is noteworthy that this osmoregulatory can also be excreted to the extracellular environment as a response to hyposalinity [ 95 ]. Furthermore, ectoine synthesis was found to be associated with many amino acids, lipids, and proteins, which ensure their structural stability [ 31 ]. Thus, this cyclic imino acid has many medical and biotechnological applications and considered an expensive ingredient that currently retails for around $1000/kg [ 96 ]. Interestingly, in halophilic environments, methanotrophic bacteria synthesize ectoine by the conversion of oxaloacetate to aspartate followed by activation of a cascade reaction catalyzed by diaminobutyric acid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA), and ectoine synthase (EctC) as shown in Figure 5 [ 97 ]. This process is regulated by the MarR-like transcriptional regulator EctR1 that is usually found in conjunction with ectABC operon [ 94 ]. An important function for ectoine and hydroxyectoine is stabilizing and protecting nucleic acids and proteins from mutations. It is a key contributor to the prevention and treatment of many illnesses that are linked to protein and nucleic acid conformation such as Alzheimer's disease (AD), Machado–Joseph disease (MJD), and spongiform encephalopathies that occur due to amyloid deformation [ 98 ]. Therefore, ectoine is important in mediating the hinderance of protein aggregation and chain elongation in these illnesses while reducing cytotoxicity [ 99 , 100 ]. Moreover, ectoine has an anti-inflammatory and hydrating nature which can prevent hostile immune responses [ 96 ]. This is achieved through reducing neutrophils activity and stabilizing epithelial cells through creating a barrier and obstructing inflammatory cell signaling initiation in the lungs, thus preventing diseases like oral mucositis (chemotherapy induced in cancer patients) and in the airways like Allergic Rhinitis (AR) and Rhinosinusitis (ARS). These hypersensitivity-related conditions are responsible for airway damage due to the increased oxidative stress caused by polluting nanoparticles in the air [ 101 ]. Topical application of ectoine solute as nasal or oral sprays have been shown to limit these conditions and to be effective as an allergy medication in order to prevent irritation [ 102 ]. Similarly, ectoine-based eye drops hydrating potential exhibit an analogous effect in controlling and treating eye inflammation which can be caused by many medically associated conditions causing eye dryness or irritation [ 103 ]. Ectoine is also employed and integrated in many skin care product formulations as it can protect skin from harmful UV-A radiation (due to its DNA stabilizing capability) while soothing skin irritations through a barrier to trap moisture and protect skin from damage [ 104 ]. Lastly, ectoine has an important role in the regeneration of body tissue and healing ulcers, treating conditions, such as vascular leak syndrome (VLS) and neurodermatitis [ 105 ]. 3.8. Carotenoids Carotenoids occur as a group of pigmented biomolecules that help in photosynthesis in a wide range of organisms such as plants, fungi, and bacteria. Carotenoids are divided into two classes: carotenes and xanthophylls. The former are biological precursors for the formation of vitamin A1 and retinol while the latter provide protection against excess absorbed light energy, therefore acting as a shield for organisms against photo-oxidative stress [ 106 ]. Carotenoids play an important role in biomedicine due to their antioxidant activity and oxygen reducing ability which endow a protective function to several organs such as the heart and pancreas. For instance, the presence of lycopene has been correlated with heart health and the prevention of cervical intraepithelial neoplasia [ 107 ] and myocardial infarction [ 108 ]. Furthermore, carotenoids has been found to regulate gene expression which in turn affects cell growth and overall immunity [ 109 ]. Cohort studies have demonstrated an association between high carotenoid intake and a reduced occurrence of breast, cervical, ovarian, colorectal cancers, cardiovascular, and eye diseases [ 110 ]. The Methylomonas genus of methanotrophic bacteria has been found to produce one form of xanthophylls, namely lycopene. Lycopene consists of a 40 carbon chain and is present in lipophilic mediums such as membranes and lipoproteins commonly found in type I methanotrophic bacteria [ 111 ]. This xanthophyll derivative is a well-known antioxidant that protects the cell against reactive oxygen species (ROS) and oxidative damage and is often associated with various chronic illnesses, including cancer and cardiovascular diseases. Likewise, studies have shown that a drop in lycopene levels in blood serum as one ages has been considered to have a role in the incidence of prostatic, cervical, and pancreatic cancers [ 110 ]. Furthermore, lycopene plays a pivotal role in protecting against coronary atherosclerosis and myocardial infraction by being a part of low-density lipoproteins (LDL) and promoting immunity against free radicals damage [ 112 ]. Due to the aforementioned functional role of carotenoids, they have been employed in the drug industry and cosmetics, since they are biological precursors for vitamin A production, an fundamental nutrient in biological functions comprising vision, reproduction, and immunity [ 113 ]. As well, carotenoids are used as additives in the form of lutein and astaxanthin which exhibit anti-inflammatory and neuroprotective effect [ 114 ]. The administration of a mixture of lutein, β-cryptoxanthin, lycopene, zeaxanthin, and fucoxanthin have been found to inhibit function of the p16 and p73 oncogenes [ 115 ]. Different types of carotenoids are used in treatment of several illnesses such as diabetes, Parkinson's, Alzheimer's ( Table 1 ). 4. Outlook and Practical Implications Many microbial organisms inhabiting wastewater treatment plants have the potential to be used in biomedical applications including genera of Methanotrophs , Nitrosomonas , Pseudomonas , and Bacillus . In addition to important bacterial isolates found in WWTPs, there are also algal species such as Chlorella , Dunaliella , Sargassum , and Enteromorpha that are able to produce extracts like isoflavones, pigments, phenolics, carotenoids, polysaccharides, vitamins, and minerals. Advancements in the extraction and purification of bioactive compounds has led to its employment as a main ingredient in developing drug delivery systems, hormonal therapy, and biocompatible medical materials. [ 118 ]. In addition to the variety of applications in pharmaceutical and biomedicines of these extracts, WWTPs are also rich with bacterial strains such as E. coli that possess the ability to produce therapeutic recombinant proteins using bacterial expression systems [ 119 ]. Consequently, many previously untreatable disorders have been treated using genetic engineering of prokaryotic expression machinery such as diabetes, obesity, sexual dysfunction, and psychological disorders and have been guided by the biosynthesis and purification of many bioactive compounds such as insulin, neurotransmitters [ 120 ] and synthetic hormones [ 121 ]. Moreover, advancements in the dermatological industry have been made due to the emergence of biological ingredients such as Kojic acid, retinols, nitric oxide, penicillin and terbinafine, which have proven their efficacy in the treatment of many skin related conditions such as hypo/hyperpigmentation, psoriasis, injuries, bacterial and fungal infections, respectively. The antimicrobial, anti-tanning, texture enhancing, anti-wrinkling, skin sensitizers, soothing, repairing, and regenerating properties of these molecules have contributed to their extensive employment in skin care products in the last two decades [ 122 , 123 ]. Similar to pharmaceutical and dermatological industries, biotechnological advancements in the field of biomedicines have also been reported. Many bacterial and algal species have been used to produce different high-value nanoparticles that have applications in imaging, diagnostics, as antimicrobial agents and novel nano-based technologies such as surgical nanobots used in non-invasive procedures, prosthetics and tissue engineering [ 124 ]. Here, we reviewed an example of a WWTP isolate, methanotrophic bacteria and their bioproducts, for potential applications biomedicine. Biomolecules purified from methanotrophs have demonstrated application in the manufacturing of implants, scaffolds, drugs, dental casting, sutures for surgical procedures, prosthetic, supplements, vaccines, drug additives, and castings. In addition to their use in diagnostics, these biomolecules are also useful in the treatment of conditions such as, arthritis, cancer, autoimmune diseases, Wilson disease, Alzheimer's disease, fatty liver disease, and BRAF-positive cancers, a wide variety of infections, oxidative damage of many organs, vascular leak syndrome, neurodermatitis, bleeding, and wounds, as well as the prevention of cancer, liver cirrhosis, neurological tissue damage, skin conditions, diabetes, coronary heart disease, and osteoporosis. 5. Conclusions There is an interplay between advances in biomedicine and biotechnology and other fields of study including microbiology, agriculture, and engineering. Taking advantage of available tools and assets that exist in industrial systems is an opportune approach for next-generation solutions. The relationship between the biomedical industry and water treatment facilities may appear tenuous at first glance; however, they are intercorrelated, and the exploration of unexploited resources such as methanotrophic bacteria in WWTPs can lead to novel biomedical advances. There is both great value and significant potential for biotechnological and biomedical applications stemming from bioproducts produced by methanotrophic bacteria found in WWTPs. While this review covered some examples of products recovered from methanotrophic bacteria, there are many other potentially beneficial products that can recovered from wastewater facilities that have important applications in biomedicine and biotechnology.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10731662/

Recent Advances in the Detection of Food Toxins Using Mass Spectrometry

Edibles are the only source of nutrients and energy for humans. However, ingredients of edibles have undergone many physicochemical changes during preparation and storage. Aging, hydrolysis, oxidation, and rancidity are some of the major changes that not only change the native flavor, texture, and taste of food but also destroy the nutritive value and jeopardize public health. The major reasons for the production of harmful metabolites, chemicals, and toxins are poor processing, inappropriate storage, and microbial spoilage, which are lethal to consumers. In addition, the emergence of new pollutants has intensified the need for advanced and rapid food analysis techniques to detect such toxins. The issue with the detection of toxins in food samples is the nonvolatile nature and absence of detectable chromophores; hence, normal conventional techniques need additional derivatization. Mass spectrometry (MS) offers high sensitivity, selectivity, and capability to handle complex mixtures, making it an ideal analytical technique for the identification and quantification of food toxins. Recent technological advancements, such as high-resolution MS and tandem mass spectrometry (MS/MS), have significantly improved sensitivity, enabling the detection of food toxins at ultralow levels. Moreover, the emergence of ambient ionization techniques has facilitated rapid in situ analysis of samples with lower time and resources. Despite numerous advantages, the widespread adoption of MS in routine food safety monitoring faces certain challenges such as instrument cost, complexity, data analysis, and standardization of methods. Nevertheless, the continuous advancements in MS-technology and its integration with complementary techniques hold promising prospects for revolutionizing food safety monitoring. This review discusses the application of MS in detecting various food toxins including mycotoxins, marine biotoxins, and plant-derived toxins. It also explores the implementation of untargeted approaches, such as metabolomics and proteomics, for the discovery of novel and emerging food toxins, enhancing our understanding of potential hazards in the food supply chain. 1 Introduction Serious health hazards and outbreaks resulting from food spoilage are major concerns of food safety worldwide. 1 Due to significant biological activity and poor detection by conventional testing techniques, food toxins among other contaminants constitute a serious risk to the public's health. 2 , 3 Food toxins are compounds that can contaminate different food products during manufacturing, processing, transit, or storage. 4 According to Fletcher and Netzel, these poisons can come from a variety of sources including fungi, bacteria, algae, plants, and animals. 5 Mycotoxins produced by molds, marine biotoxins from toxic algal blooms, and plant-derived toxins like alkaloids and glycoalkaloids are all well-known examples of food toxins. 6 , 7 Mycotoxins are mainly produced by toxigenic fungal species belonging to the genera of Fusarium , Aspergillus , and Penicillium . 8 These mycotoxins pose a challenge to food safety because they can contaminate food products even when good storage and processing protocols are employed for food safety. Mycotoxin contamination accounts for the major cause of food borne diseases as reported by the World Health Organization (WHO). Recent studies performed by the European Commission revealed that 80% of the samples were contaminated with at least one mycotoxin. 9 Perusal of literature revealed that most of the mycotoxins are chemically and thermally stable; therefore, they can survive under storage, processing, and even cooking. 10 It has been observed that crops that are stored for more than a few days become probable targets for the growth of fungi and mycotoxin formation. These mycotoxins can affect a variety of food commodities such as dried fruits, coffee, spices, nuts, cereals, oil seeds, fruits, spices, cocoa, beans, etc. 11 Apart from mycotoxins, other microorganisms like bacteria, algae, and even plants also produce such metabolites that are equally toxic and lethal. Bacteria contamination is mainly attributed to poor hygiene and cleanliness and common uncleaned and poorly cooked meat and vegetable-based foods, especially fermented foods. Nem Chua fermented food prepared from pork sausage in Vietnam has the possibility for Staphylococcus aureus contamination, and New Zealand mussel ( Perna canaliculus ) traditional fermented food of New Zealand is usually contaminated with Clostridium botulinum . 12 Likewise, aquatic and marine food like Shellfish, fish, and even water are contaminated with algal biotoxins. 13 In the case of plants, some normal metabolites produced for various purposes like natural defense and stress tolerance act as toxins for other organisms. Cyanogenic glycosides in almonds, and summer fruits, furocoumarins in citrus fruits, and lectins in beans are some of the common examples of phytotoxins. 14 Besides, some newly emerging chemicals include perchlorate, flame retardants, halo compounds, packaging materials, petrochemicals residues, healthcare products traces, and microplastics. 15 The use of contaminated cereals, grapes, and barley used to produce wine and beer products and their consumption are the main causes of toxicological effects in human beings ( Table 1 ). Table 1 Various Food Toxins from Various Sources Class Toxin's name Source Effect Ref Mycotoxins Aflatoxin Aspergillus flavus and A. parasiticus Liver failure, cirrhosis ( 16 ) Lysergic acid (ergot alkaloids) Claviceps purpurea Ergotism, vasoconstriction, uterine contraction ( 17 ) Fumonisins B1 and B2 Fusarium verticillioides and Fusarium proliferatum disruption of sphingolipid metabolism, leuko-encephalomalacia ( 18 ) Ochratoxin A Aspergillus and Penicillium Carcinogenic, immunotoxic mutagenic, nephrotoxic, and teratogenic ( 19 ) Patulin Aspergillus , Byssochlamysand Penicillium Teratogenic, carcinogenic and mutagenic ( 20 ) Zearalenone Fusarium graminearum , F. culmorum , F. crookwellense , F. poae , F. semitectum , and F. equiseti Hepatotoxicity, immunotoxicity, reproductive toxicity ( 21 ) Tentoxin Alternaria Genotoxic, mutagenic, and carcinogenic ( 22 ) Bacterial toxins Cholera toxins Vibrio cholerae diarrhea ( 23 ) Enterotoxins Staphylococcus epidermidis Toxic shock syndrome ( 24 ) Shiga toxins Escherichia coli Gastrointestinal complications ( 25 ) Botulinum toxins Clostridium botulinum Neurotoxic ( 26 ) Cereulide Bacillus cereus Dysfunction of liver, pancreatic islet, intestines, brain, ( 27 ) Marine biotoxins Saxitoxin Cyanobacteria and dinoflagellates Neurotoxin, paralysis ( 28 ) domoic acid Diatoms Neurotoxin ( 29 ) Azaspiracid Azadiniumpoporum Diarrheic shellfish poisoning ( 30 ) Brevetoxin Karenia brevis Immunotoxicity ( 31 ) okadaic acid Halichondriamelanodocia and Halichondriaokadai Diarrhea, nausea ( 32 ) Plant-based toxins Cyanogenic glycosides Almonds, cassava, pome fruit, stone fruit Tissue damage ( 33 ) Furocoumarins Citrus fruits Skin cancer ( 34 ) Ptaquiloside Bracken ferns Carciogenic ( 35 ) Dehydropyrrolizidine Cyanoglossum , Senecio , Echium , Crotalaria , Heliotropium , Symphytum , Trichodesma Carcinogenic ( 36 ) The use of contaminated cereals, grapes, and barley for the production of wine and beer products is the cause of toxicological effects in human beings. Consumption of contaminated meat and milk-based products is another route for these toxins to enter the human food chain. Further, the abusive use of drugs in livestock, animal waste pollution, and the use of industrial wastewater for irrigation are also responsible factors for the entry of these contaminants into the food chain. 37 Conventional methods for the detection of food toxin include immunoassays and chromatographic techniques, which, while effective, have certain limitations. 38 Immunoassays, for instance, can be sensitive but may give false results if structurally related compounds are present in the testing matrix. 10 Chromatographic techniques, on the other hand, require complex sample preparation and longer analysis times. 39 In contrast, significant advancements in analytical techniques have revolutionized the field of food safety, and one such breakthrough is the application of mass spectrometry (MS) for the rapid and sensitive detection of food toxins. It is highly sensitive and provides selectivity and capability to handle complex mixtures, making it an ideal tool for the detection and characterization of food toxins. 40 , 41 This led to the selection of the MS-based approach as the first choice tool among researchers, regulatory agencies, and food industries to ensure the safety and quality of the food supply chain. 41 − 43 Recent advances and innovations in instrumentation, such as the development of high-resolution mass spectrometry (HRMS) and tandem mass spectrometry (MS/MS), have significantly improved sensitivity and selectivity, allowing for the detection of food toxins at ultralow levels. 44 Additionally, several ionization methods allow rapid in situ analysis, reducing the time and resources required for analysis. 45 , 46 Further, advancement in the field of untargeted metabolomics and several web-based repositories of metabolites allows for the detection of not only the known toxin but also the unknown variants of toxins, broadening our understanding of potential hazards in the food supply chain. 47 , 48 The current review summarizes the overview of available detection techniques of food toxins and then further elaborates on the MS-based approaches, their benefits and drawbacks, and how they are used in various food matrices. We will also go over the difficulties in applying MS-based techniques to routine food safety monitoring as well as the potential of this technology to protect public health and global food security. 2 Conventional Methods for the Detection of Food Toxins There are several specific and well-established conventional methods used on a regular basis for food safety assessment and regulation. These methods are often specific, but they may have limitations in terms of sensitivity, speed, and ability to detect a wide range of toxins ( Figure 1 ). Some of the most common conventional methods for the detection of food toxins are mentioned below. Figure 1 Schematic representation of various methods used for the detection of food toxins. 2.1 Immunoassays The most commonly used immunoassay methods in the field of food toxin detection are enzyme-linked immunosorbent assays (ELISA) and lateral flow immunoassays. These methods are very rapid and help with easy detection. Samples are allowed to interact with either labeled enzymes or antibodies, thus helping in the detection. However, compounds with similar core structures or nontoxic analogs can provide false positive results. Phycotoxins like Okadaic acid, Yessotoxin, Pectenotoxin, Azaspiracid, Cyclic imines, Palytoxin, Domoic acid, Saxitoxin, Microcystin, and Cylindrospermospsin; mycotoxins like Aflatoxin B1, Deoxynivalenol, Fumonisin B1 Zearalenone, and T-2; and bacterial toxins like Clostridium perfringens α, β, and ε toxin, Staphylococcal enterotoxins A, B, C, and E, botulinum toxins, and Escherichia coli enterotoxins 49 − 51 are detected using immunoassay methods. 2.2 Chromatographic Techniques Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC) are commonly used to separate and quantify food toxins. Among various seafood-originated toxins, Domoic acid, paralytic shellfish toxins, and Aflatoxin B1 can be easily detected by HPLC and TLC. 52 − 55 Among all, TLC is a simple technique in which food toxins are chromatographed on a plate layered with a thin layer of stationary phase using different mobile phase solvents, aiding the separation and visualization of the toxins. On the other hand, HPLC and GC are more advanced ways to separate toxins depending on various principles, enabling us to separate and quantify different food toxins. HPLC and GC analysis needs often include a complex protocol for sample preparation, which is one of the major limitations in the application of these techniques. 2.3 Spectroscopic-Based Techniques: UV–visible and Fluorescence Spectroscopy Each toxin possesses a different core structure with different motifs and thus absorbs light at a particular wavelength, enabling its detection if monitored at their respective wavelength. This principle is harnessed for the detection of food toxins such as aflatoxins and can be measured with UV-fluorescence spectroscopy. 56 It was suggested that if a sample is showing response at 400 and 550 nm with respect to 365 and 730 nm excitation wavelengths, it is supposed to be contaminated with aflatoxins. Singh et al. 57 also reported that aflatoxin B1 and ochratoxin A have maximum absorption (λ max ) at 365 and 380 nm. Both UV–visible and fluorescence spectroscopic techniques are easy to handle and cost-effective. Moreover, fluorescence spectroscopy has a high sensitivity for the detection of food toxins. Despite these advantages, there are certain toxins whose absorption and emission wavelengths may not be very selective and specific, making one of the major limitations in their detection by this technique. Other spectroscopic methods like nuclear magnetic resonance (NMR) spectroscopy are also used to elucidate the complex structure of food toxins. 58 Similarly, near infrared (NIR) spectroscopy uses NIR (14000–4000 cm –1 ) wavelength that causes vibration of C–H, O–H, N–H, and C=O bonds in the biomolecules and can be helpful for the detection of toxins in the food. Recently, this technique was used to detect Diarrhetic shellfish toxins in the mussels. 59 2.4 Biological Assays One of the most conventional methods is the direct injection of toxic samples into live animals and monitoring of their physiological response, behavior, and mortality. This assay is commonly used for the detection of marine toxins like Diarrhetic shellfish toxins in seafood. 60 , 61 However, these tests are time-consuming and costly and raise major ethical concerns. 2.5 Biochemical Assays Biochemical assays do not detect food toxins by direct measurement but rather involve the measurement of toxin-induced biochemical changes such as enzymes. For example, the inhibition of phosphatase activity is used to detect the presence of diarrhetic shellfish toxins. 62 , 63 The use of this technique is very narrow, utilizing different instruments and reagents, and cannot be applied to multiple types of toxins, thus making it less applicable for the detection of various food toxins. 2.6 Microbiological Assays Some food toxins are produced by certain microbes. Hence, assays involving the presence of the microbe are sometimes used as a proxy to detect their presence. For instance, the detection of Bacillus cereus in the food samples can point toward the presence of enterotoxins. 64 , 65 Although these assays are easy to operate and inexpensive, they lack sensitivity and specificity. 2.7 Sensor-Based Approaches Sensor-based methods of toxin detection in foods are very popular since they can be used on the site. There are many toxins such as aflatoxin B1, diarrhetic shellfish toxins, and microcystins that can be detected by sensors. 66 Examples may be biosensors and aptasensors. Undoubtedly, the use of sensors helps to screen samples for the possible presence of toxins. However, the sensors may vary in terms of sensitivity and specificity. 2.1 Immunoassays The most commonly used immunoassay methods in the field of food toxin detection are enzyme-linked immunosorbent assays (ELISA) and lateral flow immunoassays. These methods are very rapid and help with easy detection. Samples are allowed to interact with either labeled enzymes or antibodies, thus helping in the detection. However, compounds with similar core structures or nontoxic analogs can provide false positive results. Phycotoxins like Okadaic acid, Yessotoxin, Pectenotoxin, Azaspiracid, Cyclic imines, Palytoxin, Domoic acid, Saxitoxin, Microcystin, and Cylindrospermospsin; mycotoxins like Aflatoxin B1, Deoxynivalenol, Fumonisin B1 Zearalenone, and T-2; and bacterial toxins like Clostridium perfringens α, β, and ε toxin, Staphylococcal enterotoxins A, B, C, and E, botulinum toxins, and Escherichia coli enterotoxins 49 − 51 are detected using immunoassay methods. 2.2 Chromatographic Techniques Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC) are commonly used to separate and quantify food toxins. Among various seafood-originated toxins, Domoic acid, paralytic shellfish toxins, and Aflatoxin B1 can be easily detected by HPLC and TLC. 52 − 55 Among all, TLC is a simple technique in which food toxins are chromatographed on a plate layered with a thin layer of stationary phase using different mobile phase solvents, aiding the separation and visualization of the toxins. On the other hand, HPLC and GC are more advanced ways to separate toxins depending on various principles, enabling us to separate and quantify different food toxins. HPLC and GC analysis needs often include a complex protocol for sample preparation, which is one of the major limitations in the application of these techniques. 2.3 Spectroscopic-Based Techniques: UV–visible and Fluorescence Spectroscopy Each toxin possesses a different core structure with different motifs and thus absorbs light at a particular wavelength, enabling its detection if monitored at their respective wavelength. This principle is harnessed for the detection of food toxins such as aflatoxins and can be measured with UV-fluorescence spectroscopy. 56 It was suggested that if a sample is showing response at 400 and 550 nm with respect to 365 and 730 nm excitation wavelengths, it is supposed to be contaminated with aflatoxins. Singh et al. 57 also reported that aflatoxin B1 and ochratoxin A have maximum absorption (λ max ) at 365 and 380 nm. Both UV–visible and fluorescence spectroscopic techniques are easy to handle and cost-effective. Moreover, fluorescence spectroscopy has a high sensitivity for the detection of food toxins. Despite these advantages, there are certain toxins whose absorption and emission wavelengths may not be very selective and specific, making one of the major limitations in their detection by this technique. Other spectroscopic methods like nuclear magnetic resonance (NMR) spectroscopy are also used to elucidate the complex structure of food toxins. 58 Similarly, near infrared (NIR) spectroscopy uses NIR (14000–4000 cm –1 ) wavelength that causes vibration of C–H, O–H, N–H, and C=O bonds in the biomolecules and can be helpful for the detection of toxins in the food. Recently, this technique was used to detect Diarrhetic shellfish toxins in the mussels. 59 2.4 Biological Assays One of the most conventional methods is the direct injection of toxic samples into live animals and monitoring of their physiological response, behavior, and mortality. This assay is commonly used for the detection of marine toxins like Diarrhetic shellfish toxins in seafood. 60 , 61 However, these tests are time-consuming and costly and raise major ethical concerns. 2.5 Biochemical Assays Biochemical assays do not detect food toxins by direct measurement but rather involve the measurement of toxin-induced biochemical changes such as enzymes. For example, the inhibition of phosphatase activity is used to detect the presence of diarrhetic shellfish toxins. 62 , 63 The use of this technique is very narrow, utilizing different instruments and reagents, and cannot be applied to multiple types of toxins, thus making it less applicable for the detection of various food toxins. 2.6 Microbiological Assays Some food toxins are produced by certain microbes. Hence, assays involving the presence of the microbe are sometimes used as a proxy to detect their presence. For instance, the detection of Bacillus cereus in the food samples can point toward the presence of enterotoxins. 64 , 65 Although these assays are easy to operate and inexpensive, they lack sensitivity and specificity. 2.7 Sensor-Based Approaches Sensor-based methods of toxin detection in foods are very popular since they can be used on the site. There are many toxins such as aflatoxin B1, diarrhetic shellfish toxins, and microcystins that can be detected by sensors. 66 Examples may be biosensors and aptasensors. Undoubtedly, the use of sensors helps to screen samples for the possible presence of toxins. However, the sensors may vary in terms of sensitivity and specificity. 3 Mass Spectrometry-Based Detection Mass spectrometry (MS) is a powerful analytical technique used to detect and quantify various compounds including food toxins. This method can provide highly sensitive and specific results, making it a valuable tool for food safety and quality control. 67 Infectious toxins like prions and Shiga toxins can also be analyzed using mass spectrometry, where peptides are digested by proteases and then the digested proteinaceous parts are analyzed by MS. 68 The pervasive contamination of food products with mycotoxins has made monitoring their levels essential. Detection of mycotoxin biomarkers in urine provides valuable and specific data for exposure assessment to these food contaminants in order to overcome the disadvantages of the indirect approach based on food analysis. 69 Due to the diverse chemistry and occurrence of food toxins in feedstuffs and foods with complex matrices, the detection has become difficult. The primary source of error in the analysis results from inadequate sampling and inefficient extraction and cleaning procedures. Gas chromatography (GC)-MS is used to analyze volatile and semivolatile compounds, such as certain mycotoxins and pesticide residues, in a variety of dietary products. Before entering the mass spectrometer for ionization and detection, the compounds are vaporized and separated according to their volatility. 70 The principle of detection of food toxins using GC-MS includes multiple target analyte extraction using multiresidue analytical methods like QuEChERs and adsorption extraction. 71 Recently, mass spectrometry has become one of the most effective methods even for identifying specific microorganisms by using matrix-assisted laser-desorption time-of-flight (MALDI-TOF) MS, followed by recognition of MS spectra unique to that organism that create a reliable fingerprint. 72 MALDI-TOF MS-based identification of bacteria is more rapid, accurate, and cost-efficient than conventional phenotypic techniques and molecular methods. Rapid and reliable identification of food-associated bacteria is of crucial significance for product quality. In contrast to genotyping methods, it can also be readily implemented in routine analysis. Due to short turnaround periods, low sample volume requirements, and low reagent costs, MALDI-TOF MS has recently emerged as a powerful tool for the identification of food toxins or toxin-producing microorganisms. 73 Food toxins from various fields of seafood, fruits, vegetables, milk, dairy products, and oils can be detected using MALDI-TOF MS. 74 The microbial databases with unique features relevant to each microbial species are the key components and are therefore continually building up in size with the updated information on newly discovered microbial species and their annotations. Another powerful MS-based tool routinely used for food toxin detection is liquid chromatography (LC)-MS due to its advantages in terms of sensitivity and selectivity. LC–MS is widely used for the analysis of mycotoxins, alkaloids, marine toxins, glycoalkaloids, cyanogenic glycosides, and furocoumarins in food. The excellent sensitivity, even at low concentration levels, selectivity, and capacity to resolve coeluting compounds based on their molecular masses make LC–MS currently the most effective technique for the simultaneous detection of multiple regulated, unregulated, and emerging toxins in a single run. Commonly, the LC–MS methods for the quantitative determination of natural toxins are based on the use of a triple-quadrupole analyzer, tandem mass spectrometry, and multiple reaction monitoring (MRM) modes. The co-occurrence of natural toxins in combination with other chemical contaminants, such as pesticides, growth regulators, and veterinary drugs, as well as bioactive compounds (i.e., lignans, flavonoids, and phenolic compounds), in a wide variety of food matrices has increased the demand for analytical methods addressing the simultaneous determination of multiple analyte classes. LC–MS method used for the detection of food toxins includes alkaloids, furocoumarins, cyanogenic glycosides, marine toxins, and mycotoxins. 41 Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful analytical technique for the detection of elementals like heavy metals that allow multielement detection simultaneously with high speed and at very low concentrations. 75 Further, for the enhanced selectivity, inductively coupled plasma mass spectrometry (ICP-MS) and tandem mass spectrometry (MS/MS) can be utilized. In the ICP-MS technique, the sample under examination is digested by employing a suitable technique such as dry ashing, acid digestion, or microwave digestion, etc. to solubilize the analytes of interest. Further, the sample is injected into an inductively coupled plasma source which ionizes the sample and detected by MS. 76 Ion mobility spectrometry (IMS) is another advancement in analytical techniques that relies on ion mobility that is under the influence of velocity of ions and strength electric field. 77 , 78 In order to take advantage, multiple MS-based approaches can be merged and operated together, which improves the efficiency of analytical techniques. Tandem mass spectrometry (TANDEM MS), also called MS/MS, is one such approach in which samples are analyzed either by multiple mass spectrometers connected to each other or with different analyzers arranged sequentially. 79 A MS technique can also be coupled with immunoaffinity chromatography, and it is known as IAC-MS. This technique uses antibodies-based columns to acquire selectivity and also to isolate target analytes from the sample matrix. 80 The isolated target molecules are injected into the MS component, which provides high sensitivity and identification of toxins based on mass-to-charge ratio. IAC-MS provides exceptional selectivity and can be used for the detection of various types of food toxins such as mycotoxins, pesticides, veterinary residues, and allergens, etc. 81 MS-based approaches have offered diverse and advanced modules for the detection of analytes precisely. Table 2 summarizes the advantages and disadvantages of MS-based approaches under different conditions. Table 2 Variants of Mass Spectrometry (MS) and Their Advantages and Disadvantages Technique(s) Important features Advantages Disadvantages Ref Accelerator mass spectrometry (AMS) • Employed particle accelerator technology into a mass spectrometer. • Small quantity of sample is sufficient • High cost makes it less affordable ( 82 , 83 ) • Its detection range include ion currents of more abundant stable isotopes (e.g., 12C, 13C) to very rare radionuclides (e.g., 14C) • Need less time for estimation • Small sample size makes it prone to contamination Liquid chromatography mass spectrometry • Simple and robust technique for regular analysis • Wide linear dynamic range • Lower accuracy ( 84 , 85 ) • Able to detect nonvolatile compounds like sugar and proteins that cannot be detected in GC-MS • Lower detection limit • Isotopes cannot be detected • High precision and accuracy Gas chromatography mass spectrometry • Sample exposed to high temperature • Analysis is faster and selectivity, • Destructive method of analysis ( 86 − 88 ) • Used for the detection of volatile compounds from sample • Lower detection limits • Only thermolabile compounds can be detected • Nonvolatile compounds like sugar can be detected after dertivatization High resolution mass spectrometry • Good for the identification of unknown samples • Highly accurate and selective measurement • Expensive analysis ( 89 ) • Efficient for nontargeted analyses • Able to detect mass accurately with even small change is detectable • Data generated is hige and complex • Not suitable for regular analysis of known samples Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry • Traditional method for the identification of microorganisms • Able to differentiate between phenotypic, genotypic, and biochemical properties • In some cases, unable to differentiate between closely related species, e.g., E. coli , and Shigella ( 90 ) • Sample is first ionized, and segregated based on mass-to-charge ratio • Reduced analysis time • Lack sufficient spectra in database • Measurement is done by determining with time-of-flight Inductively coupled plasma mass spectrometry • Used to measure the element level in sample • Wide analytical range with lower detection limit • High cost of investment and operation ( 91 ) • Sample converted to aerosol from liquid • Need small quantity of sample • Need experts for operation • High throughput with multielement detection Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry • It is also known as SELDI-TOF-MS-based ProteinChip System • It employes chromatographic separation techniques • Low detection precision for individual proteins from complex ( 92 , 93 ) • It is modified form of MALDI-TOF • Less time-consuming and high through put system • Low mass resolution • Proteomic profiling of biological fluids Tandem mass spectrometry • Two or more MS units are interconnected with quadrupoles and TOF analyzer • Highly specific • High operational cost ( 79 , 94 , 95 ) • Effective in analyzing complex mixture • Low signal-to-noise ratio • Limited sample through put • Able to detect covalent modifications in proteins • Sensitive and reproducible 4 Recent Advancements in Food Toxin Detection Using MS Food toxins have become a serious concern for the society. The presence of various types of contamination and toxins like pesticides, herbicides, microbial metabolites, and plant-based toxins is highly detrimental to human health even at ppb concentrations when present in food, water, or animal feed. 96 Safe food is explicitly a matter of concern and is indispensable for human health. In the current scenario, an effective and sensitive detection method becomes necessary to detect contamination of food and water with chemicals and pathogenic microbes and related products. Existing conventional methods for food analysis, based on PCR, chromatography, and spectrophotometry, have shown significant reliability and accuracy; however, the cost of analysis, time consumption, and requirement of specialized personnel impede their usage for frequent monitoring of food samples (as already summarized above). Hence, there exists an upsurging thrust for innovating rapid, accurate, robust, but inexpensive alternatives for in situ and real-time detection of contamination of food samples. The primary step involved in every methodology to identify the food toxins in the sample under examination involves the extraction of food toxins from the matrix followed by purification to remove other substances that can interfere with the analysis. 97 After successful extraction of toxins, the sample is ionized into ions with ionization techniques such as electrospray ionization (ESI), chemical ionization (CI), or APCI (atmospheric pressure chemical ionization) or desorption techniques such as matrix-assisted laser desorption ionization (MALDI). These ions from the ionization chamber are accelerated followed by deflection in the magnetic field due to a difference in their masses. The beam of ions is then analyzed by the detector based on their mass-to-charge ratio. There are many types of mass analyzers available such as magnetic sector analyzers, quadrupole mass analyzers, double focusing analyzers, time-of-flight analyzers, etc. Further, MS can also distinguish between different food toxins depending upon their mass-to-charge ratio. In order to overcome the limitations associated with the conventional methods, MS can be coupled with these techniques to enhance the capabilities in complete and accurate analysis of different food toxins present in the sample. 98 GC technique relies on the comparison of retention times with known standards and also lacks in the distinction of structurally similar compounds. Thus, to enhance selectivity, identification, and elucidation of the structure of various toxins present in the sample under examination, GC is coupled with the MS. 99 HPLC technique requires optimization such as the selection of columns and mobile phases for each specific class of toxins. In addition to this, the sensitivity of HPLC is also low as that of MS techniques. HPLC techniques are more time-consuming than MS as they involve complex procedures for sample preparation. 100 We are herein summarizing various types of food toxins and the recent advancements in their detection using MS. 4.1 Mycotoxins Mycotoxins are toxic secondary metabolites produced by fungi. These toxins can accumulate in the fungal-contaminated food grains such as corn, cereals, and legumes, and upon ingestion can traverse into the food chain affecting humans and animals. 101 As per the Rapid Alert System for Food and Feed of EU (RASFF) report, mycotoxins contaminate around one-quarter of global food grain production both during pre- and postharvest. 102 This clearly indicates the severity of the mycotoxin problem in the food that we consume. In the literature, around 300 mycotoxins are reported, but only seven toxins are quite common in food worldwide such as aflatoxins (AF), trichothecenes (TC), zearalenone (ZEN), fumonisins (FB), ochratoxins (OTA), citrinin (CIT), and patulin (PAT). 101 Fungal species belonging to the genera of Aspergillus , Penicillium , and Fusarium are predominantly toxigenic and most frequently lead to cases of mycotoxin concentration. 101 AF (B1, B2, G1, and G2 produced by Aspergillus ), CIT (by Penicillium , Aspergillus , and Monascus etc.), ergot alkaloids ( Claviceps purpurea ), FB ( Fusarium sp.), ochratoxin A (produced by Penicillium and Aspergillus ), PAT ( Penicillium patulum ), TC ( Fusarium , Stachybotrys , Trichothecium , and Trichoderma sp.), and ZEN ( Fusarium graminearum ) are some of the well-known mycotoxins responsible for serious lethal reactions like cancer induction, kidney toxicity, immune suppression, stachybotryotoxicosis, turkey X syndrome, etc. 103 Another issue with these mycotoxins is their resistance and tolerance toward thermal treatment; hence, they remain active even after heating. 104 AFs are groups of potentially toxic fungal secondary metabolites reported from Aspergillus flavus , A. parasiticus , and A. nomius ( Figure 2 ). They are produced from polyketides and commonly present in cereal crops like corn, peanuts, walnuts, wheat, etc. 105 AFs can lead to chronic toxicity related to hepatic tissues, necrosis, hepatomas, periportal fibrosis, jaundice, hemorrhage, and fatty liver changes, and also exhibit teratogenicity, carcinogenicity, and immunotoxicity. 105 , 106 They were first reported in the state of Gujrat and Rajasthan, India in 1974, which resulted in the onset of hepatitis caused by A. flavus infected staple food and maize. A. flavus , A. parasiticus , A. nomius , and sometimes Emericella spp. are the main producers of AFs. 105 To date, more than 20 AFs variants have been reported, among which variant B1 is the most common and most lethal. AFs B1 and B2 are produced by A. flavus and A. parasiticus , and AF M1 is produced by A. parasiticus and can be transmitted through milk. Variants M1 and M2 are also produced by metabolism of B and B2. 16 AF B1 is metabolized by the P450 monooxygenase system and generates AF 8,9-epoxide (reactive epoxide) that induces mutations and cancer by forming DNA abducts. 106 Figure 2 General structures of some of the Aflatoxin variants. Besides Aspergillus , isolates belonging to Fusarium are also known to produce the most potent toxins, which include deoxynivalenol (DON), FBs, and ZENs ( Figure 3 ). DON belongs to the sesquiterpenoid group of trichothecenes. It mainly contaminates corn, wheat, and barley. It is also a toxic secondary metabolite that has a negative health impact on the consumer by disturbing the intestinal barrier and has immune-stimulatory as well as immune-suppression properties at low and high doses, respectively. 107 The same group also contains its acetylated derivatives named nivalenol, T-2 toxin, and HT-2 toxins. 108 After ingestion, DON is absorbed and metabolized in the intestine via DON-3S, DON-GlcA, and DOM-1. In poultry birds, DON-3S and DON-15S are eliminated via bile and urine, while in swine, it is absorbed in the upper digestive system; hence, poultry birds are the least sensitive to these toxins, and swine are most sensitive to these toxins. In humans, contaminated foods like infected meat and cereals are the most common sources. 107 Fusarium sp. is also responsible for other mycotoxins named FBs (secreted by Fusarium verticillioides and Fusarium proliferatum ). Aspergillus niger is also able to produce FBs. Such toxins are commonly reported from cereals like peanuts, maize, rye, oats, millets, and grape. 109 To date, more than 15 homologous forms of fumonisin have been reported that are referred to as A, B1, B2, B3, C, P, etc. However, B1 is the most toxic form of FB. 110 , 111 FBs are known to have carcinogenic, neurotoxic, and hepatoxic effects that cause hepatocarcinoma, defects in the neural-tube, and nephrotoxicity. 109 ZEN is a nonsteroidal, estrogenic mycotoxin produced by F. acuminatum , F. crookwellense , F. culmorum , F. cerealis , F. equiseti , F. graminearum , F. oxysporum , F. sporotrichioides , F. semitectum , and F. verticillioides . 112 It disrupts reproductive capacity by affecting mammalian folliculogenesis and impairs granulosa cell development and follicle steroidogenesis. 113 It is thermostable 114 and resistant to processing stress like milling and storage. 115 , 116 ZEN leads to kidney damage and liver injury and causes inflammation. 112 Figure 3 General structures of various mycotoxins. Ergot alkaloids are toxic secondary metabolites produced by several fungi of Clavicipitaceae ( Epichloë , Claviceps , Balansia , and Periglandula ) and Aspergillus fumigatus . The producers mainly include Epichloë endophytes , Epichloë festucae var. lolii , Epichloë coenophialum , and Claviceps purpurea . 117 They belong to compounds containing an indole group and are derived from L -tryptophan. They cause "ergotism" and their toxic effect is reflected by hyperglycemia, gastrointestinal upset, mydriasis, 118 and even endocrine disruption. 119 Besides these major mycotoxins, some other chemicals including OTA and PAT have also been reported from fungi. OTA is nephrotoxic and produced by a diverse range of fungi such as Aspergillus ochraceus , A. carbonarius , A. niger , and Penicillium verrucosum . It can also lead to renal tumors, Balkan endemic nephropathy, and chronic interstitial nephropathy. 120 Patulin is produced by Penicillium , Aspergillus , and Byssochlamys , while alternariol (AOH) and alternariol monomethyl ether (AME) are Alternaria toxins, produced by fungi of the Alternaria genus found in fruits and related products. 20 To ensure food safety with special consideration to mycotoxins, international, national, and regional agencies like the World Health Organization, Food Agriculture Organization, Codex Alimentarius Joint Expert Committee for Food Additives and Contaminants, European Food Safety Authority, GCC Standardization Organization, and Japanese Association of Mycotoxicology have determined the permission limit for mycotoxins contamination in food as well as feed. 121 Table 3 summarizes the permissible limit for various mycotoxins in food 122 − 125 and animal feed 126 , 127 as per the European Commission. Table 3 Regulatory Permission Limits for Various Mycotoxins in Food and Feed Products As Per European Commission 122 − 127 Category Food or animal feed products Permissible limit (μg/kg) Aflatoxins B1 (AFB1) Food Brazil nuts, groundnuts, hazelnuts, and oilseeds for human consumption after physical treatment 8 Almonds, apricot kernels, and pistachios for human consumption after physical treatment 12 Brazil nuts, groundnuts, hazelnuts, and oilseeds for human consumption directly (No physical treatment) 2–5 Almonds, apricot kernels, and pistachios for human consumption directly (No physical treatment) 8 Dairy products for consumption by infants, baby food, and processed cereal-based food 0.1 Spices 5 Dried fruits, and Figures for human consumption after physical treatment 5–6 Dried fruits (except Figures) for human consumption directly (No physical treatment) 2 Maize and rice (as ingredients) for human consumption after physical treatment 5 Feed Feed materials 0.02–0.05 Complete feeding stuff with the exception of 0.05 Calves, cattle, and lambs 0.005–0.01 Poultry 0.02 Complementary feeding stuff 0.005–0.05 Aflatoxins M1 (AFM1) Food Milk 0.05 Infants' dairy products, baby formula and baby milk, 0.025 Aflatoxins (AFs) total Food Almonds, apricot kernels, brazil nuts, groundnuts, hazelnuts, oilseeds, and pistachios for human consumption after physical treatment 15 Groundnuts, oilseeds, and processed products for human consumption directly (No physical treatment) or as ingredient 4 Almonds, apricot kernels, brazil nuts, hazelnuts, pistachios, and for human consumption directly (No physical treatment) 10 Spices 10 Dried fruits, and Figures for human consumption after physical treatment 10 Dried fruits (except Figures) for human consumption directly (No physical treatment) 4 Maize and rice (direct or as ingredients) for human consumption after physical treatment 10 Citrinin (CIT) Food Food supplements prepared from red yeast fermented rice 2000 Deoxynivalenol (DON) Food Unprocessed durum, maize, oats and wheat 1750 Cereals and cereal flour for direct human consumption 750 Cereal-based processed foods and baby food 200 Feed Animal feed from–cereals 8–10 Complete as well as complementary feeding stuff 0.9–5 Fumonisin (FB1+FB2) Food Unprocessed maize 4000 Maize for direct human consumption 1000 Maize-based processed food for babies and young children 200 Feed Maze based feed 60 Complete and complementary feeding stuff 5–50 Ochratoxins (OTA) Food Cereals-based unprocessed products 3–5 Cereal-based processed food and baby food, dietary foods products specially for medical purposes purposes 0.5 Beverages based on grapes 2 Coffee roasted/instant 5/10 Spices 15 Feed Cereals-based feed materials 0.25 Complete and complementary feeding stuff for poultry 0.1 Patulin (PAT) Food Fruit juices 50 Solid apple products 25 Solid apple as well as apple juice for babies and young children 10 Zearalenone (ZEN) Food Cereal products (unprocessed except maize) 100 (350) Cereals for direct human consumption 75 Maize for direct human consumption 100 Cereals and maize processed products for babies and young children 20 Feed Cereals and maze-based feed materials 2–3 HPLC and GC are the common approaches for the detection and higher accuracy and sensitivity making MS an elegant and dominant analytical tool for toxicological and metabolite analysis. 128 In addition, it also allows simultaneous detection of a diverse range of toxins together and aids in method standardization and implication to ensure the rapid evaluation of samples for food safety analysis. Areo et al. 129 have employed UHPLC–MS/MS for the detection of AFs, ZEN, and OCT A from 100 tea samples (collected from registered shops within South Africa), prepared in acetonitrile/water/acetic acid solvent by QuEChERS extraction method. The supernatant was mixed with 900 mg of anhydrous MgSO 4 , 150 mg of C18, and 150 mg of primary secondary amine that separates the organic phase. The organic phase was collected and dried under a nitrogen stream and further reconstituted in methanol/water for analysis via UHPLC–MS/MS. The method selected has very high linearity (>0.99) and precision (6–29%). AFs, i.e., AFB1, AFB2, AFG2, OCT A, and ZEN, were absent in the samples, and AFG1 was present in very low amounts 1.72–5.19 μg/kg that were also below the regulated level in food as recommended by EU Commission Regulation 1881/2006. 129 You et al. 130 have evaluated the effect of culture medium for mycotoxin accumulation by Alternaria . Secondary metabolites were characterized by nontarget analysis with HRMS. Mycotoxins produced by Alternaria were grouped into for families: alternariol monomethyl ether (AME), alternariol (AOH), altenuene (ALU), Desmethyl dehydro altenusin (DMDA), and dehydroaltenusin (DHA) families, Altertoxin-I (ATX-I) family, tentoxin (TEN) family, and tenuazonic acid (TeA) family. Culture medium greatly influenced the type of mycotoxins produced. wiz Potato Sucrose Agar medium is suitable for AOH, AME, ALU, ALT, DHA, and DMDA, while Potato Dextrose Agar supported the accumulation of ATX-I, TEN, and TeA. Table 4 summarizes some of the research for the detection of various mycotoxins with MS-based tools. Table 4 Detection of Mycotoxins by MS-Based Approach Name Sample Method Operating parameters Outcome Ref Trichothecenes T2 Wheat and maize Portable mass spectrophotometer MS parameter: heater 200 °C; sample pump 0%; mass center: m / z = 484; mass tolerance: 1; spectrum average: 10 Limit of detection 0.2 mg/kg ( 131 ) Alternaria toxin Fruits and vegetables QuEChERS extraction followed by MS analysis Triple quadrupole MS; pressure curtain gas 35 psi and collision gas 8 psi; ion spray voltage 5,500 V (positive ion mode), and −4,500 V (negative ion mode) Detection limit 1.0–5.0 μg/kg (Extraction recoveries 73.0–120%) ( 132 ) Repeatability <12.9% Mycotoxins Standard sample UHPLC-QTrap-MS/MS Injection volume: 20 μL; column temperature: 30 °C; flow rate: 0.2 mL min –1 ; mobile-phase 0.1% formic acid+ acetonitrile run time: 25 min Limit of quantification 0.005 and 13.54 ng mL –1 (Limit of detection 0.001–9.88 ng mL –1 ; 0.005; Recovery 67.5–119.8%) ( 133 ) Aflatoxin B1 and M1 Blood HPLC–MS/MS Allure PFPP column; positive electrospray ionization; source temp 500 °C LOD 0.05 to 0.2 ng/mL; accuracy 92–111% ( 134 ) Alternaria toxins Vegetable sample UPLC–MS/MS ACQUITY UPLC BEH C18 column; injection volume: 5 μL, column temperature: 40 °C; flow rate 0.4 mL min –1 , mobile phase: 0.1% formic acid water; gradient flow 10–90% Contaminated solanaceous vegetable: 41.1% ( 135 ) AME: 4.26%; AOH: 6.38%; altenuene: 6.38%; tentoxin: 42.6%; tenuazonic acid: 55.3% Aflatoxin M1 HPLC–MS/MS Quantitative daughter m / z : 273; qualitative daughter m / z : 259; collision energy 23 eV Quantification limit 1.62 ppb; detection limit 0.54 ppb; 98.5% accuracy ( 136 ) Ochratoxin A Coffee and tea UHPLC–MS/MS Triple quadrupole MS; electrospray ionization, ion source temp 300 °C; flow rate 3 L/min Sensitivity 0.30 and 0.29 ng/mL ( 137 ) Trichothecenes Oat based products U-HPLC–HRMS/MS Reverse phase column; electro-ionization detector; column temperature 40 °C; ethanol+water+formic acid gradient elution Frequency of free T2 toxin 92% ( 138 ) T2 mono glucoside 69% Alternaria toxins Rice LC–MS/MS Separation with hyper clone column and detection with C18 column; detection with negative electrospray ionization, source temperature 300 °C Limit of detection and quantification ( 139 ) AME: 0.03 and 0.09 μg/L; altenuene: 5.48 and 16.24 μg/L Alternariol monomethyl ether (AME), Alternariol (AOH), and tentoxin Standard sample LC-ESI-MS/MS LC–MS/MS with triple quadrupole; separation at 25 °C with C18 column; analysis with quadrupole MS, source temperature 450 °C; ion spray voltage 5500 V Limits of detection and quantitation: 0.7 and 3.5 ng/g recovery 80% ( 140 ) 4.2 Bacterial Toxins Bacterial contamination has shown diverse causes as Shigella mainly infect via unwashed hands, while Campylobacter and Escherichia coli are usually present in raw milk, undercooked meat and poultry products, and contaminated water. In contrast, Listeria monocytogenes and Yersinia enterocolitica are found in refrigerated food. 141 Bacterial toxins have been classified as endotoxins and exotoxins. Structurally, endotoxins have distinct structural regions, i.e., glycolipid is made up of disaccharide and fatty acids which are usually capric, lauric, myristic, palmitic, and stearic acids. These acids are buried within the outer cell membrane of the bacterium. The nucleus is the second important part, which is made up of a hexose- and heptose-based heteropolysaccharide. The glycolipid and nucleus are interconnected by the sugar acid 2-keto-3-deoxyoctanate. Endotoxins are lipopolysaccharides and are part of the outer membrane of Gram-negative bacteria. These are also identified as important determinants and antigenic parts of bacteria that aid in attachment with the host as well as in pathogenicity. Exotoxins are proteins in nature that are released by Gram-negative bacteria and disrupt cell division, causing lysis and tissue damage. 142 , 143 Exotoxins are further classified into types I, II, and III based on the mechanism of action. Toxin type I can make critical changes in the host's cells without internalizing. Superantigens secreted by Staphylococcus aureus and Streptococcus pyogenes are examples of a type I toxin. The type II group includes hemolysins, phospholipases, aerolysin, and GCAT proteins. It intrudes the host cells and creates pores to destroy the host cell's membrane. In comparison to type I and II, type III is a diverse group in terms of activity. It has a binary structure with fractions A and B. Fraction B in the toxin facilitates the binding with receptor in the host cell, while another fraction, i.e., "A" carries enzymatic activity and is responsible for the toxin effect. Anthrax toxin ( Bacillus anthracis ), Cholera toxin ( Vibrio cholerae ), and Shiga toxin ( Escherichia coli O157:H7) are some examples of exotoxins. 142 , 144 Botulinum is 150 kDa and composed of a heavy chain of 100 kDa and a light chain of 50 kDa. Heavy chain is responsible for binding to receptors on neuron surface, while light chain cleaves proteins required for nerve signal transmission, i.e., botulinum A, C, and E cleave synaptosomal-associated protein 25, while B, D, F, and G variants act on synaptobrevin-2. 72 , 145 , 146 In a similar fashion, B. anthracis produces three types of proteins or factors, e.g., lethal factor, edema factor, and protective antigen. Protective antigens split and form a fragment of 63 kDa that forms heptamers and octamers to finally find the cell surface. In addition, they also bind with lethal factors to form lethal toxin. 72 , 147 4.2.1 Detection of Bacterial Food Toxins Clostridium , Salmonella , Staphylococcus , and Listeria are some common pathogens causing foodborne infections in humans. Previously used methods, e.g., enzyme immunoassay (EIA), were fast and sensitive, but their accuracy was limited due to cross-reactivity reporting a high rate of false positives and misguided public health care personnel. Techniques based on MS are powerful and can be multiplexed for the detection of various protein toxins, e.g., toxins from Clostridium , 148 Bacillus , 88 and many more with speed, sensitivity, and accuracy. Botulinum, a neurotoxin, is produced by Clostridium botulinum in seven different serotypes (A–G). Specific detection of these toxins from different strains needs high analytical sensitivity and a MS-based approach. The enzymatic activity-based approach relies upon substrate fragments generated by these toxins which can be used as targets by MALDI-TOF MS. 72 In 2002, a peptide mass map of toxin variants A1 and B1 was prepared by targeting the trypsin digest of the toxin by van Baar and colleagues. The work was extended to C, D, E, and F in 2004. 72 In successive generations, several advancements have shown effective approaches like endopep-MS. 149 Rosen et al. have developed the endopep-MS-based method for the identification of botulinum A and E simultaneously and rapidly. 150 Both A and E identify the same target SNAP 25 protein but act on different sites. 3D structures of both types of fragments were used for differential identification. Drigo et al. 151 employed the same endoPep-MS approach for botulinum toxins C and D. The method has shown a sensitivity of 100% with specificity and accuracy of 96.08% and 97.47%, respectively. Integration of MS with other analytical methods like HPLC, GC, FPLC, etc. has improved bacterial toxin profiling. Toxoflavin and fervenulin are bacterial toxins produced by Berkholderia and Streptomyces hiroshimensis . These compounds are common contaminants in fermented corn flour, rice bran oil, distiller's yeast, sweet potato starch, Tremella fuciformis Berk., and rice noodles. These compounds are sensitive to degradation in 1% ammonia solution. UHPLC-Q-TOF/MS allowed for the detection of degradation products. The modified approach led to lower down the limits of detection of toxoflavin and fervenulin to 12 μg/kg and 24 μg/kg, respectively, with recovery of 70.1–108.7%. 152 The MS approach also employed natural phenomena of antigen–antibody interactions for the detection of toxins and related antigens. Salmonella typhi , Gram-negative enterobacteria, is responsible for typhoid fever and meningitis. The immunoreactive proteins of bacteria were used as targets to develop improved diagnostic tools with MS. An immunoaffinity-based proteomic approach was employed with IgG and IgM antibodies from typhoid patients. The approach aided in the identification of 28 immunoreactive proteins, out of which 14 were complementary to IgG, 4 for IgM, and the rest 10 for both, hence retained by respective charged columns. In context to antigenicity, 22 proteins have shown antigenicity and immunogenicity. 153 Such an approach is helpful for rapid identification, and its reproducibility and reliability can be employed for vaccine and drug development. Peptide mass fingerprinting technique (PMF) associated with MALDI TOF/MS or ESI/MS is a top-down MS protocol where proteins are directly ionized to create a fingerprint of individual proteins and applied for detection of various microbial strains. PMF of unknown organisms is compared to those existing in PMF databases or compared to the spectrum of biomarker proteins with the proteomic spectral database, using MALDI-TOF MS. Typically a mass range m / z of 2–20 kDa is used for species-level identification where ribosomal proteins representing 60–70% of microbial cells' dry weight and some housekeeping proteins are selected. 154 Thus, by comparing with extensive commercial databases, microbial contaminants can be traced to the genus, species, or strain level, and such an identification tool is conveniently adapted in diagnostic laboratories. 155 However, using biomarkers is not very common for identification since it requires prior insight into the genome sequences before creating the required databases for proteins' molecular masses. Staphylococcus aureus delta-toxin has been detected using whole-cell (WC) MALDI-TOF/MS and LC–MS to correlate the expression of delta-toxin with the status of the agr (accessory gene regulator) status. Mass spectra of pure toxin from wild type strains and mutants for agr-rnaIII gene were compared specifically at the position of the peak for delta-toxin. 156 Biosensors have taken up an important role in the accurate and fast detection of food contaminants even in very low concentrations 157 using biorecognition elements, such as antibodies, enzymes, nucleic acids, phages, etc., along with electrochemical, optic, or piezo-electric devices for the detection of food contaminants. MS-based biosensors are less prevalent as compared to optical or electrochemical ones 158 , 159 but have the potential to overcome the drawbacks of conventional models of biosensors. A multitoxin biosensor-MS was developed for the detection of multiple bacterial toxins simultaneously. Biomolecular interaction analysis-MS (BIA-MS) that used a two-step method, i.e., first bonding of toxin molecules to antibodies immobilized on a sensor chip using SPR (surface plasmon resonance) and then the bound toxin, was identified by MALDI-TOF/MS. The potential of the multiaffinity sensor chip was validated by the detection of endotoxin from Staphylococcus in mushroom and milk samples and it successfully detected multiple toxins at concentrations as low as 1 ng/mL. 160 4.3 Marine Biotoxins Marine biotoxins are natural compounds released in the marine environment by algae and phytoplankton during harmful algal blooms ( Figure 4 ). These compounds are highly toxic for consumers and not only are related to serious illness but also lead to the death of aquatic organisms and even humans. 161 Due to continuous release in the surrounding environment, these biotoxins accumulate in aquatic and marine organisms such as mollusks and fishes. Based on the chemical nature and solubility, these biotoxins are hydrophilic and lipophilic. Hydrophilic biotoxins are water-soluble and can cause amnesic shellfish poisoning, paralytic shellfish poisoning, and emerging pufferfish poisoning, while other groups of lipid-soluble biotoxins are responsible for diarrhetic shellfish poisoning and azaspiracid shellfish poisoning. There is another group of toxins with less available information that is categorized as emerging toxins and can cause unregulated ciguatera fish poisoning, cyclic imines, and neurotoxic shellfish poisoning. 162 − 165 Paralytic shellfish poisoning (PST) is a group of more than fifty-eight related compounds, produced by Alexandrium dinoflagellates of the Atlantic and Pacific coast and Mediterranean Sea. It has a tetrahydropurine skeleton among which saxitoxin (SXT) and gonyautoxin (GNT) are common. Structurally, STX has been categorized into four subgroups named carbamate, N-sulfo-carbamoyl, decarbamoyl, and hydroxylated saxitoxins. 166 The toxicity related to PST is reflected in mild as well as severe depending upon toxicity. The mild symptoms include numbness, tingling sensation around lips followed by expansion of the area, itching and prickly sensation in fingertips and toes, dizziness, headache, and nausea. Moderate and severe illness symptoms include incoherent speech, prickly sensation and stiffness in limbs, weakness, difficulty in respiration, and muscular paralysis. 161 Figure 4 General structures of various marine biotoxins. Amnesic shellfish poisoning is mainly caused by domoic acid (DA) and derivatives produced by marine diatoms of Pseudonitzschia . DA, cyclic tricarboxylic amino acid, can bind with glutamate receptors in the central nervous system due to structural analogy and result in excess stimulation, induced production of reactive oxygen species (ROS), and ultimately cell death. 167 Consumption of DA resulted in gastrointestinal ailments including abdominal cramps, diarrhea, nausea, and vomiting. Neurological symptoms may also include confusion, disorientation, paresthesia, lethargy, short-term memory loss, and in severe toxicity cases, it may also result in coma or death. 168 Diarrheic shellfish poisoning (DST) is a toxin produced by dinoflagellates of Dinophysis and Prorocentrum. It is a common type of contamination in shellfish industries due to overextended prohibitions on mussel harvesting activity. 169 The responsible toxins for DST are a group of polyether compounds recognized as okadaic acid and its derivatives (dinophysistoxin); pectenotoxin; yessotoxin and its derivatives; and azaspiracid. Okadaic acid and azaspiracid consumption resulted in abdominal pain, diarrhea, nausea, and vomiting. 161 , 170 Pectenotoxin and yessotoxin are not involved in human illness. 171 Neurotoxic Shellfish Poisoning (NST) is another type of algal toxin that causes neurological as well as gastrointestinal ailments. Brevetoxins are a kind of marine biotoxin produced by Karenia brevis (Florida red tide dinoflagellate). It is a poly(ether ladder) compound ( Figure 5 ). It causes mortality in massive fish and marine mammals. In humans, these toxins resulted in asthma-like symptoms if inhaled. 172 Neurological and toxicity symptoms of NST are paralysis, seizures, paresthesia, and coma, while gastrointestinal ailments are represented by nausea, diarrhea, vomiting, cramps, and bronchoconstriction, and extreme poisoning may also lead to death. 161 Ciguatera fish poisoning (CFT) is one of the most common foodborne illnesses caused by marine biotoxin of ciguatoxin. 173 , 174 Ciguatoxins are toxic and lipid-soluble compounds found in marine organisms. Gambiertoxins, the precursor toxins, are produced by benthic dinoflagellates of Gambierdiscus genus. These toxins are accumulated in large predatory fishes like Spanish mackerels, moray eels, barracuda, and snappers. 161 These compounds abnormally activate sodium ion channels and disrupt the cell membrane. 175 These compounds cause abdominal pain, nausea, diarrhea, vomiting, hypertension, and bradycardia along with neurological complications. 161 Figure 5 General structures of various marine biotoxin (Brevetoxin). 4.3.1 MS Analysis and Detection of Algal and Marine Biotoxins Algal and marine biotoxins are another class of heterogeneous toxins produced by algae and cyanobacteria, Dinoflagellates and diatoms produced during algal blooms in rivers, freshwater lakes, and marine aquatic systems. 176 Karunarathne et al., have found that 16,659 deaths have been reported in India between 1999 and 2018 due to poisoning. 177 In the case of sea food such as shellfish, exposure and contamination to multiple toxins are possible, hence an efficient system is able to detect diverse classes of toxins at the same time effectively. Blay et al., 178 developed a method for the detection of multiple lipophilic biotoxins including azaspiracids, dinophysistoxins, and pectenotoxins as well as negative toxins via reversed-phase LC–MS within 7 min and hydrophilic toxins such as okadaic acid, dinophysistoxin-1,2, and yessotoxin from shell fish by recording scans at 2 Hz in positive and negative scans alternatively and 1 Hz in positive mode, respectively. Hydrophilic toxins including gonyautoxins domoic acid and saxitoxin were detected with mass accuracy of less than 1 ppm error and resolving power of 100,000 for the analytes ( m / z 300–500). The limits of detection for lipophilic toxins were 0.041–0.10 μg/L ppm (positive ions), 1.6–5.1 μg/L (negative mode), and 3.4–14 μg/L for domoic acid and paralytic shellfish toxins. 178 The biggest advantage of the method is that the analytes were detected with real time samples without any interference. Aquatic water bodies have a higher possibility of having aquatic biotoxins; hence, monitoring of water in water bodies is mandatory. Estevez et al. developed a method to seawater monitoring for marine biotoxins by hydrophilic interaction liquid chromatography coupled with HRMS. The main analytes considered for the detection were saxitoxin, decarbamoyl-saxitoxin, neosaxitoxin, gonaytoxin-2,3, and tetrodotoxin due to their adverse effects on gastrointestinal and central nervous systems in humans if taken up via seafood. Samples were processed via ultrasound-assisted solid–liquid extraction with methanol to extract toxins, followed by solid phase extraction using silica cartridges. The selected toxins are polar in nature; hence, the extraction stage is crucial for analysis, and the developed method has recoveries of 15–47% in filtrate and 26–71% in particulate fraction. Simultaneously, limit of detection was also affected with source as LOD was 0.5–5 μg/L for filtrate and 3.1–62 μg/L for particulate fraction. 179 Kolrep et al. 180 conducted comparative metabolite profiling to track the metabolism of okadaic acid in the liver and the role of CYP3A4 and CYP3A5 in its detoxification. It was found that LC–MS/MS can identify the metabolites distinctly from humans and rats based on the difference in +16 (+O) and +14 (+O/–H 2 ) Da. It suggested some critical differences in the metabolism of okadaic acid in humans and rats. In continuation, it was also found that rats generated more metabolites from okadaic acid in comparison to humans in the presence of NADPH-dependent enzymes. 181 The establishment of metabolic patterns and fragments might be crucial for the identification of fingerprints for toxin identification. Table 5 elaborates on the detection of bacterial and marine biotoxins from different samples. Table 5 Detection of Bacterial and Marine Biotoxins by MS-Based Approach Name Sample Method Operating parameters Outcome Ref Enterotoxin Commercial LC–HRMS LC–MS Q-extractive mass spectrophotometer C18 reverse phase column; isocratic elution 93 signature peptides identified for enterotoxins ( 182 ) Botulinum Commercial Endopep MS Triple quadrupole mass spectrometer; Turbo Ion Spray interface; C18 column; gradient elution Toxin detection 0.1 MLD 50 and quantification 0.62 MLD 50 ( 183 ) Okadaic acid Raw and cooked food (Mussel, clam, flatfish) Tandem mass spectrometry C18 column with triple-quadruple mass spectrometer; ammonium format gradient elution; negative ionization mode LOD and accuracy 0.2–5.1 μg/k ( 184 ) Dinophysistoxin Raw and cooked food (Mussel, clam, flatfish) Tandem mass spectrometry C18 column with triple-quadruple mass spectrometer; ammonium format gradient elution; negative ionization mode LOD and accuracy 0.2–5.1 μg/k ( 184 ) Anatoxins a (ATX) and Homoanatoxin-a (HAT) Benthic-cyanobacterial-mat field samples LC–HRMS/MS Q Exactive HF Orbitrap MS; HESI-II electrospray ionization source; at 40 °C; resolution 60,000; collision energy 20 eV Toxins are in conjugated form 15% ATX and 38% HAT ( 185 ) Cyanotoxins Blue-green algae dietary supplement Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Electrospray ionization positive; source temperature 550 °C; ion spray voltage 5500 V; curtain gas 25 psi; collision gas 10 psi Quantification limits 60–300 μg kg –1 ( 186 ) Paralytic shellfish toxins Marine shellfish Hydrophilic interaction chromatography-tandem mass spectrometry Separation with HILIC-Z column; acetonitrile and ammonium formate-formic acid as the mobile phase; positive electrospray ionization; samples are cleaned with by ion-pair SPE using a porous graphitic carbon cartridge Limits of detection 1.7–13.7 μg kg –1 ; and quantitation 5.2–41.0 μg kg –1 ; recoveries 76.5–95.5% ( 187 ) Tetrodotoxin Marine shellfish Hydrophilic interaction chromatography-tandem mass spectrometry Separation with HILIC-Z column; acetonitrile and ammonium formate-formic acid as the mobile phase; positive electrospray ionization; samples are cleaned with by ion-pair SPE using a porous graphitic carbon cartridge Limits of detection 1.7–13.7 μg kg –1 ; and quantitation 5.2–41.0 μg kg –1 ; recoveries 76.5–95.5% 4.4 Phytotoxins Phytotoxins are plant-derived compounds, including alkaloids and glycoalkaloids, that are naturally produced within plants but prove harmful if they remain in food products ( Figure 6 ). These are secondary metabolites in plants and include cyanogenic glycosides, glucosinolates, glycoalkaloids, pyrrolizidine alkaloids, and lectins. 188 Based on the site, these toxins can be classified into endotoxins and exotoxins. Endotoxins may be normal metabolites that are present in cells but become harmful if consumed in higher concentrations, and these compounds are also refereed as antinutritional factors, while exotoxins are toxic metabolites that are released from cells. Based on chemical nature, these are dehydropyrrolizidine, alkaloids, ptaquiloside, corynetoxins, and phomopsins. 189 Cyanogenic glycosides (CGLs) are present in almonds, cassava, bamboo roots, sorghum, and stone fruits. These toxins are generated from proteinogenic amino acids like leucine, isoleucine, phenylalanine, tyrosine, and valine as well as nonproteinogenic amino acids like cyclopentenylglycin. CGLs are potentially toxic for humans and result in acute cyanide intoxication, high respiration rate, lower blood pressure, headache, dizziness, stomach pains, diarrhea, vomiting, and mental confusion. 188 , 189 Furocoumarins are found in many plants including carrots, celery roots, citrus fruits, parsley, and citrus plants. These compounds are responsible for gastrointestinal ailments and phototoxicity, skin reactions under UV light. 34 , 190 Lectins are reported from beans like kidney beans and can result in stomachache, diarrhea, and vomiting. 189 Figure 6 General structures of various Phytotoxins. The general scaffold for Cyanogenic glycosides, Furocoumarin and Dehydropyrrolizidine, has been depicted with providing a few examples of various compounds belonging to the class of Furocoumarin. Phytotoxins are sometimes a part of the natural defense of plants, like Pyrrolizidine alkaloids (PAls) that are produced in Asteraceae , Boraginaceae , and Fabaceae families to defend plants against herbivores as well as insects. These toxins tend to have a common 1-hydroxymethyl pyrrolizidine core that is esterified with aliphatic acids. Besides edibles from these plants, honey is one of the common products contaminated with PAls, and hence, the extract or infusion can be used as an analyte for the detection of PAls. Ten plant samples, Anchusa officinalis , Borago officinalis , Echium italicum , Eupatorium cannabinum , Heliotropiumeuropaeum , Lithospermum officinale , Petasites hybridus , Senecio vulgaris , Symphytum officinale, and Tussilago farfara from Orto botanicodellaScuola Medica Salernitana, Salerno, Italy, were collected, and aqueous extract was prepared with salting-out assisted liquid–liquid extraction. The aqueous extracts were analyzed with UPLC–MS/MS. The analysis was able to identify 88 PAs from 282 samples with an identification limit of 0.6–30 μg kg –1 and a false negative rate <1.3% (at the concentration range of 4 μg L –1 ). 191 For simultaneous detection of phytotoxins and microbial toxins, HRMS was employed, which applied to over 156 compounds inclusive of about 90 plant toxins (e.g., various alkaloids and aristolochic acids), about fifty-four mycotoxins, and 12 phytoestrogens (e.g., lignan, isoflavones, coumestans, etc.) in plant-protein samples, like cereals. MS library, created on fragmentation pattern obtained with both negative and positive ionization modes for each toxin, using ten different collision energies was used for analysis. A typical workflow was followed with generic QuEChERS-like sample preparation, followed by UPLC using suitable mobile phases that allowed the resolution of over 50 toxic alkaloids. The method performance was evaluated for its sensitivity at levels ranging from 1–100 μg/kg, and reproducibility. The quantitation obtained against the standard addition approach could meet SANTE/12682/2019 criteria for 132 toxins out of the tested 156 toxin samples. 192 The plant toxins ricin and RCA120 were detected, differentiated, and quantified by Kalb et al., 193 via MS-based methods for the EQuATox proficiency test, in ∼9 samples. They successfully identified the samples spiked with ricin or RCA120; samples spiked with a 0.414 ng/mL concentration could not be detected. Liang et al. 194 employed reversed phase LC–HRMS to detect five major phytotoxin groups including alkaloids, aromatic polyketides, flavonoids and steroids, and terpenoids at alkaline pH (>9). The developed method not only allowed the detection of 30 phytotoxins but also had forty-times higher detection sensitivity in comparison to older methods. Table 6 discusses some of the examples for phytotoxins detection using the MS-based approach. Table 6 Phytotoxins Detection from Food Samples via MS-Based Approach Name Sample Method Operating parameters Outcome Ref Toxoflavin and Fervenulin Food samples and Tremella fuciformis Berk UPLC–MS/MS Separation column: ZORBAX SB-C18 column; oven temp: 35 °C; mobile phase: 0.1% formic acid+ methanol; flow rate 0.4 mL min –1 Detection limits (μg/kg) Toxoflavin 12 ( 152 ) Fervenulin 24 Toxin level (mg/kg) Toxoflavin: 7.5; fervenulin: 3.2 Ricin Soft drinks and serum Surface-assisted laser desorption/ionization mass spectrometry Pulsed Smartbeam II 2 kHz laser; wavelength 355 nm (∼3.49 eV); frequency 1000 Hz; delayed extraction time 350 ns for proteins Limit of detection 0.5 pmol/μL ( 195 ) Pyrrolizidine Alkaloids LC–MS/MS Tea UPHPLC with Quadrupole mass spectrometer; C18 Hypersil Gold column fitted; gradient elution Total PA levels 13.4 to 286,682.2 μg/kg d.m ( 196 ) Ptaquiloside LC–MS/MS Bracken fern LC–MS/MS; C18 column; gradient elution; column temperature 35 °C; electrospray positive ionization Limits of detection 0.03 and quantification 0.09 μg/kg ( 197 ) Furanocoumarin UPLC–MS/MS Citrus sp. Nexcol C18 column; column temp 40 °C; gradient elution; positive electrospray ionization Compound recovery 94.07–114.53% ( 198 ) Amygdalin LC–MS/MS Kernels and Almonds LC–MS/MS equipped with 6500 quadruple linear ion trap (QTRAP) mass spectrometer and electrospray ionization Recovery 90–107%; limit of detection 0. Ng/g and limit of quantitation 8 and 2.5 ng/g ( 199 ) Pyrrolizidine alkaloids quadrupole orbitrap MS Honey Polar C18 column; temp 40 °C; mobile phase flow rate of 400 μL min –1 Limit of quantification 0.1–0.3 μg kg –1 ( 200 ) MS detection with positive ionization mode; scan range 250–500 m / z and 70 k (fwhm) Pyrrolizidine alkaloids quadrupole orbitrap MS Black and green tea Polar C18 column; temp 40 °C; mobile phase flow rate of 400 μL min –1 Limit of quantification 1–11.7 μg kg –1 ( 200 ) MS detection with positive ionization mode; scan range 250–500 m / z and 70 k (fwhm) Pyrrolizidine alkaloids quadrupole orbitrap MS Herbal infusion Polar C18 column; temp 40 °C; mobile phase flow rate of 400 μL min –1 Limit of quantification 0.9–2.1 μg kg –1 ( 200 ) MS detection with positive ionization mode; scan range 250–500 m / z and 70 k (fwhm) 4.5 Emerging Toxins In addition to conventional toxins already known and summarized above, there is a group of toxic chemicals that are continuously evolving, mainly due to rising pollution. In the last eight years, the list of emerging chemicals has increased day by day. Synthetic chemical toxins include microplastics, organophosphorus and polybrominated flame retardants, perfluoroalkyl compounds, food process and packaging, waste substances, and nanomaterials. 15 Besides, heavy metals, antibiotics and drug traces and metabolic intermediates, and agricultural chemicals 201 have shown bioaccumulation and have serious toxic effects if consumed, even in low concentrations. Health ailments include endocrine disruption, suppression and overexpression of the immune system, inflammation, abnormal metabolic changes, skin diseases, carcinogenesis, etc., and the toxicity relies on interaction with the cellular system and receptors. 15 , 201 , 202 With the increase in pollution and intrusion of pollutants in the food web, the toxic chemicals traced in food and edibles are increasing. Some of those chemicals exhibited bioaccumulation and become silent killers, but some are potentially lethal. These emerging pollutants include pesticides, herbicides, healthcare, cosmetic chemicals, etc. Fipronil is a wide-spectrum phenylpyrazole insecticide used to control beetles, ants, cockroaches, etc. but its entry into the food chain is alarming due to its carcinogenic nature, essentiating its prohibition by the US Environmental Protection Agency (EPA). Suitable detection methods are thereby essential to identify and quantify these contaminants before they enter the food chain. The most reliable detection method includes LC–MS/MS and GC–MS, having specific sample preparation before the analyses ( Table 7 ). 203 One such preparatory method involved a modified QuEChERS sample preparation before using a triple quadrupole MS instrument coupled to ESI for detecting fipronil and its major metabolite fipronil sulfone, at concentrations of 5 μg/kg. The use of nontargeted approaches, such as SWATH-MS (sequential window acquisition of all theoretical mass spectra), enables the sequential analysis of fipronil and other such contaminants, e.g., pesticides and polyaromatic hydrocarbons. Glyphosate (insecticide) was detected in an underivatized form by innovating new extraction methods coupled with instrumentation. The QuPPe (Quick Pesticide Preparation) method was used for sample preparation 204 followed by detection via sensitive MS instruments to achieve accurate quantitative results. LC–MS/MS was used in combination with the DMS (differential mobility separation) technique to terminate analytical interferences leading to improved signal by decreasing noise and, consequently, increasing accuracy and confidence in data. Using this method, LC–DMS–MS/MS was used for identification and quantification of pesticide contaminants in food samples. Triclosan is a well-known and common biocide agent against bacteria as well as fungi, 205 while bisphenol analogues are used in packaging and lining. 206 Morgan et al. 206 employed GC–MS to monitor the levels of triclosan and five bisphenol analogues (B, F, P, S, and Z) in 776 adult solid food samples. More than 80% of the samples were contaminated with at least one target phenol. Based on the frequencies, 59% of samples were contaminated with triclosan followed by 32% bisphenol S, and 28% bisphenol Z. The maximum concentration for triclosan was 394 ng/g. Table 7 Detection of Emerging Toxins from Food and Water Samples by MS Name Sample Method Operating parameters Outcome Ref Cypermethrin Baby food liquid chromatography coupled to quadrupole Orbitrap mass spectrometry Gradient elution; negative ionization mode; capillary temperature 300 °C and heater 305 °C Detection concentration in baby food 10.3 μg kg –1 ( 208 ) Parabens Surface water UHPLC–MS/MS LC-18 column; column temp 40 °C; gradient elution; capillary voltage −3.0 kV Limit of detection 0.04 ng L –1 and Limit of quantification 0.82 ng L –1 ( 209 ) Bisphenol Water GS–MS/MS Temperature transfer line 250 °C; ion source 230 °C and quadrupole 150 °C. solvent delay 4.5 min; electron ionization (EI) mode (70 eV) Recovery 81.8% −96.1%; limit of detection was 0.2 ng L –1 ( 210 ) Parabens water GS–MS/MS Temperature transfer line 250 °C; ion source 230 °C and quadrupole 150 °C. solvent delay 4.5 min; electron ionization (EI) mode (70 eV) Recovery 81.8% −96.1%; limit of detection was 0.2 ng L –1 ( 210 ) Triclosan water GS–MS/MS Temperature transfer line 250 °C; ion source 230 °C and quadrupole 150 °C. solvent delay 4.5 min; electron ionization (EI) mode (70 eV) Recovery 81.8% −96.1%; limit of detection was 0.2 ng L –1 ( 210 ) Neonicotinoids vegetables QuEChERS-Portable MS PDESI as ion source; ultrapure helium (≥99.999%) as carrier gas; inlet temperature 200 °C; molecular pump speed was 1375 Hz Limit of detection 2.0 ng g –1 recovery 82.2% −109.7% ( 211 ) Carbamates Vegetables QuEChERS-Portable MS PDESI as ion source; ultrapure helium (≥99.999%) as carrier gas; inlet temperature 200 °C; molecular pump speed was 1375 Hz Limit of detection 2.0 ng g –1 recovery 82.2% −109.7% ( 211 ) Phenyl Pyrazole Vegetables QuEChERS-Portable MS PDESI as ion source; ultrapure helium (≥99.999%) as carrier gas; inlet temperature 200 °C; molecular pump speed was 1375 Hz Limit of detection 2.0 ng g –1 recovery 82.2% −109.7% ( 211 ) Pesticides Fruits and vegetables QuEChERS-LC–MS Sciex QTRAP 5500 triple quadrupole MS; positive electrospray ionization; ion source temperature 550 °C 24 pesticides detected distinctly ( 212 ) Pesticides Milk LC-LTQ/Orbitrap Mass Spectrometry Separation with reverse phase C18 column; positive ionization mode; spray voltage 4 kV; auxiliary gas flow rate 10 arbitrary units; tube lens 90 V, capillary temperature 320 °C Limit of detection 0.2–8.1 μg kg –1 and quantification 0.61–24.8 μg kg –1 ( 213 ) Not only emerging toxins but also the availability of efficient and portable systems have become necessary prerequisites. Some of the recent advancements have shown the availability of portable MS systems for detection and monitoring. Maragos 131 has evaluated the potential of portable MS (APCI-MS) for the detection of T-2 toxin mycotoxin in contaminated cereal grains, wheat, and maize by APCI-MS. The sample was extracted with acetonitrile+water (84:16, v/v) followed by drying and reconstitution in ammonium formate. The MS system contains a linear ion trap mass analyzer to avoid the need of an external supply of gas or air. The device and developed method were able to detect T-2 toxin above 0.2 mg/kg from soft white and hard red wheat, and yellow dent maize. The method was more efficient and hence able to lower-down the detection limit from >0.9 mg/kg. In a similar line, FB and its isoforms were detected in maize with a portable mass spectrometer. For the detection, samples were extracted with aqueous methanol followed by cleaning up in the immunoaffinity column. Ultimately, cleaned samples were successfully analyzed with the portable MS with detection limits of 0.15 (B1), 0.19 (B2/B3), and 0.28 (total FB) mg/kg maize. The method has quantification limits of 0.33, 0.59, and 0.74 mg/kg and recoveries of 93.6% to 108.6%. Wichert et al. 207 have also reported such kind of advancements to detect proteinaceous toxins (912.5–66.5 kDa) from plants as well as microorganisms origin using paper spray-MS (PS-MS) with wipe samples of bench, glass, leaves, flooring, etc., and validated with biological toxin simulant for Staphylococcal enterotoxin B. Carbon sputtered porous polyethylene dominated conventional chromatography paper, carbon nanotube-coated paper, and polyethylene for paper spray. The method was able to distinguish the protein toxin simulant efficiently with a good signal-to-noise ratio. 4.1 Mycotoxins Mycotoxins are toxic secondary metabolites produced by fungi. These toxins can accumulate in the fungal-contaminated food grains such as corn, cereals, and legumes, and upon ingestion can traverse into the food chain affecting humans and animals. 101 As per the Rapid Alert System for Food and Feed of EU (RASFF) report, mycotoxins contaminate around one-quarter of global food grain production both during pre- and postharvest. 102 This clearly indicates the severity of the mycotoxin problem in the food that we consume. In the literature, around 300 mycotoxins are reported, but only seven toxins are quite common in food worldwide such as aflatoxins (AF), trichothecenes (TC), zearalenone (ZEN), fumonisins (FB), ochratoxins (OTA), citrinin (CIT), and patulin (PAT). 101 Fungal species belonging to the genera of Aspergillus , Penicillium , and Fusarium are predominantly toxigenic and most frequently lead to cases of mycotoxin concentration. 101 AF (B1, B2, G1, and G2 produced by Aspergillus ), CIT (by Penicillium , Aspergillus , and Monascus etc.), ergot alkaloids ( Claviceps purpurea ), FB ( Fusarium sp.), ochratoxin A (produced by Penicillium and Aspergillus ), PAT ( Penicillium patulum ), TC ( Fusarium , Stachybotrys , Trichothecium , and Trichoderma sp.), and ZEN ( Fusarium graminearum ) are some of the well-known mycotoxins responsible for serious lethal reactions like cancer induction, kidney toxicity, immune suppression, stachybotryotoxicosis, turkey X syndrome, etc. 103 Another issue with these mycotoxins is their resistance and tolerance toward thermal treatment; hence, they remain active even after heating. 104 AFs are groups of potentially toxic fungal secondary metabolites reported from Aspergillus flavus , A. parasiticus , and A. nomius ( Figure 2 ). They are produced from polyketides and commonly present in cereal crops like corn, peanuts, walnuts, wheat, etc. 105 AFs can lead to chronic toxicity related to hepatic tissues, necrosis, hepatomas, periportal fibrosis, jaundice, hemorrhage, and fatty liver changes, and also exhibit teratogenicity, carcinogenicity, and immunotoxicity. 105 , 106 They were first reported in the state of Gujrat and Rajasthan, India in 1974, which resulted in the onset of hepatitis caused by A. flavus infected staple food and maize. A. flavus , A. parasiticus , A. nomius , and sometimes Emericella spp. are the main producers of AFs. 105 To date, more than 20 AFs variants have been reported, among which variant B1 is the most common and most lethal. AFs B1 and B2 are produced by A. flavus and A. parasiticus , and AF M1 is produced by A. parasiticus and can be transmitted through milk. Variants M1 and M2 are also produced by metabolism of B and B2. 16 AF B1 is metabolized by the P450 monooxygenase system and generates AF 8,9-epoxide (reactive epoxide) that induces mutations and cancer by forming DNA abducts. 106 Figure 2 General structures of some of the Aflatoxin variants. Besides Aspergillus , isolates belonging to Fusarium are also known to produce the most potent toxins, which include deoxynivalenol (DON), FBs, and ZENs ( Figure 3 ). DON belongs to the sesquiterpenoid group of trichothecenes. It mainly contaminates corn, wheat, and barley. It is also a toxic secondary metabolite that has a negative health impact on the consumer by disturbing the intestinal barrier and has immune-stimulatory as well as immune-suppression properties at low and high doses, respectively. 107 The same group also contains its acetylated derivatives named nivalenol, T-2 toxin, and HT-2 toxins. 108 After ingestion, DON is absorbed and metabolized in the intestine via DON-3S, DON-GlcA, and DOM-1. In poultry birds, DON-3S and DON-15S are eliminated via bile and urine, while in swine, it is absorbed in the upper digestive system; hence, poultry birds are the least sensitive to these toxins, and swine are most sensitive to these toxins. In humans, contaminated foods like infected meat and cereals are the most common sources. 107 Fusarium sp. is also responsible for other mycotoxins named FBs (secreted by Fusarium verticillioides and Fusarium proliferatum ). Aspergillus niger is also able to produce FBs. Such toxins are commonly reported from cereals like peanuts, maize, rye, oats, millets, and grape. 109 To date, more than 15 homologous forms of fumonisin have been reported that are referred to as A, B1, B2, B3, C, P, etc. However, B1 is the most toxic form of FB. 110 , 111 FBs are known to have carcinogenic, neurotoxic, and hepatoxic effects that cause hepatocarcinoma, defects in the neural-tube, and nephrotoxicity. 109 ZEN is a nonsteroidal, estrogenic mycotoxin produced by F. acuminatum , F. crookwellense , F. culmorum , F. cerealis , F. equiseti , F. graminearum , F. oxysporum , F. sporotrichioides , F. semitectum , and F. verticillioides . 112 It disrupts reproductive capacity by affecting mammalian folliculogenesis and impairs granulosa cell development and follicle steroidogenesis. 113 It is thermostable 114 and resistant to processing stress like milling and storage. 115 , 116 ZEN leads to kidney damage and liver injury and causes inflammation. 112 Figure 3 General structures of various mycotoxins. Ergot alkaloids are toxic secondary metabolites produced by several fungi of Clavicipitaceae ( Epichloë , Claviceps , Balansia , and Periglandula ) and Aspergillus fumigatus . The producers mainly include Epichloë endophytes , Epichloë festucae var. lolii , Epichloë coenophialum , and Claviceps purpurea . 117 They belong to compounds containing an indole group and are derived from L -tryptophan. They cause "ergotism" and their toxic effect is reflected by hyperglycemia, gastrointestinal upset, mydriasis, 118 and even endocrine disruption. 119 Besides these major mycotoxins, some other chemicals including OTA and PAT have also been reported from fungi. OTA is nephrotoxic and produced by a diverse range of fungi such as Aspergillus ochraceus , A. carbonarius , A. niger , and Penicillium verrucosum . It can also lead to renal tumors, Balkan endemic nephropathy, and chronic interstitial nephropathy. 120 Patulin is produced by Penicillium , Aspergillus , and Byssochlamys , while alternariol (AOH) and alternariol monomethyl ether (AME) are Alternaria toxins, produced by fungi of the Alternaria genus found in fruits and related products. 20 To ensure food safety with special consideration to mycotoxins, international, national, and regional agencies like the World Health Organization, Food Agriculture Organization, Codex Alimentarius Joint Expert Committee for Food Additives and Contaminants, European Food Safety Authority, GCC Standardization Organization, and Japanese Association of Mycotoxicology have determined the permission limit for mycotoxins contamination in food as well as feed. 121 Table 3 summarizes the permissible limit for various mycotoxins in food 122 − 125 and animal feed 126 , 127 as per the European Commission. Table 3 Regulatory Permission Limits for Various Mycotoxins in Food and Feed Products As Per European Commission 122 − 127 Category Food or animal feed products Permissible limit (μg/kg) Aflatoxins B1 (AFB1) Food Brazil nuts, groundnuts, hazelnuts, and oilseeds for human consumption after physical treatment 8 Almonds, apricot kernels, and pistachios for human consumption after physical treatment 12 Brazil nuts, groundnuts, hazelnuts, and oilseeds for human consumption directly (No physical treatment) 2–5 Almonds, apricot kernels, and pistachios for human consumption directly (No physical treatment) 8 Dairy products for consumption by infants, baby food, and processed cereal-based food 0.1 Spices 5 Dried fruits, and Figures for human consumption after physical treatment 5–6 Dried fruits (except Figures) for human consumption directly (No physical treatment) 2 Maize and rice (as ingredients) for human consumption after physical treatment 5 Feed Feed materials 0.02–0.05 Complete feeding stuff with the exception of 0.05 Calves, cattle, and lambs 0.005–0.01 Poultry 0.02 Complementary feeding stuff 0.005–0.05 Aflatoxins M1 (AFM1) Food Milk 0.05 Infants' dairy products, baby formula and baby milk, 0.025 Aflatoxins (AFs) total Food Almonds, apricot kernels, brazil nuts, groundnuts, hazelnuts, oilseeds, and pistachios for human consumption after physical treatment 15 Groundnuts, oilseeds, and processed products for human consumption directly (No physical treatment) or as ingredient 4 Almonds, apricot kernels, brazil nuts, hazelnuts, pistachios, and for human consumption directly (No physical treatment) 10 Spices 10 Dried fruits, and Figures for human consumption after physical treatment 10 Dried fruits (except Figures) for human consumption directly (No physical treatment) 4 Maize and rice (direct or as ingredients) for human consumption after physical treatment 10 Citrinin (CIT) Food Food supplements prepared from red yeast fermented rice 2000 Deoxynivalenol (DON) Food Unprocessed durum, maize, oats and wheat 1750 Cereals and cereal flour for direct human consumption 750 Cereal-based processed foods and baby food 200 Feed Animal feed from–cereals 8–10 Complete as well as complementary feeding stuff 0.9–5 Fumonisin (FB1+FB2) Food Unprocessed maize 4000 Maize for direct human consumption 1000 Maize-based processed food for babies and young children 200 Feed Maze based feed 60 Complete and complementary feeding stuff 5–50 Ochratoxins (OTA) Food Cereals-based unprocessed products 3–5 Cereal-based processed food and baby food, dietary foods products specially for medical purposes purposes 0.5 Beverages based on grapes 2 Coffee roasted/instant 5/10 Spices 15 Feed Cereals-based feed materials 0.25 Complete and complementary feeding stuff for poultry 0.1 Patulin (PAT) Food Fruit juices 50 Solid apple products 25 Solid apple as well as apple juice for babies and young children 10 Zearalenone (ZEN) Food Cereal products (unprocessed except maize) 100 (350) Cereals for direct human consumption 75 Maize for direct human consumption 100 Cereals and maize processed products for babies and young children 20 Feed Cereals and maze-based feed materials 2–3 HPLC and GC are the common approaches for the detection and higher accuracy and sensitivity making MS an elegant and dominant analytical tool for toxicological and metabolite analysis. 128 In addition, it also allows simultaneous detection of a diverse range of toxins together and aids in method standardization and implication to ensure the rapid evaluation of samples for food safety analysis. Areo et al. 129 have employed UHPLC–MS/MS for the detection of AFs, ZEN, and OCT A from 100 tea samples (collected from registered shops within South Africa), prepared in acetonitrile/water/acetic acid solvent by QuEChERS extraction method. The supernatant was mixed with 900 mg of anhydrous MgSO 4 , 150 mg of C18, and 150 mg of primary secondary amine that separates the organic phase. The organic phase was collected and dried under a nitrogen stream and further reconstituted in methanol/water for analysis via UHPLC–MS/MS. The method selected has very high linearity (>0.99) and precision (6–29%). AFs, i.e., AFB1, AFB2, AFG2, OCT A, and ZEN, were absent in the samples, and AFG1 was present in very low amounts 1.72–5.19 μg/kg that were also below the regulated level in food as recommended by EU Commission Regulation 1881/2006. 129 You et al. 130 have evaluated the effect of culture medium for mycotoxin accumulation by Alternaria . Secondary metabolites were characterized by nontarget analysis with HRMS. Mycotoxins produced by Alternaria were grouped into for families: alternariol monomethyl ether (AME), alternariol (AOH), altenuene (ALU), Desmethyl dehydro altenusin (DMDA), and dehydroaltenusin (DHA) families, Altertoxin-I (ATX-I) family, tentoxin (TEN) family, and tenuazonic acid (TeA) family. Culture medium greatly influenced the type of mycotoxins produced. wiz Potato Sucrose Agar medium is suitable for AOH, AME, ALU, ALT, DHA, and DMDA, while Potato Dextrose Agar supported the accumulation of ATX-I, TEN, and TeA. Table 4 summarizes some of the research for the detection of various mycotoxins with MS-based tools. Table 4 Detection of Mycotoxins by MS-Based Approach Name Sample Method Operating parameters Outcome Ref Trichothecenes T2 Wheat and maize Portable mass spectrophotometer MS parameter: heater 200 °C; sample pump 0%; mass center: m / z = 484; mass tolerance: 1; spectrum average: 10 Limit of detection 0.2 mg/kg ( 131 ) Alternaria toxin Fruits and vegetables QuEChERS extraction followed by MS analysis Triple quadrupole MS; pressure curtain gas 35 psi and collision gas 8 psi; ion spray voltage 5,500 V (positive ion mode), and −4,500 V (negative ion mode) Detection limit 1.0–5.0 μg/kg (Extraction recoveries 73.0–120%) ( 132 ) Repeatability <12.9% Mycotoxins Standard sample UHPLC-QTrap-MS/MS Injection volume: 20 μL; column temperature: 30 °C; flow rate: 0.2 mL min –1 ; mobile-phase 0.1% formic acid+ acetonitrile run time: 25 min Limit of quantification 0.005 and 13.54 ng mL –1 (Limit of detection 0.001–9.88 ng mL –1 ; 0.005; Recovery 67.5–119.8%) ( 133 ) Aflatoxin B1 and M1 Blood HPLC–MS/MS Allure PFPP column; positive electrospray ionization; source temp 500 °C LOD 0.05 to 0.2 ng/mL; accuracy 92–111% ( 134 ) Alternaria toxins Vegetable sample UPLC–MS/MS ACQUITY UPLC BEH C18 column; injection volume: 5 μL, column temperature: 40 °C; flow rate 0.4 mL min –1 , mobile phase: 0.1% formic acid water; gradient flow 10–90% Contaminated solanaceous vegetable: 41.1% ( 135 ) AME: 4.26%; AOH: 6.38%; altenuene: 6.38%; tentoxin: 42.6%; tenuazonic acid: 55.3% Aflatoxin M1 HPLC–MS/MS Quantitative daughter m / z : 273; qualitative daughter m / z : 259; collision energy 23 eV Quantification limit 1.62 ppb; detection limit 0.54 ppb; 98.5% accuracy ( 136 ) Ochratoxin A Coffee and tea UHPLC–MS/MS Triple quadrupole MS; electrospray ionization, ion source temp 300 °C; flow rate 3 L/min Sensitivity 0.30 and 0.29 ng/mL ( 137 ) Trichothecenes Oat based products U-HPLC–HRMS/MS Reverse phase column; electro-ionization detector; column temperature 40 °C; ethanol+water+formic acid gradient elution Frequency of free T2 toxin 92% ( 138 ) T2 mono glucoside 69% Alternaria toxins Rice LC–MS/MS Separation with hyper clone column and detection with C18 column; detection with negative electrospray ionization, source temperature 300 °C Limit of detection and quantification ( 139 ) AME: 0.03 and 0.09 μg/L; altenuene: 5.48 and 16.24 μg/L Alternariol monomethyl ether (AME), Alternariol (AOH), and tentoxin Standard sample LC-ESI-MS/MS LC–MS/MS with triple quadrupole; separation at 25 °C with C18 column; analysis with quadrupole MS, source temperature 450 °C; ion spray voltage 5500 V Limits of detection and quantitation: 0.7 and 3.5 ng/g recovery 80% ( 140 ) 4.2 Bacterial Toxins Bacterial contamination has shown diverse causes as Shigella mainly infect via unwashed hands, while Campylobacter and Escherichia coli are usually present in raw milk, undercooked meat and poultry products, and contaminated water. In contrast, Listeria monocytogenes and Yersinia enterocolitica are found in refrigerated food. 141 Bacterial toxins have been classified as endotoxins and exotoxins. Structurally, endotoxins have distinct structural regions, i.e., glycolipid is made up of disaccharide and fatty acids which are usually capric, lauric, myristic, palmitic, and stearic acids. These acids are buried within the outer cell membrane of the bacterium. The nucleus is the second important part, which is made up of a hexose- and heptose-based heteropolysaccharide. The glycolipid and nucleus are interconnected by the sugar acid 2-keto-3-deoxyoctanate. Endotoxins are lipopolysaccharides and are part of the outer membrane of Gram-negative bacteria. These are also identified as important determinants and antigenic parts of bacteria that aid in attachment with the host as well as in pathogenicity. Exotoxins are proteins in nature that are released by Gram-negative bacteria and disrupt cell division, causing lysis and tissue damage. 142 , 143 Exotoxins are further classified into types I, II, and III based on the mechanism of action. Toxin type I can make critical changes in the host's cells without internalizing. Superantigens secreted by Staphylococcus aureus and Streptococcus pyogenes are examples of a type I toxin. The type II group includes hemolysins, phospholipases, aerolysin, and GCAT proteins. It intrudes the host cells and creates pores to destroy the host cell's membrane. In comparison to type I and II, type III is a diverse group in terms of activity. It has a binary structure with fractions A and B. Fraction B in the toxin facilitates the binding with receptor in the host cell, while another fraction, i.e., "A" carries enzymatic activity and is responsible for the toxin effect. Anthrax toxin ( Bacillus anthracis ), Cholera toxin ( Vibrio cholerae ), and Shiga toxin ( Escherichia coli O157:H7) are some examples of exotoxins. 142 , 144 Botulinum is 150 kDa and composed of a heavy chain of 100 kDa and a light chain of 50 kDa. Heavy chain is responsible for binding to receptors on neuron surface, while light chain cleaves proteins required for nerve signal transmission, i.e., botulinum A, C, and E cleave synaptosomal-associated protein 25, while B, D, F, and G variants act on synaptobrevin-2. 72 , 145 , 146 In a similar fashion, B. anthracis produces three types of proteins or factors, e.g., lethal factor, edema factor, and protective antigen. Protective antigens split and form a fragment of 63 kDa that forms heptamers and octamers to finally find the cell surface. In addition, they also bind with lethal factors to form lethal toxin. 72 , 147 4.2.1 Detection of Bacterial Food Toxins Clostridium , Salmonella , Staphylococcus , and Listeria are some common pathogens causing foodborne infections in humans. Previously used methods, e.g., enzyme immunoassay (EIA), were fast and sensitive, but their accuracy was limited due to cross-reactivity reporting a high rate of false positives and misguided public health care personnel. Techniques based on MS are powerful and can be multiplexed for the detection of various protein toxins, e.g., toxins from Clostridium , 148 Bacillus , 88 and many more with speed, sensitivity, and accuracy. Botulinum, a neurotoxin, is produced by Clostridium botulinum in seven different serotypes (A–G). Specific detection of these toxins from different strains needs high analytical sensitivity and a MS-based approach. The enzymatic activity-based approach relies upon substrate fragments generated by these toxins which can be used as targets by MALDI-TOF MS. 72 In 2002, a peptide mass map of toxin variants A1 and B1 was prepared by targeting the trypsin digest of the toxin by van Baar and colleagues. The work was extended to C, D, E, and F in 2004. 72 In successive generations, several advancements have shown effective approaches like endopep-MS. 149 Rosen et al. have developed the endopep-MS-based method for the identification of botulinum A and E simultaneously and rapidly. 150 Both A and E identify the same target SNAP 25 protein but act on different sites. 3D structures of both types of fragments were used for differential identification. Drigo et al. 151 employed the same endoPep-MS approach for botulinum toxins C and D. The method has shown a sensitivity of 100% with specificity and accuracy of 96.08% and 97.47%, respectively. Integration of MS with other analytical methods like HPLC, GC, FPLC, etc. has improved bacterial toxin profiling. Toxoflavin and fervenulin are bacterial toxins produced by Berkholderia and Streptomyces hiroshimensis . These compounds are common contaminants in fermented corn flour, rice bran oil, distiller's yeast, sweet potato starch, Tremella fuciformis Berk., and rice noodles. These compounds are sensitive to degradation in 1% ammonia solution. UHPLC-Q-TOF/MS allowed for the detection of degradation products. The modified approach led to lower down the limits of detection of toxoflavin and fervenulin to 12 μg/kg and 24 μg/kg, respectively, with recovery of 70.1–108.7%. 152 The MS approach also employed natural phenomena of antigen–antibody interactions for the detection of toxins and related antigens. Salmonella typhi , Gram-negative enterobacteria, is responsible for typhoid fever and meningitis. The immunoreactive proteins of bacteria were used as targets to develop improved diagnostic tools with MS. An immunoaffinity-based proteomic approach was employed with IgG and IgM antibodies from typhoid patients. The approach aided in the identification of 28 immunoreactive proteins, out of which 14 were complementary to IgG, 4 for IgM, and the rest 10 for both, hence retained by respective charged columns. In context to antigenicity, 22 proteins have shown antigenicity and immunogenicity. 153 Such an approach is helpful for rapid identification, and its reproducibility and reliability can be employed for vaccine and drug development. Peptide mass fingerprinting technique (PMF) associated with MALDI TOF/MS or ESI/MS is a top-down MS protocol where proteins are directly ionized to create a fingerprint of individual proteins and applied for detection of various microbial strains. PMF of unknown organisms is compared to those existing in PMF databases or compared to the spectrum of biomarker proteins with the proteomic spectral database, using MALDI-TOF MS. Typically a mass range m / z of 2–20 kDa is used for species-level identification where ribosomal proteins representing 60–70% of microbial cells' dry weight and some housekeeping proteins are selected. 154 Thus, by comparing with extensive commercial databases, microbial contaminants can be traced to the genus, species, or strain level, and such an identification tool is conveniently adapted in diagnostic laboratories. 155 However, using biomarkers is not very common for identification since it requires prior insight into the genome sequences before creating the required databases for proteins' molecular masses. Staphylococcus aureus delta-toxin has been detected using whole-cell (WC) MALDI-TOF/MS and LC–MS to correlate the expression of delta-toxin with the status of the agr (accessory gene regulator) status. Mass spectra of pure toxin from wild type strains and mutants for agr-rnaIII gene were compared specifically at the position of the peak for delta-toxin. 156 Biosensors have taken up an important role in the accurate and fast detection of food contaminants even in very low concentrations 157 using biorecognition elements, such as antibodies, enzymes, nucleic acids, phages, etc., along with electrochemical, optic, or piezo-electric devices for the detection of food contaminants. MS-based biosensors are less prevalent as compared to optical or electrochemical ones 158 , 159 but have the potential to overcome the drawbacks of conventional models of biosensors. A multitoxin biosensor-MS was developed for the detection of multiple bacterial toxins simultaneously. Biomolecular interaction analysis-MS (BIA-MS) that used a two-step method, i.e., first bonding of toxin molecules to antibodies immobilized on a sensor chip using SPR (surface plasmon resonance) and then the bound toxin, was identified by MALDI-TOF/MS. The potential of the multiaffinity sensor chip was validated by the detection of endotoxin from Staphylococcus in mushroom and milk samples and it successfully detected multiple toxins at concentrations as low as 1 ng/mL. 160 4.2.1 Detection of Bacterial Food Toxins Clostridium , Salmonella , Staphylococcus , and Listeria are some common pathogens causing foodborne infections in humans. Previously used methods, e.g., enzyme immunoassay (EIA), were fast and sensitive, but their accuracy was limited due to cross-reactivity reporting a high rate of false positives and misguided public health care personnel. Techniques based on MS are powerful and can be multiplexed for the detection of various protein toxins, e.g., toxins from Clostridium , 148 Bacillus , 88 and many more with speed, sensitivity, and accuracy. Botulinum, a neurotoxin, is produced by Clostridium botulinum in seven different serotypes (A–G). Specific detection of these toxins from different strains needs high analytical sensitivity and a MS-based approach. The enzymatic activity-based approach relies upon substrate fragments generated by these toxins which can be used as targets by MALDI-TOF MS. 72 In 2002, a peptide mass map of toxin variants A1 and B1 was prepared by targeting the trypsin digest of the toxin by van Baar and colleagues. The work was extended to C, D, E, and F in 2004. 72 In successive generations, several advancements have shown effective approaches like endopep-MS. 149 Rosen et al. have developed the endopep-MS-based method for the identification of botulinum A and E simultaneously and rapidly. 150 Both A and E identify the same target SNAP 25 protein but act on different sites. 3D structures of both types of fragments were used for differential identification. Drigo et al. 151 employed the same endoPep-MS approach for botulinum toxins C and D. The method has shown a sensitivity of 100% with specificity and accuracy of 96.08% and 97.47%, respectively. Integration of MS with other analytical methods like HPLC, GC, FPLC, etc. has improved bacterial toxin profiling. Toxoflavin and fervenulin are bacterial toxins produced by Berkholderia and Streptomyces hiroshimensis . These compounds are common contaminants in fermented corn flour, rice bran oil, distiller's yeast, sweet potato starch, Tremella fuciformis Berk., and rice noodles. These compounds are sensitive to degradation in 1% ammonia solution. UHPLC-Q-TOF/MS allowed for the detection of degradation products. The modified approach led to lower down the limits of detection of toxoflavin and fervenulin to 12 μg/kg and 24 μg/kg, respectively, with recovery of 70.1–108.7%. 152 The MS approach also employed natural phenomena of antigen–antibody interactions for the detection of toxins and related antigens. Salmonella typhi , Gram-negative enterobacteria, is responsible for typhoid fever and meningitis. The immunoreactive proteins of bacteria were used as targets to develop improved diagnostic tools with MS. An immunoaffinity-based proteomic approach was employed with IgG and IgM antibodies from typhoid patients. The approach aided in the identification of 28 immunoreactive proteins, out of which 14 were complementary to IgG, 4 for IgM, and the rest 10 for both, hence retained by respective charged columns. In context to antigenicity, 22 proteins have shown antigenicity and immunogenicity. 153 Such an approach is helpful for rapid identification, and its reproducibility and reliability can be employed for vaccine and drug development. Peptide mass fingerprinting technique (PMF) associated with MALDI TOF/MS or ESI/MS is a top-down MS protocol where proteins are directly ionized to create a fingerprint of individual proteins and applied for detection of various microbial strains. PMF of unknown organisms is compared to those existing in PMF databases or compared to the spectrum of biomarker proteins with the proteomic spectral database, using MALDI-TOF MS. Typically a mass range m / z of 2–20 kDa is used for species-level identification where ribosomal proteins representing 60–70% of microbial cells' dry weight and some housekeeping proteins are selected. 154 Thus, by comparing with extensive commercial databases, microbial contaminants can be traced to the genus, species, or strain level, and such an identification tool is conveniently adapted in diagnostic laboratories. 155 However, using biomarkers is not very common for identification since it requires prior insight into the genome sequences before creating the required databases for proteins' molecular masses. Staphylococcus aureus delta-toxin has been detected using whole-cell (WC) MALDI-TOF/MS and LC–MS to correlate the expression of delta-toxin with the status of the agr (accessory gene regulator) status. Mass spectra of pure toxin from wild type strains and mutants for agr-rnaIII gene were compared specifically at the position of the peak for delta-toxin. 156 Biosensors have taken up an important role in the accurate and fast detection of food contaminants even in very low concentrations 157 using biorecognition elements, such as antibodies, enzymes, nucleic acids, phages, etc., along with electrochemical, optic, or piezo-electric devices for the detection of food contaminants. MS-based biosensors are less prevalent as compared to optical or electrochemical ones 158 , 159 but have the potential to overcome the drawbacks of conventional models of biosensors. A multitoxin biosensor-MS was developed for the detection of multiple bacterial toxins simultaneously. Biomolecular interaction analysis-MS (BIA-MS) that used a two-step method, i.e., first bonding of toxin molecules to antibodies immobilized on a sensor chip using SPR (surface plasmon resonance) and then the bound toxin, was identified by MALDI-TOF/MS. The potential of the multiaffinity sensor chip was validated by the detection of endotoxin from Staphylococcus in mushroom and milk samples and it successfully detected multiple toxins at concentrations as low as 1 ng/mL. 160 4.3 Marine Biotoxins Marine biotoxins are natural compounds released in the marine environment by algae and phytoplankton during harmful algal blooms ( Figure 4 ). These compounds are highly toxic for consumers and not only are related to serious illness but also lead to the death of aquatic organisms and even humans. 161 Due to continuous release in the surrounding environment, these biotoxins accumulate in aquatic and marine organisms such as mollusks and fishes. Based on the chemical nature and solubility, these biotoxins are hydrophilic and lipophilic. Hydrophilic biotoxins are water-soluble and can cause amnesic shellfish poisoning, paralytic shellfish poisoning, and emerging pufferfish poisoning, while other groups of lipid-soluble biotoxins are responsible for diarrhetic shellfish poisoning and azaspiracid shellfish poisoning. There is another group of toxins with less available information that is categorized as emerging toxins and can cause unregulated ciguatera fish poisoning, cyclic imines, and neurotoxic shellfish poisoning. 162 − 165 Paralytic shellfish poisoning (PST) is a group of more than fifty-eight related compounds, produced by Alexandrium dinoflagellates of the Atlantic and Pacific coast and Mediterranean Sea. It has a tetrahydropurine skeleton among which saxitoxin (SXT) and gonyautoxin (GNT) are common. Structurally, STX has been categorized into four subgroups named carbamate, N-sulfo-carbamoyl, decarbamoyl, and hydroxylated saxitoxins. 166 The toxicity related to PST is reflected in mild as well as severe depending upon toxicity. The mild symptoms include numbness, tingling sensation around lips followed by expansion of the area, itching and prickly sensation in fingertips and toes, dizziness, headache, and nausea. Moderate and severe illness symptoms include incoherent speech, prickly sensation and stiffness in limbs, weakness, difficulty in respiration, and muscular paralysis. 161 Figure 4 General structures of various marine biotoxins. Amnesic shellfish poisoning is mainly caused by domoic acid (DA) and derivatives produced by marine diatoms of Pseudonitzschia . DA, cyclic tricarboxylic amino acid, can bind with glutamate receptors in the central nervous system due to structural analogy and result in excess stimulation, induced production of reactive oxygen species (ROS), and ultimately cell death. 167 Consumption of DA resulted in gastrointestinal ailments including abdominal cramps, diarrhea, nausea, and vomiting. Neurological symptoms may also include confusion, disorientation, paresthesia, lethargy, short-term memory loss, and in severe toxicity cases, it may also result in coma or death. 168 Diarrheic shellfish poisoning (DST) is a toxin produced by dinoflagellates of Dinophysis and Prorocentrum. It is a common type of contamination in shellfish industries due to overextended prohibitions on mussel harvesting activity. 169 The responsible toxins for DST are a group of polyether compounds recognized as okadaic acid and its derivatives (dinophysistoxin); pectenotoxin; yessotoxin and its derivatives; and azaspiracid. Okadaic acid and azaspiracid consumption resulted in abdominal pain, diarrhea, nausea, and vomiting. 161 , 170 Pectenotoxin and yessotoxin are not involved in human illness. 171 Neurotoxic Shellfish Poisoning (NST) is another type of algal toxin that causes neurological as well as gastrointestinal ailments. Brevetoxins are a kind of marine biotoxin produced by Karenia brevis (Florida red tide dinoflagellate). It is a poly(ether ladder) compound ( Figure 5 ). It causes mortality in massive fish and marine mammals. In humans, these toxins resulted in asthma-like symptoms if inhaled. 172 Neurological and toxicity symptoms of NST are paralysis, seizures, paresthesia, and coma, while gastrointestinal ailments are represented by nausea, diarrhea, vomiting, cramps, and bronchoconstriction, and extreme poisoning may also lead to death. 161 Ciguatera fish poisoning (CFT) is one of the most common foodborne illnesses caused by marine biotoxin of ciguatoxin. 173 , 174 Ciguatoxins are toxic and lipid-soluble compounds found in marine organisms. Gambiertoxins, the precursor toxins, are produced by benthic dinoflagellates of Gambierdiscus genus. These toxins are accumulated in large predatory fishes like Spanish mackerels, moray eels, barracuda, and snappers. 161 These compounds abnormally activate sodium ion channels and disrupt the cell membrane. 175 These compounds cause abdominal pain, nausea, diarrhea, vomiting, hypertension, and bradycardia along with neurological complications. 161 Figure 5 General structures of various marine biotoxin (Brevetoxin). 4.3.1 MS Analysis and Detection of Algal and Marine Biotoxins Algal and marine biotoxins are another class of heterogeneous toxins produced by algae and cyanobacteria, Dinoflagellates and diatoms produced during algal blooms in rivers, freshwater lakes, and marine aquatic systems. 176 Karunarathne et al., have found that 16,659 deaths have been reported in India between 1999 and 2018 due to poisoning. 177 In the case of sea food such as shellfish, exposure and contamination to multiple toxins are possible, hence an efficient system is able to detect diverse classes of toxins at the same time effectively. Blay et al., 178 developed a method for the detection of multiple lipophilic biotoxins including azaspiracids, dinophysistoxins, and pectenotoxins as well as negative toxins via reversed-phase LC–MS within 7 min and hydrophilic toxins such as okadaic acid, dinophysistoxin-1,2, and yessotoxin from shell fish by recording scans at 2 Hz in positive and negative scans alternatively and 1 Hz in positive mode, respectively. Hydrophilic toxins including gonyautoxins domoic acid and saxitoxin were detected with mass accuracy of less than 1 ppm error and resolving power of 100,000 for the analytes ( m / z 300–500). The limits of detection for lipophilic toxins were 0.041–0.10 μg/L ppm (positive ions), 1.6–5.1 μg/L (negative mode), and 3.4–14 μg/L for domoic acid and paralytic shellfish toxins. 178 The biggest advantage of the method is that the analytes were detected with real time samples without any interference. Aquatic water bodies have a higher possibility of having aquatic biotoxins; hence, monitoring of water in water bodies is mandatory. Estevez et al. developed a method to seawater monitoring for marine biotoxins by hydrophilic interaction liquid chromatography coupled with HRMS. The main analytes considered for the detection were saxitoxin, decarbamoyl-saxitoxin, neosaxitoxin, gonaytoxin-2,3, and tetrodotoxin due to their adverse effects on gastrointestinal and central nervous systems in humans if taken up via seafood. Samples were processed via ultrasound-assisted solid–liquid extraction with methanol to extract toxins, followed by solid phase extraction using silica cartridges. The selected toxins are polar in nature; hence, the extraction stage is crucial for analysis, and the developed method has recoveries of 15–47% in filtrate and 26–71% in particulate fraction. Simultaneously, limit of detection was also affected with source as LOD was 0.5–5 μg/L for filtrate and 3.1–62 μg/L for particulate fraction. 179 Kolrep et al. 180 conducted comparative metabolite profiling to track the metabolism of okadaic acid in the liver and the role of CYP3A4 and CYP3A5 in its detoxification. It was found that LC–MS/MS can identify the metabolites distinctly from humans and rats based on the difference in +16 (+O) and +14 (+O/–H 2 ) Da. It suggested some critical differences in the metabolism of okadaic acid in humans and rats. In continuation, it was also found that rats generated more metabolites from okadaic acid in comparison to humans in the presence of NADPH-dependent enzymes. 181 The establishment of metabolic patterns and fragments might be crucial for the identification of fingerprints for toxin identification. Table 5 elaborates on the detection of bacterial and marine biotoxins from different samples. Table 5 Detection of Bacterial and Marine Biotoxins by MS-Based Approach Name Sample Method Operating parameters Outcome Ref Enterotoxin Commercial LC–HRMS LC–MS Q-extractive mass spectrophotometer C18 reverse phase column; isocratic elution 93 signature peptides identified for enterotoxins ( 182 ) Botulinum Commercial Endopep MS Triple quadrupole mass spectrometer; Turbo Ion Spray interface; C18 column; gradient elution Toxin detection 0.1 MLD 50 and quantification 0.62 MLD 50 ( 183 ) Okadaic acid Raw and cooked food (Mussel, clam, flatfish) Tandem mass spectrometry C18 column with triple-quadruple mass spectrometer; ammonium format gradient elution; negative ionization mode LOD and accuracy 0.2–5.1 μg/k ( 184 ) Dinophysistoxin Raw and cooked food (Mussel, clam, flatfish) Tandem mass spectrometry C18 column with triple-quadruple mass spectrometer; ammonium format gradient elution; negative ionization mode LOD and accuracy 0.2–5.1 μg/k ( 184 ) Anatoxins a (ATX) and Homoanatoxin-a (HAT) Benthic-cyanobacterial-mat field samples LC–HRMS/MS Q Exactive HF Orbitrap MS; HESI-II electrospray ionization source; at 40 °C; resolution 60,000; collision energy 20 eV Toxins are in conjugated form 15% ATX and 38% HAT ( 185 ) Cyanotoxins Blue-green algae dietary supplement Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Electrospray ionization positive; source temperature 550 °C; ion spray voltage 5500 V; curtain gas 25 psi; collision gas 10 psi Quantification limits 60–300 μg kg –1 ( 186 ) Paralytic shellfish toxins Marine shellfish Hydrophilic interaction chromatography-tandem mass spectrometry Separation with HILIC-Z column; acetonitrile and ammonium formate-formic acid as the mobile phase; positive electrospray ionization; samples are cleaned with by ion-pair SPE using a porous graphitic carbon cartridge Limits of detection 1.7–13.7 μg kg –1 ; and quantitation 5.2–41.0 μg kg –1 ; recoveries 76.5–95.5% ( 187 ) Tetrodotoxin Marine shellfish Hydrophilic interaction chromatography-tandem mass spectrometry Separation with HILIC-Z column; acetonitrile and ammonium formate-formic acid as the mobile phase; positive electrospray ionization; samples are cleaned with by ion-pair SPE using a porous graphitic carbon cartridge Limits of detection 1.7–13.7 μg kg –1 ; and quantitation 5.2–41.0 μg kg –1 ; recoveries 76.5–95.5% 4.3.1 MS Analysis and Detection of Algal and Marine Biotoxins Algal and marine biotoxins are another class of heterogeneous toxins produced by algae and cyanobacteria, Dinoflagellates and diatoms produced during algal blooms in rivers, freshwater lakes, and marine aquatic systems. 176 Karunarathne et al., have found that 16,659 deaths have been reported in India between 1999 and 2018 due to poisoning. 177 In the case of sea food such as shellfish, exposure and contamination to multiple toxins are possible, hence an efficient system is able to detect diverse classes of toxins at the same time effectively. Blay et al., 178 developed a method for the detection of multiple lipophilic biotoxins including azaspiracids, dinophysistoxins, and pectenotoxins as well as negative toxins via reversed-phase LC–MS within 7 min and hydrophilic toxins such as okadaic acid, dinophysistoxin-1,2, and yessotoxin from shell fish by recording scans at 2 Hz in positive and negative scans alternatively and 1 Hz in positive mode, respectively. Hydrophilic toxins including gonyautoxins domoic acid and saxitoxin were detected with mass accuracy of less than 1 ppm error and resolving power of 100,000 for the analytes ( m / z 300–500). The limits of detection for lipophilic toxins were 0.041–0.10 μg/L ppm (positive ions), 1.6–5.1 μg/L (negative mode), and 3.4–14 μg/L for domoic acid and paralytic shellfish toxins. 178 The biggest advantage of the method is that the analytes were detected with real time samples without any interference. Aquatic water bodies have a higher possibility of having aquatic biotoxins; hence, monitoring of water in water bodies is mandatory. Estevez et al. developed a method to seawater monitoring for marine biotoxins by hydrophilic interaction liquid chromatography coupled with HRMS. The main analytes considered for the detection were saxitoxin, decarbamoyl-saxitoxin, neosaxitoxin, gonaytoxin-2,3, and tetrodotoxin due to their adverse effects on gastrointestinal and central nervous systems in humans if taken up via seafood. Samples were processed via ultrasound-assisted solid–liquid extraction with methanol to extract toxins, followed by solid phase extraction using silica cartridges. The selected toxins are polar in nature; hence, the extraction stage is crucial for analysis, and the developed method has recoveries of 15–47% in filtrate and 26–71% in particulate fraction. Simultaneously, limit of detection was also affected with source as LOD was 0.5–5 μg/L for filtrate and 3.1–62 μg/L for particulate fraction. 179 Kolrep et al. 180 conducted comparative metabolite profiling to track the metabolism of okadaic acid in the liver and the role of CYP3A4 and CYP3A5 in its detoxification. It was found that LC–MS/MS can identify the metabolites distinctly from humans and rats based on the difference in +16 (+O) and +14 (+O/–H 2 ) Da. It suggested some critical differences in the metabolism of okadaic acid in humans and rats. In continuation, it was also found that rats generated more metabolites from okadaic acid in comparison to humans in the presence of NADPH-dependent enzymes. 181 The establishment of metabolic patterns and fragments might be crucial for the identification of fingerprints for toxin identification. Table 5 elaborates on the detection of bacterial and marine biotoxins from different samples. Table 5 Detection of Bacterial and Marine Biotoxins by MS-Based Approach Name Sample Method Operating parameters Outcome Ref Enterotoxin Commercial LC–HRMS LC–MS Q-extractive mass spectrophotometer C18 reverse phase column; isocratic elution 93 signature peptides identified for enterotoxins ( 182 ) Botulinum Commercial Endopep MS Triple quadrupole mass spectrometer; Turbo Ion Spray interface; C18 column; gradient elution Toxin detection 0.1 MLD 50 and quantification 0.62 MLD 50 ( 183 ) Okadaic acid Raw and cooked food (Mussel, clam, flatfish) Tandem mass spectrometry C18 column with triple-quadruple mass spectrometer; ammonium format gradient elution; negative ionization mode LOD and accuracy 0.2–5.1 μg/k ( 184 ) Dinophysistoxin Raw and cooked food (Mussel, clam, flatfish) Tandem mass spectrometry C18 column with triple-quadruple mass spectrometer; ammonium format gradient elution; negative ionization mode LOD and accuracy 0.2–5.1 μg/k ( 184 ) Anatoxins a (ATX) and Homoanatoxin-a (HAT) Benthic-cyanobacterial-mat field samples LC–HRMS/MS Q Exactive HF Orbitrap MS; HESI-II electrospray ionization source; at 40 °C; resolution 60,000; collision energy 20 eV Toxins are in conjugated form 15% ATX and 38% HAT ( 185 ) Cyanotoxins Blue-green algae dietary supplement Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Electrospray ionization positive; source temperature 550 °C; ion spray voltage 5500 V; curtain gas 25 psi; collision gas 10 psi Quantification limits 60–300 μg kg –1 ( 186 ) Paralytic shellfish toxins Marine shellfish Hydrophilic interaction chromatography-tandem mass spectrometry Separation with HILIC-Z column; acetonitrile and ammonium formate-formic acid as the mobile phase; positive electrospray ionization; samples are cleaned with by ion-pair SPE using a porous graphitic carbon cartridge Limits of detection 1.7–13.7 μg kg –1 ; and quantitation 5.2–41.0 μg kg –1 ; recoveries 76.5–95.5% ( 187 ) Tetrodotoxin Marine shellfish Hydrophilic interaction chromatography-tandem mass spectrometry Separation with HILIC-Z column; acetonitrile and ammonium formate-formic acid as the mobile phase; positive electrospray ionization; samples are cleaned with by ion-pair SPE using a porous graphitic carbon cartridge Limits of detection 1.7–13.7 μg kg –1 ; and quantitation 5.2–41.0 μg kg –1 ; recoveries 76.5–95.5% 4.4 Phytotoxins Phytotoxins are plant-derived compounds, including alkaloids and glycoalkaloids, that are naturally produced within plants but prove harmful if they remain in food products ( Figure 6 ). These are secondary metabolites in plants and include cyanogenic glycosides, glucosinolates, glycoalkaloids, pyrrolizidine alkaloids, and lectins. 188 Based on the site, these toxins can be classified into endotoxins and exotoxins. Endotoxins may be normal metabolites that are present in cells but become harmful if consumed in higher concentrations, and these compounds are also refereed as antinutritional factors, while exotoxins are toxic metabolites that are released from cells. Based on chemical nature, these are dehydropyrrolizidine, alkaloids, ptaquiloside, corynetoxins, and phomopsins. 189 Cyanogenic glycosides (CGLs) are present in almonds, cassava, bamboo roots, sorghum, and stone fruits. These toxins are generated from proteinogenic amino acids like leucine, isoleucine, phenylalanine, tyrosine, and valine as well as nonproteinogenic amino acids like cyclopentenylglycin. CGLs are potentially toxic for humans and result in acute cyanide intoxication, high respiration rate, lower blood pressure, headache, dizziness, stomach pains, diarrhea, vomiting, and mental confusion. 188 , 189 Furocoumarins are found in many plants including carrots, celery roots, citrus fruits, parsley, and citrus plants. These compounds are responsible for gastrointestinal ailments and phototoxicity, skin reactions under UV light. 34 , 190 Lectins are reported from beans like kidney beans and can result in stomachache, diarrhea, and vomiting. 189 Figure 6 General structures of various Phytotoxins. The general scaffold for Cyanogenic glycosides, Furocoumarin and Dehydropyrrolizidine, has been depicted with providing a few examples of various compounds belonging to the class of Furocoumarin. Phytotoxins are sometimes a part of the natural defense of plants, like Pyrrolizidine alkaloids (PAls) that are produced in Asteraceae , Boraginaceae , and Fabaceae families to defend plants against herbivores as well as insects. These toxins tend to have a common 1-hydroxymethyl pyrrolizidine core that is esterified with aliphatic acids. Besides edibles from these plants, honey is one of the common products contaminated with PAls, and hence, the extract or infusion can be used as an analyte for the detection of PAls. Ten plant samples, Anchusa officinalis , Borago officinalis , Echium italicum , Eupatorium cannabinum , Heliotropiumeuropaeum , Lithospermum officinale , Petasites hybridus , Senecio vulgaris , Symphytum officinale, and Tussilago farfara from Orto botanicodellaScuola Medica Salernitana, Salerno, Italy, were collected, and aqueous extract was prepared with salting-out assisted liquid–liquid extraction. The aqueous extracts were analyzed with UPLC–MS/MS. The analysis was able to identify 88 PAs from 282 samples with an identification limit of 0.6–30 μg kg –1 and a false negative rate <1.3% (at the concentration range of 4 μg L –1 ). 191 For simultaneous detection of phytotoxins and microbial toxins, HRMS was employed, which applied to over 156 compounds inclusive of about 90 plant toxins (e.g., various alkaloids and aristolochic acids), about fifty-four mycotoxins, and 12 phytoestrogens (e.g., lignan, isoflavones, coumestans, etc.) in plant-protein samples, like cereals. MS library, created on fragmentation pattern obtained with both negative and positive ionization modes for each toxin, using ten different collision energies was used for analysis. A typical workflow was followed with generic QuEChERS-like sample preparation, followed by UPLC using suitable mobile phases that allowed the resolution of over 50 toxic alkaloids. The method performance was evaluated for its sensitivity at levels ranging from 1–100 μg/kg, and reproducibility. The quantitation obtained against the standard addition approach could meet SANTE/12682/2019 criteria for 132 toxins out of the tested 156 toxin samples. 192 The plant toxins ricin and RCA120 were detected, differentiated, and quantified by Kalb et al., 193 via MS-based methods for the EQuATox proficiency test, in ∼9 samples. They successfully identified the samples spiked with ricin or RCA120; samples spiked with a 0.414 ng/mL concentration could not be detected. Liang et al. 194 employed reversed phase LC–HRMS to detect five major phytotoxin groups including alkaloids, aromatic polyketides, flavonoids and steroids, and terpenoids at alkaline pH (>9). The developed method not only allowed the detection of 30 phytotoxins but also had forty-times higher detection sensitivity in comparison to older methods. Table 6 discusses some of the examples for phytotoxins detection using the MS-based approach. Table 6 Phytotoxins Detection from Food Samples via MS-Based Approach Name Sample Method Operating parameters Outcome Ref Toxoflavin and Fervenulin Food samples and Tremella fuciformis Berk UPLC–MS/MS Separation column: ZORBAX SB-C18 column; oven temp: 35 °C; mobile phase: 0.1% formic acid+ methanol; flow rate 0.4 mL min –1 Detection limits (μg/kg) Toxoflavin 12 ( 152 ) Fervenulin 24 Toxin level (mg/kg) Toxoflavin: 7.5; fervenulin: 3.2 Ricin Soft drinks and serum Surface-assisted laser desorption/ionization mass spectrometry Pulsed Smartbeam II 2 kHz laser; wavelength 355 nm (∼3.49 eV); frequency 1000 Hz; delayed extraction time 350 ns for proteins Limit of detection 0.5 pmol/μL ( 195 ) Pyrrolizidine Alkaloids LC–MS/MS Tea UPHPLC with Quadrupole mass spectrometer; C18 Hypersil Gold column fitted; gradient elution Total PA levels 13.4 to 286,682.2 μg/kg d.m ( 196 ) Ptaquiloside LC–MS/MS Bracken fern LC–MS/MS; C18 column; gradient elution; column temperature 35 °C; electrospray positive ionization Limits of detection 0.03 and quantification 0.09 μg/kg ( 197 ) Furanocoumarin UPLC–MS/MS Citrus sp. Nexcol C18 column; column temp 40 °C; gradient elution; positive electrospray ionization Compound recovery 94.07–114.53% ( 198 ) Amygdalin LC–MS/MS Kernels and Almonds LC–MS/MS equipped with 6500 quadruple linear ion trap (QTRAP) mass spectrometer and electrospray ionization Recovery 90–107%; limit of detection 0. Ng/g and limit of quantitation 8 and 2.5 ng/g ( 199 ) Pyrrolizidine alkaloids quadrupole orbitrap MS Honey Polar C18 column; temp 40 °C; mobile phase flow rate of 400 μL min –1 Limit of quantification 0.1–0.3 μg kg –1 ( 200 ) MS detection with positive ionization mode; scan range 250–500 m / z and 70 k (fwhm) Pyrrolizidine alkaloids quadrupole orbitrap MS Black and green tea Polar C18 column; temp 40 °C; mobile phase flow rate of 400 μL min –1 Limit of quantification 1–11.7 μg kg –1 ( 200 ) MS detection with positive ionization mode; scan range 250–500 m / z and 70 k (fwhm) Pyrrolizidine alkaloids quadrupole orbitrap MS Herbal infusion Polar C18 column; temp 40 °C; mobile phase flow rate of 400 μL min –1 Limit of quantification 0.9–2.1 μg kg –1 ( 200 ) MS detection with positive ionization mode; scan range 250–500 m / z and 70 k (fwhm) 4.5 Emerging Toxins In addition to conventional toxins already known and summarized above, there is a group of toxic chemicals that are continuously evolving, mainly due to rising pollution. In the last eight years, the list of emerging chemicals has increased day by day. Synthetic chemical toxins include microplastics, organophosphorus and polybrominated flame retardants, perfluoroalkyl compounds, food process and packaging, waste substances, and nanomaterials. 15 Besides, heavy metals, antibiotics and drug traces and metabolic intermediates, and agricultural chemicals 201 have shown bioaccumulation and have serious toxic effects if consumed, even in low concentrations. Health ailments include endocrine disruption, suppression and overexpression of the immune system, inflammation, abnormal metabolic changes, skin diseases, carcinogenesis, etc., and the toxicity relies on interaction with the cellular system and receptors. 15 , 201 , 202 With the increase in pollution and intrusion of pollutants in the food web, the toxic chemicals traced in food and edibles are increasing. Some of those chemicals exhibited bioaccumulation and become silent killers, but some are potentially lethal. These emerging pollutants include pesticides, herbicides, healthcare, cosmetic chemicals, etc. Fipronil is a wide-spectrum phenylpyrazole insecticide used to control beetles, ants, cockroaches, etc. but its entry into the food chain is alarming due to its carcinogenic nature, essentiating its prohibition by the US Environmental Protection Agency (EPA). Suitable detection methods are thereby essential to identify and quantify these contaminants before they enter the food chain. The most reliable detection method includes LC–MS/MS and GC–MS, having specific sample preparation before the analyses ( Table 7 ). 203 One such preparatory method involved a modified QuEChERS sample preparation before using a triple quadrupole MS instrument coupled to ESI for detecting fipronil and its major metabolite fipronil sulfone, at concentrations of 5 μg/kg. The use of nontargeted approaches, such as SWATH-MS (sequential window acquisition of all theoretical mass spectra), enables the sequential analysis of fipronil and other such contaminants, e.g., pesticides and polyaromatic hydrocarbons. Glyphosate (insecticide) was detected in an underivatized form by innovating new extraction methods coupled with instrumentation. The QuPPe (Quick Pesticide Preparation) method was used for sample preparation 204 followed by detection via sensitive MS instruments to achieve accurate quantitative results. LC–MS/MS was used in combination with the DMS (differential mobility separation) technique to terminate analytical interferences leading to improved signal by decreasing noise and, consequently, increasing accuracy and confidence in data. Using this method, LC–DMS–MS/MS was used for identification and quantification of pesticide contaminants in food samples. Triclosan is a well-known and common biocide agent against bacteria as well as fungi, 205 while bisphenol analogues are used in packaging and lining. 206 Morgan et al. 206 employed GC–MS to monitor the levels of triclosan and five bisphenol analogues (B, F, P, S, and Z) in 776 adult solid food samples. More than 80% of the samples were contaminated with at least one target phenol. Based on the frequencies, 59% of samples were contaminated with triclosan followed by 32% bisphenol S, and 28% bisphenol Z. The maximum concentration for triclosan was 394 ng/g. Table 7 Detection of Emerging Toxins from Food and Water Samples by MS Name Sample Method Operating parameters Outcome Ref Cypermethrin Baby food liquid chromatography coupled to quadrupole Orbitrap mass spectrometry Gradient elution; negative ionization mode; capillary temperature 300 °C and heater 305 °C Detection concentration in baby food 10.3 μg kg –1 ( 208 ) Parabens Surface water UHPLC–MS/MS LC-18 column; column temp 40 °C; gradient elution; capillary voltage −3.0 kV Limit of detection 0.04 ng L –1 and Limit of quantification 0.82 ng L –1 ( 209 ) Bisphenol Water GS–MS/MS Temperature transfer line 250 °C; ion source 230 °C and quadrupole 150 °C. solvent delay 4.5 min; electron ionization (EI) mode (70 eV) Recovery 81.8% −96.1%; limit of detection was 0.2 ng L –1 ( 210 ) Parabens water GS–MS/MS Temperature transfer line 250 °C; ion source 230 °C and quadrupole 150 °C. solvent delay 4.5 min; electron ionization (EI) mode (70 eV) Recovery 81.8% −96.1%; limit of detection was 0.2 ng L –1 ( 210 ) Triclosan water GS–MS/MS Temperature transfer line 250 °C; ion source 230 °C and quadrupole 150 °C. solvent delay 4.5 min; electron ionization (EI) mode (70 eV) Recovery 81.8% −96.1%; limit of detection was 0.2 ng L –1 ( 210 ) Neonicotinoids vegetables QuEChERS-Portable MS PDESI as ion source; ultrapure helium (≥99.999%) as carrier gas; inlet temperature 200 °C; molecular pump speed was 1375 Hz Limit of detection 2.0 ng g –1 recovery 82.2% −109.7% ( 211 ) Carbamates Vegetables QuEChERS-Portable MS PDESI as ion source; ultrapure helium (≥99.999%) as carrier gas; inlet temperature 200 °C; molecular pump speed was 1375 Hz Limit of detection 2.0 ng g –1 recovery 82.2% −109.7% ( 211 ) Phenyl Pyrazole Vegetables QuEChERS-Portable MS PDESI as ion source; ultrapure helium (≥99.999%) as carrier gas; inlet temperature 200 °C; molecular pump speed was 1375 Hz Limit of detection 2.0 ng g –1 recovery 82.2% −109.7% ( 211 ) Pesticides Fruits and vegetables QuEChERS-LC–MS Sciex QTRAP 5500 triple quadrupole MS; positive electrospray ionization; ion source temperature 550 °C 24 pesticides detected distinctly ( 212 ) Pesticides Milk LC-LTQ/Orbitrap Mass Spectrometry Separation with reverse phase C18 column; positive ionization mode; spray voltage 4 kV; auxiliary gas flow rate 10 arbitrary units; tube lens 90 V, capillary temperature 320 °C Limit of detection 0.2–8.1 μg kg –1 and quantification 0.61–24.8 μg kg –1 ( 213 ) Not only emerging toxins but also the availability of efficient and portable systems have become necessary prerequisites. Some of the recent advancements have shown the availability of portable MS systems for detection and monitoring. Maragos 131 has evaluated the potential of portable MS (APCI-MS) for the detection of T-2 toxin mycotoxin in contaminated cereal grains, wheat, and maize by APCI-MS. The sample was extracted with acetonitrile+water (84:16, v/v) followed by drying and reconstitution in ammonium formate. The MS system contains a linear ion trap mass analyzer to avoid the need of an external supply of gas or air. The device and developed method were able to detect T-2 toxin above 0.2 mg/kg from soft white and hard red wheat, and yellow dent maize. The method was more efficient and hence able to lower-down the detection limit from >0.9 mg/kg. In a similar line, FB and its isoforms were detected in maize with a portable mass spectrometer. For the detection, samples were extracted with aqueous methanol followed by cleaning up in the immunoaffinity column. Ultimately, cleaned samples were successfully analyzed with the portable MS with detection limits of 0.15 (B1), 0.19 (B2/B3), and 0.28 (total FB) mg/kg maize. The method has quantification limits of 0.33, 0.59, and 0.74 mg/kg and recoveries of 93.6% to 108.6%. Wichert et al. 207 have also reported such kind of advancements to detect proteinaceous toxins (912.5–66.5 kDa) from plants as well as microorganisms origin using paper spray-MS (PS-MS) with wipe samples of bench, glass, leaves, flooring, etc., and validated with biological toxin simulant for Staphylococcal enterotoxin B. Carbon sputtered porous polyethylene dominated conventional chromatography paper, carbon nanotube-coated paper, and polyethylene for paper spray. The method was able to distinguish the protein toxin simulant efficiently with a good signal-to-noise ratio. 5 Detection of Food Fraud and Food Adulteration Food adulteration and fraud have become a common practice nowadays to gain more profit. To avoid detection by current available analytical methods, new adulteration and fraud practices are becoming advanced and sophisticated making detection one of the biggest challenges for society. 214 The tracing of fraud in food products via chemical analysis has become more complex especially due to the emergence of new and unknown adulterants. 74 MS has become an indistinguishable part of testing and food authentication analysis due to its potential to trace chemical compounds based on their chemical fingerprints or chemical profiles. 215 Adulterants are used as ingredients and additives in food products for economic gain for the seller at the cost of the health of consumers. The use of bulking agents such as sulfated polysaccharides is very common in minced meat and is a fraudulent practice that is very difficult to detect. Kosek et al. 216 have evaluated rapid evaporative MS (REIMS) to detect adulterants in sausages and burgers prepared from chopped pork and chicken meat. The technique was able to detect the adulterants efficiently with 2.5% as the threshold concentration for protein additives (carrageenan) can be detected at 1% concentration. The major advantage of the system is the quick detection of adulterants in samples, which aids in preventing any major health issue from the consumption of adulterated food products. The problem becomes more serious when consumers are infants or minors. Milk is one of the common products that is supposed to provide optimum nutrients including lipids and proteins. Piras et al. 217 employed atmospheric pressure matrix-assisted laser desorption/ionization with a Q-TOF mass analyzer, which generated charged proteinaceous as well as lipids/metabolites ions to find the possible fraud with milk. The common adulteration in milk is the intermixing of milk from different sources, and the current methods were able to identify milk from camel, cow, goat, and sheep with 100% accuracy. The goat milk was analyzed for the presence of cow milk as an adulterant, and it was detected even at a lower concentration of 5% with sensitivity and specificity of 92.5% and 94.5%, respectively. The major outcome of the work is its time consumption for sample analysis, i.e., 10 s per unadulterated sample to prepare profile. The method creates differences between protein and lipid molecules in terms of the number of charged moieties as lipids are single charged, while protein moieties have multiple charges. Integration of artificial intelligence and neural network models has further improved the demand for efficiency of MS systems. Nichani et al. 218 have developed a method for the differential identification of spelt and wheat. Nontargeted LC–MS/MS along with convolutional neural network (CNN) models was developed. The employed neural network was able to learn patterns by itself and discriminated between spelt and wheat efficiently. For external validation of the model, artificial mixed spectra of spelt bread and flour, 11 untypical spelt, and six old wheat cultivars (which were not part of model training) were analyzed. The model was able to identify the nonconventional cultivars of wheat and spelt with a D value of 0.57. 218 Not only food products but also active pharma formulations and ingredients are under the threat of adulteration and fraud. One such example is where MS is used to develop a model to differentiate between wild and cultivated Cordyceps sinensis harvests. Elemental analysis coupled with stable isotope ratio MS (EA-IRMS) and GasBench II coupled to isotope ratio MS (GB-IRMS) was used with orthogonal partial least-squares discriminant analysis (OPLS-DA) to identify the significant and unique markers of stable isotope ratios in both samples. Analysis identified three stable isotope markers, i.e., δ2H, δ18O, and δ15N, and their concentrations can be used to identify fresh C. sinensis samples as well as differentiating between wild and cultivated C. sinensis . δ2H values reduced sequentially in fresh samples based on respective origins. δ18O and δ15N have shown differential patterns as lower δ18O and higher δ15N represented cultivated samples, while higher δ18O and lower δ15N represented wild samples. The analyzed cultivated and wild samples have δ18O and δ15N of 18.99%, 4.80%, and 25.72%, 2.29%, respectively. 219 6 Current Challenges and Future Prospects The methods currently in practice for toxin detection are quite advanced, comprising a variety of direct and indirect analytical techniques. 220 However, there are still a few challenges that make laboratory detection difficult, and toxins may sometimes go undetected. 221 The following are the significant challenges for toxin analysis that are commonly encountered. 6.1 Representative Sample Microbial toxins are rarely evenly distributed in foodstuff and edibles. 222 As in the case of mycotoxins, in some regions of the victuals, called "mycotoxin pockets", concentrations may be extremely high, whereas the rest of the material may be free from contamination. The materials are distributed more heterogeneously for products with larger particle sizes, such as nuts and figs. This necessitates representative sampling that accounts for the random distribution of the "hotspots" to provide an accurate view of the degree of contamination in the specimen. This can be performed by taking a large number of small, incremental samples from various locations distributed throughout the lot. 223 The selection of incremental samples from the bulk is crucial to give all morsel particles a chance of being selected, thus reducing the statistical bias. Besides the number of samples, sample type and its processing are equally important as in some cases surface swab is sufficient, while others need extraction followed by processing like digestion of target sample as reported in the case of protein-based toxins. Kalb et al. employed endopep-MS to identify botulinum A, B, E, and F from food samples by optimizing the fingerprint peptides generated. 149 Hence, detection technology must be optimized to compete with emerging challenges. 6.2 Sample Preparation Sample preparation is a highly complex process, with various pitfalls. Every step encountered introduces a level of variability that aggregates and contributes to total variability within single analytical data. It has been generally observed that nearly 1/3 of the variability is attributed to sample preparation. On the other hand, a much smaller amount of variation is contributed by the analytical method being employed. Even with the best analytical equipment, sample preparation is critical. 224 , 225 By adhering to the key factors of sample preparation (size reduction, sampling size, and uniformity), the root–mean–square value can be kept significantly within 5–10%, increasing the prediction result's accuracy. 6.3 Low Concentration Even at low concentration levels, toxins can be highly toxic. 226 Different classes of toxins have different levels of toxic effects on the target. Glycoalkaloids (potato) and isoflavones (clover) have shown low toxicity, linamarin (cassava) and coniin (hemlock) are somewhat toxic, while ricin (castor beans) and cyanotoxin and saxitoxin (blue–green algae) are extremely toxic. 227 Hence, the tolerable concentration range is also varied in the same proportion. The Food Safety and Standards Authority of India (FSSAI) has set limit values of 15 μg/kg in cereal products, pulses, and nuts, and 30 μg/kg in spices, whereas, for milk, the allowable range is considerably low (0.5 μg/kg). 228 The values are more stringent for the US FDA and European Union, nearly 1/3 of the FSSAI-approved figures. The analysis method needs to be extremely precise and sensitive to detect such low concentrations. The low levels of toxin concentration, often not detected, are hugely responsible for reduced production efficiency and increased susceptibility to various diseases. 229 6.4 Complex Matrix Generally, the toxin matrices found in food are fairly complex and pose a major challenge for laboratories. For example, heterogeneous matrices such as spices contain numerous interfering substances, which makes it extremely difficult to detect the toxins precisely. Also, food may not be necessarily safe, even if well-known toxins are not detected during analysis. These compounds may still be present in conjugated form in masked or bound form. 230 These modified toxins are derived from plants by conjugation and have their chemical structures altered, making the analysis more complex. Also, even though a large number of different toxins including mycotoxins, bacterial toxins, phytotoxins, etc. exist, only a few are characterized and regulated by law. 2 In reality, several toxins may be present simultaneously, and it may be challenging to find/pinpoint specific analytes in the food particles. 6.5 Portability of Instrument The normal trend in sample analysis is like the collection of samples from the site and transfer to the lab for further analysis. However, such a protocol seems obsolete when we need to characterize the sample rapidly. In such cases, a portable and handy instrument with easily transferable and ready-to-use techniques is required. Some of the recent research has shown the pay for portable MS-based detection systems for the food analysis and characterization of toxins like PS-MS. 207 However, more advancement needed with the miniaturization of instruments with rapid detection and high accuracy is required. 6.6 Cost A variety of factors contribute to the cost of toxin analysis. They include facilities, sophisticated analytical instruments, reagents, and logistics. Additionally, the number of tests needed for representative sampling to negate the misreporting of toxins in bulk material also contributes to the testing cost. Although effective sampling is one of the most critical factors in mycotoxin analysis, it is the costliest, and surprisingly, innovations in this area have not taken place rapidly. 231 Reducing the time and cost requirements through representative sampling while increasing accuracy is the need of the hour. A balance needs to be achieved between the cost per sample and the number of runs essential to generate the most confident results that will ensure lower downtime with precise results. 232 6.7 Multiple Toxins Contamination Naturally, all the food materials are prone to be contaminated with multiple toxins due to the presence of diverse microbial contaminants simultaneously. The method should be effective and efficient to detect all of the toxins together even in minute quantity. A MS-based approach made it possible to allow the detection of diverse range of toxins. Lattanzio et al. 233 have reported the detection of multiple aflatoxins including B1, B2, G1, G2, ochratoxin A, fumonisins B1 and B2, deoxynivalenol, zearalenone, T-2, and HT-2 toxins simultaneously from maize via LC–MS. In a similar line, Cheng et al. 234 have also reported the detection of 15 toxic alkaloids from vegetables and meat samples using double layer pipet tip magnetic dispersive solid phase extraction method that used polyamidoamine-functionalized magnetic carbon nanotubes. Extracted samples were characterized with ultrafast liquid chromatography-tandem quadrupole mass spectrometry (UFLC–MS/MS) coupled with DPT-MSPE method. The system has shown high recovery efficiency with toxin recovery of up to 125% for meat as well as vegetable dishes. In food samples, fungal contamination is a common phenomenon; hence, it is the prime target for most of the work. Wang et al. 235 and Nualkaw et al. 236 have employed UPLC–MS for the detection of mycotoxins from animal feed like dairy product, poultry, and animal feed. Lee et al. 184 reported the presence of okadaic acid, dinophysistoxin-1, dinophysistoxin-2, and dinophysistoxin-3 in raw as well as cooked mussels using LC–tandem mass spectrometry. The method has shown detection and quantification limit of 0.2–5.1 μg/kg with accuracy and precision of 80.5–109.8% and 0.9–20.1%, respectively. Albero et al. 237 have also identified mycotoxins in aquaculture feed by LC–MS/MS. The method employed ultrasound-assisted extraction followed by LC–MS/MS, which identified 15 mycotoxins together that also included enniatins (EENB and ENNB1), beauvericin, and fumonisin B2. In some cases, the toxins are present in modified or bounded form (masked form), which hinder its detection by conventional methods. As mentioned by Berthiller et al., 238 mycotoxins have high probability of masking due to food processing which changes its structural as well as behavior and obstruct the appearance of common characteristics of respective toxins. Masked mycotoxins have been detected in extractable conjugated and nonextractable varieties (usually to unavailability of toxins in extracted samples, bound mycotoxins are not accessible for analysis and need chemical or enzymatic treatment prior detection). 238 Fiby et al. 239 reported the presence of Fusarium mycotoxin like deoxynivalenol in native as well as masked form (DON-3-glucoside "D3 G ", 3-acetyl-DON "3ADON", or 15-acetyl-DON "15ADON") in cereals. The presence was detected with a stable-isotope dilution liquid chromatography–tandem mass spectrometry-based approach that relies on labeling of toxins enzymatic byproducts with 13C. The method efficiently detected D3G (76–98%), DON (86–103%), 15ADON (68–100%), and 3ADON (63–96%). 239 Zhang et al. 240 employed ultrahigh-performance liquid chromatography–HRMS to detect 82 mycotoxins and categorize into 8 classes by Python program developed with "Fragmentation pattern screener (FPScreener)" and nontarget screening rules. Pascari et al. 241 combined QuEChERS with liquid chromatography–triple quadrupole mass spectrometry for the detection of ZEN from oat flour. For the identification, oat and wheat flours were treated with amylolytic enzymes (α-amylase and amyloglucosidase), similar to the one used in the cereal-based baby food production process that reduced the β-zearalenol (β-ZEL) and β-ZEL-14-sulfate by 40% within 90 min and allowed the specific detection of ZEN-sulfate derivates from cereals. 6.1 Representative Sample Microbial toxins are rarely evenly distributed in foodstuff and edibles. 222 As in the case of mycotoxins, in some regions of the victuals, called "mycotoxin pockets", concentrations may be extremely high, whereas the rest of the material may be free from contamination. The materials are distributed more heterogeneously for products with larger particle sizes, such as nuts and figs. This necessitates representative sampling that accounts for the random distribution of the "hotspots" to provide an accurate view of the degree of contamination in the specimen. This can be performed by taking a large number of small, incremental samples from various locations distributed throughout the lot. 223 The selection of incremental samples from the bulk is crucial to give all morsel particles a chance of being selected, thus reducing the statistical bias. Besides the number of samples, sample type and its processing are equally important as in some cases surface swab is sufficient, while others need extraction followed by processing like digestion of target sample as reported in the case of protein-based toxins. Kalb et al. employed endopep-MS to identify botulinum A, B, E, and F from food samples by optimizing the fingerprint peptides generated. 149 Hence, detection technology must be optimized to compete with emerging challenges. 6.2 Sample Preparation Sample preparation is a highly complex process, with various pitfalls. Every step encountered introduces a level of variability that aggregates and contributes to total variability within single analytical data. It has been generally observed that nearly 1/3 of the variability is attributed to sample preparation. On the other hand, a much smaller amount of variation is contributed by the analytical method being employed. Even with the best analytical equipment, sample preparation is critical. 224 , 225 By adhering to the key factors of sample preparation (size reduction, sampling size, and uniformity), the root–mean–square value can be kept significantly within 5–10%, increasing the prediction result's accuracy. 6.3 Low Concentration Even at low concentration levels, toxins can be highly toxic. 226 Different classes of toxins have different levels of toxic effects on the target. Glycoalkaloids (potato) and isoflavones (clover) have shown low toxicity, linamarin (cassava) and coniin (hemlock) are somewhat toxic, while ricin (castor beans) and cyanotoxin and saxitoxin (blue–green algae) are extremely toxic. 227 Hence, the tolerable concentration range is also varied in the same proportion. The Food Safety and Standards Authority of India (FSSAI) has set limit values of 15 μg/kg in cereal products, pulses, and nuts, and 30 μg/kg in spices, whereas, for milk, the allowable range is considerably low (0.5 μg/kg). 228 The values are more stringent for the US FDA and European Union, nearly 1/3 of the FSSAI-approved figures. The analysis method needs to be extremely precise and sensitive to detect such low concentrations. The low levels of toxin concentration, often not detected, are hugely responsible for reduced production efficiency and increased susceptibility to various diseases. 229 6.4 Complex Matrix Generally, the toxin matrices found in food are fairly complex and pose a major challenge for laboratories. For example, heterogeneous matrices such as spices contain numerous interfering substances, which makes it extremely difficult to detect the toxins precisely. Also, food may not be necessarily safe, even if well-known toxins are not detected during analysis. These compounds may still be present in conjugated form in masked or bound form. 230 These modified toxins are derived from plants by conjugation and have their chemical structures altered, making the analysis more complex. Also, even though a large number of different toxins including mycotoxins, bacterial toxins, phytotoxins, etc. exist, only a few are characterized and regulated by law. 2 In reality, several toxins may be present simultaneously, and it may be challenging to find/pinpoint specific analytes in the food particles. 6.5 Portability of Instrument The normal trend in sample analysis is like the collection of samples from the site and transfer to the lab for further analysis. However, such a protocol seems obsolete when we need to characterize the sample rapidly. In such cases, a portable and handy instrument with easily transferable and ready-to-use techniques is required. Some of the recent research has shown the pay for portable MS-based detection systems for the food analysis and characterization of toxins like PS-MS. 207 However, more advancement needed with the miniaturization of instruments with rapid detection and high accuracy is required. 6.6 Cost A variety of factors contribute to the cost of toxin analysis. They include facilities, sophisticated analytical instruments, reagents, and logistics. Additionally, the number of tests needed for representative sampling to negate the misreporting of toxins in bulk material also contributes to the testing cost. Although effective sampling is one of the most critical factors in mycotoxin analysis, it is the costliest, and surprisingly, innovations in this area have not taken place rapidly. 231 Reducing the time and cost requirements through representative sampling while increasing accuracy is the need of the hour. A balance needs to be achieved between the cost per sample and the number of runs essential to generate the most confident results that will ensure lower downtime with precise results. 232 6.7 Multiple Toxins Contamination Naturally, all the food materials are prone to be contaminated with multiple toxins due to the presence of diverse microbial contaminants simultaneously. The method should be effective and efficient to detect all of the toxins together even in minute quantity. A MS-based approach made it possible to allow the detection of diverse range of toxins. Lattanzio et al. 233 have reported the detection of multiple aflatoxins including B1, B2, G1, G2, ochratoxin A, fumonisins B1 and B2, deoxynivalenol, zearalenone, T-2, and HT-2 toxins simultaneously from maize via LC–MS. In a similar line, Cheng et al. 234 have also reported the detection of 15 toxic alkaloids from vegetables and meat samples using double layer pipet tip magnetic dispersive solid phase extraction method that used polyamidoamine-functionalized magnetic carbon nanotubes. Extracted samples were characterized with ultrafast liquid chromatography-tandem quadrupole mass spectrometry (UFLC–MS/MS) coupled with DPT-MSPE method. The system has shown high recovery efficiency with toxin recovery of up to 125% for meat as well as vegetable dishes. In food samples, fungal contamination is a common phenomenon; hence, it is the prime target for most of the work. Wang et al. 235 and Nualkaw et al. 236 have employed UPLC–MS for the detection of mycotoxins from animal feed like dairy product, poultry, and animal feed. Lee et al. 184 reported the presence of okadaic acid, dinophysistoxin-1, dinophysistoxin-2, and dinophysistoxin-3 in raw as well as cooked mussels using LC–tandem mass spectrometry. The method has shown detection and quantification limit of 0.2–5.1 μg/kg with accuracy and precision of 80.5–109.8% and 0.9–20.1%, respectively. Albero et al. 237 have also identified mycotoxins in aquaculture feed by LC–MS/MS. The method employed ultrasound-assisted extraction followed by LC–MS/MS, which identified 15 mycotoxins together that also included enniatins (EENB and ENNB1), beauvericin, and fumonisin B2. In some cases, the toxins are present in modified or bounded form (masked form), which hinder its detection by conventional methods. As mentioned by Berthiller et al., 238 mycotoxins have high probability of masking due to food processing which changes its structural as well as behavior and obstruct the appearance of common characteristics of respective toxins. Masked mycotoxins have been detected in extractable conjugated and nonextractable varieties (usually to unavailability of toxins in extracted samples, bound mycotoxins are not accessible for analysis and need chemical or enzymatic treatment prior detection). 238 Fiby et al. 239 reported the presence of Fusarium mycotoxin like deoxynivalenol in native as well as masked form (DON-3-glucoside "D3 G ", 3-acetyl-DON "3ADON", or 15-acetyl-DON "15ADON") in cereals. The presence was detected with a stable-isotope dilution liquid chromatography–tandem mass spectrometry-based approach that relies on labeling of toxins enzymatic byproducts with 13C. The method efficiently detected D3G (76–98%), DON (86–103%), 15ADON (68–100%), and 3ADON (63–96%). 239 Zhang et al. 240 employed ultrahigh-performance liquid chromatography–HRMS to detect 82 mycotoxins and categorize into 8 classes by Python program developed with "Fragmentation pattern screener (FPScreener)" and nontarget screening rules. Pascari et al. 241 combined QuEChERS with liquid chromatography–triple quadrupole mass spectrometry for the detection of ZEN from oat flour. For the identification, oat and wheat flours were treated with amylolytic enzymes (α-amylase and amyloglucosidase), similar to the one used in the cereal-based baby food production process that reduced the β-zearalenol (β-ZEL) and β-ZEL-14-sulfate by 40% within 90 min and allowed the specific detection of ZEN-sulfate derivates from cereals. 7 Future Perspective The past few decades have witnessed significant advancements in analytical techniques and technologies to detect and manage the problem of mycotoxin analysis. The global mycotoxin testing market is estimated to grow at a cumulative annual growth rate (CAGR) of 7.8%, which corresponds to a market of $1.4 billion by 2026. 242 As the number indicates, the accelerated need for food safety has significantly accelerated the mycotoxin testing market. Needless to say, effective sampling, method performance, cost, and rapid detection are of paramount importance for successful mycotoxin testing. However, inexpensive and fast tests with low precision would lead to misclassification through inaccurate results and impact the business decisions of the producers. In light of this, the existing techniques must be refined/tuned for higher precision while active R&D is pursued to develop new methods that can detect multiple toxins simultaneously with high sensitivity (at regulatory levels) with minimal cost and runtime. One option could be developing miniaturized MS, which could be used in a fashion similar to the screening devices used for COVID detection at airports and train stations. The spectrometers are generally efficient and reliable and allow for rapid detection and solid sample detection through thermal absorption. One such example is mycotoxin detection in milled wheat by an MS developed by M/s BaySpec Inc. 243 The portable device could detect even ppm (1.4) levels of the contaminant within less than a minute's time. Recent studies have highlighted that mass-sensitive microarray (MSMA)-based biosensors could be a promising tool for rapidly detecting mycotoxin material. 244 The device consists of a mass-sensitive transducer based on solidly mounted resonator (SMR) technology with a specific integrated circuit. This technology allows small molecular weight toxins (up to 3 toxins simultaneously) to be rapidly detected with high sensitivity (for a single sample) within less than 10 min using mycotoxin analysis as a model example. The authors argued that with further upgrade, many more analytes (∼32) could be detected on the devices from a single sample, and with automation, the analysis was performed and printed without the requirement of any operator. The device could be an ideal system for multiplex analysis of mycotoxins; however, significant improvements must be made before it can be considered for field deployment in the analysis of real-time samples.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302679/

Obituary: Mark Thomas Fisher, Ph.D. June 21,1954–September 4, 2018

Author Contributions The author confirms being the sole contributor of this work and has approved it for publication. Conflict of Interest Statement The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest Statement The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094993/

The challenge of emerging and re-emerging infectious diseases

Infectious diseases have for centuries ranked with wars and famine as major challenges to human progress and survival. They remain among the leading causes of death and disability worldwide. Against a constant background of established infections, epidemics of new and old infectious diseases periodically emerge, greatly magnifying the global burden of infections. Studies of these emerging infections reveal the evolutionary properties of pathogenic microorganisms and the dynamic relationships between microorganisms, their hosts and the environment. Main Emerging infections (EIs) can be defined as "infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range" 1 . EIs have shaped the course of human history and have caused incalculable misery and death. In 1981, a new disease — acquired immune deficiency syndrome (AIDS) — was first recognized. As a global killer, AIDS now threatens to surpass the Black Death of the fourteenth century and the 1918–1920 influenza pandemic, each of which killed at least 50 million people 2 , 3 . Of the 'newly emerging' and 're-emerging/resurging' diseases that have followed the appearance of AIDS ( Fig. 1 ), some have been minor curiosities, such as the 2003 cases of monkeypox imported into the United States 4 , whereas others, such as severe acute respiratory syndrome (SARS), which emerged in the same year 5 , have had a worldwide impact. The 2001 anthrax bioterrorist attack in the United States 6 falls into a third category: 'deliberately emerging' diseases. EIs can be expected to remain a considerable challenge for the foreseeable future. Here we examine the nature and scope of emerging and re-emerging microbial threats, and consider methods for their control. We emphasize that emergence results from dynamic interactions between rapidly evolving infectious agents and changes in the environment and in host behaviour that provide such agents with favourable new ecological niches. Figure 1 Global examples of emerging and re-emerging infectious diseases, some of which are discussed in the main text. Red represents newly emerging diseases; blue, re-emerging/resurging diseases; black, a 'deliberately emerging' disease. Adapted, with permission, from ref. 23 . Global burden of infectious diseases About 15 million (>25%) of 57 million annual deaths worldwide are estimated to be related directly to infectious diseases; this figure does not include the additional millions of deaths that occur as a consequence of past infections (for example, streptococcal rheumatic heart disease), or because of complications associated with chronic infections, such as liver failure and hepatocellular carcinoma in people infected with hepatitis B or C viruses 7 ( Fig. 2 ). Figure 2 Leading causes of death worldwide. About 15 million (>25%) of 57 million annual deaths worldwide are the direct result of infectious disease. Figures published by the World Health Organization (see http://www.who.int/whr/en and ref. 7 ). The burden of morbidity (ill health) and mortality associated with infectious diseases falls most heavily on people in developing countries 8 , and particularly on infants and children (about three million children die each year from malaria and diarrhoeal diseases alone 7 ). In developed nations, infectious disease mortality disproportionately affects indigenous and disadvantaged minorities 9 . Emerging infections in historical context EIs have been familiar threats since ancient times. They were once identified by terms such as λoιµóς ( loimos ) 10 , and later as 'pestilences', 'pestes', 'pests' and 'plagues'. Many examples can be cited in addition to the Black Death and the 1918 influenza pandemic, such as certain biblical pharaonic plagues and the unidentified Plague of Athens, which heralded the end of Greece's Golden Age 11 . The Age of Discovery, starting in the fifteenth century, was a particularly disastrous period with regard to the spread of infectious diseases. Importation of smallpox into Mexico caused 10–15 million deaths in 1520–1521, effectively ending Aztec civilization 12 , 13 . Other Amerindian and Pacific civilizations were destroyed by imported smallpox and measles 13 , 14 , 15 , 16 , 17 . Historians have referred to these events as apocalypses 16 and even as genocide 15 . For centuries, mankind seemed helpless against these sudden epidemics. But the establishment of the germ theory and the identification of specific microbes as the causative agents of a wide variety of infectious diseases 18 , 19 , 20 led to enormous progress, notably the development of vaccines and ultimately of antimicrobials 20 . In fact, the era of the identification of microbes had barely begun 18 when optimists at the end of the nineteenth century predicted the eradication of infectious diseases 21 . By the 1950s, which witnessed the widespread use of penicillin, the development of polio vaccines and the discovery of drugs for tuberculosis, complacency had set in 22 , and in 1967, the US Surgeon General stated that "the war against infectious diseases has been won" 23 . Some experts remained sceptical, aware of recurrent lessons from history. They were less persuaded by successes than alarmed by failures such as the lack of progress against infections in the developing world and the global spread of antimicrobial resistance. Richard Krause, then the director of the US National Institute of Allergy and Infectious Diseases, warned in 1981 (ref. 24 ) that microbial diversity and evolutionary vigour were still dynamic forces threatening mankind. As Krause was completing his book The Restless Tide 24 , AIDS — one of history's most devastating pandemics — was already insidiously emerging. The emergence of AIDS led to renewed appreciation of the inevitability and consequences of the emergence of infectious diseases 25 , 26 , 27 , 28 , 29 , 30 , 31 . In the past 25 years, some of the factors that resulted in AIDS have also led to re-emergences of historically important diseases such as cholera, diphtheria, trench fever and plague. Many re-emergences have been catalysed by wars, loss of social cohesion, and natural disasters such as earthquakes and floods, indicating the importance not only of microbial and viral factors, but also of social and environmental determinants 25 , 26 , 27 , 28 , 29 , 30 , 31 . Newly emerging and newly recognized infections The classification of EIs as 'newly emerging', 're-emerging/resurging' or 'deliberately emerging' is useful because the underlying causes of emergence and the optimal prevention or control responses frequently differ between the groups. Newly emerging infections are those that have not previously been recognized in man. Many diverse factors contribute to their emergences (see Box 1 ); these include microbial genetic mutation and viral genetic recombination or reassortment, changes in populations of reservoir hosts or intermediate insect vectors, microbial switching from animal to human hosts, human behavioural changes (notably human movement and urbanization), and environmental factors. These numerous microbial, host and environmental factors interact to create opportunities for infectious agents to evolve into new ecological niches, reach and adapt to new hosts, and spread more easily between them. The AIDS model Any discussion of recent EIs must begin with the human immunodeficiency virus (HIV) that causes AIDS. HIV has so far infected more than 60 million people worldwide 33 . Before jumping to humans an estimated 60–70 years ago 34 , perhaps as a consequence of the consumption of 'bush meat' from non-human primates, HIV-1 and HIV-2 had ample opportunity to evolve in hosts that were genetically similar to man (the chimpanzee, Pan troglodytes , and the sooty mangabey, Cercocebus atys ). But HIV/AIDS might never have emerged had it not been for disruptions in the economic and social infrastructure in post-colonial sub-Saharan Africa. Increased travel, the movement of rural populations to large cities, urban poverty and a weakening of family structure all promoted sexual practices, such as promiscuity and prostitution, that facilitate HIV transmission 34 , 35 , 36 , 37 . Such complex interactions between infectious agents, hosts and the environment are not unique to the epidemiology of HIV/AIDS. The examples cited below further illustrate how changes in population density, human movements and the environment interact to create ecological niches that facilitate microbial or viral adaptation. Dead-end transmission of zoonotic and vector-borne diseases Some infectious agents that have adapted to non-human hosts can jump to humans but, unlike HIV, are not generally transmitted from person to person, achieving only 'dead end' transmission. Infections in animals that are transmitted to humans (zoonoses), and those transmitted from one vertebrate to another by an arthropod vector (vector-borne diseases), have repeatedly been identified as ranking among the most important EIs 25 , 26 . Examples include the arenavirus haemorrhagic fevers (Argentine, Bolivian, Venezuelan and Lassa haemorrhagic fevers) and hantavirus pulmonary syndrome (HPS). Viruses in these groups have co-evolved with specific rodent species whose contact with humans has increased as a result of modern environmental and human behavioural factors. Farming, keeping domestic pets, hunting and camping, deforestation and other types of habitat destruction all create new opportunities for such infectious agents to invade human hosts 25 , 26 , 27 , 28 , 29 , 30 , 31 . The first epidemic of HPS, detected in the southwestern region of the United States in 1993 (ref. 38 ), resulted from population booms of the deer mouse Peromyscus maniculatis , in turn caused by climate-related and recurrent proliferation of rodent food sources. Increased rodent populations and eventual shortages of food drove expanded deer mouse populations into homes, exposing people to virus-containing droppings. The 1998–1999 Malaysian Nipah virus epidemic 39 further illustrates the influence of human behaviours and environmental perturbations on newly emerging human infections. Pigs crammed together in pens located in or near orchards attracted fruit bats whose normal habitats had been destroyed by deforestation and whose droppings contained the then-unknown paramyxovirus. Virus aerosolization caused infection of pigs, with overcrowding leading to explosive transmission rates and ultimately to infections in pig handlers. Variant Creutzfeldt–Jakob disease (vCJD) is another example of a zoonotic disease emerging in humans. vCJD is caused by the human-adapted form of the prion associated with the emerging epizootic (large-scale animal outbreak) of bovine spongiform encephalopathy (BSE) 40 , commonly known as mad cow disease. The ongoing BSE epizootic/vCJD epidemic, primarily affecting Great Britain, probably resulted from the now-abandoned practice of supplementing cattle feed with the pulverized meat and bones of previously slaughtered cattle. BSE itself is suspected to have emerged because of even earlier use of cattle feed containing the agent of sheep scrapie, a prion disease recognized by farmers more than 250 years ago 41 . Alarmingly, the new BSE prion has become uncharacteristically promiscuous: unlike most known prions, it readily infects multiple species in addition to humans. This suggests the possibility of further emerging diseases associated with prions with currently unknown transmissibility to humans 40 . The recent reports of variant strains of the BSE prion 42 suggest that the BSE agent could be a more serious threat than other animal prions. Environmentally persistent organisms Infectious agents indirectly transmitted to or between humans by way of human-modified environments account for other emerging zoonoses, as well as certain non-zoonotic diseases, which are discussed below. For example, legionnaires' disease, first identified in 1976, is caused by Legionella pneumophila , whose emergence as a human pathogen might not have occurred were it not for the environmental niche provided by air-conditioning systems 26 . Campylobacter jejuni and Shiga-toxin-producing Escherichia coli ( E. coli O157:H7 and other agents of haemolytic–uraemic syndrome) infect agricultural animals, gaining access to humans through food, milk, water or direct animal contact. Other enteric pathogens, such as the vibrios causing classical cholera (re-emerging; see below) and serogroup O139 cholera, and the zoonotic protozoa Cryptosporidium parvum and Cyclospora cayetanensis 26 , seem to have come from environmental or animal organisms that have adapted to human-to-human 'faecal–oral' transmission through water. Old microbes cause new diseases Some EIs come from microorganisms that once caused familiar diseases, but which now cause new or previously uncommon diseases. Streptococcus pyogenes caused a fatal pandemic of scarlet and puerperal fevers between 1830 and 1900 (ref. 44 ). Scarlet fever, then the leading cause of death in children, is now rare, but has been largely supplemented by other streptococcal complications such as streptococcal toxic shock syndrome, necrotizing fasciitis and re-emergent rheumatic fever 45 . When new microbes are discovered, their emergences as disease-causing pathogens may be delayed. For example, in 1883, Robert Koch was unable to show that the newly discovered Koch–Weeks bacillus caused serious disease. More than a century later, a fatal EI dubbed Brazilian purpuric fever was linked to virulent clonal variants of Haemophilus influenzae biogroup aegyptius (the Koch–Weeks bacillus) 46 . Although the bases of emergences of new and more severe diseases caused by S. pyogenes and H. influenzae biogroup aegyptius are not fully known, in both cases complex microbial genetic events are suspected. The distinctive clonal variants associated with severe H. influenzae biogroup aegyptius disease have been shown by PCR (polymerase chain reaction)-based subtractive genome hybridization to contain not only a unique plasmid, but also unique chromosomal regions, some of which are encoded by bacteriophages 47 . This research has narrowed the search for virulence determinants to unique proteins, some of which may have been acquired from other organisms by horizontal gene transfer. Streptococcus pyogenes has been studied more extensively, but the basis of severe disease emergence seems to be more complex than for H. influenzae biogroup aegyptius. Many factors associated with streptococcal virulence have been identified in strains bearing the M1 surface protein as well as in other M protein strains, among them bacteriophage-encoded superantigen toxins and a protein known as sic (streptococcal inhibitor of complement), which seems to be strongly selected by human host mucosal factors. Several lines of evidence suggest that changes in streptococcal virulence reflect genetic changes associated with phage integration, large-scale chromosomal rearrangements and possibly the shuffling of virulence cassettes (clusters of genes responsible for pathogenicity), followed by rapid human spread and immune selection 48 , 49 . Microbial agents and chronic diseases Infectious agents that are associated with chronic diseases are one of the most challenging categories of newly emerging (or at least newly appreciated) infections. Examples include the associations of hepatitis B and C with chronic liver damage and hepatocellular carcinoma, of certain genotypes of human papillomaviruses with cancer of the uterine cervix, of Epstein–Barr virus with Burkitt's lymphoma (largely in Africa) and nasopharyngeal carcinoma (in China), of human herpesvirus 8 with Kaposi sarcoma, and of Helicobacter pylori with gastric ulcers and gastric cancer 50 , 51 , 52 . Some data even suggest infectious aetiologies for cardiovascular disease and diabetes mellitus 53 , major causes of death and disability worldwide. Other associations between infectious agents and idiopathic chronic diseases will inevitably be found. Re-emerging and resurging infections Re-emerging and resurging infections are those that existed in the past but are now rapidly increasing either in incidence or in geographical or human host range. Re-emergence is caused by some of the factors that cause newly emerging infectious diseases, such as microbial evolutionary vigour, zoonotic encounters and environmental encroachment. Re-emergences or at least cyclical resurgences of some diseases may also be climate-related — for example, the El Niño/Southern Oscillation (ENSO) phenomenon is associated with resurgences of cholera and malaria 54 . Geographical spread of infections The impact of both new and re-emerging infectious diseases on human populations is affected by the rate and degree to which they spread across geographical areas, depending on the movement of human hosts or of the vectors or reservoirs of infections. Travel has an important role in bringing people into contact with infectious agents 55 . An increase in travel-associated importations of diseases was anticipated as early as 1933, when commercial air travel was still in its infancy 56 . This has since been demonstrated dramatically by an international airline hub-to-hub pandemic spread of acute haemorrhagic conjunctivitis in 1981 (ref. 57 ), by epidemics of meningococcal meningitis associated with the Hajj, and more recently by the exportation of epidemic SARS (a newly emerging disease) from Guangdong Province, China, to Hong Kong, and from there to Beijing, Hanoi, Singapore, Toronto and elsewhere 5 ( Fig. 3 ). The persistent spread of HIV along air, trucking, drug-trafficking and troop-deployment routes is a deadly variation on this theme 35 , 36 , 37 . Figure 3 Probable cases of severe acute respiratory syndrome (SARS) with onset of illness from 1 November 2002 to 31 July 2003. Cases are given by country. SARS-related deaths are indicated in parentheses. A total of 8,096 cases (and 774 deaths) are presented. Figures published by the World Health Organization (see http://www.who.int/csr/sars/country/en ). Malaria Plasmodium falciparum malaria was neglected for several decades, but is now among the most important re-emerging diseases worldwide ( Fig. 2 ). Years of effective use of dichlorodiphenyltrichloroethane (DDT) led to the abandonment of other mosquito-control programmes, but the insecticide fell into disuse because of mosquito resistance and concerns about the insecticide's potentially harmful effects on humans and wildlife. Consequently, malaria has re-emerged, and the situation has been worsened by the development of drug resistance to chloroquine and mefloquine 58 . Research efforts focus on the development of vaccines 59 and new drugs, and on re-establishing public health measures such as the use of bed nets. Tuberculosis Tuberculosis is one of the most deadly re-emerging diseases ( Fig. 2 ). The discovery of isoniazid and other drugs initially led to effective tuberculosis cures, empty sanitoria and the dismantling of public health control systems in developed nations. Consequently, by the 1980s, when tuberculosis had re-emerged in the era of HIV/AIDS, local and state health departments in the United States lacked field, laboratory and clinical staff and so had to reinvent tuberculosis-control programmes 25 . The remarkable re-emergence of tuberculosis was fuelled by the immune deficiencies of people with AIDS, which greatly increases the risk of latent Mycobacterium tuberculosis infections progressing to active disease, and being transmitted to others. Inadequate courses of anti-tuberculosis therapy compound the problem, leading to the emergence and spread of drug-resistant and multidrug-resistant strains 60 , and a need for more expensive treatment strategies such as directly observed therapy. It has been known for over a century that tuberculosis is a disease of poverty, associated with crowding and inadequate hygiene. The continuing expansion of global populations living in poverty makes tuberculosis more difficult to control. Drug-resistant microbes Drug resistance, another factor causing microbial and viral re-emergence, may result from mutation (for example, in the case of viruses and M. tuberculosis ), or from bacterial acquisition of extraneous genes through transformation or infection with plasmids. Sequential emergences of Staphylococcus aureus that are resistant to sulpha drugs (1940s), penicillin (1950s), methicillin (1980s) and to vancomycin in 2002 (ref. 61 ) — a last line of antibiotic defence for some multiply drug-resistant bacteria — are troubling. Nosocomial Enterococcus faecalis became fully resistant to vancomycin by 1988, and then apparently transferred vanA resistance genes to co-infecting staphylococci 61 . Methicillin-resistant staphylococci are now being isolated from livestock that have been fed with growth-promoting antibiotics 62 , possibly contributing to resistance problems in humans. Many other important microbes have also become effective 'resistors', among them Streptococcus pneumoniae and Neisseria gonorrhoeae 63 . Opportunistic re-emerging infections Immune deficiency associated with AIDS, and with chemotherapy for cancer, immune-mediated diseases and transplantation, has contributed to an enormous global increase in the numbers of immunosuppressed people over the past few decades (probably more than 1% of the world's population), setting the stage for the re-emergence of many opportunistic infections. HIV, which has infected more than 60 million people globally 33 , is the largest single cause of human immune deficiency and markedly increases vulnerability to a wide range of opportunistic pathogens, including Pneumocystis carinii , various fungi, tuberculosis, protozoa and herpesviruses 64 . Breakthroughs in cancer therapy and in immunosuppressive therapies used to treat immune-mediated diseases and for transplantation 65 , 66 can also leave patients susceptible to opportunistic infections. Human organ transplantation adds a further risk of infection with undetected pathogens in donor tissues, and transplantation of animal organs introduces the risk of transmission to humans of animal microbes 67 . Re-emerging zoonotic and vector-borne diseases The emergence of zoonotic and vector-borne diseases can also be associated with human behaviours and environmental perturbation. In 2003, monkeypox — an endemic infection of African rodents — crossed the Atlantic with exported pets, which were then shipped from Texas to infect people throughout the US Midwest 4 . Lyme disease, caused by Borrelia burgdorferi , re-emerged in the United States as a result of suburban expansion, which brought people into increasing contact with deer, deer mice and ticks. Similarly, tick-borne encephalitis re-emerged in Russia when weekend getaways (dachas) drew city dwellers into contact with forest ticks. The simultaneous 1999 emergences of encephalitis due to West Nile virus in the United States and in Russia 68 , 69 reflect abundances of eclectic vector mosquitoes and avian hosts in these locations. Both were probably connected to endemic sites by virus carriage in migratory birds and travellers. The remarkable geographical spread of West Nile virus in the five years since its introduction into the Western Hemisphere reflects an unfortunate confluence of viral promiscuity and ecological diversity 70 . Although humans are dead-end hosts for West Nile virus, the risk of infection is greatly increased by marked zoonotic viral amplification and persistence in the environment. Unlike most viruses, which tend to be fairly narrowly adapted to specific hosts, West Nile virus is known to infect more than 30 North American mosquito species, which together transmit infection to at least 150 North American bird species, many of which migrate to new and distant locations, spreading the virus to rural and urban ecosystems throughout North and Central America 70 . Although West Nile virus is now a major epidemiological concern in the developed world, dengue remains the most significant and widespread flavivirus disease to have emerged globally 71 . A 2001–2002 epidemic in Hawaii — fortunately without fatalities — is a reminder that dengue has also re-emerged in locations once considered to be dengue-free. Usually transmitted by Aedes aegypti mosquitoes, dengue has recently been transmitted by Aedes albopictus — a vector switch of potential significance with respect to dengue re-emergence 71 . In the Americas, including many US southern states, A. albopictus has been spreading into areas where A. aegypti mosquitoes are not found, and persisting for longer seasonal periods, putting tens of millions more people at risk of dengue infection. Dengue re-emergence is further complicated by disturbing increases in a serious and formerly rare form of the disease, dengue haemorrhagic fever (dengue shock syndrome being its highly fatal form). These severe complications are thought to result from the evolution of dengue viruses to escape high population immunity, seen in increased viral virulence and human immunopathogenesis due to antibody-dependent enhancement of viral infection 72 . Cholera is also of interest, not only as an important cause of mortality, but also because of the complexity of factors that determine its re-emergence. Both virulent and avirulent strains of these zoonotic bacteria are maintained in the environment and are rapidly evolving in association with phyto- and zooplankton, algae and crustaceans. Such environmental strains seem to act as reservoirs for human virulence genes (for example, genes for the phage-encoded cholera toxin and the toxin-coregulated pilus (TCP) factor associated with attachment), and to undergo gene transfer events that lead to new strains containing further virulence gene combinations. These result in periodic cholera emergences that cause epidemics and pandemics 73 . Thus, although the disease we know as cholera has appeared to be clinically and epidemiologically stable at least since the third pandemic (in the 1840s), modern evidence suggests that such apparent stability masks aggressive bacterial evolution in complex natural environments. Influenza A Influenza A viruses, which are endemic gastrointestinal viruses of wild waterfowl, have evolved elaborate mechanisms to jump species into domestic fowl, farm animals and humans. Periodic gene segment reassortments between human and animal viruses produce important antigenic changes, referred to as 'shifts'. These can lead to deadly pandemics, as occurred in 1888, 1918, 1957 and 1968 (refs 74 , 75 ). In intervening years, shifted viruses undergo continual but less dramatic antigenic changes called 'drifts', which allow them partially to escape human immunity raised by previously circulating influenza viruses. Influenza drift is an evolutionary success story for the virus. Influenza A has a seemingly inexhaustible repertoire of mutational possibilities at several critical epitopes surrounding the viral haemagglutinin site that attaches to human cells. It remains something of a mystery how zoonotic influenza viruses mix with each other and with human strains to acquire the additional properties of human virulence and human-to-human transmissibility. Before 1997, mild cases of human disease associated with avian influenza viruses were occasionally reported 76 . These events have become more frequent, sometimes resulting in severe cases of disease and death. Avian influenza has recently made dead-end jumps to humans — for example, the 1997 Hong Kong outbreak of the newly emergent H5N1 influenza, the 2003 H7N7 epidemic in the Netherlands, the 2003–2004 H5N1 and H7N3 epizootics in Asia and elsewhere, and occasional cases of H9N2 disease ( Fig. 4 ). Meanwhile, back-switches of human H3N2 viruses have emerged in pigs, from which both doubly mixed (pig–human) and triply mixed (pig–human–avian) viruses 74 , 75 have been isolated. Such enzootic/zoonotic mixing is suspected to have occurred in the influenza pandemic of 1918–1920, which was caused by an H1N1 virus with an avian-like receptor-binding site 77 . The predicted virulence genes of this virus are now being sought from 85-year-old pathology specimens and from frozen corpses 78 . The implications of interspecies genetic mixing for future influenza pandemics are troubling. Although much remains speculative about how influenza viruses emerge and spread, it seems clear that the process is driven by prolific and complex viral evolution (genetic reassortment and mutational 'drift'), interspecies mixing and adaptation, and ecological factors that bring humans into contact with animals and each other. By whatever means new influenza virus pandemic strains emerge, they eventually reach a critical threshold of human transmission beyond which epidemic and pandemic spread follows mathematically predictable patterns. Figure 4 Documented human infections with avian influenza viruses, 1997–2004. Sporadic cases of mild human illness associated with avian influenza viruses were reported before 1997. See http://www.who.int/csr/disease/influenza/en and ref. 76 . The dynamics and determinants of such epidemic development have been studied since the nineteenth century for several infectious diseases. For influenza, both historical and prospective epidemics have been described or predicted using deterministic and stochastic mathematical models, often with surprising accuracy when compared with actual epidemic data. More complicated mathematical models that describe how diseases spread by means other than person-to-person aerosol transmission have generally been less successful in describing and predicting epidemics, but have nonetheless been helpful in planning public health responses to epidemics caused by HIV 79 , vCJD 80 and other diseases. Mathematical modelling is also used to determine the impact of emerging epidemics. For example, it has been difficult to estimate overall influenza mortality because fatal infections are often neither diagnosed nor accurately recorded in hospital records and death certificates, especially in the elderly. Recent epidemiological attempts to obtain improved influenza mortality estimates from seasonal excess mortality data 81 have indicated that influenza mortality may be greater than was previously suspected, because influenza deaths are frequently coded under seemingly unrelated categories such as cardiovascular diseases. The same approaches also show that other influenza-like deaths may actually be due to other agents, such as respiratory syncytial virus (RSV), a common childhood virus that in the past decade has emerged as a major cause of adult mortality 81 . Deliberately emerging infections Deliberately emerging microbes are those that have been developed by man, usually for nefarious use. The term 'deliberately emerging' refers to both naturally occurring microbial agents such as anthrax 6 , and to bioengineered microorganisms such as those created by the insertion of genetic virulence factors that produce or exacerbate disease. Deliberately emerging microbes include microorganisms or toxins produced in a form that would cause maximal harm because of ease of dissemination, enhanced infectivity or heightened pathogenicity 82 . Bioterrorism and biowarfare As concepts, bioterrorism and biowarfare are probably not new. The alleged catapulting of plague-ridden corpses over enemy walls in the 1346 siege of Caffa (the modern Crimean port of Feodosia, Ukraine) and the dispatch of smallpox-impregnated blankets to Indians by British officers in the Seven Years War (1754–1763) have frequently been cited as examples of bioterrorism or biowarfare 83 , 84 . Two modern attacks have been well documented. In 1984, an Oregon religious cult spiked restaurant salad bars with Salmonellae in an attempt to sway a local election 85 . A 2001 anthrax attack 6 , in which a terrorist mailed anthrax-spore-filled letters to prominent figures, including two US senators, resulted in illness in at least 18 people and the death of five of these individuals. Public alarm was elevated by the knowledge that Bacillus anthracis is a common and easily obtainable enzootic and soil organism found in laboratories worldwide, and that scientific technology had increased its lethality: the spores had been weaponized by being concentrated, finely milled and packed with a dispersal agent to increase their capacity to disseminate 82 . The United States, the United Kingdom, the Soviet Union and other nations once had sophisticated offensive bioweapons programmes that included the production of weaponized anthrax spores 82 . Soviet scientists continued to produce large quantities of organisms adapted for biowarfare and bioterror — among them the agents of smallpox, plague, tularaemia and Marburg virus — for several years after their signing of the Bioweapons and Toxins Treaty Convention in 1972, which forbade such activities 82 . By 1987, the Soviet programme was annually producing 5,000 tonnes of weaponized anthrax spores, packing them into warheads and other delivery devices 82 . Before the 2001 anthrax attacks 6 , the US scientific community had for several years been bolstering its biodefence research capacity. The anthrax attacks greatly accelerated this expansion as part of a national defence plan, which includes efforts to provide a knowledge base for the development of effective countermeasures against agents of bioterror, such as diagnostics, therapeutics and vaccines, and to translate this knowledge into the production and delivery of such measures 86 . Bioterror agents have been grouped into three categories of risk 87 . The six category A agents (anthrax, smallpox, plague, tularaemia, viral haemorrhagic fevers and clostridial botulinum toxin) are given top priority because they are highly lethal and readily deployed as weapons. Category B and C agents include food-borne and water-borne organisms that incapacitate but usually do not kill. Meeting the challenge of emerging infections Infectious diseases will continue to emerge and re-emerge, leading to unpredictable epidemics and difficult challenges to public health and to microbiology and allied sciences. Surveillance and response, the key elements in controlling EIs, be they naturally occurring or deliberately engineered, depend on rapid clinical diagnosis and detection and containment in populations and in the environment. Globally, such efforts are coordinated by the World Health Organization, which recently led a multifaceted effort to successfully contain the global SARS outbreak of 2002–2003 (ref. 88 ). In the United States, such efforts are led by the US Centers for Disease Control and Prevention (CDC) 89 , which along with state and local health departments and other agencies have been making significant strides in national surveillance–response capacity. The enormous influx of US government-funded research resources (largely through the National Institutes of Health) and public health resources (mainly through the CDC, and state and local public health agencies) in response to the increased threat of a bioterrorist attack 86 will fortify the response capabilities related to all EIs. However, it is clear that surveillance and other activities that traditionally fall within the domain of public health are not in themselves sufficient to adequately address the problem of EIs. Of critical importance are basic, translational and applied research efforts to develop advanced countermeasures such as surveillance tools, diagnostic tests, vaccines and therapeutics 86 . Genomics, proteomics and advances in nanotechnology 90 are increasingly being exploited in diagnostic, therapeutic and microbial research applications, and in rational drug and vaccine design. Direct and computational structural determination 91 , prediction of protein–protein interactions between microorganisms and drugs, and sophisticated bioinformatics techniques support research in all of the above areas. These technologies have led to numerous advances in real-world utility against EIs, most notably in the development of more than 20 antiretroviral drugs that can effectively suppress HIV replication. Where they are available and properly used in HIV-infected individuals, these medications have dramatically reduced HIV morbidity and mortality 92 . Gene- and protein-based microarrays can be used to detect pathogen signals, to monitor resistance to anti-infective agents, to characterize host gene responses to recent infections, and to facilitate the development of new drugs and vaccines 93 . Basic and applied research together have provided promising new vaccine platforms, such as recombinant proteins, immunogenic peptides, naked DNA vaccines, viral vectors of extraneous genes encoding immunogenic proteins (including chimaeras), replicons and pseudovirions 94 . Many novel vaccine candidates are now being developed against EIs such as HIV, Ebola virus, West Nile virus, dengue, the SARS coronavirus, tuberculosis and malaria. Of particular note are novel tuberculosis vaccines that recently entered clinical trials — the first time in more than 60 years that new approaches to vaccination for tuberculosis have been assessed in humans 95 . Chimaeric flavivirus vaccines for West Nile virus, dengue and Japanese encephalitis virus are effective in animal models and are in various stages of clinical testing 95 . Our growing understanding of the human immune system is also helping to accelerate vaccine development. This is especially true in the case of innate immune responses, which are evolutionarily older, less specific and faster-acting than the adaptive responses that have been the traditional targets of vaccines 96 . As we learn more about innate immunity and its relationship with the adaptive immune system, opportunities to create more effective vaccine adjuvants will emerge. For example, synthetic DNA sequences that contain repeated CpG motifs mimic the stimulatory activity that bacterial DNA fragments exert on the innate immune system. These sequences show promise as vaccine adjuvants that accelerate and augment immune responses 97 . We can anticipate more progress of this kind as we continue to delineate the complex interactions between innate and adaptive immune responses. The sequencing of the human genome, the genomes of six other animals, including the mouse, and those of microbial vectors and microbes themselves (for example, P. falciparum and its mosquito vector, Anopheles gambiae ), have elevated microbiology to a whole-genome level. The ability to sequence microbial genomes in a few days 98 or less, and to examine host–vector–microbe interactions at both the genome level and at the tertiary protein structural level, will help us to understand the molecular mechanisms that underlie the pathogenesis of infectious disease and host defences, including resistance and immune evasion. These advances will facilitate the development of new countermeasures. Other fertile areas of research include the use of geographical information systems 99 and satellite imaging to support field study and epidemic prevention (for example, predicting HPS and Rift Valley fever epidemics in indigenous areas by satellite imagery of water and vegetation related to animal reservoir and vector prevalence). Underlying disease emergence are evolutionary conflicts between rapidly evolving and adapting infectious agents and their slowly evolving hosts. These are fought out in the context of accelerating environmental and human behavioural alterations that provide new ecological niches into which evolving microbes can readily fit. It is essential that broadly based prevention strategies, as well as new and improved countermeasures (that is, surveillance tools, diagnostics, therapeutics and vaccines), be continually tested, refined and upgraded, requiring a strengthened relationship between public health and basic and clinical sciences. The challenge presented by the ongoing conflict between pathogenic microorganisms and man has been well summarized by a noted champion of the war on EIs, Joshua Lederberg: "The future of microbes and mankind will probably unfold as episodes of a suspense thriller that could be entitled Our Wits Versus Their Genes " 29 . The global scientific and public health communities must confront this reality not only with wit, but also with vision and sustained commitment to meet a perpetual challenge. Box 1: Factors involved in the emergence of infectious diseases Selected factors contribute to the emergence/re-emergence of infectious diseases 25 , 26 . These factors, which frequently differ for 'newly emerging', 're-emerging/resurging' and 'deliberately emerging' diseases, include genetic, biological, and social, political and economic factors. • Microbial adaptation and change • Human susceptibility to infection • Climate and weather • Changing ecosystems • Human demographics and behaviour • Economic development and land use • International travel and commerce • Technology and industry • Breakdown of public health measures • Poverty and social inequality • War and famine • Lack of political will • Intent to harm Box 1: Factors involved in the emergence of infectious diseases Selected factors contribute to the emergence/re-emergence of infectious diseases 25 , 26 . These factors, which frequently differ for 'newly emerging', 're-emerging/resurging' and 'deliberately emerging' diseases, include genetic, biological, and social, political and economic factors. • Microbial adaptation and change • Human susceptibility to infection • Climate and weather • Changing ecosystems • Human demographics and behaviour • Economic development and land use • International travel and commerce • Technology and industry • Breakdown of public health measures • Poverty and social inequality • War and famine • Lack of political will • Intent to harm

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC348865/

Effect of Extracellular Products of Pseudoalteromonas atlantica on the Edible Crab Cancer pagurus

Previous studies have shown that injection of extracellular products (ECP) of Pseudoalteromononas atlantica isolated from shell disease-infected edible crabs ( Cancer pagurus ) into healthy crabs causes rapid death. In this study we examined the nature of the active lethal factor(s) in ECP. Injection of ECP into crabs caused a rapid decline in the total number of circulating hemocytes (blood cells), and the crabs died within 60 to 90 min. The individuals that died showed eyestalk retraction, limb paralysis, and lack of antennal sensitivity, suggesting that the active factor(s) targeted the nervous system. Histopathological investigations showed that affected crabs had large aggregates of hemocytes in the gills, and there was destruction of the tubules in the hepatopancreas. The active factor in ECP was not sensitive to heat treatment (100°C for 30 min) and proteinase K digestion. As lipopolysaccharide (LPS) was a potential candidate for the lethal factor, it was purified from whole P. atlantica bacteria or ECP and subsequently injected into crabs. These crabs had all of the external symptoms observed previously with ECP, such as limb paralysis and eyestalk retraction, and they died within 90 min after challenge, although no significant decline in the number of circulating hemocytes was observed. Similarly, in vitro incubation of hemocytes with purified LPS (1 to 20 μg) from P. atlantica did not result in the clumping reaction observed with ECP but did result in a degranulation reaction and eventual cell lysis. Injection of crabs with Escherichia coli or Pseudomonas aeruginosa LPS (1 μg g of body weight −1 ) did not cause any of the characteristic symptoms observed following exposure to P. atlantica LPS. No mortality of crabs followed the injection of E. coli LPS, but P. aeruginosa LPS caused ca. 80% mortality at 2 h after injection. Overall, these results show that the main virulence factor of P. atlantica for edible crabs is LPS either alone or in combination with other heat-stable factors.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716618/

A comprehensive guide for studying inflammasome activation and cell death

Inflammasomes are multimeric heterogenous mega-Dalton protein complexes that play key roles in the host innate immune response to infection and sterile insults. Assembly of the inflammasome complex following infection or injury begins with the oligomerization of the upstream inflammasome-forming sensor and proceeds through a multistep process of well-coordinated events and downstream effector functions. Together, these steps allow for elegant experimental readouts to reliably assess the successful activation of the inflammasome complex and cell death. Here, we describe a comprehensive protocol that details several in vitro (in bone-marrow derived macrophages) and in vivo (in mice) strategies for activating the inflammasome and explain how to subsequently assess multiple downstream effects in parallel to unequivocally establish the activation status of the inflammasome and the cell death pathways. Our workflow assesses inflammasome activation via the formation of the ASC speck, cleavage of caspase-1 and gasdermin D, release of IL-1β, IL-18, caspase-1, and lactate dehydrogenase from the cell, and real-time analysis of cell death by imaging. Analyses take up to approximately 24 hours to complete. Overall, our multifaceted approach provides a comprehensive and consistent protocol for assessing inflammasome activation and cell death.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821973/

Leaf-Encapsulated Vaccines: Agroinfiltration and Transient Expression of the Antigen Staphylococcal Endotoxin B in Radish Leaves

Transgene introgression is a major concern associated with transgenic plant-based vaccines. Agroinfiltration can be used to selectively transform nonreproductive organs and avoid introgression. Here, we introduce a new vaccine modality in which Staphylococcal enterotoxin B (SEB) genes are agroinfiltrated into radishes ( Raphanw sativus L.), resulting in transient expression and accumulation of SEB in planta . This approach can simultaneously express multiple antigens in a single leaf. Furthermore, the potential of high-throughput vaccine production was demonstrated by simultaneously agroinfiltrating multiple radish leaves using a multichannel pipette. The expression of SEB was detectable in two leaf cell types (epidermal and guard cells) in agroinfiltrated leaves. ICR mice intranasally immunized with homogenized leaves agroinfiltrated with SEB elicited detectable antibody to SEB and displayed protection against SEB-induced interferon-gamma (IFN- γ ) production. The concept of encapsulating antigens in leaves rather than purifying them for immunization may facilitate rapid vaccine production during an epidemic disease. 1. Introduction Transgenic plants have emerged as a promising technology to generate recombinant biopharmaceutical proteins and vaccines [ 1 , 2 ]. Plants produce full-length mammalian proteins that appear to be processed correspondingly to their native counterpart with appropriate folding, assembly, and posttranslational modifications [ 3 ]. Although stably transformed transgenic plants have been widely created to deliver edible vaccines [ 4 , 5 ] and have proven success in clinical trials [ 6 , 7 ], the fact that transgenes are permanently incorporated into the genomes of transgenic plants raises many concerns, such as the environmental release of genetically modified plants and the possibility of transgene introgression into nonmodified counterparts [ 8 ]. In addition, immunization with edible vaccines derived from transgenic plants may carry a risk of inducing oral tolerance due to immunization with multidoses within a long period of time. Transient expression of recombinant proteins in leaf tissue avoids transgene introgression and provides a fast platform for protein production without an effort-exhaustive process to generate stably transformed transgenic plants [ 9 ]. Currently, there are at least four approaches to transforming and inducing transient expression in plants: (1) delivery of "naked" DNA by particle bombardment [ 10 ], (2) infection with modified viral vectors [ 6 , 10 , 11 ], (3) agroinfiltration of plant tissues with Agrobacteria [ 10 , 12 ], and (4) polyethylene glycol- (PEG-) mediated gene transfer and electroporation of protoplasts [ 13 ]. Agroinfiltration accommodates transforming plants with large genes encoding complex proteins, such as antibodies. Moreover, agroinfiltration-induced transient expression can yield high levels of recombinant protein [ 14 ]. Vacuum and syringe infiltration are two major methods of promoting agroinfiltration and expressing proteins/antigens in plants [ 15 , 16 ]. Unlike the vacuum infiltration, syringe infiltration can be easily applied for infiltrating multiple antigens on the same leaf. Syringe infiltration, where a needle-less syringe is placed at the surface of a leaf and used to push a suspension of A. tumefaciens into the leaf interior, provides a high level of control over which tissues are transformed. In contrast to agroinfiltration, the efficiency of particle bombardment using a gene gun is relatively low since transgenes are successfully delivered to only few target cells [ 14 ]. Furthermore, transient expression using plant virus infection shows many disadvantages, such as biosafety and construct-size limitation [ 2 ]. Protoplast transformation involves a care-intensive, complicated procedure of isolating protoplasts from leaf mesophylls. Protoplasts can also respond differently from intact cells and may not be suitable for certain types of expression analysis [ 17 ]. Staphylococcal enterotoxin B (SEB) is one of the several toxins produced by Staphylococcus aureus bacteria [ 18 , 19 ]. The toxin commonly causes outbreaks of food poisoning. Also, SEB has been studied as a potential biological warfare agent because it can easily be aerosolized, is very stable, and can cause shortness of breath, widespread systemic damage, and even shock and death when inhaled at very high dosages [ 20 – 22 ]. Molecularly, SEB acts as a superantigen, binding to class II major histocompatibility complex proteins and stimulating T cells to induce inflammation and cytokine (e.g., tumor necrosis factor alpha and interferon-gamma (IFN- γ )) release [ 23 ]. Considering the toxicity and potential weaponization of SEB, there is an urgent need to have anti-SEB vaccines that can be produced in an effortless and timely manner during SEB outbreaks. Here, we generate SEB vaccines by agroinfiltrating SEB genes into radish leaves. Intranasal immunization of mice with SEB-expressing leaves in conjunction with adjuvant cholera toxin (CT) elicited systemic antibodies to SEB and offered protective immunity against SEB-induced IFN- γ production. We also demonstrate that two different antigens (SEB and a tetanus toxin C fragment (TetC)) can be simultaneously agroinfiltrated and transiently expressed within the same leaf. Notably, we here highlight the concept of stamping antigens onto leaves to generate vaccines by using agroinfiltration. The technique shows that agroinfiltration can be used to rapidly induce transient expression of antigens in leaf tissue, which can be used for immunization in a way that eliminates complicated purification procedures commonly associated with recombinant antigens. This work illustrates that agroinfiltrated/stamped leaves can not only act as bioreactors for antigen production but may also serve as capsulated vaccines containing one or more antigens for patient immunization. 2. Materials and Methods 2.1. Plant Materials Japanese radish sprouts (Kaiware-daikon) ( Raphanus sativus L.) and lettuce ( Lactuca sativa ) were obtained from a commercial supplier (ICREST International, JCP, Carson, CA). Japanese radish sprouts that were 9 cm in length with two leaflets were used. Arabidopsis thaliana seeds were kindly provided by Professor Nigel Crawford at University of California, San Diego. All plants were grown at room temperature under a 23-watt fluorescent bulb (Philips, Portland, OR) and were sprayed with water daily. 2.2. Vector Construction and Agrobacterium tumefaciens Transformation The methods of vector construction and transformation were according to a modified protocol described in our previous publication. Briefly, the binary vector pBI121 carrying the reporter GUS driven by the CaMV 35S promoter was used [ 24 , 25 ]. A forward primer (5′-GATTCTAGAATGGAGAGTCAACCAGATCCTAAACCAGA-3′) and a reverse primer (5′-TCGCCCGGGCGCTTTTTCTTTGTCGTAAGATAAACTTC-3′) were utilized for polymerase chain reaction (PCR) to amplify the open reading frame of detoxified SEB cDNA with three mutations (National Center for Biotechnology Information (NCBI) accession number M11118) [ 26 ]. A forward primer (5′-GGATCTAGAATGGAAAATCTGGATTGTTGGG-3′) and a reverse primer (5′-AATCCCGGGCGGTCGTTGGTCCAACCTTC-3′) were added into a PCR reaction to amplify the TetC cDNA (NCBI accession number AM412776). PCR products were cloned into polylinker sites of pBI121 vectors to generate 35S:: SEB-GUS and 35S:: TetC-GUS constructs [ 25 ]. These two constructs were then transformed into Agrobacterium tumefaciens strain LBA4404 according to a liquid nitrogen freeze-thaw method. 2.3. Agroinfiltration of 35S::SEB-GUS and 35S::TetC-GUS Constructs into Radish Leaves A single colony of A. tumefaciens transformants was cultured in 2 ml of YEP media (10 mg/ml Bacto™ Tryptone (DIFCO, Detroit, MI), 10 mg/ml yeast extract (DIFCO, Detroit, MI), and 5 mg/ml NaCl (Sigma, St. Louis, MO; pH 7.5)) containing 50 μ g/ml kanamycin and streptomycin at 28°C until optical density (OD) at 600 nm (OD 600 ) reached 0.5. Nontransformed Agrobacterium served as a negative control. For syringe infiltration, as previously described [ 25 ], 0.1 ml of Agrobacterium bacterial suspension (5 × 10 7 CFU) was injected into the wounded lower epidermis site for five days. For high-throughput agroinfiltration, six radish leaves were concurrently infiltrated with 0.1 ml of bacterial suspension containing the 35S:: SEB - GUS construct using a multichannel pipette with open (2.2 mm diameter) tips. The infiltrated leaves were next placed in a dish containing wet cloths and incubated overnight. 2.4. Histochemical GUS Assays Agroinfiltrated leaves were stained using a histochemical GUS assay solution consisting of 0.1 M NaPO 4 (pH 7.0), 0.5 mM K 3 Fe(CN) 6 , 0.5 mM K 4 Fe(CN) 6 , 0.1% ( v / v ) Triton X-100, and 0.05% ( w / v ) X-Gluc (Sigma, St. Louis, MO) [ 27 ]. Leaves were submerged in the staining solution and incubated at 37°C in the dark overnight. After incubation, leaves were removed from the staining solution and immersed in a stop solution containing 42.5% ( v / v ) ethanol, 10% ( v / v ) formaldehyde, and 5% ( v / v ) acetic acid [ 28 ]. Stained leaves were embedded in OCT compound (Miles Inc., Diagnostics Division, Elkhart, IN) and cut with a glass knife on a cryogenic ultramicrotome (7 μ m thick). Fresh-mounted OCT sections were examined under bright-field microscopy (Olympus America, Inc., Melville, NY). 2.5. Intranasal Immunization with Homogenized Leaves Containing Recombinant SEB Our previous study indicated that intranasal immunization of mice with ground leaves expressing CAMP factor elicits detectable antibodies to P. acnes CAMP factor, indicating that intranasal administration of whole plant leaves may be a new regimen for vaccination [ 25 ]. In the study, female ICR (Institute of Cancer Research) mice (3 to 6 weeks old; Harlan, Indianapolis, IN) were utilized for intranasal immunization. Intranasal immunization holds the potential to induce a mucosal immune response that recapitulates the natural SEB infection across the respiratory tract [ 29 ]. All mice used in the study were maintained in accordance to institutional IACUC guidelines. The central areas (25 mm 2 ) of five radish leaves expressing SEB-GUS or GUS alone were excised using a sterile scalpel. Leaf sections were then pooled and homogenized under liquid nitrogen followed by addition of 700 μ l ddH 2 O and then sterilized by an ultraviolet crosslinker (Spectronics, Westbury, NY) at 7000 J/m 2 for 30 min. Inactivation of sterilized Agrobacterium was confirmed by their inability to form colonies on YEP agar plates (data not shown). Twenty-five microliter homogenized leaves containing either SEB-GUS or GUS alone (as a negative control) mixed with a CT adjuvant (Sigma-Aldrich, St. Louis, MO) which has been used to boost the mucosal immunogenicity (5 μ g/25 μ l of ground leaf materials as described below) were then intranasally inoculated into the nasal cavities of ICR mice (25 μ l of ground leaf materials). Three boosts at the same dose were performed at 1, 2, and 4 weeks after the first immunization [ 30 ]. 2.6. Western Blotting Twenty μ g of homogenized leaves expressing either SEB-GUS or GUS alone were loaded into a 10% SDS-PAGE for antigen detection. After electrophoretically transferring SDS-PAGE to nitrocellulose membranes, the membranes were incubated with mouse monoclonal anti-SEB antibody (1 : 1000 dilution) (Toxin Technology, Sarasota, FL). To detect the production of antibodies in immunized mice, recombinant SEB (15 μ g) (Toxin Technology, Sarasota, FL) was subject to a 10% SDS-PAGE and transferred to a nitrocellulose membrane which was subsequently immunoreacted to four-week serum (1 : 500 dilution) obtained from mice immunized with whole leaf containing SEB-GUS. Immunoglobulin G (IgG) antibodies were detected with anti-mouse horseradish peroxidase-conjugated IgG (1 : 5000 dilution, Promega, Madison, WI). A Western Lighting™ Chemiluminescence kit (PerkinElmer, Boston, MA) was used to visualize the peroxidase activity. 2.7. Titration of Antibodies The antibody titer of SEB was quantified by ELISA. Eight mice were used per group. Sera were collected 4 weeks after first immunization with L-GUS or L-SEB-GUS. Purified recombinant SEB (0.1 μ g/well) was diluted with PBS buffer and coated onto a 96-well ELISA plate (Corning, Lowell, MA) at 4°C overnight. The plate was washed with PBS containing 0.05% ( w / v ) Tween-20 and blocked with PBS containing 1% ( w / v ) bovine-serum albumin and 0.05% ( w / v ) Tween-20 for 2 h at room temperature. Pooled antisera obtained from eight immunized mice with L-GUS or L-SEB-GUS were serially diluted by 10-fold and separately added to the wells and incubated for 2 h. A goat anti-mouse IgG-HRP conjugate (Promega, Madison, WI) (1 : 5000 dilution) was added and incubated for 2 h before washing. HRP activity was determined with an OptEIA™ Reagent Set (BD Biosciences). The OD of each well was measured at 490 nm. The endpoint was defined as the dilution of sera producing the same OD at 490 nm as a 1/100 dilution of preimmune sera. Sera negative at the lowest dilution tested were assigned endpoint titers of 100. The data was presented as geometric mean endpoint ELISA titers as previously described [ 31 ]. 2.8. Measurement of SEB-Induced IFN- γ Production in Immunized Mice Naïve mice and immunized mice after the third boost were challenged intranasally with recombinant SEB (40 μ g/mouse) for overnight. Eight mice were used per group. After trachea cannulation, the lungs were lavaged twice with 0.5 ml of phosphate-buffered saline, and BAL fluids were pooled. After centrifugation at 1300g, IFN- γ in fluids pooled from eight mice per group was measured by an ELISA kit as directed by the manufacturer (BD Biosciences, San Diego, CA) [ 31 ]. 2.1. Plant Materials Japanese radish sprouts (Kaiware-daikon) ( Raphanus sativus L.) and lettuce ( Lactuca sativa ) were obtained from a commercial supplier (ICREST International, JCP, Carson, CA). Japanese radish sprouts that were 9 cm in length with two leaflets were used. Arabidopsis thaliana seeds were kindly provided by Professor Nigel Crawford at University of California, San Diego. All plants were grown at room temperature under a 23-watt fluorescent bulb (Philips, Portland, OR) and were sprayed with water daily. 2.2. Vector Construction and Agrobacterium tumefaciens Transformation The methods of vector construction and transformation were according to a modified protocol described in our previous publication. Briefly, the binary vector pBI121 carrying the reporter GUS driven by the CaMV 35S promoter was used [ 24 , 25 ]. A forward primer (5′-GATTCTAGAATGGAGAGTCAACCAGATCCTAAACCAGA-3′) and a reverse primer (5′-TCGCCCGGGCGCTTTTTCTTTGTCGTAAGATAAACTTC-3′) were utilized for polymerase chain reaction (PCR) to amplify the open reading frame of detoxified SEB cDNA with three mutations (National Center for Biotechnology Information (NCBI) accession number M11118) [ 26 ]. A forward primer (5′-GGATCTAGAATGGAAAATCTGGATTGTTGGG-3′) and a reverse primer (5′-AATCCCGGGCGGTCGTTGGTCCAACCTTC-3′) were added into a PCR reaction to amplify the TetC cDNA (NCBI accession number AM412776). PCR products were cloned into polylinker sites of pBI121 vectors to generate 35S:: SEB-GUS and 35S:: TetC-GUS constructs [ 25 ]. These two constructs were then transformed into Agrobacterium tumefaciens strain LBA4404 according to a liquid nitrogen freeze-thaw method. 2.3. Agroinfiltration of 35S::SEB-GUS and 35S::TetC-GUS Constructs into Radish Leaves A single colony of A. tumefaciens transformants was cultured in 2 ml of YEP media (10 mg/ml Bacto™ Tryptone (DIFCO, Detroit, MI), 10 mg/ml yeast extract (DIFCO, Detroit, MI), and 5 mg/ml NaCl (Sigma, St. Louis, MO; pH 7.5)) containing 50 μ g/ml kanamycin and streptomycin at 28°C until optical density (OD) at 600 nm (OD 600 ) reached 0.5. Nontransformed Agrobacterium served as a negative control. For syringe infiltration, as previously described [ 25 ], 0.1 ml of Agrobacterium bacterial suspension (5 × 10 7 CFU) was injected into the wounded lower epidermis site for five days. For high-throughput agroinfiltration, six radish leaves were concurrently infiltrated with 0.1 ml of bacterial suspension containing the 35S:: SEB - GUS construct using a multichannel pipette with open (2.2 mm diameter) tips. The infiltrated leaves were next placed in a dish containing wet cloths and incubated overnight. 2.4. Histochemical GUS Assays Agroinfiltrated leaves were stained using a histochemical GUS assay solution consisting of 0.1 M NaPO 4 (pH 7.0), 0.5 mM K 3 Fe(CN) 6 , 0.5 mM K 4 Fe(CN) 6 , 0.1% ( v / v ) Triton X-100, and 0.05% ( w / v ) X-Gluc (Sigma, St. Louis, MO) [ 27 ]. Leaves were submerged in the staining solution and incubated at 37°C in the dark overnight. After incubation, leaves were removed from the staining solution and immersed in a stop solution containing 42.5% ( v / v ) ethanol, 10% ( v / v ) formaldehyde, and 5% ( v / v ) acetic acid [ 28 ]. Stained leaves were embedded in OCT compound (Miles Inc., Diagnostics Division, Elkhart, IN) and cut with a glass knife on a cryogenic ultramicrotome (7 μ m thick). Fresh-mounted OCT sections were examined under bright-field microscopy (Olympus America, Inc., Melville, NY). 2.5. Intranasal Immunization with Homogenized Leaves Containing Recombinant SEB Our previous study indicated that intranasal immunization of mice with ground leaves expressing CAMP factor elicits detectable antibodies to P. acnes CAMP factor, indicating that intranasal administration of whole plant leaves may be a new regimen for vaccination [ 25 ]. In the study, female ICR (Institute of Cancer Research) mice (3 to 6 weeks old; Harlan, Indianapolis, IN) were utilized for intranasal immunization. Intranasal immunization holds the potential to induce a mucosal immune response that recapitulates the natural SEB infection across the respiratory tract [ 29 ]. All mice used in the study were maintained in accordance to institutional IACUC guidelines. The central areas (25 mm 2 ) of five radish leaves expressing SEB-GUS or GUS alone were excised using a sterile scalpel. Leaf sections were then pooled and homogenized under liquid nitrogen followed by addition of 700 μ l ddH 2 O and then sterilized by an ultraviolet crosslinker (Spectronics, Westbury, NY) at 7000 J/m 2 for 30 min. Inactivation of sterilized Agrobacterium was confirmed by their inability to form colonies on YEP agar plates (data not shown). Twenty-five microliter homogenized leaves containing either SEB-GUS or GUS alone (as a negative control) mixed with a CT adjuvant (Sigma-Aldrich, St. Louis, MO) which has been used to boost the mucosal immunogenicity (5 μ g/25 μ l of ground leaf materials as described below) were then intranasally inoculated into the nasal cavities of ICR mice (25 μ l of ground leaf materials). Three boosts at the same dose were performed at 1, 2, and 4 weeks after the first immunization [ 30 ]. 2.6. Western Blotting Twenty μ g of homogenized leaves expressing either SEB-GUS or GUS alone were loaded into a 10% SDS-PAGE for antigen detection. After electrophoretically transferring SDS-PAGE to nitrocellulose membranes, the membranes were incubated with mouse monoclonal anti-SEB antibody (1 : 1000 dilution) (Toxin Technology, Sarasota, FL). To detect the production of antibodies in immunized mice, recombinant SEB (15 μ g) (Toxin Technology, Sarasota, FL) was subject to a 10% SDS-PAGE and transferred to a nitrocellulose membrane which was subsequently immunoreacted to four-week serum (1 : 500 dilution) obtained from mice immunized with whole leaf containing SEB-GUS. Immunoglobulin G (IgG) antibodies were detected with anti-mouse horseradish peroxidase-conjugated IgG (1 : 5000 dilution, Promega, Madison, WI). A Western Lighting™ Chemiluminescence kit (PerkinElmer, Boston, MA) was used to visualize the peroxidase activity. 2.7. Titration of Antibodies The antibody titer of SEB was quantified by ELISA. Eight mice were used per group. Sera were collected 4 weeks after first immunization with L-GUS or L-SEB-GUS. Purified recombinant SEB (0.1 μ g/well) was diluted with PBS buffer and coated onto a 96-well ELISA plate (Corning, Lowell, MA) at 4°C overnight. The plate was washed with PBS containing 0.05% ( w / v ) Tween-20 and blocked with PBS containing 1% ( w / v ) bovine-serum albumin and 0.05% ( w / v ) Tween-20 for 2 h at room temperature. Pooled antisera obtained from eight immunized mice with L-GUS or L-SEB-GUS were serially diluted by 10-fold and separately added to the wells and incubated for 2 h. A goat anti-mouse IgG-HRP conjugate (Promega, Madison, WI) (1 : 5000 dilution) was added and incubated for 2 h before washing. HRP activity was determined with an OptEIA™ Reagent Set (BD Biosciences). The OD of each well was measured at 490 nm. The endpoint was defined as the dilution of sera producing the same OD at 490 nm as a 1/100 dilution of preimmune sera. Sera negative at the lowest dilution tested were assigned endpoint titers of 100. The data was presented as geometric mean endpoint ELISA titers as previously described [ 31 ]. 2.8. Measurement of SEB-Induced IFN- γ Production in Immunized Mice Naïve mice and immunized mice after the third boost were challenged intranasally with recombinant SEB (40 μ g/mouse) for overnight. Eight mice were used per group. After trachea cannulation, the lungs were lavaged twice with 0.5 ml of phosphate-buffered saline, and BAL fluids were pooled. After centrifugation at 1300g, IFN- γ in fluids pooled from eight mice per group was measured by an ELISA kit as directed by the manufacturer (BD Biosciences, San Diego, CA) [ 31 ]. 3. Results and Discussion 3.1. Agroinfiltration, Transient Expression, and Encapsulation of β -Glucuronidase (GUS) Protein in a Model Plant and Two Edible Crops Many plants, including Arabidopsis , a model plant, are able to express proteins [ 32 ] via either stable genetic or transient transformation [ 33 ]. Agrobacterium has been utilized as a vector to deliver foreign DNA and induce transient expression of recombinant proteins in various plants [ 34 ]. In this study, Arabidopsis and two edible crops, lettuce and radish, were used as platforms to transiently express GUS and/or antigens. Leaves of these plants were bombarded with Agrobacterium harboring a 35S:: GUS construct via a pressure infiltration. Five days postinfiltration, spatial expression of GUS within the leaves was detected by histochemical GUS staining. Infiltration of radish, lettuce, and Arabidopsis leaves with A. tumefaciens harboring 35S::GUS constructs resulted in GUS expression in all three plants. Control infiltrations, in which A. tumefaciens lacking 35S::GUS constructs was used, did not yield detectable GUS expression ( Figure 1 ). These results confirm the versatility of agroinfiltration for inducing transient expression of transgenes in a variety of plants. Radishes, being edible and easily grown, were used for all following transient expression experiments. As presented in Figure 1 , GUS can be agroinfiltrated and transiently expressed in Arabidopsis , lettuce, and radish, demonstrating A. tumefaciens ' broad host range [ 35 ]. The Japanese radish ( Raphanus sativus L. ) is an edible leaf vegetable that is grown and consumed throughout the world. Recently, it has been reported that the Japanese radish is the vegetable with the highest per capita consumption within the Brassicaceae family. Moreover, it is rich in antioxidant constituents that can potentially prevent several human diseases [ 36 ]. Due to its easy growth and edibility, Japanese radish was selected for transient expression of GUS and/or SEB-GUS. Histochemical GUS assays demonstrated that GUS expression is detectable in radish five days after agroinfiltration ( Figure 1 ). Detection of GUS activity using 4-methylumbelliferyl-D-glucuronide (4-MUG) as a substrate indicated that the amount of GUS expression was dramatically elevated to the 0.45 U/mg five days after agroinfiltration [ 25 ], which may predict the kinetics or amount of transient protein expression in agroinfiltrated leaves although in planta transient transgene expression has not been well quantified [ 37 ]. 3.2. Agroinfiltration of SEB-GUS into Radish Leaves SEB has been categorized as a biological threat agent in bioterrorism and epidemic outbreaks of food poisoning. Development of a modality that can produce vaccines against SEB in a quick and undemanding way may be an effective strategy to block the SEB spread. In this study, the action of agroinfiltration stamping was displayed by means of pressure infiltration of leaves with an Agrobacterium -loaded syringe ( Figure 2(a) ). Infiltration of radish leaves with Agrobacterium containing a 35S:: SEB - GUS construct resulted in recombinant SEB-GUS encapsulation within leaves, as indicated by GUS histochemical staining in the central part of the leaf ( Figure 2(b) , SEB-GUS). Control leaves agroinfiltrated with A. tumefaciens lacking the 35S:: SEB-GUS construct did not exhibit any staining ( Figure 2(b) ). Infiltrating each leaflet of a single radish leaf with different Agrobacterium transformants, specifically, one containing a 35S:: SEB - GUS construct another containing 35S:: TetC - GUS , allowed a single radish leaf to express two different antigens (SEB and TetC) with distinct spatial encapsulation of the antigens within the leaf ( Figure 2(b) , SEB-GUS + TetC-GUS), demonstrating the simplicity of using agroinfiltration stamping to create a bivalent vaccine in plants [ 38 ]. The throughput of syringe infiltration was increased by using a multichannel pipette to infiltrate six harvested radish leaves in parallel ( Figure 2(c) ). A. tumefaciens either harboring or lacking the 35S:: SEB-GUS construct was loaded into tips on the multichannel pipette and pressure infiltrated into leaves in a manner similar to that used with syringes. SEB-GUS was detected in the leaves agroinfiltrated with the 35S:: SEB - GUS construct, as indicated by histochemical staining ( Figure 2(d) ). In this study, we emphasized the concept of using agroinfiltration stamping to transiently express and encapsulate antigens in radish leaves. The agroinfiltration stamping was illustrated by applying pressure infiltration of A. tumefaciens suspension into leaf tissue, accomplished with either a syringe or a multichannel pipette ( Figure 2 ), avoided more complicated techniques like microparticle bombardment [ 39 ], which requires gene guns [ 40 ] and coating DNA on gold particles. Unlike agroinfiltration stamping, microparticle bombardment will thus make it difficult to simultaneously transfer multiple antigens into a single leaf as well as to bombard antigens in a high-throughput manner. Agroinfiltration is an efficient method for inducing transient expression of multiple antigen transgenes in plant tissue. The concept of high-throughput agroinfiltration system in the study could be applied for producing high level and variety of antigens in the future [ 41 ]. Moreover, agroinfiltration can provide milligram amounts of a recombinant protein within a week [ 42 ]. This is an important issue because it dramatically accelerates the development of plant lines producing recombinant therapeutics. Importantly, agroinfiltration may even prove suitable for preclinical trials without the need for production of stably transformed plants [ 14 ]. 3.3. Cellular Distribution of SEB-GUS Transient Expression in Radish Leaves To examine the cellular distributions of GUS and SEB expression, Tissue-Tek Optimal Cutting Temperature- (OCT-) embedded tissue sections of agroinfiltrated radish leaves were stained with 5-bromo-4-chloro-3-indolyl- β -D-glucuronic acid cyclohexylammonium salt (X-Gluc). No GUS expression was detected when leaves were infiltrated with nontransformant A. tumefaciens (control). GUS expression (indicated by a blue precipitate after X-Gluc treatment) was condensed in the wounded area of radish leaves infiltrated with A. tumefaciens carrying a 35S:: GUS construct ( Figure 3(a) ). The GUS or SEB-GUS was detectable in epidermal cells, but predominantly expressed in guard cells in the wounded area agroinfiltrated with 35S:: GUS or 35S:: SEB - GUS constructs, respectively ( Figure 3(b) ). GUS expression was used as an indicator for SEB expression since constructs were designed to have the SEB coding sequence upstream of the GUS coding sequence in SEB-GUS fusions. Additionally, SEB-GUS expression was detected by a Western blot analysis. Proteins in agroinfiltrated radish leaves were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and reacted with a mouse monoclonal anti-SEB antibody. A band at 96 kDa corresponding to the expression of a SEB- (28 kDa) GUS (68 kDa) fusion protein appeared for leaves infiltrated with A. tumefaciens carrying a 35S: SEB:GUS construct ( Figure 4 ). Although several protein bands were recognized by a mouse monoclonal anti-SEB antibody, the 96 kDa band is not detected in leaves infiltrated with nontransformant Agrobacterium (control). Future work will extract SEB-GUS from infiltrated leaves [ 43 ] and conduct Western blot analysis to validate the expression of SEB in leaves. Data from Figures 3 and 4 indicate that SEB-GUS was expressed and encapsulated in radish leaves after agroinfiltration. Through advances in molecular and genetic techniques, protein expression in plants has been optimized for high-level production [ 44 ]. Recently, synthesis of codon-optimized bacteria gene in plants is powerful and common [ 45 ]. It is conceivable that pathogens and radish sprouts have very different tRNA pools. Thus, synthesis of a codon-optimized gene ought to enhance the production of in plant cells [ 46 ]. Moreover, transient expression levels can be elevated by using the cauliflower mosaic virus (CaMV) 35S promoter to drive transgene expression in plants [ 47 ]. Previous studies demonstrated the cell type-specific expression of a CaMV 35S-GUS gene in transgenic plants [ 48 ]. Here, we showed that epidermal cells and guard cells in CaMV 35S-GUS-transformed radish leaves expressed GUS most readily ( Figure 3 ), which is consistent with GUS expression patterns seen in transgenic tobacco leaves [ 49 ]. 3.4. SEB Immunogenicity and Protective Immunity against IFN- γ Production The functionality of SEB-GUS encapsulated in radish leaves as a vaccine was tested. Without purifying SEB from leaves, whole leaves infiltrated with A. tumefaciens carrying a 35S:: SEB - GUS (L-SEB-GUS) or a 35S:: GUS (L-GUS) construct were ground in sterile water, ultraviolet-inactivated, and mixed with cholera toxin, a common adjuvant used for intranasal immunization [ 50 ]. The ground leaves were subsequently inoculated into nasal cavities of ICR mice for intranasal immunization. The anti-SEB-GUS antibodies were measurable by a Western blot assay in mouse serum four weeks after intranasal immunization with leaves containing SEB-GUS ( Figure 5(a) ). Data from enzyme-linked immunosorbent assay (ELISA) indicated that mice immunized with L-SEB-GUS elicited antibody to SEB ( Figure 5(b) ). No antibodies against SEB were detected in mice immunized with GUS alone. This result demonstrates that SEB expressed in radish leaves can act as a vaccine to confer immunity against SEB. It has been reported that levels of IFN- γ in bronchoalveolar lavage (BAL) fluids dramatically increase in mice during SEB-induced inflammation [ 20 ]. We intranasally inoculated naïve mice with 40 μ g of recombinant SEB or the same volume of phosphate-buffered saline (PBS). The challenge of recombinant SEB significantly augmented the production of IFN- γ in BAL fluids ( Figure 5(c) ). To assess the protective effects of SEB vaccines encapsulated in radish leaves, we next intranasally challenged SEB into mice and measured the change of IFN- γ levels in BAL fluids. In mice that had previously been inoculated with leaves containing only GUS, BAL fluid IFN- γ levels were 2345.49 ± 64.65 pg/ml after being challenged with SEB. However, in mice that had previously been inoculated with leaves containing SEB-GUS, IFN- γ levels in BAL fluid dropped to 586.18 ± 30.69 pg/ml ( Figure 5(c) ). This result illustrates that SEB vaccine encapsulated in radish leaves confers protection against SEB-induced IFN- γ production. Recently, a number of studies have demonstrated the capability of agroinfiltration to generate recombinant proteins as antigens [ 51 , 52 ]. These studies focused on increasing recombinant protein yields for purification [ 53 ]. Indeed, the antigenicity of proteins relies not only on the protein amounts but also on the protein structures. However, low amounts of protein can provide sufficiently high immunogenicity [ 54 ]. In this study, we used homogenized radish leaves expressing SEB, rather than purified recombinant SEB, for immunization. The production of SEB antibodies in immunized mice ( Figure 5(a) ) demonstrated that agroinfiltration and in planta transient expression of SEB is sufficient for leaf tissue to exhibit SEB immunogenicity. Notably, the use of minimally prepared homogenized leaves containing SEB as vaccines can eliminate sophisticated procedures for antigen purification. In fact, agroinfiltration is adding lipopolysaccharide (LPS) from the Agrobacterium , which in itself may be a molecule capable of impacting the immune responses [ 55 ]. Further works should focus on performing control data for the LPS responses like using SEB from non-LPS sources as a control and comparing its immune response to that from LPS sources. Furthermore, using Western blot and ELISA assays, antibodies against SEB were detectable in mice immunized with homogenized leaves expressing SEB without the addition of an exogenous CT adjuvant (data not shown). This result supports other evidence indicating that leaves contain natural adjuvants such as phyto-saponins [ 56 ]. Unfortunately, these immunized mice are unable to suppress SEB-induced IFN- γ production (data not shown). Conversely, intranasal immunization of mice with SEB-expressing leaves in conjunction with adjuvant CT not only elicited systemic antibodies to SEB but also offered protective immunity against SEB-induced IFN- γ production although it was shown that CT may induce Bell's palsy [ 57 ]. Thus, other safe mucosal adjuvants should be analyzed in the future. GUS has been shown to be an immunogenic protein [ 58 ]. In addition, several leaf proteins are antigenic in mice as well [ 59 ]. The immunogenicities of GUS and radish proteins in mice immunized with whole leaves containing GUS are undetermined in this study. However, in comparison with immunizations using leaves containing SEB-GUS, mice immunized with leaves containing only GUS elicited high levels of IFN- γ after SEB challenge ( Figure 5(b) ), suggesting that the background of GUS and leaf proteins present in leaves did not inhibit or confound SEB immunogenicity. 3.1. Agroinfiltration, Transient Expression, and Encapsulation of β -Glucuronidase (GUS) Protein in a Model Plant and Two Edible Crops Many plants, including Arabidopsis , a model plant, are able to express proteins [ 32 ] via either stable genetic or transient transformation [ 33 ]. Agrobacterium has been utilized as a vector to deliver foreign DNA and induce transient expression of recombinant proteins in various plants [ 34 ]. In this study, Arabidopsis and two edible crops, lettuce and radish, were used as platforms to transiently express GUS and/or antigens. Leaves of these plants were bombarded with Agrobacterium harboring a 35S:: GUS construct via a pressure infiltration. Five days postinfiltration, spatial expression of GUS within the leaves was detected by histochemical GUS staining. Infiltration of radish, lettuce, and Arabidopsis leaves with A. tumefaciens harboring 35S::GUS constructs resulted in GUS expression in all three plants. Control infiltrations, in which A. tumefaciens lacking 35S::GUS constructs was used, did not yield detectable GUS expression ( Figure 1 ). These results confirm the versatility of agroinfiltration for inducing transient expression of transgenes in a variety of plants. Radishes, being edible and easily grown, were used for all following transient expression experiments. As presented in Figure 1 , GUS can be agroinfiltrated and transiently expressed in Arabidopsis , lettuce, and radish, demonstrating A. tumefaciens ' broad host range [ 35 ]. The Japanese radish ( Raphanus sativus L. ) is an edible leaf vegetable that is grown and consumed throughout the world. Recently, it has been reported that the Japanese radish is the vegetable with the highest per capita consumption within the Brassicaceae family. Moreover, it is rich in antioxidant constituents that can potentially prevent several human diseases [ 36 ]. Due to its easy growth and edibility, Japanese radish was selected for transient expression of GUS and/or SEB-GUS. Histochemical GUS assays demonstrated that GUS expression is detectable in radish five days after agroinfiltration ( Figure 1 ). Detection of GUS activity using 4-methylumbelliferyl-D-glucuronide (4-MUG) as a substrate indicated that the amount of GUS expression was dramatically elevated to the 0.45 U/mg five days after agroinfiltration [ 25 ], which may predict the kinetics or amount of transient protein expression in agroinfiltrated leaves although in planta transient transgene expression has not been well quantified [ 37 ]. 3.2. Agroinfiltration of SEB-GUS into Radish Leaves SEB has been categorized as a biological threat agent in bioterrorism and epidemic outbreaks of food poisoning. Development of a modality that can produce vaccines against SEB in a quick and undemanding way may be an effective strategy to block the SEB spread. In this study, the action of agroinfiltration stamping was displayed by means of pressure infiltration of leaves with an Agrobacterium -loaded syringe ( Figure 2(a) ). Infiltration of radish leaves with Agrobacterium containing a 35S:: SEB - GUS construct resulted in recombinant SEB-GUS encapsulation within leaves, as indicated by GUS histochemical staining in the central part of the leaf ( Figure 2(b) , SEB-GUS). Control leaves agroinfiltrated with A. tumefaciens lacking the 35S:: SEB-GUS construct did not exhibit any staining ( Figure 2(b) ). Infiltrating each leaflet of a single radish leaf with different Agrobacterium transformants, specifically, one containing a 35S:: SEB - GUS construct another containing 35S:: TetC - GUS , allowed a single radish leaf to express two different antigens (SEB and TetC) with distinct spatial encapsulation of the antigens within the leaf ( Figure 2(b) , SEB-GUS + TetC-GUS), demonstrating the simplicity of using agroinfiltration stamping to create a bivalent vaccine in plants [ 38 ]. The throughput of syringe infiltration was increased by using a multichannel pipette to infiltrate six harvested radish leaves in parallel ( Figure 2(c) ). A. tumefaciens either harboring or lacking the 35S:: SEB-GUS construct was loaded into tips on the multichannel pipette and pressure infiltrated into leaves in a manner similar to that used with syringes. SEB-GUS was detected in the leaves agroinfiltrated with the 35S:: SEB - GUS construct, as indicated by histochemical staining ( Figure 2(d) ). In this study, we emphasized the concept of using agroinfiltration stamping to transiently express and encapsulate antigens in radish leaves. The agroinfiltration stamping was illustrated by applying pressure infiltration of A. tumefaciens suspension into leaf tissue, accomplished with either a syringe or a multichannel pipette ( Figure 2 ), avoided more complicated techniques like microparticle bombardment [ 39 ], which requires gene guns [ 40 ] and coating DNA on gold particles. Unlike agroinfiltration stamping, microparticle bombardment will thus make it difficult to simultaneously transfer multiple antigens into a single leaf as well as to bombard antigens in a high-throughput manner. Agroinfiltration is an efficient method for inducing transient expression of multiple antigen transgenes in plant tissue. The concept of high-throughput agroinfiltration system in the study could be applied for producing high level and variety of antigens in the future [ 41 ]. Moreover, agroinfiltration can provide milligram amounts of a recombinant protein within a week [ 42 ]. This is an important issue because it dramatically accelerates the development of plant lines producing recombinant therapeutics. Importantly, agroinfiltration may even prove suitable for preclinical trials without the need for production of stably transformed plants [ 14 ]. 3.3. Cellular Distribution of SEB-GUS Transient Expression in Radish Leaves To examine the cellular distributions of GUS and SEB expression, Tissue-Tek Optimal Cutting Temperature- (OCT-) embedded tissue sections of agroinfiltrated radish leaves were stained with 5-bromo-4-chloro-3-indolyl- β -D-glucuronic acid cyclohexylammonium salt (X-Gluc). No GUS expression was detected when leaves were infiltrated with nontransformant A. tumefaciens (control). GUS expression (indicated by a blue precipitate after X-Gluc treatment) was condensed in the wounded area of radish leaves infiltrated with A. tumefaciens carrying a 35S:: GUS construct ( Figure 3(a) ). The GUS or SEB-GUS was detectable in epidermal cells, but predominantly expressed in guard cells in the wounded area agroinfiltrated with 35S:: GUS or 35S:: SEB - GUS constructs, respectively ( Figure 3(b) ). GUS expression was used as an indicator for SEB expression since constructs were designed to have the SEB coding sequence upstream of the GUS coding sequence in SEB-GUS fusions. Additionally, SEB-GUS expression was detected by a Western blot analysis. Proteins in agroinfiltrated radish leaves were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and reacted with a mouse monoclonal anti-SEB antibody. A band at 96 kDa corresponding to the expression of a SEB- (28 kDa) GUS (68 kDa) fusion protein appeared for leaves infiltrated with A. tumefaciens carrying a 35S: SEB:GUS construct ( Figure 4 ). Although several protein bands were recognized by a mouse monoclonal anti-SEB antibody, the 96 kDa band is not detected in leaves infiltrated with nontransformant Agrobacterium (control). Future work will extract SEB-GUS from infiltrated leaves [ 43 ] and conduct Western blot analysis to validate the expression of SEB in leaves. Data from Figures 3 and 4 indicate that SEB-GUS was expressed and encapsulated in radish leaves after agroinfiltration. Through advances in molecular and genetic techniques, protein expression in plants has been optimized for high-level production [ 44 ]. Recently, synthesis of codon-optimized bacteria gene in plants is powerful and common [ 45 ]. It is conceivable that pathogens and radish sprouts have very different tRNA pools. Thus, synthesis of a codon-optimized gene ought to enhance the production of in plant cells [ 46 ]. Moreover, transient expression levels can be elevated by using the cauliflower mosaic virus (CaMV) 35S promoter to drive transgene expression in plants [ 47 ]. Previous studies demonstrated the cell type-specific expression of a CaMV 35S-GUS gene in transgenic plants [ 48 ]. Here, we showed that epidermal cells and guard cells in CaMV 35S-GUS-transformed radish leaves expressed GUS most readily ( Figure 3 ), which is consistent with GUS expression patterns seen in transgenic tobacco leaves [ 49 ]. 3.4. SEB Immunogenicity and Protective Immunity against IFN- γ Production The functionality of SEB-GUS encapsulated in radish leaves as a vaccine was tested. Without purifying SEB from leaves, whole leaves infiltrated with A. tumefaciens carrying a 35S:: SEB - GUS (L-SEB-GUS) or a 35S:: GUS (L-GUS) construct were ground in sterile water, ultraviolet-inactivated, and mixed with cholera toxin, a common adjuvant used for intranasal immunization [ 50 ]. The ground leaves were subsequently inoculated into nasal cavities of ICR mice for intranasal immunization. The anti-SEB-GUS antibodies were measurable by a Western blot assay in mouse serum four weeks after intranasal immunization with leaves containing SEB-GUS ( Figure 5(a) ). Data from enzyme-linked immunosorbent assay (ELISA) indicated that mice immunized with L-SEB-GUS elicited antibody to SEB ( Figure 5(b) ). No antibodies against SEB were detected in mice immunized with GUS alone. This result demonstrates that SEB expressed in radish leaves can act as a vaccine to confer immunity against SEB. It has been reported that levels of IFN- γ in bronchoalveolar lavage (BAL) fluids dramatically increase in mice during SEB-induced inflammation [ 20 ]. We intranasally inoculated naïve mice with 40 μ g of recombinant SEB or the same volume of phosphate-buffered saline (PBS). The challenge of recombinant SEB significantly augmented the production of IFN- γ in BAL fluids ( Figure 5(c) ). To assess the protective effects of SEB vaccines encapsulated in radish leaves, we next intranasally challenged SEB into mice and measured the change of IFN- γ levels in BAL fluids. In mice that had previously been inoculated with leaves containing only GUS, BAL fluid IFN- γ levels were 2345.49 ± 64.65 pg/ml after being challenged with SEB. However, in mice that had previously been inoculated with leaves containing SEB-GUS, IFN- γ levels in BAL fluid dropped to 586.18 ± 30.69 pg/ml ( Figure 5(c) ). This result illustrates that SEB vaccine encapsulated in radish leaves confers protection against SEB-induced IFN- γ production. Recently, a number of studies have demonstrated the capability of agroinfiltration to generate recombinant proteins as antigens [ 51 , 52 ]. These studies focused on increasing recombinant protein yields for purification [ 53 ]. Indeed, the antigenicity of proteins relies not only on the protein amounts but also on the protein structures. However, low amounts of protein can provide sufficiently high immunogenicity [ 54 ]. In this study, we used homogenized radish leaves expressing SEB, rather than purified recombinant SEB, for immunization. The production of SEB antibodies in immunized mice ( Figure 5(a) ) demonstrated that agroinfiltration and in planta transient expression of SEB is sufficient for leaf tissue to exhibit SEB immunogenicity. Notably, the use of minimally prepared homogenized leaves containing SEB as vaccines can eliminate sophisticated procedures for antigen purification. In fact, agroinfiltration is adding lipopolysaccharide (LPS) from the Agrobacterium , which in itself may be a molecule capable of impacting the immune responses [ 55 ]. Further works should focus on performing control data for the LPS responses like using SEB from non-LPS sources as a control and comparing its immune response to that from LPS sources. Furthermore, using Western blot and ELISA assays, antibodies against SEB were detectable in mice immunized with homogenized leaves expressing SEB without the addition of an exogenous CT adjuvant (data not shown). This result supports other evidence indicating that leaves contain natural adjuvants such as phyto-saponins [ 56 ]. Unfortunately, these immunized mice are unable to suppress SEB-induced IFN- γ production (data not shown). Conversely, intranasal immunization of mice with SEB-expressing leaves in conjunction with adjuvant CT not only elicited systemic antibodies to SEB but also offered protective immunity against SEB-induced IFN- γ production although it was shown that CT may induce Bell's palsy [ 57 ]. Thus, other safe mucosal adjuvants should be analyzed in the future. GUS has been shown to be an immunogenic protein [ 58 ]. In addition, several leaf proteins are antigenic in mice as well [ 59 ]. The immunogenicities of GUS and radish proteins in mice immunized with whole leaves containing GUS are undetermined in this study. However, in comparison with immunizations using leaves containing SEB-GUS, mice immunized with leaves containing only GUS elicited high levels of IFN- γ after SEB challenge ( Figure 5(b) ), suggesting that the background of GUS and leaf proteins present in leaves did not inhibit or confound SEB immunogenicity. 4. Conclusion The argoinfiltration stamping was exploited as a novel modality to generate monovalent or bivalent vaccines. Argoinfiltrating gene (SEB and TetC) into radish leaves provides a simple approach for transiently expressing and encapsulating antigens in leaf tissue. This approach avoids the issue of transgene introgression and offers means to generate vaccines in a rapid manner. Moreover, the coexpression of antigens could be applied for analyzing multiple immunological responses to provide new means of vaccine manufacture and delivery without the complicated codelivery procedure following mixing of many expressed antigens. Increased awareness about the prospects of global epidemics and bioterrorism has motivated the development of techniques to create inexpensive vaccines on a rapid, massive scale if necessary [ 60 ]. As shown with SEB, transient expression of antigens in plant tissue offers one such method of rapid production. Intranasal immunization with minimally prepared homogenized leaves containing recombinant antigens eliminates the cost and time requirements of antigen purification and avoids the intrinsic problems associated with needle injections. Also, intranasal immunization of mice with ground leaves expressing SEB elicits detectable antibodies to S. aurues SEB. However, it had been reported that vaccination via an intranasal route can cause facial nerve paralysis [ 61 ]. Therefore, the safety of intranasal administration is worthy to be investigated since the human respiratory tract is not exposed to plant leaves on a routine basis [ 7 ]. In addition, the concept of encapsulating proteins/antigens in the leaves instead of purifying them for immunization may benefit vaccine production in the developing countries where cold chain facilities are lacking and emerge as a commercially viable approach for urgent vaccine development. Conflicts of Interest The authors declare no conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069233/

The delicate balance in genetically engineering live vaccines

Contemporary vaccine development relies less on empirical methods of vaccine construction, and now employs a powerful array of precise engineering strategies to construct immunogenic live vaccines. In this review, we will survey various engineering techniques used to create attenuated vaccines, with an emphasis on recent advances and insights. We will further explore the adaptation of attenuated strains to create multivalent vaccine platforms for immunization against multiple unrelated pathogens. These carrier vaccines are engineered to deliver sufficient levels of protective antigens to appropriate lymphoid inductive sites to elicit both carrier-specific and foreign antigen-specific immunity. Although many of these technologies were originally developed for use in Salmonella vaccines, application of the essential logic of these approaches will be extended to development of other enteric vaccines where possible. A central theme driving our discussion will stress that the ultimate success of an engineered vaccine rests on achieving the proper balance between attenuation and immunogenicity. Achieving this balance will avoid over-activation of inflammatory responses, which results in unacceptable reactogenicity, but will retain sufficient metabolic fitness to enable the live vaccine to reach deep tissue inductive sites and trigger protective immunity. The breadth of examples presented herein will clearly demonstrate that genetic engineering offers the potential for rapidly propelling vaccine development forward into novel applications and therapies which will significantly expand the role of vaccines in public health.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989367/

Mucosal Adjuvants For Vaccines To Control Upper Respiratory Infections In The Elderly

Influenza virus and Streptococcus pneumoniae are two major pathogens that lead to significant morbidity and mortality in the elderly. Since both pathogens enter the host via the mucosa, especially the upper respiratory tract (URT), it is essential to elicit pathogen-specific secretory IgA (SIgA) antibody (Ab) responses at mucosal surfaces for defense of the elderly. However, as aging occurs, alterations in the mucosal immune system of older individuals result in a failure to induce SIgA Abs for protection from these infections. To overcome mucosal immunosenescence, we have developed a mucosal dendritic cell targeting, novel double adjuvant system which we show to be an attractive and effective immunological modulator. This system induces a more balanced Th1- and Th2- type cytokine response which supports both mucosal SIgA and systemic IgG1 and IgG2a Ab responses. Thus, adaptation of this adjuvant system to nasal vaccines for influenza virus and S. pneumoniae could successfully provide protection by supporting pathogen-specific SIgA Ab responses in the URT in the mouse model of aging. In summary, a double adjuvant system is considered to be an attractive and potentially important strategy for the future development of mucosal vaccines for the elderly.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3495862/

Anthrax Lethal Factor Cleaves Mouse Nlrp1b in Both Toxin-Sensitive and Toxin-Resistant Macrophages

Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin are likely due to polymorphisms other than those at the LF cleavage sites. Introduction Anthrax lethal toxin (LT) consists of a receptor-binding protein, protective antigen (PA), and a protease, lethal factor (LF). LT is a major virulence factor, and its injection alone is sufficient to induce the vascular collapse associated with anthrax disease in animal models (for review see [1] ). Rodent macrophages from certain inbred strains are rapidly lysed by LT. Susceptibility to LT is controlled by alleles encoding variants of the NOD-like receptor (NLR) Nlrp1b in mice [2] , and its ortholog in rats [3] . Nlrp proteins are intracellular pattern recognition receptors that detect a variety of signals associated with pathogens and other dangers to the cell [4] . While a wide range of stimuli are recognized by intracellular sensors such as Nlrp3 [4] , the only known activator of rodent Nlrp1 is LT. Treatment of macrophages and dendritic cells from some inbred rodent strains such as Balb/cJ (mice) and Fischer (rats) to LT results in Nlrp1/Nlrp1b-mediated activation of caspase-1 and subsequent cleavage of IL-1β and IL-18, initiating an immune response concurrent with rapid cell death (pyroptosis) [2] , [5] . Macrophages from other rodent strains such as C57BL/6J and NOD/LtJ (mice) and Lewis (rats) express Nlrp1 variants which are not activated by LT and these cells are resistant to the toxin [2] , [5] . Our laboratory recently demonstrated that LT sensitivity in rats is determined by polymorphisms at residues 40-48 in the N-terminus of rat Nlrp1 [6] . LF cleaves this site in rat Nlrp1 of inbred strains harboring LT-sensitive macrophages, but not the Nlrp1 of LT-resistant strains [6] . Mice harbor three Nlrp1 paralogs in their genomes, but the existing data indicate that only Nlrp1b controls LT sensitivity [2] . Mouse Nlrp1b proteins are highly polymorphic [2] , unlike the situation in rats, where Nlrp1 is almost identical in all strains except in the small region in the N-terminus noted above. Sequencing Nlrp1b from 18 inbred mouse strains identified five different Nlrp1b protein sequences, two associated with LT sensitivity and three with resistance [2] . The proteins encoded by four of the five different Nlrp1b alleles have >88% homology to one another. Especially intriguing is the high degree of homology between proteins encoded by Nlrp1b allele 1 (expressed in Balb/cJ and numerous other strains with LT-sensitive macrophages), Nlrp1b allele 3 (expressed in NOD/LtJ and AKR/J mice, which harbor LT-resistant macrophages) and Nlrp1b allele 5 (expressed in CAST/EiJ mice, which harbor LT-sensitive macrophages). We wished to determine whether the differential responses of these highly homologous proteins were determined by LF cleavage of Nlrp1b, as it is in rats [6] . We report here that Nlrp1b expressed in both Balb/cJ macrophages (LT-sensitive) as well as Nlrp1b from NOD/LtJ macrophages (LT-resistant) are cleaved by LT within the N-terminal region, at the same sites. These results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin, is likely due to polymorphisms that render the protein inactive in a manner independent of LT cleavage. 10.1371/journal.pone.0049741.g001 Figure 1 Nlrp1 protein alignments and constructs. (A). Alignment of amino acid sequences from the N-terminus of mouse Nlrp1b and rat Nlrp1 proteins. Sequences shown are those of 4 mouse and 2 rat strains, including strains having macrophages that are either sensitive (S) or resistant (R) to LT. The previously identified LT cleavage site after residue 44 in rat CDF Nlrp1 is indicated by an arrow. The red box indicates the region of mouse sequence shown in (B). (B) Nlrp1b constructs used in this study with focus on N-terminal regions containing putative LF cleavage sites. The top two constructs represent the full-length HA-tagged Nlrp1b proteins from the LT-sensitive Balb/cJ (BALB) and the LT-resistant NOD/LtJ (NOD) macrophages, which were expressed in HT1080 cells. Full length NOD Nlrp1b is shorter (1172 aa) than BALB Nlrp1b due to a region downstream of the leucine rich repeat domain that is missing in this protein. The next four constructs represent proteins where aa 3-118 of Nlrp1b were expressed and purified from E. coli as N-terminal GST-tagged proteins. These proteins also contain a C-terminal His6 tag (not represented in figure). In the sequence alignments, residues identical to those in the construct listed above are indicated by quotation marks ("). Putative LF cleavage sites based on previously described motifs are drawn as vertical dotted lines below filled arrows. The MEK4 cleavage site is also aligned with both putative Nlrp1b cleavage sites. The last two sequences are those of constructs having two key lysine residues substituted with alanine. Results and Discussion Our earlier studies identified an LF cleavage site within the N-terminus of rat Nlrp1. Toxin cleavage at this site leads to macrophage pyroptosis [6] . An alignment of rat Nlrp1 sequences of Fischer (CDF, LT-sensitive) and Lewis (LEW, LT-resistant) rats, as well as Nlrp1b sequences from four mouse strains is shown in Figure 1A . The alignment shows that the LF cleavage site within rat Nlrp1 lies in an inserted sequence that is absent in mouse Nlrp1b proteins. Perplexingly, unlike the situation in rats, the Balb/cJ (BALB, LT-sensitive) and NOD/LtJ (NOD, LT-resistant) Nlrp1b sequences, present in mice having macrophages of opposing sensitivities to LT, are identical over the first 55 amino acids of the protein. Examination of the BALB (S) and NOD (R) Nlrp1b sequences revealed two nearby potential LF cleavage sites having characteristics like those of the established cleavage sites identified in rat Nlrp1, mitogen activated protein kinase kinase 4 (MEK4), and other LF substrates [6] – [9] (Red box in Figure 1A and Figure 1B ). Nlrp1 proteins are expressed endogenously at low levels that are difficult to detect via Western blotting. Therefore we expressed full-length N-terminally hemagglutinin (HA) epitope-tagged rat Nlrp1 (CDF-S) and mouse Nlrp1b (BALB-S and NOD-R) proteins in HT1080 human fibroblasts by stable transfection. Immunoprecipitation (IP) with anti-HA antibodies showed expression of full-length HA-tagged Nlrp1 proteins, as well as multiple C-terminally truncated variants ( Figure 2 ). As previously reported [6] , LF cleavage of HA-tagged rat Nlrp1 (CDF) expressed in fibroblasts produced a 6-kDA HA-antibody reactive cleavage fragment ( Figure 2A ). In a new result, we found that LF cleaved the HA-tagged BALB (S) Nlrp1b protein, producing a (slightly smaller) ∼5-kDa HA-antibody reactive cleavage fragment ( Figure 2A ), suggesting that the BALB (S) Nlrp1b cleavage site is slightly upstream of the rat Nlrp1 insertion sequence which contains the cleavage site. This would also mean that this cleavage site would lie within a region of identical sequence for both BALB (S) and NOD (R) Nlrp1b. 10.1371/journal.pone.0049741.g002 Figure 2 Cleavage of full length rat and mouse Nlrp1b proteins by LF. (A) IP (anti-HA pulldown) followed by anti-HA Western blotting of lysates from HT1080 cells expressing HA-tagged mouse Nlrp1b (BALB) or rat Nlrp1(CDF) proteins following treatment with LF (1 µg/ml) for 15 min or 2 h. Cleavage of CDF Nlrp1 leads to appearance of a 6-kDa HA-reactive band and cleavage of BALB Nlrp1b leads to a slightly smaller fragment. (B) IP (anti-HA pulldown) followed by anti-HA Western blotting of lysates from HT1080 cells expressing HA-tagged Nlrp1b proteins or control vector following treatment with LF (1 µg/ml, 30 min). Anti-HA cross-reactive bands not marked as HA-Nlrp1 also appear in vector-transfected controls. (C) Comparison of size of cleavage fragments generated after cleavage of BALB and NOD HA-tagged Nlrp1b (using conditions same as 2B), indicating the smaller size of the fragment generated following cleavage of the BALB protein (Western representative of five similar experiments). 10.1371/journal.pone.0049741.g003 Figure 3 Cleavage of mouse BALB118 and NOD118 Nlrp1 fusion proteins by LF. (A, B) In vitro cleavage of N-terminally 6His-GST-tagged aa 3-118 of Nlrp1b proteins. Purified proteins (0.53 mg/ml or 0.94 mg/ml, in A and B, respectively) were treated with the indicated molar ratios of LF, or with a 1∶10 molar ratio of the mutant LF E687C (LFm), for 4 h prior to SDS gel electrophoresis and Coomasie staining. F1 and F2 refer to two fragments generated following LF treatment. (C) GST-tagged or double alanine mutant variants (0.44-0.66 mg/ml) were treated with 33 µg/ml LF or LFm for 4 h prior to SDS gel electrophoresis and Coomassie staining. We also found that LF cleaves the HA-NOD (R) Nlrp1b protein ( Figure 2B ), as might be expected given its sequence identity to the HA-BALB (S) protein. Careful assessment in repeated experiments of the fragments generated by LF cleavage of these Nlrp1b proteins showed that the BALB cleavage fragment was slightly smaller than the NOD cleavage fragment, suggesting cleavage at two unique sites within the N-terminal regions of these proteins ( Figure 2C ). This finding suggested that one or more of the six polymorphisms immediately downstream of the potential LF cleavage sites (with the following amino acid changes: R56K, R67K, L79P, C85Y, I93V, V101I) may influence the site at which LF cleaves these proteins. 10.1371/journal.pone.0049741.g004 Figure 4 Caspase-1 activation in bone marrow-derived mouse macrophages. LPS-primed (1 µg/ml, 2 h) bone marrow-derived macrophages from Balb/cJ or NOD/LtJ mice were treated with LT (1 µg/ml) for 60 or 80 min, or with nigericin at indicated doses for 20 min. Cell lysates were analyzed by Western blotting for IL-1β, and the same samples were probed with caspase-1 p10 antibody to detect caspase-1 cleavage. To identify the exact cleavage sites, the first 118 aa of BALB (S) and NOD (R) Nlrp1b were expressed and purified as GST fusions (designated BALB118 and NOD118) ( Figure 1B , lines 3 and 4). Both BALB118 and NOD118 were cleaved by LF ( Figure 3A and 3B ). Interestingly, in repeated experiments we noted that NOD118 appeared to be more efficiently cleaved by LF than BALB118 (data not shown). This result corresponds to a similar phenomenon seen in cell lysates from HT1080 cells expressing full-length HA-Nlrp1b proteins, where NOD (R) Nlrp1b was cleaved more efficiently than BALB (S) Nlrp1b (data not shown). Furthermore, in analyses of the canonical cleavage pathway, where HA-tagged Nlrp1b proteins were immunoprecipitated from cells after delivery of LF to the cytosol by PA, NOD Nlrp1b protein appeared to be more efficiently cleaved ( Figure S1 ). Mass spectrometry analyses of BALB118 and NOD118 following cleavage by LF yielded masses of 30,486 and 31,257, indicating that LF cleaves between the two lysine-leucine bonds (after K38 and K44) ( Figure 1B , arrows). Both cleavage fragments were found following LF treatment of both proteins, albeit with differing prevalence in multiple independent cleavage runs (data not shown). Thus, it appears probable that the two fragments of 5-6 kDa observed following cleavage of the full length Nlrp1b proteins in cell lysates result from cleavage at the K38 and K44 cleavage sites, and that BALB (allele 1) Nlrp1b is preferentially cleaved at the first site while NOD (allele 3) Nlrp1b is preferentially cleaved at the second site. Substitution of the lysines at both sites with alanines ( Figure 1B , lines 5, 6) abrogated LF-mediated cleavage of the BALB118 and NOD118 proteins ( Figure 3C ). Since the data presented above suggests that Nlrp1b of NOD macrophages would be cleaved by LF delivered to the cytosol, the question arises as to why these cells do not undergo pyroptosis. One possible defect in downstream events could be a failure to activate caspase-1. However, we found that nigericin activated the Nlrp3 inflammasome to elicit efficient activation of caspase-1 and subsequent IL-1β cleavage in both NOD and BALB bone marrow-derived macrophages ( Figure 4 ), indicating that the caspase-1 activation pathway was fully functional. NOD macrophages also had no defect in flagellin-mediated Nlrc4 inflammasome activation (data not shown). As expected, LT activated caspase-1 in BALB (S) but not in NOD (R) macrophages ( Figure 4 ). A possible reason for these findings is that NOD macrophages may be deficient in protein's that are important to inflammasome assembly in an Nlrp1b-dependent manner, which does not impact Nlrp3-mediated caspase-1 activation. While this manuscript was in preparation, Frew et al. reported that C-terminal autoproteolysis of Nlrp1b within the FIIND (function-to-find) domain is required for inflammasome activation and that this proteolytic processing was absent in allele 3 (NOD) Nlrp1b due to a single polymorphism, V988D [10] . The site for C-terminal autoproteolysis in Nlrp1 has been identified [11] . Differentially C-terminally truncated variants of NOD (R) Nlrp1b, and BALB (S) Nlrp1b were also present in our expression system ( Figure 2 ). Frew et al. were able to eliminate proteolytic processing of BALB (allele 1) Nlrp1b by introducing the V988D substitution (from NOD, allele 3), and this mutation prevented the protein from being activated by LT [10] . Intriguingly, the authors were unable to restore LF responsiveness to the LT-nonresponsive NOD (R) Nlrp1b protein even after restoration of its autoproteolytic processing. These results, in combination with the findings reported here, suggest that the resistance of NOD (R) Nlrp1b to LF is not due to absence of a required LF cleavage event, or simply due to a deficiency in C-terminal autoproteolysis. It is possible, but unlikely, that preferential LF cleavage of NOD (R) Nlrp1b at residue K44, instead of K38, likely due to the presence of downstream polymorphisms altering folding in the N-terminus of this protein, is the reason for the defect in activation of this protein. It seems more likely that polymorphisms in other domains of this protein render it nonresponsive to LT. The truncated domain downstream of the leucine rich repeats in NOD Nlrp1b may result in altered conformation and folding of this protein in a manner that interferes with its unfolding to allow dimerization or caspase-1 recruitment. Thus, even when autoproteolysis at the C-terminus is restored and LT cleaves the N-terminus efficiently, the protein may be unable to act as an inflammasome platform. The deciphering of the mechanism for resistance to LT requires further experimentation. We propose, however, that cleavage of the N-terminus of both mouse and rat Nlrp1 proteins by LF may be required for activation of the inflammasome by LT, although it may be insufficient in the absence of other processing events. Materials and Methods Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All bone marrow harvests were performed in accordance to protocols approved by the NIAID Animal Care and Use Committee. Materials PA, LF, and LF E687C purification from avirulent Bacillus anthracis strains has been described [12] . Concentrations of LT correspond to the concentration of each toxin component (i.e., 1 µg/ml LT has 1 µg/ml PA and 1 µg/ml LF). GST-fusion proteins of BALB118 and NOD118 (described below) were expressed from pGEX-KG vectors in Escherichia coli BL21(DE3) and purified in a two-step process on glutathione-Sepharose and nickel chelate columns using standard purification protocols. High affinity anti-HA (cat# 11867423001, Roche Diagnostics, Indianapolis, IN), anti-IL-1β (cat# AF-401-NA, R&D Systems, Minneapolis, MN) and various IR-dye conjugated secondary antibodies (Licor Biosciences, Lincoln, NE and Rockland Immunochemicals, Gilbertsville, PA) were purchased. Nigericin was purchased from Calbiochem (San Diego, CA). Cell Culture and Transfections Cell culture and transfection methods have been previously described [6] . For stable transfections, Nlrp1b-expressing cell lines were derived by selection with hygromycin B (500 µg/ml; Invitrogen) for 15 days. Western blot with anti-HA antibody was performed to identify expression levels. Bone marrow-derived macrophages (BMDMs) were generated from marrow obtained from Balb/cJ or Nod/LtJ mice (Jackson Laboratories, Bar Harbor, ME) as previously described [13] . Constructs cDNA sequences for BALB and NOD mNlrp1b were synthesized by GeneArt Life Technologies (Grand Island, NY) and were cloned into the pIREShyg3 vector using Nhe1 and Xma1 sites. BALB118 and NOD118 sequences were synthesized by GeneArt Life Technologies and cloned along with added C-terminal His6 tags into the pGEX-KG vector using BamHI and EcoRI sites. Mutagenesis was performed using the QuikChange system (Agilent Technologies, La Jolla, CA) and sequencing was performed by Macrogen (Rockville, MD). Cleavage Assays To assess Nlrp1 cleavage in cell lysates, cells were grown to confluence and lysed in sucrose buffer (250 mM sucrose, 10 mM HEPES, 0.05 M EDTA, 0.2% Nonidet-P40) containing ZnCl 2 (1 µM) and NaCl (5 mM), followed by LF treatment at 37°C for varying times. Alternatively, cells were first treated with LT at 1 µg/ml for 5 h (canonical cleavage), followed by lysis in sucrose buffer containing 5 ng/ml LF inhibitor PT-168541-1 (gift of Alan Johnson, Panthera Biopharma). Cleavage reactions were analyzed by Western blot (WB) or immunoprecipitatoin (IP) followed by WB. For in vitro cleavage assays with purified proteins, BALB118 and NOD118 were incubated for varying times at 37°C with purified LF at varying concentrations in the presence of ZnCl 2 (1 µM) and NaCl (5 mM). Samples were separated on an 8-25% SDS-PAGE gel using the PhastSystem (GE Life Sciences, Piscataway, NJ) and visualized by Coomassie staining. Western Blots and Immunoprecipitation WB were performed using either anti-HA (1∶1000), anti-caspase-1 (1∶200), or anti-IL-1β (1∶2,500) and proteins were detected using the Odyssey Infrared Imaging System (Licor Biosciences). For IP, anti-HA antibody (Roche Diagnostics) was added to cell lysates (5-15 µg/ml) and samples were continuously mixed by rotation at 4°C for 1 h, followed by Protein A/G agarose (Santa Cruz Biotechnology) addition and continued overnight 4°C incubation with rotation. Beads were centrifuged at 4,000 rpm for 2 min and washed with 10 mM HEPES three times prior to elution of proteins using SDS loading buffer (10% SDS, 0.6 M DTT, 30% glycerol, 0.012% bromophenol blue, at 90°C, 5 min). Mass Spectrometry The molecular masses of the BALB118 and NOD118 proteins and their cleavage products were determined by liquid chromatography-electrospray mass spectrometry using an HP/Agilent 1100 MSD instrument (Hewlett Packard, Palo Alto, CA) at the NIDDK core facility, Bethesda, MD. Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All bone marrow harvests were performed in accordance to protocols approved by the NIAID Animal Care and Use Committee. Materials PA, LF, and LF E687C purification from avirulent Bacillus anthracis strains has been described [12] . Concentrations of LT correspond to the concentration of each toxin component (i.e., 1 µg/ml LT has 1 µg/ml PA and 1 µg/ml LF). GST-fusion proteins of BALB118 and NOD118 (described below) were expressed from pGEX-KG vectors in Escherichia coli BL21(DE3) and purified in a two-step process on glutathione-Sepharose and nickel chelate columns using standard purification protocols. High affinity anti-HA (cat# 11867423001, Roche Diagnostics, Indianapolis, IN), anti-IL-1β (cat# AF-401-NA, R&D Systems, Minneapolis, MN) and various IR-dye conjugated secondary antibodies (Licor Biosciences, Lincoln, NE and Rockland Immunochemicals, Gilbertsville, PA) were purchased. Nigericin was purchased from Calbiochem (San Diego, CA). Cell Culture and Transfections Cell culture and transfection methods have been previously described [6] . For stable transfections, Nlrp1b-expressing cell lines were derived by selection with hygromycin B (500 µg/ml; Invitrogen) for 15 days. Western blot with anti-HA antibody was performed to identify expression levels. Bone marrow-derived macrophages (BMDMs) were generated from marrow obtained from Balb/cJ or Nod/LtJ mice (Jackson Laboratories, Bar Harbor, ME) as previously described [13] . Constructs cDNA sequences for BALB and NOD mNlrp1b were synthesized by GeneArt Life Technologies (Grand Island, NY) and were cloned into the pIREShyg3 vector using Nhe1 and Xma1 sites. BALB118 and NOD118 sequences were synthesized by GeneArt Life Technologies and cloned along with added C-terminal His6 tags into the pGEX-KG vector using BamHI and EcoRI sites. Mutagenesis was performed using the QuikChange system (Agilent Technologies, La Jolla, CA) and sequencing was performed by Macrogen (Rockville, MD). Cleavage Assays To assess Nlrp1 cleavage in cell lysates, cells were grown to confluence and lysed in sucrose buffer (250 mM sucrose, 10 mM HEPES, 0.05 M EDTA, 0.2% Nonidet-P40) containing ZnCl 2 (1 µM) and NaCl (5 mM), followed by LF treatment at 37°C for varying times. Alternatively, cells were first treated with LT at 1 µg/ml for 5 h (canonical cleavage), followed by lysis in sucrose buffer containing 5 ng/ml LF inhibitor PT-168541-1 (gift of Alan Johnson, Panthera Biopharma). Cleavage reactions were analyzed by Western blot (WB) or immunoprecipitatoin (IP) followed by WB. For in vitro cleavage assays with purified proteins, BALB118 and NOD118 were incubated for varying times at 37°C with purified LF at varying concentrations in the presence of ZnCl 2 (1 µM) and NaCl (5 mM). Samples were separated on an 8-25% SDS-PAGE gel using the PhastSystem (GE Life Sciences, Piscataway, NJ) and visualized by Coomassie staining. Western Blots and Immunoprecipitation WB were performed using either anti-HA (1∶1000), anti-caspase-1 (1∶200), or anti-IL-1β (1∶2,500) and proteins were detected using the Odyssey Infrared Imaging System (Licor Biosciences). For IP, anti-HA antibody (Roche Diagnostics) was added to cell lysates (5-15 µg/ml) and samples were continuously mixed by rotation at 4°C for 1 h, followed by Protein A/G agarose (Santa Cruz Biotechnology) addition and continued overnight 4°C incubation with rotation. Beads were centrifuged at 4,000 rpm for 2 min and washed with 10 mM HEPES three times prior to elution of proteins using SDS loading buffer (10% SDS, 0.6 M DTT, 30% glycerol, 0.012% bromophenol blue, at 90°C, 5 min). Mass Spectrometry The molecular masses of the BALB118 and NOD118 proteins and their cleavage products were determined by liquid chromatography-electrospray mass spectrometry using an HP/Agilent 1100 MSD instrument (Hewlett Packard, Palo Alto, CA) at the NIDDK core facility, Bethesda, MD. Supporting Information Figure S1 Canonical cleavage of full length mouse Nlrp1b proteins by LT. HT1080 cells expressing HA-tagged mouse Nlrp1b (BALB or NOD) proteins were first treated with LF+PA (1 µg/ml, each) for 3 h. IP (anti-HA pulldown) was then performed on lysates followed by anti-HA Western blotting. (TIF) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC93069/

Bacillus Spore Inactivation Methods Affect Detection Assays

Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis , the causative agent of anthrax. Assays for detection in the laboratory often employ inactivated preparations of spores or nonpathogenic simulants. This study uses several common biodetection platforms to detect B. anthracis spores that have been inactivated by two methods and compares those data to detection of spores that have not been inactivated. The data demonstrate that inactivation methods can affect the sensitivity of nucleic acid- and antibody-based assays for the detection of B. anthracis spores. These effects should be taken into consideration when comparing laboratory results to data collected and assayed during field deployment.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783214/

THE SMALL ACID SOLUBLE PROTEINS (SASP α and SASP β) OF BACILLUS WEIHENSTEPHANENSIS AND B. MYCOIDES GROUP 2 ARE THE MOST DISTINCT AMONG THE B. CEREUS GROUP

The Bacillus cereus group includes Bacillus anthracis , Bacillus cereus , Bacillus thuringiensis , Bacillus mycoides and Bacillus weihenstephanensis . The small acid-soluble spore protein (SASP) β has been previously demonstrated to be among the biomarkers differentiating B. anthracis and B. cereus; SASP β of B. cereus most commonly exhibits one or two amino acid substitutions when compared to B. anthracis. SASP α is conserved in sequence among these two species. Neither SASP α nor β for B. thuringiensis , B. mycoides and B. weihenstephanensis have been previously characterized as taxonomic discriminators. In the current work molecular weight (MW) variation of these SASPs were determined by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) for representative strains of the 5 species within the B. cereus group. The measured MWs also correlate with calculated MWs of translated amino acid sequences generated from whole genome sequencing projects. SASP α and β demonstrated consistent MW among B. cereus , B. thuringiensis, and B. mycoides strains (group 1). However B. mycoides (group 2) and B. weihenstephanensis SASP α and β were quite distinct making them unique among the B. cereus group. Limited sequence changes were observed in SASP α (at most 3 substitutions and 2 deletions) indicating it is a more conserved protein than SASP β (up to 6 substitutions and a deletion). Another even more conserved SASP, SASP α-β type, was described here for the first time.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9203521/

Robust antibacterial activity of functionalized carbon nanotube- levofloxacine conjugate based on in vitro and in vivo studies

A new nano-antibiotic was synthesized from the conjugation of multi-walled carbon nanotubes with levofloxacin (MWCNT-LVX) through covalent grafting of drug with surface-modified carbon nanotubes in order to achieve an effective, safe, fast-acting nano-drug with the minimal side effects. This study is the first report on the evaluation of in vitro cell viability and antibacterial activity of nano-antibiotic along in addition to the in vivo antibacterial activity in a burn wound model. The drug-loading and release profile at different pH levels was determined using an ultraviolet–visible spectrometer. MWCNT-LVX was synthesized by a simple, reproducible and cost-effective method for the first time and characterized using various techniques, such as scanning electron microscope, transmission electron microscopy, and Brunauer–Emmett–Teller analysis, and so forth. The noncytotoxic nano-antibiotic showed more satisfactory in vitro antibacterial activity against Staphylococcus aureus compared to Pseudomona aeruginosa . The novel synthetic nano-drug possessed high loading capacity and pH-sensitive release profile; resultantly, it exhibited very potent bactericidal activity in a mouse S. aureus wound infection model compared to LVX. Based on the results, the antibacterial properties of the drug enhanced after conjugating with surface-modified MWCNTs. The nano-antibiotic has great industrialization potential for the simple route of synthesis, no toxicity, proper drug loading and release, low effective dose, and strong activity against wound infections. In virtue of unique properties, MWCNTs can serve as a controlled release and delivery system for drugs. The easy penetration to biological membranes and barriers can also increase the drug delivery at lower doses compared to the main drug alone, which can lead to the reduction of its side effects. Hence, MWCNTs can be considered a promising nano-carrier of LVX in the treatment of skin infections. Introduction The introduction of antibiotics induced a radical decline in the rate of morbidity and mortality at the beginning of the twentieth century 1 . However, inappropriate and excessive use of antibiotics caused the prevalence of antibiotic tolerance and resistance in the past decades 2 . The global crisis of antimicrobial resistance currently threatens both the effective prevention and treatment of a wide range of bacterial infectious diseases 3 . With no effective antibiotics, the medical world can once again return to the "pre-antibiotic era". Consequently, the production of novel antibacterial agents or the application of innovative strategies to improve the pharmacokinetic and reduce resistance to available antibacterial drugs is of paramount importance. Discovering new drugs is time-consuming and laborious. However, enhancing the efficacy of the existing antibacterial agents can accelerate the process of drug development through different techniques such as the combination therapies or the application of nanotechnology-based drug delivery systems 4 , 5 . The nanomaterials have attracted particular interest from various research groups due to their ideal physical and chemical properties, proper drug targeting efficiency, enhanced uptake, and suitable bio-distribution 5 . Recently, the application of nano-carriers has aroused considerable attention as the main strategy for improving antibacterial drug delivery, especially for the treatment of resistant infections 6 . Carbon nanotubes (CNTs) are known as one of the most promising nano-carriers in biomedical applications due to their unique intrinsic properties, including hollow structure, high surface area to volume ratios, easy surface modification, ideal compatibility, and remarkable cell membrane penetration 7 , 8 . However, the hydrophobic nature, poor dispersibility, and toxicity effects of multi-walled carbon nanotubes (MWCNTs) can limit their applications in biomedical studies 9 . In order to overcome these drawbacks, the surface chemistry can be modified using different functionalization approaches and also the dispersity and biocompatibility need to be enhanced under physiological conditions 9 , 10 . Moreover, the decreased toxicity of the functionalized MWCNTs has been confirmed in previous research studies 4 , 11 . Oxidation of MWCNTs with strong acids is known to be among the most promising and frequently employed approaches for surface modification 10 . This technique produces the carboxylic acid-functionalized MWCNTs (MWCNT-COOH) as efficient nano-platforms to immobilize multiple molecules by covalent bonds, hydrogen bonds, or π-π stacking interactions 12 , 13 . The functionalized MWCNTs with large surface areas possess a high loading capacity for various ligands such as peptides, proteins, nucleic acids, and drugs 14 . Moreover, they can be taken up by various tissues and cells without causing any damage, and depending on the types of surface functional groups, the loaded ligands can also be transferred 15 . The cellular internalization of CNTs contains three different mechanisms: endocytosis, phagocytosis, and direct translocation across the plasma membrane 15 . Studies have affirmed the effects of coating on the surface of CNTs in their internalization into mammalian cells 14 , 15 . The chemically modified MWCNTs have been also extensively studied for their various biological activities, including antifungal, antibacterial, antiviral, and potent anticancer activities 16 – 18 . The crisis of both antibiotic tolerance and resistance encouraged many researchers to investigate antibacterial drug delivery systems by MWCNTs 19 – 22 . Also, the evaluation of the antibacterial effects of the carriers themselves (MWCNTs) and their functionalized forms was noticed by research groups. A number of investigations have confirmed that the functionalization of MWCNTs improves their dispersion in aqueous media, resulting in the enhanced antibacterial activity, e.g. functionalizing with amino acids (arginine and lysine) 23 and surfactants (polysorbates, sodium dodecylbenzene sulfonate, and hexadecyltrimethylammonium bromide) 24 , 25 . Azizi-Lalabadi et al. have reviewed the antimicrobial activity of carbon nanomaterials and reported that MWCNTs with the surface factors (–OH and –COOH) do not display any bacteriostatic properties 26 . However, some studies have presented the proper bacteriostatic effects of the composites, including MWCNTs and silver nanoparticles against S. aureu s, P. aeruginosa and Escherichia coli , which can related to a powerful synergistic effect between MWCNTs and silver nanoparticles 26 . They also introduced the low-density polyethylene-based nanocomposites containing MWCNTs with antimicrobial activity against E. coli 26 . Nonetheless, the study of Ding and coworkers displayed the higher antibacterial activity of individually dispersed oxidized MWCNTs compared to aggregated raw MWCNTs 27 . Their study confirmed the importance of dispersion of MWCNTs in the antibacterial effect of CNTs. The antimicrobial activity of pristine and functionalized MWCNTs with mono-, di-, and triethanolamine (MEA, DEA, and TEA) against multiple bacterial species demonstrated the direct impact of functional group type on antimicrobial properties of mentioned nanotubes (MWCNT-TEA > MWCNT-DEA > MWCNT-MEA > pristine MWCNT) 28 . The results of these surveys are powerful evidence approving the key role of functional groups in antimicrobial activities of MWCNTs. The functionalization of MWCNTs with an antibiotic can also ameliorate its antibacterial activity. Spizzirri et al. have synthesized bioconjugates using gelatin, MWCNTs, and fluoroquinolones and demonstrated that the hybrid nanomaterials greatly enhance antimicrobial activities against Klebsiella pneumoniae and E. coli 29 . Selecting a suitable antibiotic in the drug delivery system has particular importance and can promote antimicrobial activity by a suitable synergistic effect between drug and nano-carrier. Levofloxacin (LVX) is a broad-spectrum, third-generation fluoroquinolone antibiotic with demonstrated activity against Gram-positive, Gram-negative, and anaerobic bacteria, which is mostly used for the treatment of many types of infections such as respiratory tract infections, post-inhalational anthrax, bacterial conjunctivitis, genitourinary infections, and skin and skin structure infections (SSSIs) 30 , 31 . SSSIs are an important issue in healthcare with a significant burden in infectious disease which is responsible to high rate of hospitalization. LVX with its once daily dose, great safety profile and easy transition to oral therapy is an proper selection to treat the full gamut of skin and skin structure infections. Levofloxacin, as a safe and effective medicine, received FDA approval in the United States in 1996 31 . LVX inhibits bacterial DNA gyrase and topoisomerase IV, two critical enzymes for the transcription, replication, and repair of bacterial DNA 32 . This antibiotic is considered a preferable drug for the management of burn-associated infections 33 . However, due to its widespread application, resistant bacterial strains began to emerge under numerous clinical conditions 34 . In developing countries, burns are recognized as one of the most common injuries with a high mortality rate 35 . Burns weaken the immune system and make the patient more susceptible to various infections as damaged skin cannot protect the body against microbes 36 . Therefore, a proper wound care is very important for reducing the risk of infection. The combination therapy with systemic and topical antibiotics, in comparison to systemic antibiotics alone, is more effective in the prevention of wound infection. Notably, topical antibiotics have the advantage of delivering high concentrations of antibiotics to the affected area, whereas systemic antibiotics need adsorption and then distribution throughout the body 37 . Due to blood vessel damage, systemic antibiotics cannot pass through the bloodstream and penetrate burn wounds 38 . Hence, the application of topical antibiotics is more common than systemic antibiotics 39 . The current innovative study aimed to design and synthesize a new nano-antibiotic based on the covalent functionalization of MWCNTs with levofloxacin. For the enhancement of the aqueous dispersity and biocompatibility of nano-antibiotics, MWCNTs were connected to LVX by the amino-PEG2-amine linker (Fig. 1 ). The in vitro loading, release profile, and in vitro antibacterial activity of nano-drug against S. aureus and P. aeruginosa were evaluated, as well. Moreover, the cytotoxicity of nano-antibiotic was assessed on the fibroblast cell line using the MTT assay. In the end, the topical delivery of the antibiotic against the S. aureus skin infection mouse model was examined. Figure 1 The covalent functionalization of MWCNTs with amino-PEG2-amine linker and levofloxacin. Abbreviations: MWCNTs: Multi-walled carbon nanotubes; LVX: Levofloxacin; PEG: Polyethylene glycol. Results Fourier-transform infrared (FTIR) spectroscopy Figure 2 exhibits the FTIR spectra of all samples. Figure 2 a demonstrates a strong band at 1629 cm −1 that was assigned to the C=O stretching vibrations in MWCNT-COOH. Accordingly, this could be attributed to the presence of carbonyl groups and was a proof of the successful oxidation of carbon nanotubes. The absorption bands at 1695 and 779 cm −1 could be ascribed to the C=O and C–Cl stretching vibrations, respectively, in MWCNT-COCl (Fig. 2 b), which validated the successful chlorination of MWCNT-COOH. After the amidation reaction (Fig. 2 c), the carbonyl stretching vibrations in MWCNT-NH 2 appeared at 1625 and 1679 cm −1 , which confirmed the elimination of acyl chloride and the formation of amide group (–CONH). Based on Fig. 2 c and d, the bands in the range of 3417 and 3412 cm −1 were related to the stretching vibration of the primary and secondary amines in MWCNT-NH 2 and MWCNT-LVX, respectively. The peaks at 3412, 1626, and 1110 cm −1 were respectively associated with the N–H, C=O, and C–N stretching vibrations in MWCNT-LVX. The peak at 2917 cm −1 corresponded to the methylene group from the polyethylene glycols (PEG)-linker. All FTIR findings validated the successful functionalization of MWCNTs. Figure 2 FTIR spectra of the samples: ( a ) MWCNT-COOH, ( b ) MWCNT-COCl, ( c ) MWCNT-NH 2 , and ( d ) MWCNT-LVX. Abbreviations: MWCNT: Multi-walled carbon nanotube; LVX: Levofloxacin; MWCNT-COOH: Carboxylic acid-functionalized MWCNTs; MWCNT-COCl: Acyl chloride-functionalized MWCNT; MWCNT-NH 2 : Amine-functionalized MWCNTs; MWCNT-LVX: Levofloxacin-loaded functionalized MWCNTs. The MWCNT-LVX data were compared with the standard spectrum of LVX and MWCNT-NH 2 carrier, and their changes were noticed. Briefly, the bands of LVX were reported as follows: 3268 cm −1 (OH), 2959–2803 cm −1 (CH 2 , CH 3 ), 1722 cm −1 (C=O acid, stretching vibration of the COOH), 1622 cm −1 (C=O ring), and 839 cm −1 (C–F). Aromatic hydrocarbons showed absorptions in the regions 1585 and 1404 cm −1 40 . The observation of a relative increase in the carbonyl band's intensity at 1626 and 1679 cm −1 confirms the formation of new amide bonds, MWCNT-NH 2 and LVX. Their interaction was also resulted in a broad band at 3412 cm −1 (3100–3600 cm −1 region), which can be related to –OH stretching vibration of LVX and –NH– stretching vibration of amide groups. The existence of a weak bond at 1715 cm −1 can be assigned to the carbonyl band of LVX. The mentioned peaks (3412 and 1715 cm −-1 ) in drug-loaded formulation may be related to the non-covalent interaction of the drug with the nano-carrier (MWCNT-NH 2 ). Out-of-plane wagging at 650 cm -1 is the characteristic of primary amines. Raman analysis and X-ray diffraction (XRD) patterns Figure 3 a shows the Raman spectra of MWCNT-COOH and MWCNT-LVX. Two main peaks in the Raman spectra were appeared at 1344.20 and 1570.97 cm −1 , known as D and G bonds, respectively. The D band is related to disordered carbon atoms of MWCNTs corresponding to SP 3 hybridization, and the G band shows the SP 2 hybridization of carbon atoms in the graphene sheets. Area ratio of the D and G bonds (I D /I G ) can be used to assess the amount of defects in the nanoparticle structure. I D /I G ratio increased for MWCNT-LVX (I D /I G = 1.168), which affirms the successful conversion of MWCNT-COOH to MWCNT-LVX. In the absence of amorphous carbon, the increase of I D is related to the elevation of carbon containing SP 3 hybridized and implies the successful functionalization reaction. Figure 3 ( a ) Raman spectroscopy of MWCNT-COOH, and MWCNT-LVX; ( b ) XRD pattern of MWCNT-COOH, MWCNT-NH 2 , and MWCNT-LVX. Abbreviations: MWCNT: Multi-walled carbon nanotube; LVX: Levofloxacin; MWCNT-COOH: Carboxylic acid-functionalized MWCNTs; MWCNT-NH 2 : Aamine-functionalized MWCNTs; MWCNT-LVX: Levofloxacin-loaded functionalized MWCNTs. Figure 3 b displays XRD spectroscopy patterns of MWCNT-COOH, MWCNT-NH 2 , and MWCNT-LVX. The XRD patterns of the oxidized CNTs and the functionalized CNTs have diffraction peaks at corresponding positions, indicating that MWCNT-NH 2 and MWCNT-LVX still have the same tubular structure compared to MWCNT-COOH without any change in the lattice spacing. The intense peak at θ = 26° is indexed as the (002) reflection of the hexagonal graphite structure and the peak around 43° is resulted to the (100) graphitic planes. The XRD pattern clearly confirms the formation of nano-carrier (MWCNT-NH 2 ) due to decreased intensity of peaks. The diffraction pattern of MWCNT-LVX showed the broad peak at 26° was deconvoluted into two peaks; the first peak centered at 2θ = 26.04°, and the other peak centered at 26.34°, clearly confirms the formation of nano-drug. Field emission scanning electron microscopy (FE-SEM) The surface morphology of the modified and functionalized MWCNTs was investigated using FE-SEM. The FE-SEM image of MWCNT-COOH indicated its smooth, long, tortuous, and agglomerated structures (Fig. 4 -Ia). However, the MWCNT-LVX showed a lower degree of entanglement and agglomeration in addition to rugged appearances compared to MWCNT-LVX. This observation is indicative of defect creation in nanotubes wherein the functional groups and drug were loaded, resulting in the appearance of irregular and branched sites on the MWCNTs surface (Fig. 4 -Ib and Ic). Figure 4 -Id validated fully coated surfaces, good dispersion, and well-organized assembly of MWCNTs-LVX. Figure 4 ( I ) FE-SEM images: ( a ) MWCNT-COOH, and ( b – d ) MWCNT-LVX with different magnifications: ( b ) 1 μm; 10.00 KX, ( c ) 200 nm; 20.00 KX, ( d ) 200 nm; 50.00 KX. ( II ) EDX mappings of the MWCNT-LVX, showing the distribution of C, N, O and F elements. ( III ) ( a ) FE-SEM image of MWMWCNT-LVX mapping analysis, ( b ) corresponding elemental mapping of C, N, O, and F elements. Abbreviations: FE-SEM: Field emission scanning electron microscopy; MWCNT: Multi-walled carbon nanotube; LVX: Levofloxacin; MWCNT-COOH: Carboxylic acid-functionalized MWCNTs; MWCNT-LVX: Levofloxacin-loaded functionalized MWCNTs. Energy dispersive X-ray spectroscopy (EDX) EDX analysis was performed to quantify the components of MWCNT-LVX. In Fig. 4 -II, EDX revealed strong signals for carbon (C) and small fractions of oxygen (O), which might be due to the acidic groups on MWCNT-COOH. The presence of both nitrogen (N) and fluorine (F) signals could also be attributed to amino-PEG2-amine linker and LVX and confirmed successful drug loading. Additionally, qualitative information about the distribution of various chemical elements in the nano-drug is presented in Fig. 4 -IIIa and b. Thereafter, the elemental map analysis revealed a good dispersion of C, N, O, and F atoms on the surface of MWCNT-LVX. Figure 5 TEM image of the samples: ( I ) MWCNT-COOH, and ( II ) MWCNT-LVX with different magnifications (nm). Abbreviations: TEM: Transmission electron microscopy; MWCNT: Multi-walled carbon nanotube; LVX: Levofloxacin; MWCNT-COOH: Carboxylic acid-functionalized MWCNTs; MWCNT-LVX: Levofloxacin-loaded functionalized MWCNTs. Figure 6 ( a ) Standard curve, absorbance at 290 nm vs. various concentrations of levofloxacin detected with UV–vis spectrometer; ( b ) pH responsive levofloxacin release profile of MWCNT-LVX within 144 h at various pHs (5.5, 7.4, and 10.5); ( c ) Drug release data fitted to various kinetic models (zero order, first order, Higuchi , and Korsmeyer–Peppas) obtained within 144 h at various pHs (5.5, 7.4, and 10.5). Abbreviations: MWCNT: Multi-walled carbon nanotube; LVX: Levofloxacin; MWCNT-LVX: Levofloxacin-loaded functionalized MWCNTs. Figure 7 Effects of different doses of MWCNT-LVX, MWCNT-NH 2 , and MWCNT-COOH on cell viability. Data are shown as mean ± standard deviation from six separate experiments (n = 6). * and $ indicate statistically significant differences in cell viability. All deviations were taken as statistically significant if p  95%) relative to the amidated and oxidized CNTs, which can be justified by proper surface modifications in MWCNT- LVX conjugate. Cell viability at different concentrations of MWCNT-LVX (31.25, 62.50, 125, and 250 μg/mL) was in the narrow range of 95.61–96.60%. These results confirmed the noncytotoxic effect of nano-antibiotic. In vivo therapeutic efficacy of MWCNT-LVX Figure 8 presents the bactericidal effects of MWCNT-LVX on a burn infection model caused by S. aureus . Accordingly, MWCNT-LVX at 312.5, 156.25, and 78.125 μg/mL concentrations showed no growth of bacterial colonies throughout the treatment. This outcome could be attributed to the efficiency of the drug delivery system in MWCNT-LVX compared to the LVX-treated group (Fig. 8 a). The bacterial growth inhibition is more noticeable in the images of Petri disks (Fig. 8 b). Statistical analysis showed that MWCNT-LVX (at all three doses) significantly suppressed the bacterial count in the infected burn wound compared to the control group ( p  95%) relative to the amidated and oxidized CNTs, which can be justified by proper surface modifications in MWCNT- LVX conjugate. Cell viability at different concentrations of MWCNT-LVX (31.25, 62.50, 125, and 250 μg/mL) was in the narrow range of 95.61–96.60%. These results confirmed the noncytotoxic effect of nano-antibiotic. In vivo therapeutic efficacy of MWCNT-LVX Figure 8 presents the bactericidal effects of MWCNT-LVX on a burn infection model caused by S. aureus . Accordingly, MWCNT-LVX at 312.5, 156.25, and 78.125 μg/mL concentrations showed no growth of bacterial colonies throughout the treatment. This outcome could be attributed to the efficiency of the drug delivery system in MWCNT-LVX compared to the LVX-treated group (Fig. 8 a). The bacterial growth inhibition is more noticeable in the images of Petri disks (Fig. 8 b). Statistical analysis showed that MWCNT-LVX (at all three doses) significantly suppressed the bacterial count in the infected burn wound compared to the control group ( p  90%, mean diameter ~ 20–30 nm, length ~ 10–30 μm, SSA > 110 m 2 /g) were purchased from US Research Nanomaterials (USA). Nitric acid, hydrogen peroxide 30%, sulfuric acid, sodium hydroxide, Mueller Hinton agar (MHA), and Mueller Hinton broth (MHB) were purchased from Merck Company (Darmstadt, Germany). Thionyl chloride, levofloxacin, 1,8-diamino-3,6-dioxaoctane, triethylamine, tetrahydrofuran (THF), 2-(1 H -benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (Germany). Oxidized MWCNTs as ~ 6% w/w were prepared based on literature 86 , 87 . A microplate reader (ELx808, BioTek, USA) was also applied to measure the absorbance. FT-IR spectra were recorded using a Perkin Elmer Spectrum Two FT-IR Spectrometer, USA. Raman measurements were performed using a XploRA plus Raman microscope with a 532 nm laser (Horiba, Japan). The XRD patterns were recorded on an X-ray diffractometer (PW1730, Philips, Netherlands) with Cuk α radiation (λ equal to 1.54056 à ) radiation. FE-SEM (ZEISS-Sigma VP model, Germany) was applied to examine the morphology of the samples. An energy-dispersive X-ray (EDX, Oxford Instruments, UK) analysis was performed to determine the elemental composition of MWCNT- LVX. The distribution pattern of structural elements was also determined by elemental mapping images. Morphology and structural transformations of the samples were investigated by TEM (ZEISS-EM10C-100 kV model, Germany). The adsorption and desorption isotherms of nitrogen were measured using the BELSORP-mini II apparatus (MicrotracBEL, Japan). The elemental analysis (2400 series II CHNS elemental analyzer, Perkin-Elmer Co., USA) determined the carbon, hydrogen, and nitrogen contents. Ethical considerations In this study, the in vivo experiments and animal care were approved by the Biomedical Research Ethics of Lorestan University of Medical Sciences (Ethics Code: IR.LUMS.REC.1397.199). All methods were carried out in accordance with the animal welfare guidelines and regulations 88 . All experiments were reported in conformity with ARRIVE guideline 2.0. Functionalization of MWCNTs The functionalization of oxidized carbon nanotube (MWCNT-COOH) was performed according to a previous method with some modifications 89 . MWCNT-COOH (200 mg) along with excess thionyl chloride (30 mL) as a reagent and solvent were sonicated for 30 min within an ultrasonic bath, and then the reaction mixture was refluxed at 80 °C for 24 h. The obtained product, acyl chloride-functionalized MWCNT (MWCNT-COCl) was then filtered under vacuum by a 0.2 μm porous polytetrafluoroethylene (PTFE) membrane filter (Whatman) and washed with dry THF (3 × 50 mL) to remove the excess of thionyl chloride. The corresponding acyl chloride without further purification was immediately mixed with 1,8-diamino-3,6-dioxaoctane (2.5 mL) in 75 mL of dry THF and refluxed at 80 °C for 48 h to praper amine-functionalized MWCNT (MWCNT-NH 2 ). Subsequently, the mixture was cooled to room temperature and filtered under vacuum on a 0.2 μm PTFE filter. Finally, the resulting precipitate (MWCNT-NH 2 ) was washed with dry THF (3 × 50 mL) and then dried in a vacuum oven for 4 h at 50 °C (Fig. 9 ). Synthesis of nano-antibiotic (MWCNT-LVX) A volume of 200 mg of LVX, 86 mg of TBTU, and trimethylamine (0.118 mL) in ethyl acetate (25 mL) were stirred under argon atmosphere for 1 h. Thereafter, 100 mg of MWCNT-NH 2 was added to the mixture, sonicated for 1 h within an ultrasonic bath, magnetically stirred for 24 h at room temperature, and then filtered under vacuum on 0.2 μm PTFE filter. The solid product (MWCNT-LVX) was washed with ethyl acetate (3 × 50 mL) and methanol (3 × 50 mL) and then dried in a vacuum oven at 60 °C for 8 h 90 . Loading LVX on PEGylated MWCNTs The calibration curve of LVX was plotted based on the maximum wavelength of (λ Max ) 290 nm using a UV–Vis spectrophotometer (R 2 = 0.9981, y = 29.182x + 0.045). Next, MWCNT-LVX and its blank sample (without loaded drug) were prepared similarly according to the synthesis procedure of nano-antibiotic as mentioned above. After 24 h, both the reaction mixtures were centrifuged at 2000 rpm (CMF 15KR, Tigra, Poland) for three times until the supernatant became colorless. The supernatant was filtered using a 0.2 μm PTFE membrane filter, and solid residues were then washed with ethyl acetate and methanol (50 × 3 mL each). Then the total volume of filtrate plus wash solutions were collected and measured. The absorbance of the solution was determined at 290 nm using a UV–Vis spectrophotometer (Cecil CE 1021, UK). Finally, the drug entrapment efficiency (EE) and LE were obtained using the following formulas 91 : \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\% { ext{Entrapment}}\;{ ext{efficiency}}\left( {\% { ext{EE}}} ight) = rac{{\left[ {{ ext{Drug}}} ight]{ ext{total}} - \left[ {{ ext{Drug}}} ight]{ ext{supernant}}}}{{\left[ {{ ext{Drug}}} ight]{ ext{total}}}} imes 100$$\end{document} % Entrapment efficiency % EE = Drug total - Drug supernant Drug total × 100 \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\% { ext{Entrapment}}\;{ ext{efficiency}}\left( {\% { ext{LE}}} ight) = rac{{{ ext{Weight}}\;{ ext{of}}\;{ ext{loaded}}\;{ ext{drug}}\;{ ext{in}}\;{ ext{MWCNTs}}}}{{{ ext{Weight}}\;{ ext{of}}\;{ ext{total}}\;{ ext{MWCNTs}}}}$$\end{document} % Entrapment efficiency % LE = Weight of loaded drug in MWCNTs Weight of total MWCNTs In vitro drug release test To investigate the drug release of MWCNT-LVX under acidic, neutral, and basic environments, PBS was prepared at pHs 5.5, 7.4, and 10.5. Nano-drug (2.5 mg) was dispersed in dialysis bags (12–14 kDa MWCO, 23 mm flat width, Sigma-Aldrich) containing 1 mL of PBS. Accordingly, each dialysis bag was separately immersed in 50 mL of PBS (pHs 5.5, 7.4, 10.5) and then stirred at room temperature at a speed of 80 rpm. At various time intervals between 2 and 144 h, 1 mL of each sample was taken out to test LVX concentration at 290 nm (Cecil CE 1021, UK). After each sampling time, 1 mL of fresh buffer was replaced to maintain a constant initial volume 92 . The drug release kinetics were determined using specific mathematical models and plotted according to zero order (cumulative % of drug released vs. time), first order (log cumulative % of drug remain), Higuchi model (cumulative % of drug released vs. square root of time), and Korsmeyer–Peppas model (log cumulative % of drug released vs. log time) equations 93 . In vitro antimicrobial activity of MWCNT-LVX The antibacterial activity of nano-antibiotic was determined using MIC and MBC tests according to Clinical Laboratory Standard Institute (CLSI) 94 . Two different strains of pathogenic bacteria, including S. aureus (ATCC 25,923) and P. aeruginosa (ATCC 27,853), were prepared from Microbial Collection (Pasteur Institute of Iran, Tehran) and cultured on blood agar at 37 °C overnight to obtain single and pure colonies. Subsequently, the optical density (OD) of bacterial suspensions were measured in the range of 600–625 nm using a UV–Vis spectrophotometer to attain 0.5 McFarland turbidity standards and then diluted (~ 10 6 CFU/mL). The aqueous suspensions (10 mg/mL) of MWCNT-LVX, MWCNT-NH 2 , and MWCNT-COOH, in addition to LVX solution (1 mg/mL) were prepared. The test compounds (100 μL) were added to the first row of 96-well plates containing 100 μL of MHB, then mixed and transferred to the second row. This procedure was repeated till the last well, from which 100 μL was removed. Finally, 100 μL of the microbial suspension was added to all wells. After incubation at 35 °C for 18 and 36 h, the turbidity of the wells were assessed, and the MIC was defined as the lowest concentration with no visible bacterial growth. For the MBC test, 100 μL of the MIC well was streaked on the MHA plates and incubated at 35 °C for 18 h. The MBC value was defined as the lowest bactericidal concentration without any bacterial colony growth on the agar plate. These tests were separately performed for both strains at different pHs 5.5, 7.4, and 10.5 in triplicate. Cell viability assay Cell viability was evaluated on mouse fibroblast cell line L929 (NCBI C161 was obtained from the National Cell Bank of Iran [NCBI], Pasteur Institute of Iran) using MTT colorimetric assay 95 . The cells were grown in RPMI 1640 medium (Gibco, Waltham, MA) containing 10% fetal bovine serum (FBS) and incubated in 90% humidified atmosphere with 5% CO 2 at 37 °C. Briefly, 1 × 10 4 cells/well in 100 μL of RPMI1640 medium were seeded onto 96-well tissue culture plates and incubated at 37 °C for 24 h, for the cells adhesion. Afterward, the culture medium was replaced with 90 μL of the samples (MWCNT-LVX [250, 125, 62.5, and 31.25 μg/mL]), MWCNT-NH 2 [250 μg/mL], or MWCNT-COOH [250 μg/mL]) and 10 μL of FBS. The control wells contained only RPMI1640 and FBS. After 48 h, the supernatants were changed with 100 μL of MTT solution (0.5 mg/mL), and the plates were incubated at 37 °C for 4 h. Then, the reaction solutions were removed, and the formazan crystals dissolved in isopropanol (100 μL). The plate was finally incubated on a shaker for 15 min, and the absorbance was measured at 570 nm using a microplate reader. This experiment was performed in sextuplicate, and the cell viability was calculated using the following expression: \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\% { ext{Viability}} = \left( { rac{{{ ext{mean}}\;{ ext{OD}}\;{ ext{of}}\;{ ext{sample}}}}{{{ ext{mean}}\;{ ext{OD}}\;{ ext{of}}\;{ ext{control}}}}} ight) imes 100$$\end{document} % Viability = mean OD of sample mean OD of control × 100 In vivo antimicrobial activity of MWCNT-LVX A total of 25 female NMRI strain mice (aged almost eight weeks with bodyweight of about 30–35 g) were purchased from the Pasteur Institute of Iran and kept for one week under standard conditions (24 ± 2 °C and 52% humidity) with adequate food and water. Mice were randomly divided into five equal groups, including three treatment groups and two control groups. Afterward, the S. aureus (ATCC 25,923) suspensions (approximately 10 5 CFU/mL) were cultured on blood agar. Mice were then anesthetized intraperitoneally [ketamine/xylazine, (5/1 mg/kg)]. Subsequently, their dorsal hair was shaved, cleaned, and disinfected with 70% (v/v) ethanol. Burn wounds (the second-degree) were created by a cylindrical metal rod (10 mm diameter, 50 g weight) which was heated to 100 °C and then pressed for 5 s on the dorsal thoracic region in the low part of the mouse body about 1 cm away from the vertebral column in the right side. The criteria to diagnosis of second degree burn were determined by major following indications including alteration of skin colour to brown, skin roughness and ruffling on the surrounding the burn wound. The injured mice were immediately placed in separate cages. After 1 h, all burn wounds were inoculated with 100 μL of the bacterial suspension (10 5 CFU/mL) and treated with 100 μL of LVX (0.488 μg/mL, positive control) and different concentrations of MWCNT-LVX aqueous solutions (312.5, 156.25, and 78.125 μg/mL) at 1 h post-infection. MWCNT-LVX solutions were dispersed completely by sonication before topical administration. The volume of the whole solution was transferred to the wound in two steps (2 × 50 μL) without any wastage. The group with no treatment was considered as a negative control. After 24 h, the mice were humanely killed, and the burned skin lesions were removed using sterile surgicassors and homogenized in 1 mL of sterile PBS. The tissue samples were serially diluted six-fold, and then all six dilutions were cultured on blood agar plates. After incubation for 24 h at 37 °C, the number of colonies was counted, and the results were expressed as the mean ± standard deviation of CFU/mL per skin sample 96 . Statistical analysis Statistical analysis was performed using SPSS software (version 22). Data were analyzed by one-way analysis of variance (ANOVA), Shapiro–Wilk and Kolmogorov–Smirnov tests followed by Tukey post-hoc test. Data were reported as a mean value with its standard deviation indicated (mean ± SD), and p -values ≤ 0.05 were considered statistically significant. Materials and instrument MWCNTs (purity > 90%, mean diameter ~ 20–30 nm, length ~ 10–30 μm, SSA > 110 m 2 /g) were purchased from US Research Nanomaterials (USA). Nitric acid, hydrogen peroxide 30%, sulfuric acid, sodium hydroxide, Mueller Hinton agar (MHA), and Mueller Hinton broth (MHB) were purchased from Merck Company (Darmstadt, Germany). Thionyl chloride, levofloxacin, 1,8-diamino-3,6-dioxaoctane, triethylamine, tetrahydrofuran (THF), 2-(1 H -benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (Germany). Oxidized MWCNTs as ~ 6% w/w were prepared based on literature 86 , 87 . A microplate reader (ELx808, BioTek, USA) was also applied to measure the absorbance. FT-IR spectra were recorded using a Perkin Elmer Spectrum Two FT-IR Spectrometer, USA. Raman measurements were performed using a XploRA plus Raman microscope with a 532 nm laser (Horiba, Japan). The XRD patterns were recorded on an X-ray diffractometer (PW1730, Philips, Netherlands) with Cuk α radiation (λ equal to 1.54056 à ) radiation. FE-SEM (ZEISS-Sigma VP model, Germany) was applied to examine the morphology of the samples. An energy-dispersive X-ray (EDX, Oxford Instruments, UK) analysis was performed to determine the elemental composition of MWCNT- LVX. The distribution pattern of structural elements was also determined by elemental mapping images. Morphology and structural transformations of the samples were investigated by TEM (ZEISS-EM10C-100 kV model, Germany). The adsorption and desorption isotherms of nitrogen were measured using the BELSORP-mini II apparatus (MicrotracBEL, Japan). The elemental analysis (2400 series II CHNS elemental analyzer, Perkin-Elmer Co., USA) determined the carbon, hydrogen, and nitrogen contents. Ethical considerations In this study, the in vivo experiments and animal care were approved by the Biomedical Research Ethics of Lorestan University of Medical Sciences (Ethics Code: IR.LUMS.REC.1397.199). All methods were carried out in accordance with the animal welfare guidelines and regulations 88 . All experiments were reported in conformity with ARRIVE guideline 2.0. Functionalization of MWCNTs The functionalization of oxidized carbon nanotube (MWCNT-COOH) was performed according to a previous method with some modifications 89 . MWCNT-COOH (200 mg) along with excess thionyl chloride (30 mL) as a reagent and solvent were sonicated for 30 min within an ultrasonic bath, and then the reaction mixture was refluxed at 80 °C for 24 h. The obtained product, acyl chloride-functionalized MWCNT (MWCNT-COCl) was then filtered under vacuum by a 0.2 μm porous polytetrafluoroethylene (PTFE) membrane filter (Whatman) and washed with dry THF (3 × 50 mL) to remove the excess of thionyl chloride. The corresponding acyl chloride without further purification was immediately mixed with 1,8-diamino-3,6-dioxaoctane (2.5 mL) in 75 mL of dry THF and refluxed at 80 °C for 48 h to praper amine-functionalized MWCNT (MWCNT-NH 2 ). Subsequently, the mixture was cooled to room temperature and filtered under vacuum on a 0.2 μm PTFE filter. Finally, the resulting precipitate (MWCNT-NH 2 ) was washed with dry THF (3 × 50 mL) and then dried in a vacuum oven for 4 h at 50 °C (Fig. 9 ). Synthesis of nano-antibiotic (MWCNT-LVX) A volume of 200 mg of LVX, 86 mg of TBTU, and trimethylamine (0.118 mL) in ethyl acetate (25 mL) were stirred under argon atmosphere for 1 h. Thereafter, 100 mg of MWCNT-NH 2 was added to the mixture, sonicated for 1 h within an ultrasonic bath, magnetically stirred for 24 h at room temperature, and then filtered under vacuum on 0.2 μm PTFE filter. The solid product (MWCNT-LVX) was washed with ethyl acetate (3 × 50 mL) and methanol (3 × 50 mL) and then dried in a vacuum oven at 60 °C for 8 h 90 . Loading LVX on PEGylated MWCNTs The calibration curve of LVX was plotted based on the maximum wavelength of (λ Max ) 290 nm using a UV–Vis spectrophotometer (R 2 = 0.9981, y = 29.182x + 0.045). Next, MWCNT-LVX and its blank sample (without loaded drug) were prepared similarly according to the synthesis procedure of nano-antibiotic as mentioned above. After 24 h, both the reaction mixtures were centrifuged at 2000 rpm (CMF 15KR, Tigra, Poland) for three times until the supernatant became colorless. The supernatant was filtered using a 0.2 μm PTFE membrane filter, and solid residues were then washed with ethyl acetate and methanol (50 × 3 mL each). Then the total volume of filtrate plus wash solutions were collected and measured. The absorbance of the solution was determined at 290 nm using a UV–Vis spectrophotometer (Cecil CE 1021, UK). Finally, the drug entrapment efficiency (EE) and LE were obtained using the following formulas 91 : \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\% { ext{Entrapment}}\;{ ext{efficiency}}\left( {\% { ext{EE}}} ight) = rac{{\left[ {{ ext{Drug}}} ight]{ ext{total}} - \left[ {{ ext{Drug}}} ight]{ ext{supernant}}}}{{\left[ {{ ext{Drug}}} ight]{ ext{total}}}} imes 100$$\end{document} % Entrapment efficiency % EE = Drug total - Drug supernant Drug total × 100 \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\% { ext{Entrapment}}\;{ ext{efficiency}}\left( {\% { ext{LE}}} ight) = rac{{{ ext{Weight}}\;{ ext{of}}\;{ ext{loaded}}\;{ ext{drug}}\;{ ext{in}}\;{ ext{MWCNTs}}}}{{{ ext{Weight}}\;{ ext{of}}\;{ ext{total}}\;{ ext{MWCNTs}}}}$$\end{document} % Entrapment efficiency % LE = Weight of loaded drug in MWCNTs Weight of total MWCNTs In vitro drug release test To investigate the drug release of MWCNT-LVX under acidic, neutral, and basic environments, PBS was prepared at pHs 5.5, 7.4, and 10.5. Nano-drug (2.5 mg) was dispersed in dialysis bags (12–14 kDa MWCO, 23 mm flat width, Sigma-Aldrich) containing 1 mL of PBS. Accordingly, each dialysis bag was separately immersed in 50 mL of PBS (pHs 5.5, 7.4, 10.5) and then stirred at room temperature at a speed of 80 rpm. At various time intervals between 2 and 144 h, 1 mL of each sample was taken out to test LVX concentration at 290 nm (Cecil CE 1021, UK). After each sampling time, 1 mL of fresh buffer was replaced to maintain a constant initial volume 92 . The drug release kinetics were determined using specific mathematical models and plotted according to zero order (cumulative % of drug released vs. time), first order (log cumulative % of drug remain), Higuchi model (cumulative % of drug released vs. square root of time), and Korsmeyer–Peppas model (log cumulative % of drug released vs. log time) equations 93 . In vitro antimicrobial activity of MWCNT-LVX The antibacterial activity of nano-antibiotic was determined using MIC and MBC tests according to Clinical Laboratory Standard Institute (CLSI) 94 . Two different strains of pathogenic bacteria, including S. aureus (ATCC 25,923) and P. aeruginosa (ATCC 27,853), were prepared from Microbial Collection (Pasteur Institute of Iran, Tehran) and cultured on blood agar at 37 °C overnight to obtain single and pure colonies. Subsequently, the optical density (OD) of bacterial suspensions were measured in the range of 600–625 nm using a UV–Vis spectrophotometer to attain 0.5 McFarland turbidity standards and then diluted (~ 10 6 CFU/mL). The aqueous suspensions (10 mg/mL) of MWCNT-LVX, MWCNT-NH 2 , and MWCNT-COOH, in addition to LVX solution (1 mg/mL) were prepared. The test compounds (100 μL) were added to the first row of 96-well plates containing 100 μL of MHB, then mixed and transferred to the second row. This procedure was repeated till the last well, from which 100 μL was removed. Finally, 100 μL of the microbial suspension was added to all wells. After incubation at 35 °C for 18 and 36 h, the turbidity of the wells were assessed, and the MIC was defined as the lowest concentration with no visible bacterial growth. For the MBC test, 100 μL of the MIC well was streaked on the MHA plates and incubated at 35 °C for 18 h. The MBC value was defined as the lowest bactericidal concentration without any bacterial colony growth on the agar plate. These tests were separately performed for both strains at different pHs 5.5, 7.4, and 10.5 in triplicate. Cell viability assay Cell viability was evaluated on mouse fibroblast cell line L929 (NCBI C161 was obtained from the National Cell Bank of Iran [NCBI], Pasteur Institute of Iran) using MTT colorimetric assay 95 . The cells were grown in RPMI 1640 medium (Gibco, Waltham, MA) containing 10% fetal bovine serum (FBS) and incubated in 90% humidified atmosphere with 5% CO 2 at 37 °C. Briefly, 1 × 10 4 cells/well in 100 μL of RPMI1640 medium were seeded onto 96-well tissue culture plates and incubated at 37 °C for 24 h, for the cells adhesion. Afterward, the culture medium was replaced with 90 μL of the samples (MWCNT-LVX [250, 125, 62.5, and 31.25 μg/mL]), MWCNT-NH 2 [250 μg/mL], or MWCNT-COOH [250 μg/mL]) and 10 μL of FBS. The control wells contained only RPMI1640 and FBS. After 48 h, the supernatants were changed with 100 μL of MTT solution (0.5 mg/mL), and the plates were incubated at 37 °C for 4 h. Then, the reaction solutions were removed, and the formazan crystals dissolved in isopropanol (100 μL). The plate was finally incubated on a shaker for 15 min, and the absorbance was measured at 570 nm using a microplate reader. This experiment was performed in sextuplicate, and the cell viability was calculated using the following expression: \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$\% { ext{Viability}} = \left( { rac{{{ ext{mean}}\;{ ext{OD}}\;{ ext{of}}\;{ ext{sample}}}}{{{ ext{mean}}\;{ ext{OD}}\;{ ext{of}}\;{ ext{control}}}}} ight) imes 100$$\end{document} % Viability = mean OD of sample mean OD of control × 100 In vivo antimicrobial activity of MWCNT-LVX A total of 25 female NMRI strain mice (aged almost eight weeks with bodyweight of about 30–35 g) were purchased from the Pasteur Institute of Iran and kept for one week under standard conditions (24 ± 2 °C and 52% humidity) with adequate food and water. Mice were randomly divided into five equal groups, including three treatment groups and two control groups. Afterward, the S. aureus (ATCC 25,923) suspensions (approximately 10 5 CFU/mL) were cultured on blood agar. Mice were then anesthetized intraperitoneally [ketamine/xylazine, (5/1 mg/kg)]. Subsequently, their dorsal hair was shaved, cleaned, and disinfected with 70% (v/v) ethanol. Burn wounds (the second-degree) were created by a cylindrical metal rod (10 mm diameter, 50 g weight) which was heated to 100 °C and then pressed for 5 s on the dorsal thoracic region in the low part of the mouse body about 1 cm away from the vertebral column in the right side. The criteria to diagnosis of second degree burn were determined by major following indications including alteration of skin colour to brown, skin roughness and ruffling on the surrounding the burn wound. The injured mice were immediately placed in separate cages. After 1 h, all burn wounds were inoculated with 100 μL of the bacterial suspension (10 5 CFU/mL) and treated with 100 μL of LVX (0.488 μg/mL, positive control) and different concentrations of MWCNT-LVX aqueous solutions (312.5, 156.25, and 78.125 μg/mL) at 1 h post-infection. MWCNT-LVX solutions were dispersed completely by sonication before topical administration. The volume of the whole solution was transferred to the wound in two steps (2 × 50 μL) without any wastage. The group with no treatment was considered as a negative control. After 24 h, the mice were humanely killed, and the burned skin lesions were removed using sterile surgicassors and homogenized in 1 mL of sterile PBS. The tissue samples were serially diluted six-fold, and then all six dilutions were cultured on blood agar plates. After incubation for 24 h at 37 °C, the number of colonies was counted, and the results were expressed as the mean ± standard deviation of CFU/mL per skin sample 96 . Statistical analysis Statistical analysis was performed using SPSS software (version 22). Data were analyzed by one-way analysis of variance (ANOVA), Shapiro–Wilk and Kolmogorov–Smirnov tests followed by Tukey post-hoc test. Data were reported as a mean value with its standard deviation indicated (mean ± SD), and p -values ≤ 0.05 were considered statistically significant.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7783500/

Medical Mistrust and Enduring Racism in South Africa

In this essay, I argue that exploring institutional racism also needs to examine interactions and communications between patients and providers. Exchange between bioethicists, social scientists, and life scientists should emphasize the biological effects—made evident through health disparities—of racism . I discuss this through examples of patient–provider communication in fertility clinics in South Africa and the ongoing COVID-19 pandemic to emphasize the issue of mistrust between patients and medical institutions. Health disparities and medical mistrust are interrelated problems of racism in healthcare provision.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6025988/

Tumor stroma–targeted antibody-drug conjugate triggers localized anticancer drug release

Although nonmalignant stromal cells facilitate tumor growth and can occupy up to 90% of a solid tumor mass, better strategies to exploit these cells for improved cancer therapy are needed. Here, we describe a potent MMAE-linked antibody-drug conjugate (ADC) targeting tumor endothelial marker 8 (TEM8, also known as ANTXR1), a highly conserved transmembrane receptor broadly overexpressed on cancer-associated fibroblasts, endothelium, and pericytes. Anti-TEM8 ADC elicited potent anticancer activity through an unexpected killing mechanism we term DAaRTS (drug activation and release through stroma), whereby the tumor microenvironment localizes active drug at the tumor site. Following capture of ADC prodrug from the circulation, tumor-associated stromal cells release active MMAE free drug, killing nearby proliferating tumor cells in a target-independent manner. In preclinical studies, ADC treatment was well tolerated and induced regression and often eradication of multiple solid tumor types, blocked metastatic growth, and prolonged overall survival. By exploiting TEM8 + tumor stroma for targeted drug activation, these studies reveal a drug delivery strategy with potential to augment therapies against multiple cancer types.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10905725/

Synthesis and Trace-Level Quantification of Mutagenic and Cohort-of-Concern Ciprofloxacin Nitroso Drug Substance-Related Impurities (NDSRIs) and Other Nitroso Impurities Using UPLC-ESI-MS/MS—Method Optimization Using I-Optimal Mixture Design

Globally, the pharmaceutical industry has been facing challenges from nitroso drug substance-related impurities (NDSRIs). In the current study, we synthesized and developed a rapid new UPLC-MS/MS method for the trace-level quantification of ciprofloxacin NDSRIs and a couple of N-nitroso impurities simultaneously. (Q)-SAR methodology was employed to assess and categorize the genotoxicity of all ciprofloxacin N -nitroso impurities. The projected results were positive, and the cohort of concern (CoC) for all three N-nitroso impurities indicates potential genotoxicity. AQbD-driven I-optimal mixture design was used to optimize the mixture of solvents in the method. The chromatographic resolution was accomplished using an Agilent Poroshell 120 Aq-C18 column (150 mm × 4.6 mm, 2.7 μm) in isocratic elution mode with 0.1% formic acid in a mixture of water, acetonitrile, and methanol in the ratio of 475:500:25 v/v/v at a flow rate of 0.5 mL/min. Quantification was carried out using triple quadrupole mass detection with electrospray ionization (ESI) in a multiple reaction monitoring technique. The finalized method was validated successfully, affording ICH guidelines. All N -nitroso impurities revealed excellent linearity over the concentration range of 0.00125–0.0250 ppm. The Pearson correlation coefficient of each N -nitroso impurity was >0.999. The method accuracy recoveries ranged from 93.98 to 108.08% for the aforementioned N -nitrosamine impurities. Furthermore, the method was effectively applied to quantify N -nitrosamine impurities simultaneously in commercially available formulated samples, with its efficiency recurring at trace levels. Thus, the current method is capable of determining the trace levels of three N -nitroso ciprofloxacin impurities simultaneously from the marketed tablet dosage forms for commercial release and stability testing. 1 Introduction Ciprofloxacin (COX) is chemically 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid and the molecular formula and molecular weight are C 17 H 18 FN 3 O 3 and 331.34 Da, respectively. It is an antibiotic agent and belongs to the class of fluoroquinolones. It is slightly soluble in ethanol and methanol but insoluble in ether, acetone, and chloroform. It is used to treat the urinary tract and pneumonia bacterial infections. 1 In addition to treating bacterial infections, it can be used to treat joint, bone, skin, sexually transmitted infections, typhoid fever, lower respiratory tract infections, plague, salmonella, and anthrax. COX is an appropriate treatment for patients with predisposing factors for Gram-negative infections and is also used for chronic bacterial prostatitis. 2 , 3 FDA-approved COX ophthalmic solution is used for treating corneal ulcers and conjunctivitis caused by susceptible strains. COX pharmacokinetics exhibited interethnic variability, with Asians exhibiting an increased bioavailability compared to Mexicans and Caucasians. It is the most extensively utilized fluoroquinolone antibiotic in treating Pseudomonas aeruginosa . 4 However, in spite of the significant amount of research designed to develop COX powder for inhalation, no formulations are commercially available. 5 − 7 An antibacterial effect of COX-coated poly(lactic acid)-based 3D discs was demonstrated against Escherichia coli . It is, therefore, possible in the future to evaluate the antibacterial activity of polylactic acid-based COX-containing 3D products. 8 , 9 In recent years, pharmaceutical companies and regulatory agencies have placed increasing emphasis on controlling genotoxic and cohort-of-concern impurities in drug substances and products. During the synthetic process, certain potential genotoxic impurities (PGIs) can emerge from byproducts, reagents, and intermediates of the drug substance and drug products. 10 Developing a highly sensitive analytical method for routine analysis is relatively stimulating since acceptable intake limits for these impurities are much lower. Furthermore, there are sensitivity concerns related to GTI assessment in the low-ppm range and reactivity or stability issues, which cause additional problems. 11 , 12 N -Nitrosamines, which can refer to any compound bearing an N - nitroso functional group, have been labeled as a cohort of concern owing to their potential to cause cancer in humans when consumed in a significant amount over an extended period of time. A number of factors and conditions can lead to the production of nitrosamine impurities in active pharmaceutical ingredients (APIs). 13 Some common conditions include using nitrosating agents in the presence of secondary or tertiary amines during the same manufacturing step or different steps, using starting materials and intermediates contaminated by processes or using raw materials containing residual nitrosamines. In-depth studies have shown that there are many more factors at stake in the formation of nitrosamine impurities besides the presence of nitrites and amines during manufacturing. 14 − 16 Several APIs and impurities are accountable for being nitrosated owing to the variety of potential routes for forming nitrosamines, either during the synthetic route of the API, during drug product manufacturing, or during finished formulation packaging and storage. Recently, the United States Food and Drug Administration (USFDA) disclosed that some drug products contain nitrosamine impurities, such as API-derived complex nitrosamines, also known as nitrosamine drug substance-related impurities (NDSRIs), 17 which are a sort of nitrosamines with structural similarities to the API and can be stimulated during the drug product's storage or through the manufacturing of the drug substances. It has also been demonstrated through research that nitrite impurities present in excipients at parts per million levels can lead to the occurrence of NDSRIs. The presence of nitrite impurities in a number of commonly used excipients, including water, has been found to contribute to the occurrence of NDSRIs in specific drug products to a greater extent . 18 − 20 A product recall is the result of careful pharmacovigilance, which is integral to drug regulation. It is one of the most serious reasons for product recalls to detect unacceptable levels of carcinogenic impurities. 21 Some batch tablets of Chantix (varenicline) were voluntarily recalled by Pfizer in August 2021, owing to the existence of N-nitroso -varenicline detected at the above-established acceptable daily intake (ADI). 22 Similarly, some batches of irbesartan tablets and irbesartan combination hydrochlorothiazide tablets were recalled by Lupin Pharmaceuticals Inc. in October 2021 due to the presence of N-nitroso -irbesartan higher than the specification limit. 23 In March 2022, Pfizer recalled five lots of quinapril tablets with the Accupril brand due to the elevated levels of the N-nitroso -quinapril. 24 Also, in March 20, 2022, Sandoz, Inc. recalled 13 lots of orphenadrine citrate 100 mg strength extended-release tablets due to the presence of the N -nitroso-orphenadrine impurity 25 and Pfizer Canada recalled the Inderal-LA (propranolol hydrochloride) different strengths of extended capsules due to the presence of N - nitroso propranolol. 26 Based on their higher potency, the animal carcinogenicity established for this structural class, such as N -nitrosamine impurities, is classified as a "cohort of concern (CoC)″. According to the International Agency for Research on Cancer (IARC), these impurities are categorized into probable or possible carcinogens. Considering a lack of compound-specific limits and the futile process for establishing acceptable intake limits (AIs) over NDSRIs, 27 health regulatory bodies such as the USFDA, the International Conference on Harmonization (ICH), and the European Medicines Agency (EMA) have proposed a limit of 18 ng/day as a precautionary measure based on robust rodent carcinogenicity data from structurally related surrogates. 28 − 30 The chemical reactions that the nitrosamine commences into a biological transformation are stimulated by the N - nitroso functional group. The nitrosamine is metabolically triggered by cytochrome P450 enzymes and forms alkyl diazonium ions through the hydroxylation of the α-carbon atom adjacent to the N-nitroso group. The alkyl diazonium ion can interact with DNA. 31 COX N -nitrosamine piperizine (secondary amine) is the accountable moiety to form the N -nitrosamine and forms an alkyl diazonium ion. The possible pathway of the N -nitroso group to form an alkyl diazonium ion and its interaction with DNA is shown in Figure 1 . Figure 1 Possible pathway of the N-nitroso group interaction with DNA via the cyclic alkyl diazonium ion. Modifying the synthetic pathway, comprehending the entire process of manufacturing, and removing nitrosamine formation sources are all recommended for achieving regulatory compliance. The presence of nitrosamines in pharmaceutical products has emerged as a significant concern for regulatory authorities aiming to mitigate the potential carcinogenic and mutagenic impacts on patients. 32 The three-step mitigation initiatives were proposed by regulatory agencies. The initial approach is derived from scientific research that demonstrates the preventive effects of commonly employed antioxidants on the production of nitrosamines. Another technique is based on the observation that the occurrence of nitrosamines can be reliably anticipated in acidic environments. As a result, under basic or neutral circumstances, the reaction kinetics are significantly reduced, and the final step is that the presence of nitrosamines is minimized. 33 , 34 Global Substance Registration System (GSRS) is a system for registering ingredients in medicines. The system was developed through collaboration between the National Center for Advancing Translational Sciences (NCATS), the EMA, and the FDA health informatics division. The United States Pharmacopeia (USP) is collaborating with the FDA and the NCATS to use GSRS's expertise to address cutting-edge and emerging informatics needs. A total of 41.4% of APIs and 30.2% of API impurities are predicted to be nitrosamine precursors based on data from the GSRS database. 35 After removing tertiary amines as nitrosamine precursors, it is, however, acknowledged that 14.7% of APIs and 12.8% of API impurities can serve as precursors for nitrosamine. In recent years, quality by design (QbD) -centered design of experiments (DoEs) have gained extreme reputation in the optimization, screening, and robustness of analytical method parameters. A traditional method development strategy is either a one-variable-at-a-time (OVAT) or trial-and-error (TAE) approach. There is, however, no information disclosed about the interaction results of two or more variables in either of these approaches. To understand the multiple variable interaction effects and to identify probable risks and failures, a statistical quality-by-design approach is useful. 36 A simple centroid mixture design (SCMD) is a type of experimental design often used in analytical method development to optimize the conditions for a particular analytical method. SCMD is useful in optimizing the method conditions from a mixture of components to identify a suitable ratio for the intended use. A set of factors or variables that can affect the capability of an analytical method are identified, including the type of reagents and concentrations, pH, temperature, and sample preparation conditions. 37 In centroid design, the experimental conditions are chosen based on a simplex-centroid design, which is a type of design that involves selecting a central point (the centroid) within a simplex, which is a geometric shape defined by the range of each factor being studied. 38 The design involves creating a simplex, which is a triangle in a multidimensional space with each vertex representing a pure component. The centroid of the simplex represents the center of the design space, which is typically the average of the pure components. A detailed literature survey indicates that as of now, no research articles have been reported on the synthesis, separation, and trace-level quantification of N -nitroso COX impurities. In the current study, we aimed to synthesize the COX-NDSRI and a couple of COX N -nitroso impurities, assess the genotoxicity using (Q)-SAR models, and develop a simple isocratic UPLC-MS/MS method and optimize using a quality by design-based design of experiments for quantifying trace-level N -nitroso COX impurities. The chemical structures of COX, COX-NDSRI, and COX- N -nitroso impurity-1 and COX- N -nitroso impurity-2 are shown in Figure 2 . Figure 2 Chemical Structures of (a) COX API, (b) COX NDSRI, (c) COX N- nitroso impurity-1, and (d) COX N- nitroso impurity-2. 2 Materials and Methods 2.1 Software The chemical structures of COX and COX- N -nitroso impurities were depicted using ChemDraw Professional 15.0. Both Derek version: Derek Nexus: 6.2.0, Knowledge: Derek KB 2022 1.0, Knowledge version: 1.0 with Knowledge Date: 06 January 6, 2022, and Sarah version: Sarah Nexus: 3.2.0, Nexus version: Nexus: 1.9, Model: Sarah Model 2022.1, were the two main (Q) SAR methods used. Bacteria are the species, and in vitro, mutagenicity is the end point. MassLynx software version 4.2 was used to control all UPLC and MS acquisition and processing parameters. Design Expert software version 13 (Stat-Ease Inc., Minneapolis) was utilized for the design of experiments and optimization data for the I-optimal centroid design. All acquisition and process parameters of LC and MS were measured using Masslynx software version 4.2 (Waters Corporation, Milford, Massachusetts). 2.2 Reagents The reference standard for COX (purity = 99.63%) was procured from M/S Synpure laboratories (Hyderabad, India). The COX-NDSRI (purity = 99.56%), N- nitroso impurity-1 (purity = 99.58%), and N -nitroso impurity-2 (purity = 99.68%) were synthesized. COX, COX- NDSRI, N -nitroso impurity-1, and N -nitroso impurity-2 were characterized by NMR and MS. Formic acid was procured from Fisher Scientific (Waltham, MA). LC-MS-grade acetonitrile and methanol were acquired from Merck Life Sciences (Mumbai, India). Millipore Millex-GV hydrophilic PVDF 0.22 μm filters were procured from Millipore (Burlington, Massachusetts). Throughout the analysis, Millipore Milli-Q high-purity water from the purification system was used (Bedford, MA). An analytical balance from Mettler-Toledo (Model: XPE205, Columbus, Ohio) was utilized for weighing the impurity standard. The centrifuge was obtained from Eppendorf (Model:5810R, Hemburg, Germany). 2.3 Mobile Phase Preparation To a 1000 mL dried and clean flask were added 475 mL of Milli-Q water, 500 mL of acetonitrile, and 25 mL of methanol to add 1 mL of formic acid and mix thoroughly and ultrasonicate for degassing. 2.4 Diluent To a 1000 mL dried and clean flask were added 200 mL of Milli-Q water and 800 mL of acetonitrile to add 1 mL of formic acid and mix thoroughly and ultrasonicate for degassing. 0.1% formic acid in water and acetonitrile composition in the ratio of 20:80 v/v was used as a diluent for the preparation of standards and sample solutions. 2.5 Standard Impurity Diluted Stock Solution Preparation The suitable amounts of each impurity were dissolved in a diluent solution to prepare a stock mixture of COX N -nitroso impurities (1000 ng/mL). The stock solution was further diluted to prepare a 10 ng/mL diluted stock solution. 2.6 Sample Preparation A sample solution of 2 mg/mL was prepared by accurately weighing about 800 mg of COX into a 10 mL volumetric flask, dissolving in 5 mL of diluent, and sonicating for about 15 min to dissolve completely. The flask was equilibrated to room temperature and made up to the mark with the diluent. The resultant sample solution was filtered through 0.22 μm Millex-GV hydrophilic PVDF syringe filters. 2.7 Formulation Tablet Sample Preparation 10 tablets of three commercially available branded tablets of COX were ground separately using a mortar and pestle to a fine powder. An equivalent of 800 mg of COX powder was weighed into a 10 mL volumetric flask with addition of 5 mL of diluent and sonicated for 30 min to dissolve. It was made up with the diluent to the mark after bringing the flask to room temperature and centrifuged for 10 min at 4500 rpm. The resultant supernatant sample solution was filtered through 0.22 μm Millipore Millex-GV hydrophilic PVDF syringe filters. 2.8 UPLC-MS/MS Operating Conditions The chromatographic investigation was accomplished using a Waters Acquity H -Class UHPLC system equipped with a quaternary solvent manager (QSM), sample manager- Flow Through Needle (FTN), column manager with eCord, and TUV/PDA detector. MRM analysis was conducted employing a Waters Xevo TQ-XS MS system with an ESI ion source. An Agilent Poroshell 120 Aq-C18 column (150 mm × 4.6 mm, 2.7 μm) was used to achieve the chromatographic separations. With an acquisition duration of 8 min and a flow rate of 0.5 mL/min with isocratic mode, the mobile phase consisted of 0.1% formic acid in water, acetonitrile, and methanol in the composition of 475:500:25 (v/v/v). While the autosampler temperature was controlled at 15 °C, the column temperature was kept at 35 °C with 10 μL injection volume. The highly sensitive and specific analytical technique multiple reaction monitoring (MRM) has become increasingly important in clinical research, drug development, and metabolomics research. MRM is highly selective for targeted analytes. By the selection of precursor and product ions specific to the analyte of interest, MRM can discriminate the targeted analyte from other molecules. The MRM technique is capable of detecting and quantifying the impurities accurately at very low concentrations. 39 It helps us to ensure that the final product meets regulatory requirements. In the current study, the MRM technique with electrospray ionization positive mode was utilized for the detection and quantification of all three N-nitroso impurities. The m / z values of 361.16/331.12 for N-nitroso COX, 375.16/345.12 for N-nitroso impurity-1, and 319.10/289.12 for N-nitroso impurity-2 were selected for the detection and quantification in MRM mode. The ESI source parameters, specifically capillary voltage (kV), desolvation gas flow, and desolvation temperature, were maintained at 3.5 kV, 900 L/h, and 450 °C, respectively. The cone voltage (kV) was set as 32 for both N-nitroso COX and N-nitroso impurity-1 and 25 for N-nitroso impurity-2. A collision energy of 20 eV was used for both N -nitroso COX and N -nitroso impurity-1 with 18 eV used for N -nitroso impurity-2. The MRM and ESI source parameters are listed in Table 1 . The first 3 min of the sample solution was bypassed from the mass detector; since the COX peak elutes about 3 min to avoid its interference on trace-level nitrosamine impurity quantification and mass detector contamination. Table 1 Optimized MRM Mass Spectrometry Conditions for N -Nitroso COX Impurities in Electron Spray Positive Ion Mode compound precursor ion ( m / z ) product ion ( m / z ) spectral window (min) collision energy (eV) cone voltage (V) capillary voltage (kV) N -nitroso COX impurity-1 375.16 345.12 3.40–3.65 20 32 3.50 COX-NDSRI 361.16 331.12 4.25–4.60 20 32 3.50 N -nitroso COX impurity-2 319.10 289.12 7.10–7.50 18 25 3.50 2.1 Software The chemical structures of COX and COX- N -nitroso impurities were depicted using ChemDraw Professional 15.0. Both Derek version: Derek Nexus: 6.2.0, Knowledge: Derek KB 2022 1.0, Knowledge version: 1.0 with Knowledge Date: 06 January 6, 2022, and Sarah version: Sarah Nexus: 3.2.0, Nexus version: Nexus: 1.9, Model: Sarah Model 2022.1, were the two main (Q) SAR methods used. Bacteria are the species, and in vitro, mutagenicity is the end point. MassLynx software version 4.2 was used to control all UPLC and MS acquisition and processing parameters. Design Expert software version 13 (Stat-Ease Inc., Minneapolis) was utilized for the design of experiments and optimization data for the I-optimal centroid design. All acquisition and process parameters of LC and MS were measured using Masslynx software version 4.2 (Waters Corporation, Milford, Massachusetts). 2.2 Reagents The reference standard for COX (purity = 99.63%) was procured from M/S Synpure laboratories (Hyderabad, India). The COX-NDSRI (purity = 99.56%), N- nitroso impurity-1 (purity = 99.58%), and N -nitroso impurity-2 (purity = 99.68%) were synthesized. COX, COX- NDSRI, N -nitroso impurity-1, and N -nitroso impurity-2 were characterized by NMR and MS. Formic acid was procured from Fisher Scientific (Waltham, MA). LC-MS-grade acetonitrile and methanol were acquired from Merck Life Sciences (Mumbai, India). Millipore Millex-GV hydrophilic PVDF 0.22 μm filters were procured from Millipore (Burlington, Massachusetts). Throughout the analysis, Millipore Milli-Q high-purity water from the purification system was used (Bedford, MA). An analytical balance from Mettler-Toledo (Model: XPE205, Columbus, Ohio) was utilized for weighing the impurity standard. The centrifuge was obtained from Eppendorf (Model:5810R, Hemburg, Germany). 2.3 Mobile Phase Preparation To a 1000 mL dried and clean flask were added 475 mL of Milli-Q water, 500 mL of acetonitrile, and 25 mL of methanol to add 1 mL of formic acid and mix thoroughly and ultrasonicate for degassing. 2.4 Diluent To a 1000 mL dried and clean flask were added 200 mL of Milli-Q water and 800 mL of acetonitrile to add 1 mL of formic acid and mix thoroughly and ultrasonicate for degassing. 0.1% formic acid in water and acetonitrile composition in the ratio of 20:80 v/v was used as a diluent for the preparation of standards and sample solutions. 2.5 Standard Impurity Diluted Stock Solution Preparation The suitable amounts of each impurity were dissolved in a diluent solution to prepare a stock mixture of COX N -nitroso impurities (1000 ng/mL). The stock solution was further diluted to prepare a 10 ng/mL diluted stock solution. 2.6 Sample Preparation A sample solution of 2 mg/mL was prepared by accurately weighing about 800 mg of COX into a 10 mL volumetric flask, dissolving in 5 mL of diluent, and sonicating for about 15 min to dissolve completely. The flask was equilibrated to room temperature and made up to the mark with the diluent. The resultant sample solution was filtered through 0.22 μm Millex-GV hydrophilic PVDF syringe filters. 2.7 Formulation Tablet Sample Preparation 10 tablets of three commercially available branded tablets of COX were ground separately using a mortar and pestle to a fine powder. An equivalent of 800 mg of COX powder was weighed into a 10 mL volumetric flask with addition of 5 mL of diluent and sonicated for 30 min to dissolve. It was made up with the diluent to the mark after bringing the flask to room temperature and centrifuged for 10 min at 4500 rpm. The resultant supernatant sample solution was filtered through 0.22 μm Millipore Millex-GV hydrophilic PVDF syringe filters. 2.8 UPLC-MS/MS Operating Conditions The chromatographic investigation was accomplished using a Waters Acquity H -Class UHPLC system equipped with a quaternary solvent manager (QSM), sample manager- Flow Through Needle (FTN), column manager with eCord, and TUV/PDA detector. MRM analysis was conducted employing a Waters Xevo TQ-XS MS system with an ESI ion source. An Agilent Poroshell 120 Aq-C18 column (150 mm × 4.6 mm, 2.7 μm) was used to achieve the chromatographic separations. With an acquisition duration of 8 min and a flow rate of 0.5 mL/min with isocratic mode, the mobile phase consisted of 0.1% formic acid in water, acetonitrile, and methanol in the composition of 475:500:25 (v/v/v). While the autosampler temperature was controlled at 15 °C, the column temperature was kept at 35 °C with 10 μL injection volume. The highly sensitive and specific analytical technique multiple reaction monitoring (MRM) has become increasingly important in clinical research, drug development, and metabolomics research. MRM is highly selective for targeted analytes. By the selection of precursor and product ions specific to the analyte of interest, MRM can discriminate the targeted analyte from other molecules. The MRM technique is capable of detecting and quantifying the impurities accurately at very low concentrations. 39 It helps us to ensure that the final product meets regulatory requirements. In the current study, the MRM technique with electrospray ionization positive mode was utilized for the detection and quantification of all three N-nitroso impurities. The m / z values of 361.16/331.12 for N-nitroso COX, 375.16/345.12 for N-nitroso impurity-1, and 319.10/289.12 for N-nitroso impurity-2 were selected for the detection and quantification in MRM mode. The ESI source parameters, specifically capillary voltage (kV), desolvation gas flow, and desolvation temperature, were maintained at 3.5 kV, 900 L/h, and 450 °C, respectively. The cone voltage (kV) was set as 32 for both N-nitroso COX and N-nitroso impurity-1 and 25 for N-nitroso impurity-2. A collision energy of 20 eV was used for both N -nitroso COX and N -nitroso impurity-1 with 18 eV used for N -nitroso impurity-2. The MRM and ESI source parameters are listed in Table 1 . The first 3 min of the sample solution was bypassed from the mass detector; since the COX peak elutes about 3 min to avoid its interference on trace-level nitrosamine impurity quantification and mass detector contamination. Table 1 Optimized MRM Mass Spectrometry Conditions for N -Nitroso COX Impurities in Electron Spray Positive Ion Mode compound precursor ion ( m / z ) product ion ( m / z ) spectral window (min) collision energy (eV) cone voltage (V) capillary voltage (kV) N -nitroso COX impurity-1 375.16 345.12 3.40–3.65 20 32 3.50 COX-NDSRI 361.16 331.12 4.25–4.60 20 32 3.50 N -nitroso COX impurity-2 319.10 289.12 7.10–7.50 18 25 3.50 3 Results and Discussion The reference standards for the small and potent nitrosamines such as N-nitroso- di isopropylamine (NDIPA), N-nitroso-dimethylamine (NDMA), N-nitroso- N -methyl-4-aminobutyric acid (NMBA), N-ethyl-N-nitroso-2-propanamine (NEIPA), N-nitroso-methyl phenylamine (NMPA), N-nitroso- di -n-propylamine (NDPA), N-nitroso-di- n -butylamine (NDBA), and N-nitroso-diethylamine (NDEA) are available in compendial databases, including USP and Ph. Eur./EDQM. In the case of NDSRI, the situation is quite different from the aforementioned small nitrosamines. So far, NDSRI compendial standards are not available with any pharmacopoeias. Due to the increase in business opportunities and demand for commercial standards, suppliers have been covering more commercial standards over the past few months. However, the list of NDSRIs is quite large; therefore, it requires a lot more time to cover all of them. 40 Hence, we synthesized the COX-NDSRI- and COX-related N-nitrosamine impurities from the COX drug substance. 3.1 Synthesis of COX Impurities and Corresponding N-Nitroso Impurities 3.1.1 Synthesis of Methyl 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylate (Compound 2 ) To a stirred solution of 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (compound 1) (1 g, 0.003 mol, 1.0 equiv) in 15 mL of methanol cooled to 0 °C was added a catalytic amount of H 2 SO 4 (0.1 mL); then, the temperature was increased to 70 °C and maintained for 16 h. The reaction mixture was diluted with 150 mL of ethyl acetate and washed with a saturated NaHCO 3 solution. The organic layer was dried over Na 2 SO 4 and concentrated. The obtained crude was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 2 was eluted in EtOAc:hexane (30:70). The obtained yield was 85% (0.88 g). [M + H] 346.12. 1 H NMR (500 MHz, DMSO-d 6 ): δ(ppm) 8.44(s, 1H), 7.76 (d, J = 14.0 Hz, 1H), 7.42 (d, J = 7.5 Hz, 1H), 3.73 (s, 3H), 3.67 (m, 1H), 3.16 (t, J = 4.5 Hz, 4H), 2.90 (t, J = 4.5 Hz, 4H), 1.25 (m, 2H), 1.11 (m, 2H). 3.1.2 Synthesis of 1-Cyclopropyl-6-fluoro-7-(piperazin-1-yl)-2,3-dihydroquinolin-4(1H)-one (Compound 3 ) To a stirred solution of 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid-compound 1 (1g, 0.003 mol, 1.0 equiv) in t -BuOH (10 vol) cooled to 0 °C was added H 2 SO 4 (0.29 g, 0.003 mol, 1.0 equiv), and then, the temperature was raised to 90 °C and maintained for 12 h. The reaction mixture was quenched with saturated NaHCO 3 solution and extracted with EtOAc (2 × 100 mL); the organic layer was washed with brine solution and dried over Na 2 SO 4 and concentrated. The obtained crude was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 3 was eluted in EtOAc:hexane(30:70). The obtained yield was 76% (0.66 g). [M + H] 290.16. 1 H NMR (500 MHz, DMSO-d 6 ): δ(ppm) 7.28 (d, 1H), 6.72 (d, J = 13.5 Hz, 1H), 3.42 (t, J = 7.0 Hz, 2H), 3.14 (t, J = 4.5 Hz, 4H), 2.90 (t, J = 4.5 Hz, 4H), 2.45 (t, J = 3.0 Hz, 2H), 2.35 (m, 1H), 0.87 (m, 2H), 0.62 (m, 2H). 3.1.3 Synthesis of N-Nitroso COX (Compound 1a ), Methyl 1-Cyclopropyl-6-fluoro-7-(4-nitroso piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Compound 2a ), and 1-Cyclopropyl-6-fluoro-7-(4-nitroso piperazin-1-yl)-2,3-dihydroquinolin-4(1H)-one (Compound 3a ) To a stirred solution of amine compounds 1–3 (1.0 equiv) in water (10 mL) cooled to 0 °C were added NaNO 2 (2.0 equiv) portion-wise slowly and acetic acid (1.0 equiv) and stirred overnight at RT (room temperature). Ice-cold water was added to the reaction mass and extracted with EtOAc (2 × 100 mL). The organic layer was dried in Na 2 SO 4 and concentrated. The obtained crude compound 1a was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 1a was eluted in EtOAc:hexane (70:30). The obtained yield was 68% (0.74 g). [M + H] 361.16. 1 H NMR (500 MHz, DMSO- d 6 ): δ(ppm) 15.17 (s, 1H),8.68 (s, 1H), 7.98 (d, J = 13.0 Hz,1H), 7.63 (d, J = 7.5 Hz,1H), 4.48 (t, J = 5.0 Hz, 2H), 3.98 (t, J = 5.0 Hz, 4H), 3.84 (m, 1H), 3.64 (t, J = 5.0 Hz, 2H), 3.42 (t, J = 5.0 Hz, 2H), 1.34 (m, 2H), 1.21 (m, 2H). The obtained crude compound 2a was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 2a was eluted in EtOAc:hexane (35:65). The obtained yield was 65% (0.42 g). [M + H] 375.04. 1 H NMR (500 MHz, DMSO- d 6 ): δ(ppm) 8.46 (s, 1H), 7.82 (d, J = 13.0 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H), 4.46 (t, J = 5.0 Hz, 2H), 3.97 (t, J = 5.0 Hz, 4H), 3.74 (m, 1H), 3.64 (t, J = 5.0 Hz, 2H),3.62 (s, 3H), 3.28 (t, J = 5.0 Hz, 2H), 1.28 (m, 2H), 1.25 (m, 2H). The obtained crude compound 3a was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 3a was eluted in EtOAc:hexane (20:80). The obtained yield was 70% (0.38 g). [M + H] 319.07. 1 H NMR (500 MHz, CDCl3): δ(ppm) 7.59 (d, J = 13.0 Hz,1H), 6.76 (d, J = 7.0 Hz, 1H), 4.47 (t, J = 5.0 Hz, 2H), 4.04 (t, J = 5.5 Hz, 2H), 3.51 (t, J = 6.5 Hz, 2H), 3.46 (t, J = 5.5 Hz, 2H), 3.21 (t, J = 5.5 Hz, 2H), 2.63 (t, 6.5 Hz, 2H), 2.32 (m, 1H), 0.92 (m, 2H), 0.89 (m, 2H). Synthetic schemes of impurities and corresponding N-nitroso impurities are shown in Figure 3 . Figure 3 Synthetic scheme of (A) COX impurities and corresponding N-n itroso impurities and (B) reaction mechanism of the formation N- N = O functional group. The NMR data clearly showed that in the formation of the N -nitroso group from cyclic secondary amine-(−NH), the splitting patterns of the piperizine ring protons were changed significantly, indicating the formation of N-nitroso (−N-N=O). The corresponding NMR and mass spectra are shown in the Supporting Information file (Figures S1–S3). 3.2 (Q)-SAR Prediction Data Quantitative structure–activity relationship ((Q)-SAR) predictions are an important part of hazard characterization as per ICH M7(R1) classification. In this case, genotoxicity might be evaluated in accordance with the warning structure(s) of substances used in toxicological research. (Q)-SAR methods with a high throughput capability can generate predictions for hundreds of impurity compounds in a very short amount of time. Two reciprocal (Q)-SAR methodologies are often used to determine the bacterial reverse mutation test results: expert rule-based and statistics-based. 41 − 43 COX, N-nitroso COX, and N-nitroso impurity structures were placed in Nexus 2.5.0. The ICH classification model was utilized for the classification as per ICH M7 guidelines. The obtained results are given in Table 2 . According to the established results of three N -nitroso impurities, Derek is plausible, while Sarah is positive and CoC. Table 2 (Q)-SAR Prediction Summary for Genotoxicity of Three N- Nitroso Impurities TD50 (threshold dose or median toxic dose) values of N -nitrosamines "Lhasa" are reimplementations of the original "Gold" TD50 values mentioned in the carcinogenic potency database. As compared to simple N -methyl, N-ethyl - N -nitrosamines (NDEA-TD50 = 0.0265 mg/kg/day or NDMA-TD50 = 0.096 mg/kg/day), the carcinogenicity and mutagenicity of nitrosamines with larger alkyl sides or cycloalkyl rings do not show the highest carcinogenic potency. Piperazine is the accountable moiety for N-nitroso COX formation and other N- nitroso COX-related impurities. N - Nitroso piperazine had TD 50 = 8.78 mg/kg/day (Gold) and 6.04 mg/kg/day (Lhasa). 44 As noted in ICH M7, the "CoC" group cannot be regulated by the TTC limit due to its higher potency than most carcinogenic compounds. The maximum daily dose (MDD) for COX was 1500 mg/day. 45 Consequently, the acceptable intake of the COX-NDSRI content is 0.012 ppm ((18 ng/day)/(1500 mg/day)) as per ICH M7 guidelines. Therefore, 0.012 ppm can be considered a specification limit, and the LOQ level is defined as 10% of the specification limit, i.e., 0.0125 ppm. 3.3 Chromatographic Method Development We aimed to develop an accurate, precise, and selective LC-MS/MS method to separate and simultaneously quantify three N-nitroso impurities in COX and its formulations. For initial method development, preparing the mixture solution of all compounds (COX, COX-NDSRI, COX N-nitroso impurity-1, and COX N-nitroso impurity-2) equivalent to 1 mg/mL solution was used for the study of chromatographic separation, and the LC-MS method event flow state was set to waste in order to prevent the MS contamination. A few chromatographic columns were evaluated to obtain a good peak symmetry and separation of the components. However, the poor peak shapes and coelution of N -nitroso COX impurity-1 and N -nitroso COX were observed while utilizing the ACQUITY BEH C18 (100 mm × 2.1 mm, 1.7 μm) column. Similarly, while using Symmetry C18 (100 mm × 2.1 mm, 1.7 μm) column, both N-nitroso COX impurity-1 and N -nitroso COX were coeluted due to having similar structural features and retention times, thus potentially causing inaccurate quantification of N -nitroso COX impurity-1 and N-nitroso COX. An Agilent Poroshell Aq-C18, 150 mm × 3.0 mm, 2.7 μm, column was found to be suitable with good applicability in terms of acceptable resolution, good peak shape, and the response of the analytes. The COX is eluting prior to all components from the sample; therefore, it is simpler to exclude the introduction of COX to the mass detector. The flow state in the MS method acquisition parameters was set to divert the eluent of 0.0–3.1 min to waste and the rest of the eluent into the mass spectrometer to detect the peaks of interest. The composed mobile phase applied was 0.1% formic acid aqueous solution, 0.01 mol/L ammonium acetate, and 0.1% trifluoroacetic acid aqueous solution and was used for initial screening, and it was found that the 0.1% formic acid in aqueous solution was suitable for the analysis. The chromatography efficiency was evaluated with different diluents. However, based on the solubility of all N -nitroso impurities chosen, the diluent was 0.1% formic acid in water and acetonitrile (20:80) v/v. Different sonication times were evaluated and finalized at 15 min for sample preparation, bearing in mind the solubility and extraction efficiency. The filter study was also evaluated against the unfiltered centrifuged solution of COX, and the three N -nitrosamine peak areas are comparable. COX and three N- nitrosamine impurities are not retained by the Millex GV PVDF filters. Therefore, the filter is appropriate for this study without retaining COX and its genotoxic impurities. The initial method development trials to separate COX-NDSRI from COX and other N -nitroso impurities with 0.1% formic acid in water and acetonitrile found inadequate separation with the longer chromatographic run time. Hence, a small amount of methanol, along with water and acetonitrile, is introduced into the mobile phase. The isocratic elution mode was used with a mobile phase composition of 0.1% formic acid in the mixture of water, acetonitrile, and methanol in the ratio of 500:450:50 v/v/v and found better peak shape, resolution, and response. The pump flow rate was set at 0.5 mL/min, and the column temperature was maintained at 35 °C. Since the isocratic mobile phase with three major components, such as water, acetonitrile, and methanol, is used in the mobile phase, using a mixture design to optimize the mobile phase compositions using a systematic QbD approach is appropriate. 3.4 Method Optimization Using Mixture Design The I-Optimal mixture design tool was utilized for the optimization of water, acetonitrile, and methanol components (mL/L) in the mobile-phase composition in order to achieve better resolution between COX and N-nitroso COX impurities. The three-component mixture design is depicted by a symmetrical triangle in a two-dimensional space. The design of experiments (DoEs) was mainly employed to discriminate and enhance the critical method parameters (CMPs). To understand the critical significance of the analytical method, a mixture design was employed to find CMPs and each of their unique interaction effects. The designated CMPs were identified based on the initial experiments and the adherences, the percentage of water in the mobile phase (CMP-A), the percentage of acetonitrile in the mobile phase (CMP-B), and the percentage of methanol in the mobile phase (CMP-C). The critical quality attributes (CQAs) are identified as the resolution between COX and N -nitroso COX impurity-1 (R1), the resolution between N -Nitroso COX impurity-1 and N-nitroso COX (R2), and the retention time of the late-eluting N -nitroso COX impurity-2 (R3). CQAs are measurable as numerical variables and are quantitative. According to the USP guideline , 46 chromatographic parameters are altered from lower to higher. The I-Optimal mixture design with three components, two replicates, and three lack-of-fit points, a total of 11 experiments, was chosen. The DoE study employed the standard solution that contained 2 mg/mL COX and each N -nitroso COX impurity. The experimental designs with variables (CMPs) and responses (CQAs) are shown in Table 3 . Table 3 Experimental Designs with Variables (CMPs) and Replies (CQAs) component 1 component 2 component 3 response 1 response 2 response 3 run A: water (mL/L) B: acetonitrile (mL/L) C: methanol (mL/L) R 1 R 2 R 3 (min) 1 512 463 25 14.63 10.19 9.87 2 525 438 37 17.18 11.51 11.59 3 512 425 63 16.06 11.14 11.09 4 550 425 25 19.2 12.4 12.66 5 512 425 63 16.32 11.31 11.31 6 475 462 63 12.99 9.1 8.92 7 487 475 38 13.65 9.38 9.13 8 512 463 25 14.84 10.31 9.65 9 475 500 25 11.95 7.24 7.08 10 487 437 76 13.2 9.96 9.81 11 475 425 100 12.79 9.67 9.64 The chosen model was analyzed, and the ANOVA test demonstrated that the model p -values are significant with <0.05 for all responses. Similarly, the lack-of-fit P values are not significant, with >0.05 for all responses. The model and lack-of-fit P values demonstrate that the model is suitable for the current study. The design model was examined and navigated using the adjusted R 2 , predicted R 2 , regression coefficient ( R 2 ), and adequate precision. The data seemed to be excellent, as evidenced by the closeness of the values between the predicted R 2 (0.9292, 0.8326, and 0.9414 for responses R 1, R 2, and R 3, respectively) and adjusted R 2 values (0.9463, 0.9830, and 0.9627 for responses R 1, R 2, and R 3, respectively), which was obtained to be <0.2. A ratio of more than 4 is enviable when measuring the signal-to-noise ratio with allowable precision. The perceived ratio (26.93, 37.62, and 34.52 for responses R 1, R 2, and R 3) shows that an ample signal and design can be used to traverse the design space. Data from the ANOVA, including P values, significance level, model R 2 , predicted R 2 , and modified R 2 , are shown in Table 4 . Table 4 Summary of ANOVA Results response P values significance level model lack of fit model lack of fit R 2 adjusted R 2 predicted R 2 adeq precision R 1 <0.0001 0.0786 significant not Significant 0.9570 0.9463 0.9292 26.9320 R 2 <0.0001 0.1882 significant not Significant 0.9915 0.9830 0.8326 37.6232 R 3 <0.0001 0.1955 significant not Significant 0.9702 0.9627 0.9414 34.5227 To distinguish the interactions between the variables and the effect of factors, trace (Piepel) plots and numerical plots were examined for each response. The model graphs revealed that response R 1 increases with the increase of percentage water composition (CMP-A) and decreases with the increase in the percentage of acetonitrile (CMP-B) and the percentage of methanol (CMP-C) in mobile-phase composition. R 2 increases with the increase of the water composition (CMP-A), decreases with the increase in the percentage of acetonitrile (CMP-B), and decreases with the percentage of methanol (CMP-C) in the mobile-phase composition. Similarly, R 3 increases with the increase in water composition (CMP-A) and decreases with the increase in the percentage of acetonitrile (CMP-B). R 3 remains unchanged with an increase in the percentage of methanol (CMP-C) in the mobile-phase composition. Normal plots, predicted vs actual plots, trace plots, and triangle 2D contour graphs are shown in Figure 4 . Figure 4 Normal plots, predicted vs actual plots, trace plots, and triangle 2D contour graphs R 1: resolution between COX and N-nitroso COX impurity-1, R 2: resolution between N-nitroso COX impurity-1, and N-nitroso COX, and R 3: retention time of N-nitroso COX impurity-2. To optimize the responses R 1, R 2, and R 3, the best optimum chromatographic conditions have been found using the numerical and graphical optimization approaches, as shown in Figures 5 and 6 . Figure 5 Numerical optimization plots. Figure 6 Surface plots and overlay plot. The water (CMP-A), the acetonitrile (CMP-B), and the methanol (CMP-C) compositions in the mobile phase were determined as 475, 500, and 25 mL/L, respectively, and the corresponding responses were 11.84, 7.32, and 7.36 min. The retention times of COX, N-nitroso impurity-1, COX-NDSRI, and N - nitroso impurity-2 were observed at 2.16, 3.42, 4.20, and 6.86 min, respectively, and the corresponding typical chromatogram is shown in Figure 7 . The diverted valve switching technique was used to avoid the introduction of highly concentrated COX (80 mg/mL) to the mass detector to protect the mass spectrometer. Figure 7 Typical chromatogram of separation of COX-N-nitroso impurities. 3.5 Method Validation The current optimized LC-MS/MS method was successfully validated in compliance with ICH guidelines, 47 USP , 48 and published journals 49 , 50 and guidelines. The method validation was verified in terms of specificity, linearity, limit of detection, limit of quantitation, method and intermediate precision, accuracy, robustness, and solution stability. 3.5.1 Specificity In the current method, the specificity of the analytical method was demonstrated by the ability of the LC-MS chromatographic system to distinguish between the diluent and individual impurities. The specificity was assessed by observing the retention times of COX, COX-NDSRI, N-nitroso impurity-1, and N-nitroso impurity-2, along with diluent solutions subjected to LC/MS analysis. The results exposed that all peaks were well separated, and no coeluting peaks were detected at the retention times of COX NDSRI, N-nitroso COX impurity-1, and N-nitroso impurity-2, thus enabling the precise and accurate quantification of the above-mentioned N-nitroso impurities in the COX drug substance as well as in drug products. The typical MRM chromatograms indicating method specificity can be found in Figure 8 a–e. Figure 8 MRM chromatograms of (a) diluent blank, (b) placebo, (c) COX N -nitroso impurity-1, (d) COX-NDSRI, and (e) COX N -nitroso impurity-2. 3.5.2 Determination of the Limit of Detection (LOD) and Limit of Quantitation (LOQ) The values of the LOD and LOQ of COX NDSRI, COX N-nitroso impurity-1, and N- nitroso impurity-2 were estimated by using the signal-to-noise ratio method (S/N) of 3 and 10, respectively. The LOD and LOQ were determined by preparing the known concentrations of standard solutions, which were injected into LC-MS spectrometry. The repeatability (precision) of LOQ was also performed by six replicate injections of these N-nitroso impurities and the % RSD value. The measured value of LOD and LOQ for all N -nitroso impurities was 0.03 and 0.1 ng/mL, respectively. The calculated results are shown in Table 5 , and the typical LOQ MRM chromatograms are shown in Figure 9 , respectively. Figure 9 Typical LOQ MRM chromatograms of (a) COX N -nitroso impurity-1, (b) COX-NDSRI, and (c) COX N -nitroso impurity-2. Table 5 Summary of Method Validation Results results method validation parameter COX-NDSRI COX- N -Nitroso imp-1 COX- N -Nitroso imp-2 specificity should be no interference from the diluent no interference no interference no interference precision % content ( n = 6, % RSD < 10.0) 0.81 0.76 0.69 intermediate precision % content ( n = 6, % RSD < 10.0) analyst-1 0.95 1.20 1.45 analyst-2 1.05 1.10 1.30 limit of detection (LOD) LOD (ng/mL) 0.03 0.03 0.03 S/N value (≥3) 6.16 3.79 4.89 limit of quantitation (LOQ) LOQ (ng/mL) 0.10 0.10 0.10 S/N value (≥10) 33.67 17.19 21.19 LOQ precision % content ( n = 6, % RSD 0.999) 0.9996 0.9994 0.9992 accuracy in pure COX (%) ( n = 3, average percentage) the level at 0.00125 ppm mean ± SD 94.80 ± 0.65 95.10 ± 0.65 96.90 ± 0.65 the level at 0.0125 ppm mean ± SD 98.24 ± 0.27 99.14 ± 0.49 98.94 ± 0.89 the level at 0.025 ppm mean ± SD 105.78 ± 0.14 107.18 ± 0.34 107.08 ± 0.36 accuracy in formulated COX (%) ( n = 3, average percentage) the level at 0.00125 ppm mean ± SD 93.98 ± 0.49 95.70 ± 0.65 96.30 ± 0.65 the level at 0.0125 ppm mean ± SD 98.94 ± 0.32 97.54 ± 0.27 98.14 ± 0.17 the level at 0.025 ppm mean ± SD 100.58 ± 0.14 107.18 ± 0.14 108.08 ± 0.14 solution stability peak area (0–48 h, % difference with initial < 10.0) 1.48 1.63 1.12 3.5.3 Linearity The linearity of the method was investigated over the concentration range between 0.1 and 2.0 ng/mL, translating from 0.00125 ppm (1.2 ppb) to 0.025 ppm (25 ppb) with respect to 80 mg/mL sample concentration for all three N- nitroso impurities. In the construction of a set of calibration standards, a diluted stock solution (10 ng/mL) was prepared as described in Section 2.5 . Furthermore, it was diluted to produce the following final concentrations: 0.1, 0.5, 0.8, 1.0, 1.2, 1.5, and 2.0 ng/mL (0.0012, 0.006, 0.01, 0.012, 0.015, 0.019, and 0.025 ppm (μg/g), correspondingly). The method linearity for all N - nitroso impurities was examined at seven distinct concentrations varying from 0.012 to 0.025 ppm (μg/g). The slope (a), intercept (b), and Pearson correlation coefficient (r) were determined by using the linear regression equation with least squares. Calculation of the linear regression equation was performed between peak area and analyte concentration. These results denote a good correlation between the peak areas and the concentrations of all three N -nitroso impurities with data summarized in Table 5 . 3.5.4 Precision In the current method, precision was evaluated by determining the % RSD of the contents of all three N-n itroso impurities from six replicate injections at a concentration of the specification-level spiked solution. Furthermore, intermediate precision is also measured on a different day by a different analyst by determining the % RSD of the contents of all three N-nitroso impurities from a total of 12 replicate injections of spiked solution at the specification level. The results are revealed in Table 5 . 3.5.5 Accuracy and Recovery Study The accuracy of the method was estimated by the triplicate preparation at three levels, 0.0012, 0.012, and 0.025 ppm, to the pure and formulated samples of COX by using the standard addition method. One preparation for each concentration was injected in triplicate. The acceptance criterion for recovery was 80–120%. The obtained recoveries were between 93.98 and 108.08% ( Table 5 ). 3.5.6 Robustness Study The robustness of the method states "the capability of an analytical method to remain unaltered by even small changes in method conditions". During routine use, it provides an indication of its consistency. The flow rate (mL/min), column temperature (°C), collision gas flow (L/Hr), and desolvation temperature (°C) were changed in order to assess the robustness of the current method. The spiked sample solution with all three N-nitroso impurities at the specification level (0.0125 ppm) to the COX drug substance at the concentration of 80 mg/mL was injected into LCMS for the method robustness evaluation. The optimized flow rate was altered by ±0.01 mL/min from the actual flow rate of 0.5 mL/min, the column temperature (°C) was altered by ±1.0 °C from the actual 35 °C, the collision gas flow was altered by ±50 L/Hr from the actual 900 L/Hr, and the desolvation gas temperature was altered by ±50 °C from the actual 450 °C. The optimized UPLC-MS/MS method was designed to be consistent; as a result, the absolute percentage difference in content with respect to nominal conditions for all three N-nitroso impurities was not greater than 3.12 under any altered conditions. The robustness results are shown in Table 6 . Table 6 Summarized Results of the Robustness Study content (%) % absolute difference with respect to the nominal condition altered condition change COX-NDSRI COX- N -Nitroso impurity-1 COX- N -Nitroso impurity-2 COX-NDSRI COX- N -Nitroso impurity-1 COX- N -Nitroso impurity-2 flow rate (mL/min) 0.45 0.0123 0.0127 0.0124 1.60 1.60 3.12 0.50 0.0125 0.0129 0.0128 0.55 0.0124 0.0126 0.0126 0.80 2.32 1.56 column temp (°C) 33.0 0.0124 0.0125 0.0125 0.80 3.12 2.34 35.0 0.0125 0.0129 0.0128 37.0 0.0123 0.0127 0.0124 1.60 1.56 3.12 collision gas flow (L/h) 850 0.0127 0.0127 0.0126 1.60 1.56 1.56 900 0.0125 0.0129 0.0128 950 0.0124 0.0128 0.0125 0.80 0.77 2.34 desolvation temp (°C) 400 0.0124 0.0128 0.0124 0.80 0.77 3.12 450 0.0125 0.0129 0.0128 500 0.0126 0.0127 0.0125 0.80 1.56 2.34 3.5.7 Solution Stability COX and three N-nitroso COX impurities were tested for solution stability by leaving spiked and unspiked samples in capped LC vials for 48 h at 25 °C in an autosampler. We determined the concentration of each impurity against a freshly prepared standard solution, and none of the N-nitroso impurities showed any significant changes. Hence, we concluded that the impurities in the sample solution remained stable at ambient temperature (25 °C) for at least 48 h. The % absolute difference was calculated with respect to the peak areas of the freshly prepared COX and all three N-nitroso impurities. 3.6 Developed Optimized Method and Their Pharmaceutical Application Five different commercially available formulation samples were investigated using our validated UPLC-MS/MS method to quantify the above-mentioned N-nitrosamine impurities accurately. The estimated amount of COX-NDSRI was in the range of 0.015–0.045 ppm. The test concentration of COX and the COX formulation was 80 mg/mL in triplicate determinations. Both COX N-nitroso impurity-1 and N-nitroso impurity-2 were not detected in all five batch formulation samples. 3.1 Synthesis of COX Impurities and Corresponding N-Nitroso Impurities 3.1.1 Synthesis of Methyl 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylate (Compound 2 ) To a stirred solution of 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (compound 1) (1 g, 0.003 mol, 1.0 equiv) in 15 mL of methanol cooled to 0 °C was added a catalytic amount of H 2 SO 4 (0.1 mL); then, the temperature was increased to 70 °C and maintained for 16 h. The reaction mixture was diluted with 150 mL of ethyl acetate and washed with a saturated NaHCO 3 solution. The organic layer was dried over Na 2 SO 4 and concentrated. The obtained crude was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 2 was eluted in EtOAc:hexane (30:70). The obtained yield was 85% (0.88 g). [M + H] 346.12. 1 H NMR (500 MHz, DMSO-d 6 ): δ(ppm) 8.44(s, 1H), 7.76 (d, J = 14.0 Hz, 1H), 7.42 (d, J = 7.5 Hz, 1H), 3.73 (s, 3H), 3.67 (m, 1H), 3.16 (t, J = 4.5 Hz, 4H), 2.90 (t, J = 4.5 Hz, 4H), 1.25 (m, 2H), 1.11 (m, 2H). 3.1.2 Synthesis of 1-Cyclopropyl-6-fluoro-7-(piperazin-1-yl)-2,3-dihydroquinolin-4(1H)-one (Compound 3 ) To a stirred solution of 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid-compound 1 (1g, 0.003 mol, 1.0 equiv) in t -BuOH (10 vol) cooled to 0 °C was added H 2 SO 4 (0.29 g, 0.003 mol, 1.0 equiv), and then, the temperature was raised to 90 °C and maintained for 12 h. The reaction mixture was quenched with saturated NaHCO 3 solution and extracted with EtOAc (2 × 100 mL); the organic layer was washed with brine solution and dried over Na 2 SO 4 and concentrated. The obtained crude was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 3 was eluted in EtOAc:hexane(30:70). The obtained yield was 76% (0.66 g). [M + H] 290.16. 1 H NMR (500 MHz, DMSO-d 6 ): δ(ppm) 7.28 (d, 1H), 6.72 (d, J = 13.5 Hz, 1H), 3.42 (t, J = 7.0 Hz, 2H), 3.14 (t, J = 4.5 Hz, 4H), 2.90 (t, J = 4.5 Hz, 4H), 2.45 (t, J = 3.0 Hz, 2H), 2.35 (m, 1H), 0.87 (m, 2H), 0.62 (m, 2H). 3.1.3 Synthesis of N-Nitroso COX (Compound 1a ), Methyl 1-Cyclopropyl-6-fluoro-7-(4-nitroso piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Compound 2a ), and 1-Cyclopropyl-6-fluoro-7-(4-nitroso piperazin-1-yl)-2,3-dihydroquinolin-4(1H)-one (Compound 3a ) To a stirred solution of amine compounds 1–3 (1.0 equiv) in water (10 mL) cooled to 0 °C were added NaNO 2 (2.0 equiv) portion-wise slowly and acetic acid (1.0 equiv) and stirred overnight at RT (room temperature). Ice-cold water was added to the reaction mass and extracted with EtOAc (2 × 100 mL). The organic layer was dried in Na 2 SO 4 and concentrated. The obtained crude compound 1a was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 1a was eluted in EtOAc:hexane (70:30). The obtained yield was 68% (0.74 g). [M + H] 361.16. 1 H NMR (500 MHz, DMSO- d 6 ): δ(ppm) 15.17 (s, 1H),8.68 (s, 1H), 7.98 (d, J = 13.0 Hz,1H), 7.63 (d, J = 7.5 Hz,1H), 4.48 (t, J = 5.0 Hz, 2H), 3.98 (t, J = 5.0 Hz, 4H), 3.84 (m, 1H), 3.64 (t, J = 5.0 Hz, 2H), 3.42 (t, J = 5.0 Hz, 2H), 1.34 (m, 2H), 1.21 (m, 2H). The obtained crude compound 2a was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 2a was eluted in EtOAc:hexane (35:65). The obtained yield was 65% (0.42 g). [M + H] 375.04. 1 H NMR (500 MHz, DMSO- d 6 ): δ(ppm) 8.46 (s, 1H), 7.82 (d, J = 13.0 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H), 4.46 (t, J = 5.0 Hz, 2H), 3.97 (t, J = 5.0 Hz, 4H), 3.74 (m, 1H), 3.64 (t, J = 5.0 Hz, 2H),3.62 (s, 3H), 3.28 (t, J = 5.0 Hz, 2H), 1.28 (m, 2H), 1.25 (m, 2H). The obtained crude compound 3a was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 3a was eluted in EtOAc:hexane (20:80). The obtained yield was 70% (0.38 g). [M + H] 319.07. 1 H NMR (500 MHz, CDCl3): δ(ppm) 7.59 (d, J = 13.0 Hz,1H), 6.76 (d, J = 7.0 Hz, 1H), 4.47 (t, J = 5.0 Hz, 2H), 4.04 (t, J = 5.5 Hz, 2H), 3.51 (t, J = 6.5 Hz, 2H), 3.46 (t, J = 5.5 Hz, 2H), 3.21 (t, J = 5.5 Hz, 2H), 2.63 (t, 6.5 Hz, 2H), 2.32 (m, 1H), 0.92 (m, 2H), 0.89 (m, 2H). Synthetic schemes of impurities and corresponding N-nitroso impurities are shown in Figure 3 . Figure 3 Synthetic scheme of (A) COX impurities and corresponding N-n itroso impurities and (B) reaction mechanism of the formation N- N = O functional group. The NMR data clearly showed that in the formation of the N -nitroso group from cyclic secondary amine-(−NH), the splitting patterns of the piperizine ring protons were changed significantly, indicating the formation of N-nitroso (−N-N=O). The corresponding NMR and mass spectra are shown in the Supporting Information file (Figures S1–S3). 3.1.1 Synthesis of Methyl 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylate (Compound 2 ) To a stirred solution of 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (compound 1) (1 g, 0.003 mol, 1.0 equiv) in 15 mL of methanol cooled to 0 °C was added a catalytic amount of H 2 SO 4 (0.1 mL); then, the temperature was increased to 70 °C and maintained for 16 h. The reaction mixture was diluted with 150 mL of ethyl acetate and washed with a saturated NaHCO 3 solution. The organic layer was dried over Na 2 SO 4 and concentrated. The obtained crude was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 2 was eluted in EtOAc:hexane (30:70). The obtained yield was 85% (0.88 g). [M + H] 346.12. 1 H NMR (500 MHz, DMSO-d 6 ): δ(ppm) 8.44(s, 1H), 7.76 (d, J = 14.0 Hz, 1H), 7.42 (d, J = 7.5 Hz, 1H), 3.73 (s, 3H), 3.67 (m, 1H), 3.16 (t, J = 4.5 Hz, 4H), 2.90 (t, J = 4.5 Hz, 4H), 1.25 (m, 2H), 1.11 (m, 2H). 3.1.2 Synthesis of 1-Cyclopropyl-6-fluoro-7-(piperazin-1-yl)-2,3-dihydroquinolin-4(1H)-one (Compound 3 ) To a stirred solution of 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid-compound 1 (1g, 0.003 mol, 1.0 equiv) in t -BuOH (10 vol) cooled to 0 °C was added H 2 SO 4 (0.29 g, 0.003 mol, 1.0 equiv), and then, the temperature was raised to 90 °C and maintained for 12 h. The reaction mixture was quenched with saturated NaHCO 3 solution and extracted with EtOAc (2 × 100 mL); the organic layer was washed with brine solution and dried over Na 2 SO 4 and concentrated. The obtained crude was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 3 was eluted in EtOAc:hexane(30:70). The obtained yield was 76% (0.66 g). [M + H] 290.16. 1 H NMR (500 MHz, DMSO-d 6 ): δ(ppm) 7.28 (d, 1H), 6.72 (d, J = 13.5 Hz, 1H), 3.42 (t, J = 7.0 Hz, 2H), 3.14 (t, J = 4.5 Hz, 4H), 2.90 (t, J = 4.5 Hz, 4H), 2.45 (t, J = 3.0 Hz, 2H), 2.35 (m, 1H), 0.87 (m, 2H), 0.62 (m, 2H). 3.1.3 Synthesis of N-Nitroso COX (Compound 1a ), Methyl 1-Cyclopropyl-6-fluoro-7-(4-nitroso piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Compound 2a ), and 1-Cyclopropyl-6-fluoro-7-(4-nitroso piperazin-1-yl)-2,3-dihydroquinolin-4(1H)-one (Compound 3a ) To a stirred solution of amine compounds 1–3 (1.0 equiv) in water (10 mL) cooled to 0 °C were added NaNO 2 (2.0 equiv) portion-wise slowly and acetic acid (1.0 equiv) and stirred overnight at RT (room temperature). Ice-cold water was added to the reaction mass and extracted with EtOAc (2 × 100 mL). The organic layer was dried in Na 2 SO 4 and concentrated. The obtained crude compound 1a was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 1a was eluted in EtOAc:hexane (70:30). The obtained yield was 68% (0.74 g). [M + H] 361.16. 1 H NMR (500 MHz, DMSO- d 6 ): δ(ppm) 15.17 (s, 1H),8.68 (s, 1H), 7.98 (d, J = 13.0 Hz,1H), 7.63 (d, J = 7.5 Hz,1H), 4.48 (t, J = 5.0 Hz, 2H), 3.98 (t, J = 5.0 Hz, 4H), 3.84 (m, 1H), 3.64 (t, J = 5.0 Hz, 2H), 3.42 (t, J = 5.0 Hz, 2H), 1.34 (m, 2H), 1.21 (m, 2H). The obtained crude compound 2a was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 2a was eluted in EtOAc:hexane (35:65). The obtained yield was 65% (0.42 g). [M + H] 375.04. 1 H NMR (500 MHz, DMSO- d 6 ): δ(ppm) 8.46 (s, 1H), 7.82 (d, J = 13.0 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H), 4.46 (t, J = 5.0 Hz, 2H), 3.97 (t, J = 5.0 Hz, 4H), 3.74 (m, 1H), 3.64 (t, J = 5.0 Hz, 2H),3.62 (s, 3H), 3.28 (t, J = 5.0 Hz, 2H), 1.28 (m, 2H), 1.25 (m, 2H). The obtained crude compound 3a was purified by silica gel (100–200 mesh) column chromatography with gradient elution mode, and compound 3a was eluted in EtOAc:hexane (20:80). The obtained yield was 70% (0.38 g). [M + H] 319.07. 1 H NMR (500 MHz, CDCl3): δ(ppm) 7.59 (d, J = 13.0 Hz,1H), 6.76 (d, J = 7.0 Hz, 1H), 4.47 (t, J = 5.0 Hz, 2H), 4.04 (t, J = 5.5 Hz, 2H), 3.51 (t, J = 6.5 Hz, 2H), 3.46 (t, J = 5.5 Hz, 2H), 3.21 (t, J = 5.5 Hz, 2H), 2.63 (t, 6.5 Hz, 2H), 2.32 (m, 1H), 0.92 (m, 2H), 0.89 (m, 2H). Synthetic schemes of impurities and corresponding N-nitroso impurities are shown in Figure 3 . Figure 3 Synthetic scheme of (A) COX impurities and corresponding N-n itroso impurities and (B) reaction mechanism of the formation N- N = O functional group. The NMR data clearly showed that in the formation of the N -nitroso group from cyclic secondary amine-(−NH), the splitting patterns of the piperizine ring protons were changed significantly, indicating the formation of N-nitroso (−N-N=O). The corresponding NMR and mass spectra are shown in the Supporting Information file (Figures S1–S3). 3.2 (Q)-SAR Prediction Data Quantitative structure–activity relationship ((Q)-SAR) predictions are an important part of hazard characterization as per ICH M7(R1) classification. In this case, genotoxicity might be evaluated in accordance with the warning structure(s) of substances used in toxicological research. (Q)-SAR methods with a high throughput capability can generate predictions for hundreds of impurity compounds in a very short amount of time. Two reciprocal (Q)-SAR methodologies are often used to determine the bacterial reverse mutation test results: expert rule-based and statistics-based. 41 − 43 COX, N-nitroso COX, and N-nitroso impurity structures were placed in Nexus 2.5.0. The ICH classification model was utilized for the classification as per ICH M7 guidelines. The obtained results are given in Table 2 . According to the established results of three N -nitroso impurities, Derek is plausible, while Sarah is positive and CoC. Table 2 (Q)-SAR Prediction Summary for Genotoxicity of Three N- Nitroso Impurities TD50 (threshold dose or median toxic dose) values of N -nitrosamines "Lhasa" are reimplementations of the original "Gold" TD50 values mentioned in the carcinogenic potency database. As compared to simple N -methyl, N-ethyl - N -nitrosamines (NDEA-TD50 = 0.0265 mg/kg/day or NDMA-TD50 = 0.096 mg/kg/day), the carcinogenicity and mutagenicity of nitrosamines with larger alkyl sides or cycloalkyl rings do not show the highest carcinogenic potency. Piperazine is the accountable moiety for N-nitroso COX formation and other N- nitroso COX-related impurities. N - Nitroso piperazine had TD 50 = 8.78 mg/kg/day (Gold) and 6.04 mg/kg/day (Lhasa). 44 As noted in ICH M7, the "CoC" group cannot be regulated by the TTC limit due to its higher potency than most carcinogenic compounds. The maximum daily dose (MDD) for COX was 1500 mg/day. 45 Consequently, the acceptable intake of the COX-NDSRI content is 0.012 ppm ((18 ng/day)/(1500 mg/day)) as per ICH M7 guidelines. Therefore, 0.012 ppm can be considered a specification limit, and the LOQ level is defined as 10% of the specification limit, i.e., 0.0125 ppm. 3.3 Chromatographic Method Development We aimed to develop an accurate, precise, and selective LC-MS/MS method to separate and simultaneously quantify three N-nitroso impurities in COX and its formulations. For initial method development, preparing the mixture solution of all compounds (COX, COX-NDSRI, COX N-nitroso impurity-1, and COX N-nitroso impurity-2) equivalent to 1 mg/mL solution was used for the study of chromatographic separation, and the LC-MS method event flow state was set to waste in order to prevent the MS contamination. A few chromatographic columns were evaluated to obtain a good peak symmetry and separation of the components. However, the poor peak shapes and coelution of N -nitroso COX impurity-1 and N -nitroso COX were observed while utilizing the ACQUITY BEH C18 (100 mm × 2.1 mm, 1.7 μm) column. Similarly, while using Symmetry C18 (100 mm × 2.1 mm, 1.7 μm) column, both N-nitroso COX impurity-1 and N -nitroso COX were coeluted due to having similar structural features and retention times, thus potentially causing inaccurate quantification of N -nitroso COX impurity-1 and N-nitroso COX. An Agilent Poroshell Aq-C18, 150 mm × 3.0 mm, 2.7 μm, column was found to be suitable with good applicability in terms of acceptable resolution, good peak shape, and the response of the analytes. The COX is eluting prior to all components from the sample; therefore, it is simpler to exclude the introduction of COX to the mass detector. The flow state in the MS method acquisition parameters was set to divert the eluent of 0.0–3.1 min to waste and the rest of the eluent into the mass spectrometer to detect the peaks of interest. The composed mobile phase applied was 0.1% formic acid aqueous solution, 0.01 mol/L ammonium acetate, and 0.1% trifluoroacetic acid aqueous solution and was used for initial screening, and it was found that the 0.1% formic acid in aqueous solution was suitable for the analysis. The chromatography efficiency was evaluated with different diluents. However, based on the solubility of all N -nitroso impurities chosen, the diluent was 0.1% formic acid in water and acetonitrile (20:80) v/v. Different sonication times were evaluated and finalized at 15 min for sample preparation, bearing in mind the solubility and extraction efficiency. The filter study was also evaluated against the unfiltered centrifuged solution of COX, and the three N -nitrosamine peak areas are comparable. COX and three N- nitrosamine impurities are not retained by the Millex GV PVDF filters. Therefore, the filter is appropriate for this study without retaining COX and its genotoxic impurities. The initial method development trials to separate COX-NDSRI from COX and other N -nitroso impurities with 0.1% formic acid in water and acetonitrile found inadequate separation with the longer chromatographic run time. Hence, a small amount of methanol, along with water and acetonitrile, is introduced into the mobile phase. The isocratic elution mode was used with a mobile phase composition of 0.1% formic acid in the mixture of water, acetonitrile, and methanol in the ratio of 500:450:50 v/v/v and found better peak shape, resolution, and response. The pump flow rate was set at 0.5 mL/min, and the column temperature was maintained at 35 °C. Since the isocratic mobile phase with three major components, such as water, acetonitrile, and methanol, is used in the mobile phase, using a mixture design to optimize the mobile phase compositions using a systematic QbD approach is appropriate. 3.4 Method Optimization Using Mixture Design The I-Optimal mixture design tool was utilized for the optimization of water, acetonitrile, and methanol components (mL/L) in the mobile-phase composition in order to achieve better resolution between COX and N-nitroso COX impurities. The three-component mixture design is depicted by a symmetrical triangle in a two-dimensional space. The design of experiments (DoEs) was mainly employed to discriminate and enhance the critical method parameters (CMPs). To understand the critical significance of the analytical method, a mixture design was employed to find CMPs and each of their unique interaction effects. The designated CMPs were identified based on the initial experiments and the adherences, the percentage of water in the mobile phase (CMP-A), the percentage of acetonitrile in the mobile phase (CMP-B), and the percentage of methanol in the mobile phase (CMP-C). The critical quality attributes (CQAs) are identified as the resolution between COX and N -nitroso COX impurity-1 (R1), the resolution between N -Nitroso COX impurity-1 and N-nitroso COX (R2), and the retention time of the late-eluting N -nitroso COX impurity-2 (R3). CQAs are measurable as numerical variables and are quantitative. According to the USP guideline , 46 chromatographic parameters are altered from lower to higher. The I-Optimal mixture design with three components, two replicates, and three lack-of-fit points, a total of 11 experiments, was chosen. The DoE study employed the standard solution that contained 2 mg/mL COX and each N -nitroso COX impurity. The experimental designs with variables (CMPs) and responses (CQAs) are shown in Table 3 . Table 3 Experimental Designs with Variables (CMPs) and Replies (CQAs) component 1 component 2 component 3 response 1 response 2 response 3 run A: water (mL/L) B: acetonitrile (mL/L) C: methanol (mL/L) R 1 R 2 R 3 (min) 1 512 463 25 14.63 10.19 9.87 2 525 438 37 17.18 11.51 11.59 3 512 425 63 16.06 11.14 11.09 4 550 425 25 19.2 12.4 12.66 5 512 425 63 16.32 11.31 11.31 6 475 462 63 12.99 9.1 8.92 7 487 475 38 13.65 9.38 9.13 8 512 463 25 14.84 10.31 9.65 9 475 500 25 11.95 7.24 7.08 10 487 437 76 13.2 9.96 9.81 11 475 425 100 12.79 9.67 9.64 The chosen model was analyzed, and the ANOVA test demonstrated that the model p -values are significant with <0.05 for all responses. Similarly, the lack-of-fit P values are not significant, with >0.05 for all responses. The model and lack-of-fit P values demonstrate that the model is suitable for the current study. The design model was examined and navigated using the adjusted R 2 , predicted R 2 , regression coefficient ( R 2 ), and adequate precision. The data seemed to be excellent, as evidenced by the closeness of the values between the predicted R 2 (0.9292, 0.8326, and 0.9414 for responses R 1, R 2, and R 3, respectively) and adjusted R 2 values (0.9463, 0.9830, and 0.9627 for responses R 1, R 2, and R 3, respectively), which was obtained to be <0.2. A ratio of more than 4 is enviable when measuring the signal-to-noise ratio with allowable precision. The perceived ratio (26.93, 37.62, and 34.52 for responses R 1, R 2, and R 3) shows that an ample signal and design can be used to traverse the design space. Data from the ANOVA, including P values, significance level, model R 2 , predicted R 2 , and modified R 2 , are shown in Table 4 . Table 4 Summary of ANOVA Results response P values significance level model lack of fit model lack of fit R 2 adjusted R 2 predicted R 2 adeq precision R 1 <0.0001 0.0786 significant not Significant 0.9570 0.9463 0.9292 26.9320 R 2 <0.0001 0.1882 significant not Significant 0.9915 0.9830 0.8326 37.6232 R 3 <0.0001 0.1955 significant not Significant 0.9702 0.9627 0.9414 34.5227 To distinguish the interactions between the variables and the effect of factors, trace (Piepel) plots and numerical plots were examined for each response. The model graphs revealed that response R 1 increases with the increase of percentage water composition (CMP-A) and decreases with the increase in the percentage of acetonitrile (CMP-B) and the percentage of methanol (CMP-C) in mobile-phase composition. R 2 increases with the increase of the water composition (CMP-A), decreases with the increase in the percentage of acetonitrile (CMP-B), and decreases with the percentage of methanol (CMP-C) in the mobile-phase composition. Similarly, R 3 increases with the increase in water composition (CMP-A) and decreases with the increase in the percentage of acetonitrile (CMP-B). R 3 remains unchanged with an increase in the percentage of methanol (CMP-C) in the mobile-phase composition. Normal plots, predicted vs actual plots, trace plots, and triangle 2D contour graphs are shown in Figure 4 . Figure 4 Normal plots, predicted vs actual plots, trace plots, and triangle 2D contour graphs R 1: resolution between COX and N-nitroso COX impurity-1, R 2: resolution between N-nitroso COX impurity-1, and N-nitroso COX, and R 3: retention time of N-nitroso COX impurity-2. To optimize the responses R 1, R 2, and R 3, the best optimum chromatographic conditions have been found using the numerical and graphical optimization approaches, as shown in Figures 5 and 6 . Figure 5 Numerical optimization plots. Figure 6 Surface plots and overlay plot. The water (CMP-A), the acetonitrile (CMP-B), and the methanol (CMP-C) compositions in the mobile phase were determined as 475, 500, and 25 mL/L, respectively, and the corresponding responses were 11.84, 7.32, and 7.36 min. The retention times of COX, N-nitroso impurity-1, COX-NDSRI, and N - nitroso impurity-2 were observed at 2.16, 3.42, 4.20, and 6.86 min, respectively, and the corresponding typical chromatogram is shown in Figure 7 . The diverted valve switching technique was used to avoid the introduction of highly concentrated COX (80 mg/mL) to the mass detector to protect the mass spectrometer. Figure 7 Typical chromatogram of separation of COX-N-nitroso impurities. 3.5 Method Validation The current optimized LC-MS/MS method was successfully validated in compliance with ICH guidelines, 47 USP , 48 and published journals 49 , 50 and guidelines. The method validation was verified in terms of specificity, linearity, limit of detection, limit of quantitation, method and intermediate precision, accuracy, robustness, and solution stability. 3.5.1 Specificity In the current method, the specificity of the analytical method was demonstrated by the ability of the LC-MS chromatographic system to distinguish between the diluent and individual impurities. The specificity was assessed by observing the retention times of COX, COX-NDSRI, N-nitroso impurity-1, and N-nitroso impurity-2, along with diluent solutions subjected to LC/MS analysis. The results exposed that all peaks were well separated, and no coeluting peaks were detected at the retention times of COX NDSRI, N-nitroso COX impurity-1, and N-nitroso impurity-2, thus enabling the precise and accurate quantification of the above-mentioned N-nitroso impurities in the COX drug substance as well as in drug products. The typical MRM chromatograms indicating method specificity can be found in Figure 8 a–e. Figure 8 MRM chromatograms of (a) diluent blank, (b) placebo, (c) COX N -nitroso impurity-1, (d) COX-NDSRI, and (e) COX N -nitroso impurity-2. 3.5.2 Determination of the Limit of Detection (LOD) and Limit of Quantitation (LOQ) The values of the LOD and LOQ of COX NDSRI, COX N-nitroso impurity-1, and N- nitroso impurity-2 were estimated by using the signal-to-noise ratio method (S/N) of 3 and 10, respectively. The LOD and LOQ were determined by preparing the known concentrations of standard solutions, which were injected into LC-MS spectrometry. The repeatability (precision) of LOQ was also performed by six replicate injections of these N-nitroso impurities and the % RSD value. The measured value of LOD and LOQ for all N -nitroso impurities was 0.03 and 0.1 ng/mL, respectively. The calculated results are shown in Table 5 , and the typical LOQ MRM chromatograms are shown in Figure 9 , respectively. Figure 9 Typical LOQ MRM chromatograms of (a) COX N -nitroso impurity-1, (b) COX-NDSRI, and (c) COX N -nitroso impurity-2. Table 5 Summary of Method Validation Results results method validation parameter COX-NDSRI COX- N -Nitroso imp-1 COX- N -Nitroso imp-2 specificity should be no interference from the diluent no interference no interference no interference precision % content ( n = 6, % RSD < 10.0) 0.81 0.76 0.69 intermediate precision % content ( n = 6, % RSD < 10.0) analyst-1 0.95 1.20 1.45 analyst-2 1.05 1.10 1.30 limit of detection (LOD) LOD (ng/mL) 0.03 0.03 0.03 S/N value (≥3) 6.16 3.79 4.89 limit of quantitation (LOQ) LOQ (ng/mL) 0.10 0.10 0.10 S/N value (≥10) 33.67 17.19 21.19 LOQ precision % content ( n = 6, % RSD 0.999) 0.9996 0.9994 0.9992 accuracy in pure COX (%) ( n = 3, average percentage) the level at 0.00125 ppm mean ± SD 94.80 ± 0.65 95.10 ± 0.65 96.90 ± 0.65 the level at 0.0125 ppm mean ± SD 98.24 ± 0.27 99.14 ± 0.49 98.94 ± 0.89 the level at 0.025 ppm mean ± SD 105.78 ± 0.14 107.18 ± 0.34 107.08 ± 0.36 accuracy in formulated COX (%) ( n = 3, average percentage) the level at 0.00125 ppm mean ± SD 93.98 ± 0.49 95.70 ± 0.65 96.30 ± 0.65 the level at 0.0125 ppm mean ± SD 98.94 ± 0.32 97.54 ± 0.27 98.14 ± 0.17 the level at 0.025 ppm mean ± SD 100.58 ± 0.14 107.18 ± 0.14 108.08 ± 0.14 solution stability peak area (0–48 h, % difference with initial < 10.0) 1.48 1.63 1.12 3.5.3 Linearity The linearity of the method was investigated over the concentration range between 0.1 and 2.0 ng/mL, translating from 0.00125 ppm (1.2 ppb) to 0.025 ppm (25 ppb) with respect to 80 mg/mL sample concentration for all three N- nitroso impurities. In the construction of a set of calibration standards, a diluted stock solution (10 ng/mL) was prepared as described in Section 2.5 . Furthermore, it was diluted to produce the following final concentrations: 0.1, 0.5, 0.8, 1.0, 1.2, 1.5, and 2.0 ng/mL (0.0012, 0.006, 0.01, 0.012, 0.015, 0.019, and 0.025 ppm (μg/g), correspondingly). The method linearity for all N - nitroso impurities was examined at seven distinct concentrations varying from 0.012 to 0.025 ppm (μg/g). The slope (a), intercept (b), and Pearson correlation coefficient (r) were determined by using the linear regression equation with least squares. Calculation of the linear regression equation was performed between peak area and analyte concentration. These results denote a good correlation between the peak areas and the concentrations of all three N -nitroso impurities with data summarized in Table 5 . 3.5.4 Precision In the current method, precision was evaluated by determining the % RSD of the contents of all three N-n itroso impurities from six replicate injections at a concentration of the specification-level spiked solution. Furthermore, intermediate precision is also measured on a different day by a different analyst by determining the % RSD of the contents of all three N-nitroso impurities from a total of 12 replicate injections of spiked solution at the specification level. The results are revealed in Table 5 . 3.5.5 Accuracy and Recovery Study The accuracy of the method was estimated by the triplicate preparation at three levels, 0.0012, 0.012, and 0.025 ppm, to the pure and formulated samples of COX by using the standard addition method. One preparation for each concentration was injected in triplicate. The acceptance criterion for recovery was 80–120%. The obtained recoveries were between 93.98 and 108.08% ( Table 5 ). 3.5.6 Robustness Study The robustness of the method states "the capability of an analytical method to remain unaltered by even small changes in method conditions". During routine use, it provides an indication of its consistency. The flow rate (mL/min), column temperature (°C), collision gas flow (L/Hr), and desolvation temperature (°C) were changed in order to assess the robustness of the current method. The spiked sample solution with all three N-nitroso impurities at the specification level (0.0125 ppm) to the COX drug substance at the concentration of 80 mg/mL was injected into LCMS for the method robustness evaluation. The optimized flow rate was altered by ±0.01 mL/min from the actual flow rate of 0.5 mL/min, the column temperature (°C) was altered by ±1.0 °C from the actual 35 °C, the collision gas flow was altered by ±50 L/Hr from the actual 900 L/Hr, and the desolvation gas temperature was altered by ±50 °C from the actual 450 °C. The optimized UPLC-MS/MS method was designed to be consistent; as a result, the absolute percentage difference in content with respect to nominal conditions for all three N-nitroso impurities was not greater than 3.12 under any altered conditions. The robustness results are shown in Table 6 . Table 6 Summarized Results of the Robustness Study content (%) % absolute difference with respect to the nominal condition altered condition change COX-NDSRI COX- N -Nitroso impurity-1 COX- N -Nitroso impurity-2 COX-NDSRI COX- N -Nitroso impurity-1 COX- N -Nitroso impurity-2 flow rate (mL/min) 0.45 0.0123 0.0127 0.0124 1.60 1.60 3.12 0.50 0.0125 0.0129 0.0128 0.55 0.0124 0.0126 0.0126 0.80 2.32 1.56 column temp (°C) 33.0 0.0124 0.0125 0.0125 0.80 3.12 2.34 35.0 0.0125 0.0129 0.0128 37.0 0.0123 0.0127 0.0124 1.60 1.56 3.12 collision gas flow (L/h) 850 0.0127 0.0127 0.0126 1.60 1.56 1.56 900 0.0125 0.0129 0.0128 950 0.0124 0.0128 0.0125 0.80 0.77 2.34 desolvation temp (°C) 400 0.0124 0.0128 0.0124 0.80 0.77 3.12 450 0.0125 0.0129 0.0128 500 0.0126 0.0127 0.0125 0.80 1.56 2.34 3.5.7 Solution Stability COX and three N-nitroso COX impurities were tested for solution stability by leaving spiked and unspiked samples in capped LC vials for 48 h at 25 °C in an autosampler. We determined the concentration of each impurity against a freshly prepared standard solution, and none of the N-nitroso impurities showed any significant changes. Hence, we concluded that the impurities in the sample solution remained stable at ambient temperature (25 °C) for at least 48 h. The % absolute difference was calculated with respect to the peak areas of the freshly prepared COX and all three N-nitroso impurities. 3.5.1 Specificity In the current method, the specificity of the analytical method was demonstrated by the ability of the LC-MS chromatographic system to distinguish between the diluent and individual impurities. The specificity was assessed by observing the retention times of COX, COX-NDSRI, N-nitroso impurity-1, and N-nitroso impurity-2, along with diluent solutions subjected to LC/MS analysis. The results exposed that all peaks were well separated, and no coeluting peaks were detected at the retention times of COX NDSRI, N-nitroso COX impurity-1, and N-nitroso impurity-2, thus enabling the precise and accurate quantification of the above-mentioned N-nitroso impurities in the COX drug substance as well as in drug products. The typical MRM chromatograms indicating method specificity can be found in Figure 8 a–e. Figure 8 MRM chromatograms of (a) diluent blank, (b) placebo, (c) COX N -nitroso impurity-1, (d) COX-NDSRI, and (e) COX N -nitroso impurity-2. 3.5.2 Determination of the Limit of Detection (LOD) and Limit of Quantitation (LOQ) The values of the LOD and LOQ of COX NDSRI, COX N-nitroso impurity-1, and N- nitroso impurity-2 were estimated by using the signal-to-noise ratio method (S/N) of 3 and 10, respectively. The LOD and LOQ were determined by preparing the known concentrations of standard solutions, which were injected into LC-MS spectrometry. The repeatability (precision) of LOQ was also performed by six replicate injections of these N-nitroso impurities and the % RSD value. The measured value of LOD and LOQ for all N -nitroso impurities was 0.03 and 0.1 ng/mL, respectively. The calculated results are shown in Table 5 , and the typical LOQ MRM chromatograms are shown in Figure 9 , respectively. Figure 9 Typical LOQ MRM chromatograms of (a) COX N -nitroso impurity-1, (b) COX-NDSRI, and (c) COX N -nitroso impurity-2. Table 5 Summary of Method Validation Results results method validation parameter COX-NDSRI COX- N -Nitroso imp-1 COX- N -Nitroso imp-2 specificity should be no interference from the diluent no interference no interference no interference precision % content ( n = 6, % RSD < 10.0) 0.81 0.76 0.69 intermediate precision % content ( n = 6, % RSD < 10.0) analyst-1 0.95 1.20 1.45 analyst-2 1.05 1.10 1.30 limit of detection (LOD) LOD (ng/mL) 0.03 0.03 0.03 S/N value (≥3) 6.16 3.79 4.89 limit of quantitation (LOQ) LOQ (ng/mL) 0.10 0.10 0.10 S/N value (≥10) 33.67 17.19 21.19 LOQ precision % content ( n = 6, % RSD 0.999) 0.9996 0.9994 0.9992 accuracy in pure COX (%) ( n = 3, average percentage) the level at 0.00125 ppm mean ± SD 94.80 ± 0.65 95.10 ± 0.65 96.90 ± 0.65 the level at 0.0125 ppm mean ± SD 98.24 ± 0.27 99.14 ± 0.49 98.94 ± 0.89 the level at 0.025 ppm mean ± SD 105.78 ± 0.14 107.18 ± 0.34 107.08 ± 0.36 accuracy in formulated COX (%) ( n = 3, average percentage) the level at 0.00125 ppm mean ± SD 93.98 ± 0.49 95.70 ± 0.65 96.30 ± 0.65 the level at 0.0125 ppm mean ± SD 98.94 ± 0.32 97.54 ± 0.27 98.14 ± 0.17 the level at 0.025 ppm mean ± SD 100.58 ± 0.14 107.18 ± 0.14 108.08 ± 0.14 solution stability peak area (0–48 h, % difference with initial < 10.0) 1.48 1.63 1.12 3.5.3 Linearity The linearity of the method was investigated over the concentration range between 0.1 and 2.0 ng/mL, translating from 0.00125 ppm (1.2 ppb) to 0.025 ppm (25 ppb) with respect to 80 mg/mL sample concentration for all three N- nitroso impurities. In the construction of a set of calibration standards, a diluted stock solution (10 ng/mL) was prepared as described in Section 2.5 . Furthermore, it was diluted to produce the following final concentrations: 0.1, 0.5, 0.8, 1.0, 1.2, 1.5, and 2.0 ng/mL (0.0012, 0.006, 0.01, 0.012, 0.015, 0.019, and 0.025 ppm (μg/g), correspondingly). The method linearity for all N - nitroso impurities was examined at seven distinct concentrations varying from 0.012 to 0.025 ppm (μg/g). The slope (a), intercept (b), and Pearson correlation coefficient (r) were determined by using the linear regression equation with least squares. Calculation of the linear regression equation was performed between peak area and analyte concentration. These results denote a good correlation between the peak areas and the concentrations of all three N -nitroso impurities with data summarized in Table 5 . 3.5.4 Precision In the current method, precision was evaluated by determining the % RSD of the contents of all three N-n itroso impurities from six replicate injections at a concentration of the specification-level spiked solution. Furthermore, intermediate precision is also measured on a different day by a different analyst by determining the % RSD of the contents of all three N-nitroso impurities from a total of 12 replicate injections of spiked solution at the specification level. The results are revealed in Table 5 . 3.5.5 Accuracy and Recovery Study The accuracy of the method was estimated by the triplicate preparation at three levels, 0.0012, 0.012, and 0.025 ppm, to the pure and formulated samples of COX by using the standard addition method. One preparation for each concentration was injected in triplicate. The acceptance criterion for recovery was 80–120%. The obtained recoveries were between 93.98 and 108.08% ( Table 5 ). 3.5.6 Robustness Study The robustness of the method states "the capability of an analytical method to remain unaltered by even small changes in method conditions". During routine use, it provides an indication of its consistency. The flow rate (mL/min), column temperature (°C), collision gas flow (L/Hr), and desolvation temperature (°C) were changed in order to assess the robustness of the current method. The spiked sample solution with all three N-nitroso impurities at the specification level (0.0125 ppm) to the COX drug substance at the concentration of 80 mg/mL was injected into LCMS for the method robustness evaluation. The optimized flow rate was altered by ±0.01 mL/min from the actual flow rate of 0.5 mL/min, the column temperature (°C) was altered by ±1.0 °C from the actual 35 °C, the collision gas flow was altered by ±50 L/Hr from the actual 900 L/Hr, and the desolvation gas temperature was altered by ±50 °C from the actual 450 °C. The optimized UPLC-MS/MS method was designed to be consistent; as a result, the absolute percentage difference in content with respect to nominal conditions for all three N-nitroso impurities was not greater than 3.12 under any altered conditions. The robustness results are shown in Table 6 . Table 6 Summarized Results of the Robustness Study content (%) % absolute difference with respect to the nominal condition altered condition change COX-NDSRI COX- N -Nitroso impurity-1 COX- N -Nitroso impurity-2 COX-NDSRI COX- N -Nitroso impurity-1 COX- N -Nitroso impurity-2 flow rate (mL/min) 0.45 0.0123 0.0127 0.0124 1.60 1.60 3.12 0.50 0.0125 0.0129 0.0128 0.55 0.0124 0.0126 0.0126 0.80 2.32 1.56 column temp (°C) 33.0 0.0124 0.0125 0.0125 0.80 3.12 2.34 35.0 0.0125 0.0129 0.0128 37.0 0.0123 0.0127 0.0124 1.60 1.56 3.12 collision gas flow (L/h) 850 0.0127 0.0127 0.0126 1.60 1.56 1.56 900 0.0125 0.0129 0.0128 950 0.0124 0.0128 0.0125 0.80 0.77 2.34 desolvation temp (°C) 400 0.0124 0.0128 0.0124 0.80 0.77 3.12 450 0.0125 0.0129 0.0128 500 0.0126 0.0127 0.0125 0.80 1.56 2.34 3.5.7 Solution Stability COX and three N-nitroso COX impurities were tested for solution stability by leaving spiked and unspiked samples in capped LC vials for 48 h at 25 °C in an autosampler. We determined the concentration of each impurity against a freshly prepared standard solution, and none of the N-nitroso impurities showed any significant changes. Hence, we concluded that the impurities in the sample solution remained stable at ambient temperature (25 °C) for at least 48 h. The % absolute difference was calculated with respect to the peak areas of the freshly prepared COX and all three N-nitroso impurities. 3.6 Developed Optimized Method and Their Pharmaceutical Application Five different commercially available formulation samples were investigated using our validated UPLC-MS/MS method to quantify the above-mentioned N-nitrosamine impurities accurately. The estimated amount of COX-NDSRI was in the range of 0.015–0.045 ppm. The test concentration of COX and the COX formulation was 80 mg/mL in triplicate determinations. Both COX N-nitroso impurity-1 and N-nitroso impurity-2 were not detected in all five batch formulation samples. 4 Conclusions The current study synthesized the ciprofloxacin nitroso drug substance-related impurity (NDSRI) and a couple of COX-related N - nitroso impurities and identified them by using NMR and mass spectroscopic data. Two complementary (Q)-SAR methodologies were employed to assess and categorize the N-nitroso COX-related impurities, which were found to be Class 3 category with a cohort of concern, indicating potential genotoxicity. Based on the available literature and regulatory guidelines, COX-NDSRI acceptable intake was considered to be 18 ng/day; hence, it is a need to control and quantify the impurities at low levels. A QbD-based isocratic new simple, accurate, and rapid UPLC-MS/MS method was optimized for the simultaneous trace-level quantification of N -Nitroso COX impurities. This method offered precise and highly sensitive quantification of N-Nitrosamine impurities simultaneously. The method was successfully validated and presented good linearity, accuracy, repeatability, and robustness. Furthermore, the method was successfully applied to N -Nitrosamine impurity quantification in ciprofloxacin drug substance and formulated samples, recurring its high efficiency at low levels. Consequently, the current method can serve as a precise and accurate method to quantify three N -nitroso COX impurities simultaneously from the marketed tablet dosage forms for commercial release and stability sample testing. Supplementary Material ao3c05170_si_001.pdf

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608170/

Forensic genetics and genomics: Much more than just a human affair

While traditional forensic genetics has been oriented towards using human DNA in criminal investigation and civil court cases, it currently presents a much wider application range, including not only legal situations sensu stricto but also and, increasingly often, to preemptively avoid judicial processes. Despite some difficulties, current forensic genetics is progressively incorporating the analysis of nonhuman genetic material to a greater extent. The analysis of this material—including other animal species, plants, or microorganisms—is now broadly used, providing ancillary evidence in criminalistics in cases such as animal attacks, trafficking of species, bioterrorism and biocrimes, and identification of fraudulent food composition, among many others. Here, we explore how nonhuman forensic genetics is being revolutionized by the increasing variety of genetic markers, the establishment of faster, less error-burdened and cheaper sequencing technologies, and the emergence and improvement of models, methods, and bioinformatics facilities. Introduction Forensic genetics derives from a late offshoot of the big tree resulting from the conjunction between legal medicine and criminalistics (for the distinction between forensic genetics and other forensic sciences, see [ 1 – 3 ]). Its historical evolution shows substantial theoretical and technological developments and has, meanwhile, turned this discipline into a broad and independent scientific area for which it is becoming more and more difficult to identify its most remote ancestors. The evolution of modern societies substantially broadened the forensic framework by introducing new forms of resolution of disputes, allowing space for prevention, and regulating more restrictively the prosecution investigations. This means that a potentially forensic situation is the one for which 2 or more sides (individual persons or institutions) agree on the reality of the facts but do disagree on the causes or authorship (thereafter, the term "forensic" is used for these scenarios). Thus, civil litigations (and not just criminal) are common but also conflicts (which are increasing with time) that are attempted to be solved outside a formal court environment [ 4 ]. It is surprising that most of the life span of the discipline has been devoted to human genetics [e.g., 5 ], since a number of disagreements on questions intrinsically related to nonhuman materials always existed and, even when strictly human issues are at stake (such as the identification of a murderer), evidence from nonhuman sources can be crucial or are just the sole type of available evidence [e.g., 6 ]. This has been recognized by the first scientific journal explicitly devoted to forensic genetics ( Forensic Science International : Genetics ), when defining it as "The application of genetics to human and nonhuman material (in the sense of a science with the purpose of studying inherited characteristics for the analysis of inter- and intraspecific variations in populations) for the resolution of legal conflicts" [ 7 ]. Consequently, the division between human and nonhuman forensic genetics (HFG and NHFG, respectively) is not just the result of an anthropocentric historical tradition; rather, it could be derived from the different genomic architectures of the involved organisms [ 8 ]. Importantly, a number of forensically relevant questions are unthinkable in purely human terms ( Fig 1 ), and in this review, we highlight their relevance. 10.1371/journal.pgen.1006960.g001 Fig 1 Most relevant applications of the zoology, botany, microbiology, and food analysis and traceability sciences to NHFG. Diverse examples for each of these applications are shown in S1 Table and S2 Table (see also S1 Text ) and described in the section applications of NHFG. NHFG, nonhuman forensic genetics. Below, we begin by describing the commonly used methodologies, including genotyping and sequencing strategies, evolutionary frameworks, and statistical approaches. Next, we broadly describe applications of NHFG based on diverse biological sources. Finally, we discuss the future of the discipline, including needs and recommendations. Experimental methodologies in NHFG The techniques of forensic genetics originally developed for humans were rapidly adapted to other sources of genetic material. The experimental pipeline used in NHFG ( Fig 2 ) starts with a request for a genetic testing. Next, samples are collected using a sampling kit (either commercial or assembled in the laboratory) and transported to the laboratory under proper conditions. An accurate description of the biological nature of the sample is usually included, and a unique code must be assigned to each collected sample. If the request is part of a legal procedure, not only traceability but also the strict maintenance of the chain of custody (chronological documentation of the evidence) are key issues. The procedure continues in the laboratory, where the genetic material is extracted from the samples using an appropriate and validated protocol. However, certain urgent situations (e.g., bioterrorism) may require the use of methods that were not previously validated. The laboratory may have to deal with new kinds of biological material or taxonomic groups never studied before. In such cases, the laboratory has to be able to develop a valid strategy to extract DNA with sufficient quality and quantity for downstream analyses. 10.1371/journal.pgen.1006960.g002 Fig 2 Pipeline showing the main steps usually involved in processing forensic nonhuman DNA samples. The exact procedure will depend on the conditions available at each laboratory. The process starts with the evaluation of the case and sample collection (green boxes). The procedure continues in the laboratory, where the DNA is extracted from the biological source material and analyzed according to an appropriate protocol (blue boxes). The genetic information is then compared with reference databases and the results are described in a written report (red boxes). Care must be taken when extracting and storing the genetic material to maintain integrity. Storage of nonhuman evidence does not create any specific problem (except required space) that is not common to HFG, and it must be handled in the same way as human material, following the same rules in labelling, chain of custody recording, etc. However, reproducibility in NHFG is clearly a major issue, especially when dealing with wildlife and environmental materials, due to inherent sampling difficulties. Therefore, validation studies cannot be performed in the same strict sense as they are in HFG (for a guideline on these problems, see [ 9 ]). The selection of the genetic test depends on the question to be addressed (see next subsection). For instance, the sequencing of a PCR-amplified genetic region (e.g., cytochrome b [CYTB], cytochrome c oxidase I [COI], and ribosomal RNA [rRNA] genes) is often used for species identification. The identification of individuals can be achieved using a number of markers sufficient to provide high power of discrimination. In any case, it is important to check the quality of profiles or DNA sequences before analysing the results. The genetic information obtained is then compared with other genetic information (i.e., derived from reliable databases [e.g., 10 ] or reference sample[s]) considering statistical analyses. The experimental workflow ends with a report describing the technical procedures applied and the answers to the question(s) of the request. Genotyping and sequencing Genetic identification is based on polymorphic DNA markers that can provide sufficient discriminatory resolution. Traditionally, PCR-based methodologies designed to generate short amplicons, such as Rapid Amplification of Random DNA (RAPD) [ 11 ], Inter Simple Sequence Repeats (ISSR) [ 12 ], and Amplified Fragment Length Polymorphism (AFLP) [ 13 ], were applied in NHFG analyses. Two relevant forensic cases applying RADP are the analysis of plant (seed pods) DNA in a murder case in Phoenix [ 14 ], and the analysis of the outbreak of human anthrax occurred in Sverdlovsk (Ekaterinburg, Russia) [ 15 ]. However, due to their limitations, these techniques were rapidly replaced by Simple Sequence Repeats (SSRs, Short Tandem Repeats [STRs], or microsatellites) and Single Nucleotide Polymorphisms (SNPs) [e.g., 16 ]. The development of reduced-size STR amplicons (miniSTRs) can provide easy PCR amplification of degraded DNA samples, better estimation of mutation rates and allele frequencies, and construction of allelic ladders for accurate classification of alleles. Alternative methods to PCR include technologies such as nucleic acid sequence-based amplification (NASBA) and loop-mediated isothermal amplification (LAMP) [ 17 , 18 ]. Later, a more advanced post-PCR technique, high-resolution DNA melting (HRM) analysis, which is based on the detection of small differences in amplicon melting (dissociation) curves, was also considered for NHFG [e.g., 19 ]. On the other hand, for DNA barcoding, a technique widely used in species identification [e.g., 20 ], is necessary to determine the DNA base composition by targeting specific regions with the Sanger sequencing method. Recently, the arrival of next generation sequencing (NGS) has also revolutionized forensic genetics [ 21 ]. These new technologies provide clear advantages regarding high-throughput due to an extensive multiplexing capacity and parallel sequencing of millions of molecules (Multiple Parallel Sequencing, MPS), allowing a faster and more informative analysis (i.e., characterization of allelic and copy-number variation, CNV) of the genomic material in a sample. Concerning NHFG, MPS is particularly useful for the analysis of samples of complex mixtures since untargeted approaches can be used without prior knowledge about the source. MPS presents additional advantages for NHFG such as the detection of rare polymorphisms, high resolution of genetic analysis, and informative power. Methodologies for the evaluation of statistical evidence and for evolutionary analysis Advances in NHFG are also caused by the progress of bioinformatics and statistical tools. A clear example is the emergence and evolution of the bioinformatics pipeline for the assembly of reads generated in MPS [see for a review, 22 ]. We next describe 2 analytic facets of crucial importance for NHFG: the quantitative evaluation of DNA evidence in the context of identification kinship and population/species assignment (i.e., in shallow evolutionary timescales) and the evolutionary analysis of genetic data (e.g., in transmission of fast evolving pathogens). Statistical evaluation of evidence Here, we overview the use of nonhuman genetic material (NHGM) as ancillary evidence to solve classical forensic problems and in cases that fall outside the civil and criminal human authorship or responsibility. NHGM used as auxiliary evidence in litigations of human authorship NHGM can play a crucial role in the investigation of diverse criminal disputes with the aim of identifying the human individual(s) who committed a crime or is/are responsible for some liability or damage. Historically, the first contributions correspond to situations in which NHGM is used as a silent witness resulting from involuntary transfer and leading to the so-called transfer or associative evidence. This type of NHGM usage is best illustrated in criminalistics where it is increasingly important, as perpetrators are progressively avoiding carefully leaving their biological traces in the crime scene. However, they can, for example, inadvertently leave their pets' hairs at the crime scene or, inversely, to carry the victims' pet biological material [e.g., 6 ]. Although pets are exceedingly common in modern households, many more exotic situations fit with this silent witness type of NHGM use (i.e., knotgrass [ 23 ], mosses [ 24 ], oak [ 25 ], and soil DNA [ 26 ]; see S1 Table and section applications of NHFG). Besides the existing variety of applications, new developments are already at sight such as the genetic profiling of microbiomes and microbial metagenomics [e.g., 27 , 28 – 30 ], or in a not too distant future, the identification of transmitted strains of pathogens or commensals even many years after the crime (as it has been already done for viral transmissions [e.g., 31 , 32 – 36 ]). NHGM used as evidence in other litigations There are several cases for which the expert evidence is used to deal with a law/regulation infringement, irrespectively of the human authorship or responsibility (which may be investigated separately). Here, a comprehensive classification is complex, given the dynamic evolution of the applications and the diversity of ever-growing fields for which laws are constantly being issued. Nevertheless, we must note a clear difference between the applications in which the litigation treats the nonhuman in a framework similar to human cases (individualization and kinship) and a plethora of other applications. Concerning the former, both theoretical frameworks and technological platforms developed for humans can be almost directly translated. This category includes several scenarios: ( i ) an individual living being (e.g., an animal) is the direct causation of an injury to another living being or causes property damages [e.g., 37 , 38 ]; ( ii ) the genetic relationship (e.g., paternity) of a living being to another one is unsettled [e.g., 39 , 40 ]; ( iii ) the identity of the donor of a sample is under dispute (e.g., doping controls in horse races) [e.g., 41 ] ( S2 Table ). As for the latter, a wide range of examples considering NHGM as auxiliary or direct evidence in litigations are derived from diverse subdisciplines such as forensics zoology, botany, microbiology, and food analysis (see Fig 1 and section applications of NHFG). In any case, statistical analysis should provide likelihoods of observations, rather than categorical answers, and at least 2 alternative, mutually exclusive hypotheses should be formulated. Broadly speaking, statistical evaluation in NHFG can be required for 3 major scenarios: Individual identification or kinship. It involves cases such as "was a given dog the perpetrator of the attack?" or "is a given foal the offspring of a given highly prized horse?" Species identification. It involves interspecies cases such as "does the label of a processed fish product agree with the species of origin?" Subspecific assignment/identification. It involves cases related with breed, variety, or populations such as "was the attack perpetrated by a dog or by a wolf?" Regarding scenario A, similarly to what has been established for humans, autosomal STRs are preferentially considered and analysed with a Bayesian approach (in which prior odds are combined with probabilities of the genotypic observations assuming the alternative hypotheses). Nevertheless, several difficulties can arise in practice, especially when dealing with small sized and/or poorly studied populations, as in endangered species. The lack of knowledge in the population structure and sampling errors obviously has a serious impact on the confidence of the parameter estimates. The software developed in HFG for kinship and identification can be used in this scenario. For instance, the computer programs GDA [ 42 ] and GenePop [ 43 ] can be applied to test Hardy-Weinberg Equilibrium and to estimate population genetics parameters, while the program Familias [ 44 ] can be used to compute kinship likelihood ratios. Regarding scenario B, the traditional procedure consists of comparing sequences that are highly variable among species but highly conserved within species, in the so-called DNA barcoding [ 45 ]. An alternative approach relies on comparing lengths of insertion and deletion polymorphisms without requiring DNA sequencing [ 46 , 47 ]. Importantly, these approaches are only possible due to the existence and maintenance of reliable and public databases such as GenBank, EMBL, and Bold. Note that the increasing number and length of sequences existing in databases and the development of automated mechanisms to prevent misclassified sequences would allow more confidence in species identification. Finally, the statistical significance of sequence comparison should be computed [ 48 ] and reported. Regarding scenario C, the selection of the genetic marker depends on the investigated species. For metazoan, the most used markers are regions of the mitochondrial genome (and plastid for Plantae) that can provide accurate distinction between subspecies [e.g., 49 ] and autosomal regions of nuclear DNA [e.g., 50 ]. The program STRUCTURE [ 51 ] is widely used for the Bayesian assignment of an individual to a population (or subspecies). Again, the statistical evaluation should be performed and reported. NHGM used as auxiliary evidence in litigations of human authorship NHGM can play a crucial role in the investigation of diverse criminal disputes with the aim of identifying the human individual(s) who committed a crime or is/are responsible for some liability or damage. Historically, the first contributions correspond to situations in which NHGM is used as a silent witness resulting from involuntary transfer and leading to the so-called transfer or associative evidence. This type of NHGM usage is best illustrated in criminalistics where it is increasingly important, as perpetrators are progressively avoiding carefully leaving their biological traces in the crime scene. However, they can, for example, inadvertently leave their pets' hairs at the crime scene or, inversely, to carry the victims' pet biological material [e.g., 6 ]. Although pets are exceedingly common in modern households, many more exotic situations fit with this silent witness type of NHGM use (i.e., knotgrass [ 23 ], mosses [ 24 ], oak [ 25 ], and soil DNA [ 26 ]; see S1 Table and section applications of NHFG). Besides the existing variety of applications, new developments are already at sight such as the genetic profiling of microbiomes and microbial metagenomics [e.g., 27 , 28 – 30 ], or in a not too distant future, the identification of transmitted strains of pathogens or commensals even many years after the crime (as it has been already done for viral transmissions [e.g., 31 , 32 – 36 ]). NHGM used as evidence in other litigations There are several cases for which the expert evidence is used to deal with a law/regulation infringement, irrespectively of the human authorship or responsibility (which may be investigated separately). Here, a comprehensive classification is complex, given the dynamic evolution of the applications and the diversity of ever-growing fields for which laws are constantly being issued. Nevertheless, we must note a clear difference between the applications in which the litigation treats the nonhuman in a framework similar to human cases (individualization and kinship) and a plethora of other applications. Concerning the former, both theoretical frameworks and technological platforms developed for humans can be almost directly translated. This category includes several scenarios: ( i ) an individual living being (e.g., an animal) is the direct causation of an injury to another living being or causes property damages [e.g., 37 , 38 ]; ( ii ) the genetic relationship (e.g., paternity) of a living being to another one is unsettled [e.g., 39 , 40 ]; ( iii ) the identity of the donor of a sample is under dispute (e.g., doping controls in horse races) [e.g., 41 ] ( S2 Table ). As for the latter, a wide range of examples considering NHGM as auxiliary or direct evidence in litigations are derived from diverse subdisciplines such as forensics zoology, botany, microbiology, and food analysis (see Fig 1 and section applications of NHFG). In any case, statistical analysis should provide likelihoods of observations, rather than categorical answers, and at least 2 alternative, mutually exclusive hypotheses should be formulated. Broadly speaking, statistical evaluation in NHFG can be required for 3 major scenarios: Individual identification or kinship. It involves cases such as "was a given dog the perpetrator of the attack?" or "is a given foal the offspring of a given highly prized horse?" Species identification. It involves interspecies cases such as "does the label of a processed fish product agree with the species of origin?" Subspecific assignment/identification. It involves cases related with breed, variety, or populations such as "was the attack perpetrated by a dog or by a wolf?" Regarding scenario A, similarly to what has been established for humans, autosomal STRs are preferentially considered and analysed with a Bayesian approach (in which prior odds are combined with probabilities of the genotypic observations assuming the alternative hypotheses). Nevertheless, several difficulties can arise in practice, especially when dealing with small sized and/or poorly studied populations, as in endangered species. The lack of knowledge in the population structure and sampling errors obviously has a serious impact on the confidence of the parameter estimates. The software developed in HFG for kinship and identification can be used in this scenario. For instance, the computer programs GDA [ 42 ] and GenePop [ 43 ] can be applied to test Hardy-Weinberg Equilibrium and to estimate population genetics parameters, while the program Familias [ 44 ] can be used to compute kinship likelihood ratios. Regarding scenario B, the traditional procedure consists of comparing sequences that are highly variable among species but highly conserved within species, in the so-called DNA barcoding [ 45 ]. An alternative approach relies on comparing lengths of insertion and deletion polymorphisms without requiring DNA sequencing [ 46 , 47 ]. Importantly, these approaches are only possible due to the existence and maintenance of reliable and public databases such as GenBank, EMBL, and Bold. Note that the increasing number and length of sequences existing in databases and the development of automated mechanisms to prevent misclassified sequences would allow more confidence in species identification. Finally, the statistical significance of sequence comparison should be computed [ 48 ] and reported. Regarding scenario C, the selection of the genetic marker depends on the investigated species. For metazoan, the most used markers are regions of the mitochondrial genome (and plastid for Plantae) that can provide accurate distinction between subspecies [e.g., 49 ] and autosomal regions of nuclear DNA [e.g., 50 ]. The program STRUCTURE [ 51 ] is widely used for the Bayesian assignment of an individual to a population (or subspecies). Again, the statistical evaluation should be performed and reported. Evolutionary analysis of genetic data in forensic genetics Diverse organisms involved in forensic studies present short generation times and belong to large populations (as most pathogens). Therefore, relationships between queried and control samples are usually obtained under the light of evolutionary analyses since those samples most likely belong to distant generations [ 52 ]. The evolutionary analysis not only provides the identification of genetic relationships (dealing with questions like, "is the suspect the cause of the studied transmission or outbreak?" or "which individuals were infected by the suspect and which individuals were infected or coinfected from other sources?") [e.g., 31 – 36 , 53 , 54 , 55 ], but also allows the estimation of the timing of transmission events (i.e., infection date of each individual, including the individual that generated the outbreak) [e.g., 53 , 54 ]. The computational pipeline for the evolutionary analysis of genetic data in NHFG follows well-established methodologies ( Fig 3 ); however, several steps must be carefully performed. First, query, control (from local and background regions), and external (i.e., from reliable databases) sequences must be aligned. Next, population genetics statistics such as genetic diversity and genetic differentiation (i.e., between query and control sequences) can be estimated [e.g., 54 , 55 , 56 ]. The alignment can also be used to infer a phylogenetic history that depicts genetic relationships between the sample sequences and provides the timing of common ancestors (i.e., transmission events). A large number of pathogens (including those involved in most of NHFG cases, Human Immunodeficiency Virus [HIV] and Hepatitis C Virus [HCV]) evolve with processes of exchange of genetic material such as recombination [ 57 , 58 ] and horizontal gene transfer [ 59 ]. Importantly, ignoring these processes can bias phylogenetic tree inferences by generating incorrect branch lengths and topologies [ 60 , 61 ]. Therefore, under the presence of these processes, a phylogenetic network, which may have embedded a phylogenetic tree for each exchanged fragment [ 62 ], should be inferred [ 35 , 61 , 63 , 64 ]. Indeed, a substitution model of evolution that fits the data best should be selected and considered in sophisticated phylogenetic inferences (i.e., based on maximum-likelihood or Bayesian approaches) [ 61 , 65 ]. Importantly, phylogenetic approaches usually implement statistical confidence of the inferred evolutionary relationships through a bootstrap analysis [ 66 ]. In NHFG, this statistical parameter can provide a measure of the reliability of relationships between the pathogen genetic sequences of the investigated individuals. For example, a number of forensic studies based on phylogenetic inferences showed a classification of all control individuals in significantly separated clades, whereas individuals related with the studied outbreak or transmission clustered in a unique clade [e.g., 31 , 32 , 34 , 35 , 53 , 54 , 55 ]. Likelihood ratio tests can also be useful for hypothesis testing (i.e., testing if control sequences group or not with the studied outbreak) [e.g., 54 ]. 10.1371/journal.pgen.1006960.g003 Fig 3 Pipeline showing the evolutionary analysis of genetic data oriented to NHFG. Data and tasks are shown in boxes, and databases and computer frameworks are shown in circles. Population genetic parameters include measures of genetic diversity, genetic differentiation, and demographics. The phylogenetic analysis requires the previous identification of recombination and can be performed ignoring or considering a substitution model of evolution. NHFG, nonhuman forensic genetics. As noted above, the estimated time of internal nodes of the inferred phylogeny can be useful in forensic litigations by revealing the timing of infections [ 52 ]. These times can be estimated with Bayesian approaches [e.g., 67 ] accounting for longitudinal sampling (the tips are dated with the corresponding sampling times) to calibrate the (often relaxed) molecular clock and can provide accurate confidence intervals [e.g., 54 ]. Applications of NHFG NHFG is expanding to more and more biological areas due to the increasing emergence of forensic cases based on NHGM. In this section, we revise the most relevant areas of NHFG, including zoology, botany, microbiology, and food analysis and traceability. Zoology The relevance and close presence of animals in a variety of human activities explain why they are among the first targets of NHFG [ 6 , 68 – 70 ]. The number of animal species studied from a forensic genetics perspective has increased significantly, and different testing protocols have been developed for determining the identity of a sample at different biological levels such as individual, population, breed, species, or higher taxonomic classifications. The preferential DNA markers used for individual identification in animals are autosomal STRs, as established in HFG. For example, STR kits have been developed for individual identification and kinship testing in dogs [ 71 – 78 ], cats [ 79 – 81 ], horses [ 40 , 82 , 83 ], cattle [ 39 , 84 ], bears [ 85 ], deer [ 86 , 87 ], badgers [ 88 ], birds [ 89 , 90 ], and koi carps [ 91 ]. They have also been employed in resolving criminal and civil cases, such as dog or bear attacks [ 37 , 38 , 92 ], silent witnesses of crimes [ 6 ], identification of samples from sport horses [ 41 , 93 ], and in wildlife crime investigations (wildlife forensics), including big cats [ 94 ], mouflons [ 95 , 96 ], wild boars [ 97 , 98 ], and elephants [ 99 ], among others. Concerning the latter, we want to highlight the application of forensic genetics to the illegal wildlife trade (IWT), since this is one of the biggest threats to a variety of species and habitats, with a consequent loss of biodiversity [ 100 , 101 ]. In addition, IWT is a large-scale business estimated in billions of euros that generate negative socioeconomic impacts [ 100 , 101 ]. Importantly, forensic genetics plays a crucial role in wildlife law enforcement [ 101 ]. Pioneer works endured tremendous efforts trying to reach the quality standards of human genetic testing. Difficulties in developing a new genotyping system for animals are various, including the collection of representative samples (especially problematic in wild species), the access to high-quality genomic sequences (not available for several species) and obtaining funding for the experiments (often focused on human research). Therefore, some of these STR kits are still a few steps behind those developed for human identification. For example, dinucleotide repeats are still used in nonhuman DNA testing [e.g., 37 , 88 , 91 , 94 ], making it difficult to interpret sample mixtures and heterozygotes due to stutter product formation [ 102 ]. The most advanced nonhuman profiling kits are those developed for domesticated animals, including several STRs with tetranucleotide repeats [e.g., 72 , 80 , 81 , 103 ]. Indeed, sex chromosome STR markers can also be useful for NHFG, however they still remain uncharacterized for many animal species. The mammalian Y-chromosome is used for gender identification, resolving paternity and family structures with application in forensic investigations [e.g., 104 , 105 , 106 ]. The development of an X-chromosome STR kit for dogs in 2010 [ 107 ] was a promising step in this field but, unfortunately, it was not followed by similar works in other species. The determination of the sex in birds has been possible using markers located in the W and Z chromosomes [e.g., 108 , 109 , 110 ]. A few panels of autosomal SNPs have also been developed for individual identification in different animal species [ 111 – 117 ]. These genetic markers may have some technical advantages over STRs [e.g., 102 , 118 ] and can provide information about physical traits. Forensic zoology often has to deal with degraded samples. In such cases, mtDNA may be the only source of genetic information that can be used. The high copy number of mtDNA in cells increases the probability of obtaining results from degraded/low-copy DNA samples such as hair, bones, and scat [ 119 , 120 ]. Importantly, the same mtDNA sequence can be found in many individuals of a population and therefore cannot be used for individual identification. However, it can be used to exclude an individual as a source of a casework sample, and its utility has been demonstrated for a variety of animal species [e.g., 70 , 121 , 122 – 124 ]. Nevertheless, the most successful use of mtDNA in forensic zoology has been in species identification. Different mtDNA regions have been tested and validated for use in a forensic context, including CYTB [ 125 – 127 ], COI [ 128 – 130 ], and rRNA genes [ 131 , 132 ]. The procedure usually involves the sequencing of a variable region amplified with conserved PCR primers followed by database searches and phylogenetic analyses. This strategy was applied in different forensic investigations such as identification of rhinoceros horns [ 133 ], ivory [ 134 ], turtle shells [ 135 ], endangered snake species [ 136 ], tigers [ 137 ], forensically important insect species [ 138 – 140 ], illegally smuggled eggs [ 141 ], or fish and fish products [ 142 – 144 ]. A few multiplex PCR/primer extension assays to genotype mtDNA SNPs have also been developed for species and subspecies identification (i.e., tiger [ 49 ], elephant [ 145 ], and other animals [ 146 , 147 ]). While the genetic identification of an individual or a species is not problematic in most situations, defining animal breeds or geographical populations has been considerably more difficult. Most breeds had a recent origin and are often defined by a few morphological features arbitrarily defined. For instance, cat breeds are defined by phenotypic characteristics (e.g., hair length, coat patterning, and colours) that are single-gene traits. Nevertheless, most cats can be assigned to their proper breed or population of origin using genetic data [ 148 ]. A crucial aspect for some forensic cases (i.e., poaching or illegal logging) is the identification of the origin of the sample. This identification depends on the existence of genetic data in different regions (including the region of the "real" origin), enough genetic differentiation among regions and the quality of the analytical method. The recent origin, intensive inbreeding, and genetic drift make difficult-to-use neutral genetic markers for rigorous identification of breed or populations. In such ambiguous cases, genetic tests should assess the genetic variants of the morphological traits that define the breed. However, our understanding of the genetics underlying such complex traits is still very limited, although significant progress is expected [ 149 ]. A famous case of origin identification was the mad cow disease between the United States and Canada [details in 150 , 151 ], where a novel parentage testing was developed by combining prions and kinship [ 151 ]. Botany Plant evidence can provide crucial information for the reconstruction of forensically relevant events or in cases where the crime scene and autopsy reports are not compelling [ 152 ]. Conventional taxonomic identification (using morphological methods) has a reduced application since botanical forensic evidences are often very fragmented (e.g., pieces of leaves or seeds) limiting the use of dichotomous keys. However, molecular markers can be applied to identify samples, regardless of their state, morphology, and development phase. In this concern, in the last couple of decades, diverse molecular markers have been applied for the forensic identification of species and individuals (i.e., HRM coupled with specific barcodes or real-time PCR to analyze chloroplast DNA regions) [e.g., 153 , 154 ]. Importantly, second- and third-generation sequencing methodologies are providing affordable analysis of complex and degraded plant samples [ 155 , 156 ]. Forensic botany presents numerous applications such as the identification of the origin of seized illegal drugs (marijuana [ 157 ], kratom [ 158 ], or opium [ 159 ]), detection of illegal logging [ 160 , 161 ], importation and commercialization of endangered and exotic species [ 162 , 163 ], or bioterrorism (abrin and ricin attacks [ 164 ]). It can also provide useful supporting evidence in crime scene investigations, allowing us to establish a link between the victim and the suspect, placing the suspect at a crime scene or estimating the time of death [e.g., 23 , 25 , 165 – 167 ]. Microbiology Although microbes have long been recognized as important players in our daily life, present in areas such as medicine and public health, ecology, and in industrial applications, microbial forensics (MF) is still a relatively recent scientific field [ 168 , 169 ]. MF aims to identify a target microorganism and its source. Although culture in selective growth media remains as the preferred standard for characterization of microbial agents at the resolution of genus/species level, complementary detection methods based on diverse molecular markers are increasingly applied [ 170 ]. Indeed, NGS technologies profoundly improved the ability to detect microorganisms, even when present in low abundance or in degraded or mixture samples, and to differentiate at strain/isolate level, using diagnostic genomic signatures [ 171 ]. Applications of MF involve diverse areas such as biocrimes, bioterrorism, frauds, outbreaks and transmission of pathogens, or accidental release of a biological agent or a toxin [e.g., 54 , 172 ]. Additionally, the recent breakthroughs derived from NGS technologies allowed the analysis of microbial evidence to be expanded to cases related with geolocation, body fluid characterization, or postmortem interval estimation [ 168 ]. Some biological agents can be used as weapons or threats. The best well-known example is the Amerithrax case (2001), where letters laden with Bacillus anthracis spores were sent through the U.S. Postal Service to several media offices in New York and Florida and to U.S. senators in Washington [ 173 , 174 ]. In this case, DNA evidence was found in the suspect's laboratory. Under the scope of epidemiological investigation, MF also helps to determine whether a pathogen outbreak was natural or human-driven. Therefore, MF is intimately associated with epidemiological surveys, allowing studying and following disease outbreak dynamics, mainly concerning the identification of the agent or toxin, origin and natural reservoirs, genetic diversity and evolution, and possible transmission routes. Some well-known cases of the epidemiological studies are the swine-origin influenza A virus (H1N1; 2009) [ 175 ], the Haitian cholera (2010) [ 176 ], the haemolytic-uremic syndrome ( Escherichia coli O104:H4; 2011) [ 177 ], the Coronavirus Middle East respiratory syndrome (2012) [ 178 ], the avian-origin Influenza A virus (H7N9; 2013) [ 179 ], the West African Ebola virus (2013/2015) [ 180 ], the Middle Eastern poliomyelitis (2014) [ 181 ], the Portuguese Legionnaires' disease (2014) [ 182 ], and the Zika virus outbreaks [ 183 ]. Note that most of the cases indicated above applied NGS approaches to identify and study the different biological agents. Applications of MF in biocrimes also include the tracking of sexually transmitted diseases and healthcare malpractice linked to the transmission of HIV [e.g., 31 – 36 , 53 , 184 ] and HCV [e.g., 54 , 55 ]. Moreover, this discipline is also used to determine responsibilities in cases of hospital-acquired infections [e.g., 185 , 186 ] or sudden death syndrome [e.g., 187 , 188 ]. The human microbiome is starting to be a focus of interest for identification purposes. The rational is to trace human microbiomes on our skin on the surfaces and objects we interact with the potential to supplement the use of human DNA for associating people with evidence and environments. The Human Microbiome Project has significantly improved the scientific knowledge in the field [ 189 , 190 ]. Note that there are 10 times more bacteria than human cells in our body [ 191 ], and a number of them appears to be unique to each person [ 192 ], offering an opportunity for new identification biomarkers [ 193 ]. Thus, the human microbiome could be used to identify suspects [e.g., 27 , 28 , 29 ] and to estimate the postmortem interval [ 194 ]. For example, the origin of human remains from the Second World War was ascertained with the parvovirus B19V [ 195 ]. Although these are promising findings, we consider that we are still far from a foundational validation of this approach to be used in legal cases. One of the main constraints associated with the use of MF is the lack of standards and guidelines, although phylogenetic analyses have supported associations and have successfully been admitted as evidence in legal criminal cases [ 196 ]. Another limitation is the insufficiency of reference databases lacking endemic data or microorganism source tracing, reference genome sequences, metadata, and representative genetic diversity coverage [ 197 ]. Food analysis and traceability The investigation of the biological composition of food products regarding the species, variety or cultivar, and geographic origin is of major forensic interest. Such investigations are relevant for guaranteeing consumer choices according to health concerns (e.g., sensitivities or allergies), dietary preferences (e.g., vegetarian, nongenetically modified organisms), religious beliefs (e.g., halal and kosher specifications), and to detect fraudulent substitution of a given species by a similar one with lower economic value [ 198 , 199 ]. Labelling is indispensable for producers, retailers, and consumers to recognize and validate components of foodstuffs [ 200 ]. Unfortunately, labels of products often provide insufficient and erroneous information concerning the exact contents. The methodology used in food forensics is similar to that used in classical crime investigations, facing the same demands of dealing with potentially degraded DNA samples [ 201 ]. Several DNA-based methods have become remarkably valuable for protecting and certifying the quality and source of food [ 202 , 203 ]. The first studies performed in the 90's resorted to classical techniques (i.e., RADP and ISSR) but nowadays, real-time PCR [ 204 ], HRM [ 205 – 207 ], and MPS [ 208 ] are widely applied for food traceability with the advantage of quantifying each particular component in a faster and affordable procedure. These genetic markers have been applied to perform identification in a variety of food products such as olive oil [e.g., 209 , 210 ], grapevine cultivars [e.g., 211 , 212 , 213 ], composition of honey [e.g., 214 , 215 , 216 ], mushrooms [e.g., 217 , 218 , 219 ], dairy products [e.g., 220 , 221 , 222 ], seafood products [ 20 ], or meat species adulteration [ 223 ]. Additional documented cases include: i ) identification of cultivars of basmati rice [ 224 ], pome [ 225 ] and stone fruits [ 226 ], leguminosae [ 227 , 228 ], coffee [ 229 ], and tea and infusions [ 230 ]; ii ) patent misappropriation of strawberry cultivars [ 231 ]; iii ) confirmation of Protected Designation of Origin (PDO), Protected Geographical Indication (PGI), or Traditional Speciality Guaranteed (TSG) in olive [ 232 ] and grape [ 213 , 233 ] products; iv ) adulteration of traditional medicines [ 234 , 235 ] and herbs or spices [ 236 ]; v ) insufficient and erroneous food labelling, including the presence of some hidden allergens [ 237 , 238 ] or genetically modified organisms [ 239 ] (GMOs; see section Genetically modified organisms). Food microbiology Over the last 2 decades, the prevalence of foodborne diseases has drastically increased, becoming a worldwide major public health concern. Foodborne diseases are often triggered by the consumption of food or water contaminated either by pathogens (bacteria, viruses, fungi, and parasites) or derived toxins. The most common pathogens responsible for foodborne disease outbreaks are Listeria monocytogenes , Escherichia coli O157:H7, Staphylococcus aureus , Salmonella enterica , Bacillus cereus , Vibrio spp., Campylobacter jejuni , Clostridium perfringens , and Shigella dysenteriae . These pathogens are often associated with consumption of raw (e.g., fruits and vegetables) or undercooked foods (e.g., seafood, meat, and poultry) [ 240 ]. To overcome the limitations of the traditional culture of microorganisms (e.g., may disallow the cultivation of the major foodborne pathogen or may present a slow growth leading to long periods of time cultivation), DNA/RNA-based methods (i.e., STR, NASBA, LAMP, and NGS) are usually applied [e.g., 241 ]. Genetically modified organisms An area of growing interest is the detection of GMOs. The number of genetically modified plants has been growing in recent years despite the intense discussion about the benefits or damage that these organisms may have on humans and ecosystems. The detection of a GMO is carried out by targeting the genetic elements (promotors, protein-coding regions or terminators) that have been introduced artificially in the genome of the transgenic organism in order to improve a particular trait [ 242 ]. A curated list of transgenic reference sequences has been recently made available and is expected to facilitate the development of methods for testing GMOs and the implementation of regulatory policies [ 243 ]. The labelling and traceability of GMOs are important issues that are highly regulated. If the content exceeds a certain threshold, the product must be labeled accordingly. The most commonly used DNA-based methodology for GMO testing is PCR, although other techniques have been proposed [ 244 – 246 ]. The quantification of DNA targets is usually done by real-time PCR, where the copy number of the transgenic element detected is correlated to a common plant marker, allowing the determination of the GMO proportion in the sample [ 247 , 248 ]. The correct detection of genetically modified materials is of forensic relevance not only due to strict legislation regarding the labelling of food products but also due to the type of materials from which DNA has to be extracted. For example, transgenic constructs have to be identified in DNA extracted from products like corn germ, flour, pasta, corn flakes, cookies, baked products, sugars derived from corn starch, soy cream or milk (liquid or lyophilized), tofu, meat products, lecithin, and even oil. Although most of the currently available GMOs are plants, the picture is expected to change soon. The first genetically modified animal (AquAdvantage salmon) is on the verge of being approved for human consumption in different countries [ 249 ]. New methods are being developed to detect the genetically modified salmon in food products [ 250 , 251 ]. Strong legislation is expected to regulate the presence of this transgenic animal in foods and environmental samples [ 252 , 253 ] and, consequently, reliable and sensitive methods for its detection will be required by regulatory and scientific agencies worldwide [ 245 , 254 ]. Food microbiology Over the last 2 decades, the prevalence of foodborne diseases has drastically increased, becoming a worldwide major public health concern. Foodborne diseases are often triggered by the consumption of food or water contaminated either by pathogens (bacteria, viruses, fungi, and parasites) or derived toxins. The most common pathogens responsible for foodborne disease outbreaks are Listeria monocytogenes , Escherichia coli O157:H7, Staphylococcus aureus , Salmonella enterica , Bacillus cereus , Vibrio spp., Campylobacter jejuni , Clostridium perfringens , and Shigella dysenteriae . These pathogens are often associated with consumption of raw (e.g., fruits and vegetables) or undercooked foods (e.g., seafood, meat, and poultry) [ 240 ]. To overcome the limitations of the traditional culture of microorganisms (e.g., may disallow the cultivation of the major foodborne pathogen or may present a slow growth leading to long periods of time cultivation), DNA/RNA-based methods (i.e., STR, NASBA, LAMP, and NGS) are usually applied [e.g., 241 ]. Genetically modified organisms An area of growing interest is the detection of GMOs. The number of genetically modified plants has been growing in recent years despite the intense discussion about the benefits or damage that these organisms may have on humans and ecosystems. The detection of a GMO is carried out by targeting the genetic elements (promotors, protein-coding regions or terminators) that have been introduced artificially in the genome of the transgenic organism in order to improve a particular trait [ 242 ]. A curated list of transgenic reference sequences has been recently made available and is expected to facilitate the development of methods for testing GMOs and the implementation of regulatory policies [ 243 ]. The labelling and traceability of GMOs are important issues that are highly regulated. If the content exceeds a certain threshold, the product must be labeled accordingly. The most commonly used DNA-based methodology for GMO testing is PCR, although other techniques have been proposed [ 244 – 246 ]. The quantification of DNA targets is usually done by real-time PCR, where the copy number of the transgenic element detected is correlated to a common plant marker, allowing the determination of the GMO proportion in the sample [ 247 , 248 ]. The correct detection of genetically modified materials is of forensic relevance not only due to strict legislation regarding the labelling of food products but also due to the type of materials from which DNA has to be extracted. For example, transgenic constructs have to be identified in DNA extracted from products like corn germ, flour, pasta, corn flakes, cookies, baked products, sugars derived from corn starch, soy cream or milk (liquid or lyophilized), tofu, meat products, lecithin, and even oil. Although most of the currently available GMOs are plants, the picture is expected to change soon. The first genetically modified animal (AquAdvantage salmon) is on the verge of being approved for human consumption in different countries [ 249 ]. New methods are being developed to detect the genetically modified salmon in food products [ 250 , 251 ]. Strong legislation is expected to regulate the presence of this transgenic animal in foods and environmental samples [ 252 , 253 ] and, consequently, reliable and sensitive methods for its detection will be required by regulatory and scientific agencies worldwide [ 245 , 254 ]. The future of NHFG Within the enormous variety of applications, methods, and sources of NHFG, the forensic use of NHGM is still limited and faces enormous difficulties due to diverse causes. Among them, and perhaps the most important, is the sheer amount of biodiversity and the current poor knowledge about it, with an impact not only on the species identification problem but also at the intraspecific level where, for most wildlife organisms, population genetics data are nonexistent or extremely poor. This makes relevant parameters difficult to estimate with acceptable accuracy and thus inhibits solid statistical evaluation of the evidence [ 255 ]. In this concern, the impact of the International Barcode of Life project (iBOL, http://www.ibol.org/ ) on forensics has been much less than desired and several difficulties have been raised on its power, limitations, and governance [ 256 , 257 ]. In fact, most biodiversity studies do not meet classical forensic standards (demanded in forensic routine casework), due to the inherently limited sampling, references and controls. Moreover, there is a lack of agreement and concerted actions between the scientific societies aiming at the forensic use of NHGM (ISAG, International Society for Animal Genetics; ISFG, International Society for Forensic Genetics; SWSF, The Society for Wildlife Forensic Science; ISEF, International Society of Environmental Forensics) that is reflected in nonreconcilable or even contradictory recommendations and guidelines (particularly between ISFG [ 9 ] and ISAG/FAO [ 258 ]). Given the increasing use of NHFG, we do hope for some progress in joining efforts between scientific communities for a mutual benefit. On the other hand, less error-burdened, cheaper, and faster MPS, together with progress in bioinformatics frameworks and computational resources, now allow the analysis of complex samples (i.e., commingled samples with DNA from more than one contributor/species) with more accurate and reliable results [e.g., 177 , 186 ]. With third generation sequencing technologies, single DNA molecules can be analyzed individually [e.g., 259 ] and, therefore, haplotypes can be determined. These advances are expected to revolutionize NHFG. Among other examples, MPS was already applied to the identification of species for quality control in the development and authentication of herbal and traditional medicines [ 260 ] and for the discrimination of soils and other detritus from alternative environments and locations, based on the composition of the microflora, plants, metazoan, and protozoa DNA sequences [ 21 , 261 – 265 ]. As noted in MF, the implementation of MPS is particularly useful for epidemiological studies. However, more research is necessary for the improvement of libraries (i.e., reference sequences reflecting the coverage of the entire genome of diverse organisms), development of bioinformatics platforms (i.e., for decreasing memory requirements and implementing algorithms for parallel computing) and for reproducibility and assignment of general quality of the results. Moreover, current MPS technologies still present relatively high sequencing errors [e.g., 266 ] which, although could be assumed for other disciplines, may not meet forensic standards [ 267 ]. Therefore, strict MPS validation studies are mandatory but they are still very scarce, even in human applications. In this regard, it is clear that genetic analyses based on very large datasets (ideally, whole genomes) can provide high statistical confidence that can be useful for forensic cases [ 268 ]. However, systematic biases in methods applied to the analysis of large data can lead to precise but incorrect results [ 269 – 271 ]. Therefore, not only are large datasets required, less biased state-of-the-art methodologies should also be applied [ 272 ]. An example of this situation is the evolutionary analysis of genetic data. This analysis can be improved with the consideration of more complex substitution models of evolution (i.e., nonreversible and nonstationary) that can better fit the data [ 65 , 273 ]. However, these models were not implemented yet into the traditional frameworks of the phylogenetic pipeline and, to our knowledge, all existing NHFG studies have ignored them. In addition, as noted above, evolutionary processes that exchange genetic material (e.g., recombination) can bias phylogenetic tree inferences [ 60 ]. However, to our knowledge, all existing NHFG studies including phylogenetic tree inferences from pathogens that usually evolve with high recombination rates (i.e., HIV and HCV) ignored recombination [e.g., 31 – 36 , 53 , 54 , 55 ]. We strongly recommend considering these aspects in future NHFG studies. The future of NHFG is dependent on the progress in removing current limitations (i.e., funding, adapting scientific methods into court [ 274 ], taking away from HFG and dealing with much smaller documented biodiversity being more complex to achieve forensic standards), but this is an emerging field of increasing importance. The number of papers in the top forensic journals on nonhuman DNA typing topics is increasing at a rate of 15% per year, especially on IWT [ 275 ]. As mentioned by Ogden and Linacre [ 101 ], perhaps the main difficulty in this field is the large proportion of traded products originated from underdeveloped countries where wildlife trade monitoring and the ability of the enforcement agencies to act are limited. That difficulty is caused by the lack of funding since the priorities of the majority of law enforcement agencies are crimes against humans and their properties. The continuous incorporation of genomic data in reliable databases together with progress of experimental methodologies and analytical software are expected to further increase the application of NHFG. Assuming this direction, we believe that, in the future, NHFG could even overpass HFG in number of cases investigated, since the number of informative organisms is extremely large. Supporting information S1 Table Illustrative examples of scenarios using nonhuman genetic material (NHGM) for auxiliary evidence in litigations related with human identification. (PDF) Click here for additional data file. S2 Table Illustrative examples of scenarios of nonhuman genetic material (NHGM) for main evidence in litigations not related with human identification. (PDF) Click here for additional data file. S1 Text Literature cited in the supplementary material. (PDF) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8946816/

In Vitro, In Vivo, and In Silico Models of Lymphangiogenesis in Solid Malignancies

Simple Summary Lymphangiogenesis is the formation of new lymphatic vessels in physiological conditions but has also been found to be associated with pathologies. For example, it has been proven to be involved in cancer progression and metastatic dissemination through the body. Thus, it became a key element to study in the management of this widespread disease. To date, the study of lymphangiogenesis takes place at the biological (in vitro and in vivo) and computational (in silico) levels. The association of these complementary fields combined with imaging techniques constitutes a real toolbox in pathological lymphangiogenesis understanding. Abstract Lymphangiogenesis (LA) is the formation of new lymphatic vessels by lymphatic endothelial cells (LECs) sprouting from pre-existing lymphatic vessels. It is increasingly recognized as being involved in many diseases, such as in cancer and secondary lymphedema, which most often results from cancer treatments. For some cancers, excessive LA is associated with cancer progression and metastatic dissemination to the lymph nodes (LNs) through lymphatic vessels. The study of LA through in vitro, in vivo, and, more recently, in silico models is of paramount importance in providing novel insights and identifying the key molecular actors in the biological dysregulation of this process under pathological conditions. In this review, the different biological (in vitro and in vivo) models of LA, especially in a cancer context, are explained and discussed, highlighting their principal modeled features as well as their advantages and drawbacks. Imaging techniques of the lymphatics, complementary or even essential to in vivo models, are also clarified and allow the establishment of the link with computational approaches. In silico models are introduced, theoretically described, and illustrated with examples specific to the lymphatic system and the LA. Together, these models constitute a toolbox allowing the LA research to be brought to the next level. Simple Summary Lymphangiogenesis is the formation of new lymphatic vessels in physiological conditions but has also been found to be associated with pathologies. For example, it has been proven to be involved in cancer progression and metastatic dissemination through the body. Thus, it became a key element to study in the management of this widespread disease. To date, the study of lymphangiogenesis takes place at the biological (in vitro and in vivo) and computational (in silico) levels. The association of these complementary fields combined with imaging techniques constitutes a real toolbox in pathological lymphangiogenesis understanding. Abstract Lymphangiogenesis (LA) is the formation of new lymphatic vessels by lymphatic endothelial cells (LECs) sprouting from pre-existing lymphatic vessels. It is increasingly recognized as being involved in many diseases, such as in cancer and secondary lymphedema, which most often results from cancer treatments. For some cancers, excessive LA is associated with cancer progression and metastatic dissemination to the lymph nodes (LNs) through lymphatic vessels. The study of LA through in vitro, in vivo, and, more recently, in silico models is of paramount importance in providing novel insights and identifying the key molecular actors in the biological dysregulation of this process under pathological conditions. In this review, the different biological (in vitro and in vivo) models of LA, especially in a cancer context, are explained and discussed, highlighting their principal modeled features as well as their advantages and drawbacks. Imaging techniques of the lymphatics, complementary or even essential to in vivo models, are also clarified and allow the establishment of the link with computational approaches. In silico models are introduced, theoretically described, and illustrated with examples specific to the lymphatic system and the LA. Together, these models constitute a toolbox allowing the LA research to be brought to the next level. 1. Introduction Cancer is still one of the major causes of death worldwide, and the incidence of this group of diseases will continue to grow because of the increasing ageing of the population [ 1 ]. A better understanding of this malignancy and its different features is required and essential to develop treatments or at least to allow this disease to become more chronic and non-lethal. Normal and healthy cells turning malignant share common identified traits and characteristics, well known as the hallmarks of cancer [ 2 ]. Tumor-induced vasculature belongs to these proven features. Excessive angiogenesis and lymphangiogenesis (LA) are associated with cancer progression and bad prognosis [ 3 , 4 , 5 ]. Indeed, both angiogenesis and LA first enable tumor cells to have access to nutrients, oxygen, and waste disposal and then allow them to disseminate to distant parts of the body and form metastases through the circulatory system [ 6 , 7 , 8 , 9 ]. Although tumor angiogenesis has already been investigated intensively and anti-angiogenic drugs developed, interest in tumor LA is fairly recent, even though its importance has been proven many times [ 10 , 11 ]. LA (vs angiogenesis) is the formation of new lymphatic (vs. blood) vessels from pre-existing ones. Although quiescent in normal conditions due to its development being restricted almost exclusively to the embryonic or postnatal stages, lymphatic vasculature can undergo intense remodeling under pathological conditions [ 12 , 13 , 14 , 15 ]. This remodeling consists in abnormal lymphatic dysplasia, vessel dilatation, and increased permeability, which can be or not be associated with LA. Pathological LA is reported in inflammation [ 16 , 17 ], wound healing [ 18 , 19 ], graft transplant rejection [ 20 , 21 ], fibrosis [ 22 ], lymphedema, which often results from cancer treatments [ 23 , 24 ], cancer [ 25 ], etc. [ 26 , 27 ]. While LA can be beneficial during the first stage of cancer progression in draining inflamed tissues and allowing immune cells tumor antigens to circulate, it is also responsible for the metastatic spread to the lymph nodes (LNs) and thereafter the dissemination to distant organs [ 6 , 28 ]. Indeed, depending on the cancer type, the primary tumor secretes pro-angiogenic and/or pro-lymphangiogenic growth factors to promote angiogenesis and/or LA. Cancer cells can therefore use blood and/or lymphatic route(s) to disseminate and form metastases [ 29 , 30 ]. In breast, cervical, and prostate cancers and melanoma, excessive LA is associated with the cancer progression and metastatic dissemination to the LNs through the lymphatic vessels [ 31 ]. The primary tumor actually also conditions the draining LNs to host cancer cells through a pre-metastatic remodeling and the establishment of a niche even before the cancer cells reach them [ 32 ]. LN metastases have thereafter the potential to seed distant organs [ 8 , 9 , 33 , 34 ]. Confirming the importance of the lymphatic route, the targeting of the VEGF-C or VEGFR-3 for therapeutic purposes demonstrates its worth for the anti-lymphangiogenic and combined treatments under development [ 35 , 36 , 37 ]. The surgical dissection of LNs when cancerous or for prevention and diagnosis can lead to the formation of lymphedemas, characterized by localized swellings [ 38 , 39 , 40 ]. The actual existing treatments for lymphedemas (surgery, compression, and draining massages) are unfortunately not very effective [ 41 ]. A better understanding of LA through cancer research could be translated to the abnormalities of lymphatic vessels in lymphedemas and therefore be of great interest in improving the lifestyles of patients with such disability. The experimental models and biological markers of LA are of paramount importance in studying this process and how it is involved in tumorigenesis [ 42 , 43 ]. Since the discovery of valuable markers [ 44 ], increasingly accurate in vitro and in vivo models have been developed to represent LA, or at least parts of it, in a healthy or pathological context [ 45 ]. Both the in vitro and the in vivo models have their own particular characteristics, advantages, and drawbacks. By definition, a model is a simplification of the reality and cannot display all the specificities of the process under study. Highlighting only some angles, models need to be combined to investigate the whole process. Although more relevant and representative than in vitro ones, in vivo models are often more complicated to set up due to financial, timing, and ethical constraints. In vitro models are nonetheless continuously improved towards three-dimensional (3D) and multicellular configurations to better match the reality and to complement the in vivo ones. More recently, mathematical and computational models, referred as in silico, have been implemented in the context of cancer, including tumor vasculogenesis, and their contributions are not negligible when put together with the in vitro and in vivo models [ 46 , 47 ]. In addition to their remarkable integration capacity, numerical models are also able to perform large-scale screening experiments, guide scientists towards the most supposedly impactful experiments, and generate new out-of-the-box insights [ 48 , 49 ]. This paper aims at reviewing the published literature on the in vitro, in vivo, and in silico models of LA, mainly in the context of cancer progression and dissemination. First, a short reminder about this process in healthy and pathological conditions will be presented. Then, the different models, their principal features, and their pros and cons will be explained, with a clear focus on their scopes of application and the represented space–time scale. The in vitro and in vivo models are discussed first, including an overview of the in vivo imaging techniques. Subsequently, the in silico models will be reviewed. Given the unfortunately still limited use of in silico models in LA research, some technical reminders about mathematical and computational modeling are explained. Although in vitro and in vivo models precisely related to tumor LA are quite current, the correlated in silico models are less common and are therefore supplemented, in this paper, with mathematical and computational models of the lymphatic system in general. Finally, the limitations and perspectives in the field of LA modeling will be discussed. 2. Biological Reminders of Molecular and Cellular Lymphangiogenesis The lymphatic system is essential for maintaining tissue fluid homeostasis, as well as for absorbing and transporting fatty acids. It is also involved in immune response by transporting immune cells and soluble antigens from peripheral tissues towards the lymph nodes [ 50 , 51 ]. Lymphatic vessels are found in all vascularized tissues, except in bone marrow and cornea. Contrary to the blood circulation, it is an open system. The overflow of fluid is first reuptaken by initial lymphatic vessels, which are small blind-ended capillaries with an incomplete basement membrane deprived of pericytes and smooth muscle cells. In capillaries, lymphatic endothelial cells (LECs) are joined through discontinuous "button-like" cell-cell junctions, enabling fluid and other cells to enter into the lymphatic vessel lumen ( Figure 1 ). Fluids are then transported through pre- and collecting lymphatics, which are characterized by a complete basement membrane, smooth muscle cells, valves, and zipper-like junctions preventing lymph leak. After being passed through the LNs, the collecting vessels reach the thoracic duct and the lymph is returned in the bloodstream through terminal lymphatics via the subclavian veins [ 25 , 52 ]. Lymphangiogenesis is the counterpart of angiogenesis for the lymphatic vessels and is therefore the formation of new lymphatic vessels by LECs sprouting from pre-existing lymphatic vessels. This process involves LEC proliferation, migration, and survival, which are essentially driven and stimulated by lymphangiogenic actors, among which the major ones are the vascular endothelial growth factor receptor-2 and -3 (VEGFR-2, VEGFR-3), and their ligands: vascular endothelial growth factor-C and -D (VEGF-C, VEGF-D) [ 53 , 54 ]. In response to these growth factors, these cells acquire an invasive phenotype and migrate by developing long filopodia. Different cells are involved in the process of LA: cells with an invasive character, which will migrate and degrade the extracellular matrix (ECM), and cells with a proliferative character, which allow the growth of lymphatic vessels. Even if BECs and LECs present morphological similarities and a similar formation of new vessels, there are however differences which do not allow a direct transposition [ 55 ]. In particular, the endocytic receptor uPARAP is present in LECs but totally absent in BECs [ 56 ]. Many advances have been made possible on the lymphatic system and therefore on the LA by the discovery of specific markers for LECs, which are different from the blood cell ones. Among these markers, it is possible to distinguish the VEGFR-3 involved in various LEC signaling pathways [ 53 , 57 ], the lymphatic vessel endothelial hyaluronan receptor (LYVE-1) [ 58 ], the mucin-like glycoprotein podoplanin (PDPN) [ 59 ], and the prospero homeobox protein 1 (Prox1) [ 60 ] ( Figure 1 ). Prox1 is a predominant lymphatic transcription factor and therefore preferentially located in the nucleus which contributes to the LEC's fate. Despite their expression in LECs, a remaining lack of specificity exists and the perfect marker for lymphatic vessels has unfortunately not been identified yet. Indeed, given that they are also expressed in other cellular types [ 28 , 50 ], these markers are usually combined to specifically characterize LECs in situ and to isolate them for in vitro studies. Even if quiescent in normal conditions, LA takes place in several pathologies [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 26 , 27 ], including cancer [ 25 ]. While LA can be beneficial during the first stage of cancer progression, it is also responsible for metastatic spread to the LNs and thereafter the dissemination to distant organs [ 6 , 28 ]. In certain types of cancer, excessive LA is associated with cancer progression and metastatic dissemination to the LNs through the lymphatic vessels [ 31 ]. The primary tumor actually secretes pro-lymphangiogenic growth factors and conditions the draining LNs to host cancer cells through a pre-metastatic remodeling and the establishment of a niche even before the cancer cells reach them [ 32 ]. Whether the cancer cells in LNs can then seed distant metastases to other organs has been a subject of considerable debate. Certain experts viewed LN metastases as clinically inconsequential [ 61 , 62 ], whereas other experts considered them to have the potential to seed distant organs [ 8 , 9 , 33 , 34 ]. These final data/references provide a definitive proof-of-concept that metastatic cells in LNs can seed distant organs and are not only worth considering but important to treat (at least for specific cancer (sub)types). Anti-angiogenic drugs are used successfully in the clinic to treat some advanced cancers, but they are associated with side effects and the development of resistance [ 63 ]. Given that these currently used anti-angiogenic drugs do not interfere with LA, one possibility for improving cancer treatments focusing on vasculature would be to target tumor LA, in combination or not with tumor angiogenesis. VEGF-C/VEGFR-3 are two important therapeutic targets in the anti-lymphangiogenic therapies under development [ 35 , 36 ]. Moreover, encouraging data have been reported for the combination of anti-angiogenic and anti-lymphangiogenic treatments [ 37 ]. Unfortunately, very few drugs specifically targeting LA have entered clinical testing so far [ 35 , 36 ], and none of them are FDA approved yet. 3. In Vitro Models The culture of LEC results from the isolation of blood endothelial cells (BECs) and LECs from tissue. These endothelial cells (BECs and LECs) can be harvested from macrovessels such as the aorta or the lymphatic duct [ 64 ]. However, the use of this type of cells is not very meaningful in the case of lymphangiogenic studies as the new vessels come from the microvascularization and not from the macrovascularization. Another method of isolation consists in collecting capillary endothelial cells from the tissues where they are most frequently found. LECs are mainly derived from the skin, which incidentally represents the most important source of LECs used (primary LECs). Other LECs are derived from rats (RMLECs) [ 65 ] or from patient lymphangiomas. In order to discriminate BECs from LECs, immunopurification is performed using specific markers, and the cells are sorted by Fluorescent Activated Cell Sorter (FACS) or via magnetic beads. In this case, the markers related to LECs are usually CD31/podoplanin or CD31/LYVE-1. However, this discrimination method does not prevent the contamination of LECs by BECs in view of the expression profiles. Indeed, BECs can express lymphatic markers or can differentiate into LECs. To overcome this selection issue, it is possible to obtain LECs by differentiating progenitor cells (embryonic stem cells) into this specific lineage. Note that the dedifferentiation remains a notorious problem. Despite the fact that most of the used cells are primary LECs, different strategies exist to transform and immortalize them. The first strategy consists in using cells from transgenic animals. The second strategy allows the transformation of LECs into an LEC line containing human telomerase reverse transcriptase (hTERT). In vitro models are suitable for investigating the mechanisms underlying lymphangiogenesis under fixed experimental conditions. However, no in vitro models are currently capable of mimicking the entire process of LA, only parts of it. Figure 2 brings together the different models mentioned here, classifying them according to their level of complexity. Table 1 classifies the different cited models according to their pros and cons. 3.1. Two-Dimensional Cultures (2D Cultures) LECs are grown in monolayers directly on culture plates or on matrix-coated plates [ 66 ]. These LEC monocultures are the basis of the in vitro tests to study LA. Each step of this process can be investigated through different assays: proliferation, migration, invasion, adhesion, or tubulogenesis tests. Apoptosis can also be studied, as well as cell cycle. The migration tests include the Boyden chamber and the scratch assay, in which the cells are in a monolayer and then scratched. The cells are cultured in a medium containing (lymph-)angiogenesis stimulators or inhibitors [ 67 ]. Combining bioinformatics, computer modeling, and imaging approaches, Williams et al. also studied 2D in vitro migration of endothelial cells. More particularly, they investigated which siRNAS of a big dataset control the migratory response of LECs and/or BECs [ 68 ].Tubulogenesis is applied to estimate the ability of cells to form vessels/tube-like structures on a collagen matrix (or Matrigel) in the presence or absence of experimentally defined molecules [ 69 ]. A limitation of this test is that the model does not represent what happens in vivo as LA takes place from already existing vessels. These 2D LEC monocultures allow the study of independent functions but do not consider more complex biological processes, such as, for instance, endothelial cell activation at the onset of LA, branching, and the ECM remodeling. Therefore, to overcome this lack, this research is more focused on the 3D study of LA. 3.2. 3D Static Cultures Growing lymphatic capillaries in vitro as 3D structures remains an important challenge to mimic the real in vivo situation and to better understand the complex LA process [ 70 ]. Moreover, the exploration of the lymphatic system has highlighted the importance of the physical (matrix stiffness and flow) and biochemical (matrix composition, soluble factors, and cellular components) in LA. Therefore, the emergence of models including this parameter, as well as 3D study, is essential for a further understanding of LA [ 71 ]. LECs derived from human embryonic stem cells allow an easier implementation of 3D cultures and can be easily grown as embryoid bodies embedded in a matrix [ 41 ]. LEC spheroids enable the investigation of migration, proliferation, or lumen formation in a more accurate 3D environment and the interaction with a thicker and stiffened matrix [ 72 ]. The spheroids are surrounded by a type I collagen matrix permitting the study of ECM remodeling. In addition, this technique enables the use of genetically modified LECs and LA stimulators or inhibitors [ 41 ]. However, the spheroid model is not adapted for the study of mature lymphatic vessels. In addition to spheroid analysis, the lymphatic ring assay technique has been validated as a model to provide information on the mechanisms of LA. It is based on the aortic ring test, a widely used model for the study of angiogenesis. Genes and molecules involved in LA can be identified through this 3D lymphatic culture. Lymphatic vessel fragments are harvested from the murine thoracic lymph duct and embedded in a collagen gel [ 70 ]. 3.3. 3D Cultures Including Flow To get closer to in vivo models, Ng et al. have highlighted the importance of the interstitial flow on LECs in in vitro models. They developed a 3D model to mimic and approximate in vivo models by inducing artificial flow in collagen gel containing LECs [ 73 ]. The interstitial flow differentially stimulates BEC and LEC morphogenesis in vitro. In order to couple the lymphatic regeneration process by combining interstitial fluid flow events and LEC migration, Boardman et al. created a skin regeneration model implanted in mouse tails and showed that lymphatic cell migration is initiated in the direction of the flow [ 74 ]. This model opened a new door to understanding the importance of interstitial flow on LECs but also highlighted a significant point: the matrix composing the model. Indeed, Helm et al. pointed to the composition of the matrix as an important parameter to consider when modeling tissue reconstruction and in vitro 3D modeling. They were able to form and regenerate lymphatic capillaries in collagen and fibrin matrices with a certain percentage and applied interstitial flow to them [ 75 ]. Subsequently, there has been an emergence of models on interstitial flow, including the model described by Pisano et al., who multiplied the forces of flow and thus developed microfluidic systems corresponding to the size of biological systems [ 76 ]. These tools are essential and allow a more in-depth study of the lymphatic microenvironment. They have thus combined a standard Boyden chamber with a microfluidic system to simulate the mechanical actions found in vivo. Beyond the purely mechanical study, this model also allowed the study of cell transmigration through a layer of LECs. This additional information enabled the study of the role of the flow on the penetration of cancer cells through an LEC monolayer. This study adds an additional dimension, coupling the study of LA and cancer [ 76 ]. In parallel, other ex vivo models have emerged in tissue engineering and allow the further integration of new parameters. One of the first engineered models established by Marino et al. enabled the formation of lymphatic capillaries in fibrin and collagen hydrogels. This study evaluated the optimal concentration of the matrix and, once the in vitro 3D construction was completed, the model was integrated in rats and in skin grafts [ 77 ]. In the interest of investigating the microenvironment of the lymphatic system and the different mechanisms of regulation of the lymphatic network, the microfluidic system, an organ-on-a-chip, has proven to be interesting in the modeling of the microenvironment from a dynamic point of view. These microfluidic systems have been used to characterize the lymphatic barrier in normal and pathological conditions [ 76 ]. Frenkel et al. developed a 3D microfluidic lymphatic vessel model, in which they study the interaction of LA with cancer organoids [ 78 ]. To add parameters to the interstitial flow, Kim et al. used the microfluidic platform system to reconstruct lymphatic budding [ 79 , 80 ]. Gibot et al. generated an in vitro tissue-engineered 3D human lymphatic microvascular network that includes the co-culture of LECs and fibroblasts, allowing the study of 3D lymphatic vessel branching, vascular permeability, and blind-ended junctions, but also creates a reproducible and interesting system for testing LA modulators [ 69 , 80 , 81 ]. In addition, Osaki et al. developed a device containing microchannels to evaluate the effects of different drugs on LA [ 80 , 82 ]. These microarray models remain innovative and are a real tool in understanding the mechanisms of LA. However, it is important to incorporate more biological components of the microenvironment to advance the understanding of tissue interactions in normal but also in pathological conditions. More information about the best practices for engineering new microvascular networks on-chip in the context of LA can be found in the paper of Tronolone and Jain [ 83 ]. To complement these models, 3D bioprinting has made possible the development of in vitro 3D constructs of LA in a tumor context in artificial matrices. In such systems, LECs are seeded in a sacrificial bioprinting matrix with tumor cells encapsulated in a hydrogel [ 80 ]. 3.4. In Vitro Models—Discussion All these studies allow a further understanding of the importance of 3D LA modeling, but these models should be definitively improved by considering all the other parameters involved, such as the ECM, the microenvironment, and their involved cellular and molecular actors, including collagen fibers and fibroblasts. Even with these features included, these in vitro models are still preliminary and need to become more complex to get closer to the in vivo observations. Currently, the transition between in vitro and in vivo can be seen in the study of Landau et al., in which engineered constructs of human lymphatics were developed. These constructs are incorporated in vivo in mice, resulting in anastomose with the host lymphatic vessels [ 84 ]. At present, in vivo studies/models remain essential for a relevant exploration of the physiological systems, including the lymphatic one. In vitro modeling displays many advantages, including low cost, a completely controlled environment, and the absence of ethical issues. However, to study and understand cancer, which is a complex and multifactorial disease, it is essential to consider several parameters, such as the tumoral microenvironment and the inflammation. Such variables are reachable in in vivo models and make them powerful and complementary allies of in vitro in the study of tumoral processes such as LA. 3.1. Two-Dimensional Cultures (2D Cultures) LECs are grown in monolayers directly on culture plates or on matrix-coated plates [ 66 ]. These LEC monocultures are the basis of the in vitro tests to study LA. Each step of this process can be investigated through different assays: proliferation, migration, invasion, adhesion, or tubulogenesis tests. Apoptosis can also be studied, as well as cell cycle. The migration tests include the Boyden chamber and the scratch assay, in which the cells are in a monolayer and then scratched. The cells are cultured in a medium containing (lymph-)angiogenesis stimulators or inhibitors [ 67 ]. Combining bioinformatics, computer modeling, and imaging approaches, Williams et al. also studied 2D in vitro migration of endothelial cells. More particularly, they investigated which siRNAS of a big dataset control the migratory response of LECs and/or BECs [ 68 ].Tubulogenesis is applied to estimate the ability of cells to form vessels/tube-like structures on a collagen matrix (or Matrigel) in the presence or absence of experimentally defined molecules [ 69 ]. A limitation of this test is that the model does not represent what happens in vivo as LA takes place from already existing vessels. These 2D LEC monocultures allow the study of independent functions but do not consider more complex biological processes, such as, for instance, endothelial cell activation at the onset of LA, branching, and the ECM remodeling. Therefore, to overcome this lack, this research is more focused on the 3D study of LA. 3.2. 3D Static Cultures Growing lymphatic capillaries in vitro as 3D structures remains an important challenge to mimic the real in vivo situation and to better understand the complex LA process [ 70 ]. Moreover, the exploration of the lymphatic system has highlighted the importance of the physical (matrix stiffness and flow) and biochemical (matrix composition, soluble factors, and cellular components) in LA. Therefore, the emergence of models including this parameter, as well as 3D study, is essential for a further understanding of LA [ 71 ]. LECs derived from human embryonic stem cells allow an easier implementation of 3D cultures and can be easily grown as embryoid bodies embedded in a matrix [ 41 ]. LEC spheroids enable the investigation of migration, proliferation, or lumen formation in a more accurate 3D environment and the interaction with a thicker and stiffened matrix [ 72 ]. The spheroids are surrounded by a type I collagen matrix permitting the study of ECM remodeling. In addition, this technique enables the use of genetically modified LECs and LA stimulators or inhibitors [ 41 ]. However, the spheroid model is not adapted for the study of mature lymphatic vessels. In addition to spheroid analysis, the lymphatic ring assay technique has been validated as a model to provide information on the mechanisms of LA. It is based on the aortic ring test, a widely used model for the study of angiogenesis. Genes and molecules involved in LA can be identified through this 3D lymphatic culture. Lymphatic vessel fragments are harvested from the murine thoracic lymph duct and embedded in a collagen gel [ 70 ]. 3.3. 3D Cultures Including Flow To get closer to in vivo models, Ng et al. have highlighted the importance of the interstitial flow on LECs in in vitro models. They developed a 3D model to mimic and approximate in vivo models by inducing artificial flow in collagen gel containing LECs [ 73 ]. The interstitial flow differentially stimulates BEC and LEC morphogenesis in vitro. In order to couple the lymphatic regeneration process by combining interstitial fluid flow events and LEC migration, Boardman et al. created a skin regeneration model implanted in mouse tails and showed that lymphatic cell migration is initiated in the direction of the flow [ 74 ]. This model opened a new door to understanding the importance of interstitial flow on LECs but also highlighted a significant point: the matrix composing the model. Indeed, Helm et al. pointed to the composition of the matrix as an important parameter to consider when modeling tissue reconstruction and in vitro 3D modeling. They were able to form and regenerate lymphatic capillaries in collagen and fibrin matrices with a certain percentage and applied interstitial flow to them [ 75 ]. Subsequently, there has been an emergence of models on interstitial flow, including the model described by Pisano et al., who multiplied the forces of flow and thus developed microfluidic systems corresponding to the size of biological systems [ 76 ]. These tools are essential and allow a more in-depth study of the lymphatic microenvironment. They have thus combined a standard Boyden chamber with a microfluidic system to simulate the mechanical actions found in vivo. Beyond the purely mechanical study, this model also allowed the study of cell transmigration through a layer of LECs. This additional information enabled the study of the role of the flow on the penetration of cancer cells through an LEC monolayer. This study adds an additional dimension, coupling the study of LA and cancer [ 76 ]. In parallel, other ex vivo models have emerged in tissue engineering and allow the further integration of new parameters. One of the first engineered models established by Marino et al. enabled the formation of lymphatic capillaries in fibrin and collagen hydrogels. This study evaluated the optimal concentration of the matrix and, once the in vitro 3D construction was completed, the model was integrated in rats and in skin grafts [ 77 ]. In the interest of investigating the microenvironment of the lymphatic system and the different mechanisms of regulation of the lymphatic network, the microfluidic system, an organ-on-a-chip, has proven to be interesting in the modeling of the microenvironment from a dynamic point of view. These microfluidic systems have been used to characterize the lymphatic barrier in normal and pathological conditions [ 76 ]. Frenkel et al. developed a 3D microfluidic lymphatic vessel model, in which they study the interaction of LA with cancer organoids [ 78 ]. To add parameters to the interstitial flow, Kim et al. used the microfluidic platform system to reconstruct lymphatic budding [ 79 , 80 ]. Gibot et al. generated an in vitro tissue-engineered 3D human lymphatic microvascular network that includes the co-culture of LECs and fibroblasts, allowing the study of 3D lymphatic vessel branching, vascular permeability, and blind-ended junctions, but also creates a reproducible and interesting system for testing LA modulators [ 69 , 80 , 81 ]. In addition, Osaki et al. developed a device containing microchannels to evaluate the effects of different drugs on LA [ 80 , 82 ]. These microarray models remain innovative and are a real tool in understanding the mechanisms of LA. However, it is important to incorporate more biological components of the microenvironment to advance the understanding of tissue interactions in normal but also in pathological conditions. More information about the best practices for engineering new microvascular networks on-chip in the context of LA can be found in the paper of Tronolone and Jain [ 83 ]. To complement these models, 3D bioprinting has made possible the development of in vitro 3D constructs of LA in a tumor context in artificial matrices. In such systems, LECs are seeded in a sacrificial bioprinting matrix with tumor cells encapsulated in a hydrogel [ 80 ]. 3.4. In Vitro Models—Discussion All these studies allow a further understanding of the importance of 3D LA modeling, but these models should be definitively improved by considering all the other parameters involved, such as the ECM, the microenvironment, and their involved cellular and molecular actors, including collagen fibers and fibroblasts. Even with these features included, these in vitro models are still preliminary and need to become more complex to get closer to the in vivo observations. Currently, the transition between in vitro and in vivo can be seen in the study of Landau et al., in which engineered constructs of human lymphatics were developed. These constructs are incorporated in vivo in mice, resulting in anastomose with the host lymphatic vessels [ 84 ]. At present, in vivo studies/models remain essential for a relevant exploration of the physiological systems, including the lymphatic one. In vitro modeling displays many advantages, including low cost, a completely controlled environment, and the absence of ethical issues. However, to study and understand cancer, which is a complex and multifactorial disease, it is essential to consider several parameters, such as the tumoral microenvironment and the inflammation. Such variables are reachable in in vivo models and make them powerful and complementary allies of in vitro in the study of tumoral processes such as LA. 4. In Vivo Models In order to understand the complex and multifactorial process of lymphangiogenesis in cancer, in vivo models have added a dimension and a level of complexity to in vitro models ( Figure 2 ). Several in vivo LA models are available and used to study the physiological state of this process but also to understand LA in a particular pathological context, such as in cancer [ 45 ]. 4.1. Mouse As a model for human cancer, the mouse has already proven to be an unavoidable research tool thanks to the genetic and physiological homology with human tumors [ 85 ]. As a recipient for tumor cells and/or genetic engineered organisms, the mouse largely contributes to a better understanding of tumorigenesis and its associated hallmarks, including LA. The transplantation of tissue or tumor cells derived from a donor of a different species from the recipient (xenograft) is a predominant approach in cancer modeling. In these model types, human tumor cell lines can be implanted in immunodeficient mice in order to induce tumorigenesis and further study the associated features. Several studies aim to study tumor LA by implanting human tumor cell lines and investigating their impact on the lymphatic network [ 86 , 87 , 88 ]. Developmental studies of the lymphatic vasculature have already contributed to the identification of key lymphangiogenic actors. Indeed, essential agents such as VEGF-C, VEGF-D, or VEGFR-3 were found to be crucial during normal mouse development [ 89 ]. The study of eventual key lymphangiogenic factors can be performed by using the matrigel plug assay, which consists in subcutaneously implanting gel containing compounds to test [ 90 ]. Xenograft-transplanted mouse models have demonstrated their usefulness in confirming the implication of these identified molecules in LA and cancer dissemination. The importance of VEGF-C-induced LA in cancer progression was largely studied in xenograft mouse models. The overexpression of VEGF-C by solid tumors was shown to increase peritumoral and intratumoral lymphatic vessels, as well as metastasis formation [ 91 , 92 , 93 , 94 ]. Moreover, when the RipVEGF-C transgenic mouse strain was crossed with the Rip1Tag2 strain, which is known to generate non-metastatic pancreatic beta cell tumors and VEGF-C-induced LA around the pancreatic beta cells, and promote metastasis in regional lymph nodes [ 92 , 95 ]. In other comparable studies, transplanted VEGF-D-overexpressing tumor cells were shown to promote tumor LA and increase the metastasis rate via dilatation of the collecting lymphatic vessels [ 96 , 97 ]. The ability of xenografts to reproduce the tumoral cascade and to generate a remodeled and extended lymphatic network also opens the way to evaluating anti-lymphangiogenic strategies through inhibitory or blocking compound screening. Such approaches can be used in order to elucidate or confirm the pro-lymphangiogenic role of a protein and even to participate in the emergence of new potential treatments. Several studies have already demonstrated an anti-lymphangiogenic effect by using blocking antibodies [ 96 ]. Because of their predominant role in lymphatic vessel scaffolding, VEGF-C and VEGF-D axis constitute ideal targets. Anti-VEGFR-3 antibodies, as well as soluble VEGFR-3, which competes with the endogenous receptor and traps VEGF-C/-D, showed a deleterious effect on tumor LA and metastasis in transplanted mice [ 86 , 98 , 99 , 100 ]. Antibodies targeting ephrinB2, a ligand of the EphB4 receptor, displayed a lymphatic vessel number decrease in transplanted mice [ 101 ], whereas antibodies against Neuropilin-2 reduced the tumoral LA, in addition to leading to a decrease of the metastasis number in the LN and distant organs [ 102 , 103 ]. The blocking of the ANG2/TIE2 pathway demonstrated an inhibition of lung and LN metastasis via an improved endothelial cell integrity [ 87 ]. Pharmacological compounds have also demonstrated an anti-lymphatic and anti-metastatic activity in a breast cancer mouse model [ 104 ]. More recently, the efficacy of afatinib, an EGFR tyrosine kinase inhibitor, was demonstrated in a lung adenocarcinoma HCC287 xenograft mouse model, where the tumor growth was inhibited and the lymphatic densities as well as the vessel diameter were decreased [ 105 ]. Besides xenografts, syngeneic transplants also present advantages for cancer cascade investigation. In this type of experiment, murine tumor cell lines are implanted to generate a solid tumor in order to further study the mechanisms of cancer. Several murine cell lines derived from spontaneous or chemically induced cancer are described for angiogenesis, metastasis, or LA modeling. For instance, Lewis lung carcinoma, CT26 colon carcinoma, 66cl4 mammary carcinoma, and B16 melanoma cells showed their ability to induce LA in several studies [ 106 ]. These models have the advantage of using immuno-competent and transgenic mice and allow the investigation of the remodeling occurring in draining LNs after tumor cell transplants. The ear sponge assay is an easy and reproductive model. In this system, a gelatin sponge soaked with tumor cells is implanted between the two mouse ear skin layers for 2–4 weeks in order to induce primary tumor growth. This model allows the tumor-associated LA study as well as the mimicking of the metastatic cascade in tumor draining LNs, thus making possible the characterization of the remodeled sentinel LN at the pre-metastatic and metastatic state [ 107 , 108 ]. 4.2. Zebrafish Due to their physiological and genetic similarities with humans, zebrafish constitute another powerful biological tool, which has already contributed to science advancement. Its high fecundity and the low cost of maintenance make it an ideal actor for disease modeling. Indeed, thanks to its ability to grow rapidly and its transparency during the early stages of life, it is ideal for development study [ 109 ], in addition to being largely used in genomics [ 110 , 111 ]. More recently, the use of zebrafish in cancer research became the aim of several studies and reviews [ 112 , 113 ]. Indeed, its properties allow scientists to easily monitor in vivo tumor growth, to perform large drug screening, and to investigate cancer-associated features such as angiogenesis and LA [ 114 , 115 ]. Thanks to the lack of a competent adaptive immune system during the early stages of life and to the apparition of immune-deficient zebrafish strains, xenograft is also an option for cancer study [ 116 ]. Recently, Chen et al. have reported an elegant update of zebrafish xenograft models in cancer research [ 112 ]. It is possible to transplant and to monitor tumor cells both in embryonic and adult animals. Indeed, the native embryonic transparency and the generation of transgenic transparent adult zebrafishes in 2008 open the way to an easier monitoring of in vivo tumor growth and cancer cell dissemination [ 117 , 118 ]. The resulting transparency makes it possible to clearly visualize the transplanted xenograft and to track labelled fluorescent tumor cells as well as extracellular vesicles in vivo [ 119 , 120 ]. Due to constant technical progress, zebrafish also became a powerful tool for in vivo imaging of blood and lymphatic development [ 118 , 121 ]. The generation of transgenic zebrafish lines that express fluorescent labeled vasculature enabled high-resolution real-time imaging of vessels [ 122 ]. In the zebrafish model, tumor neovascularization is so far the most studied. The implication of blood vessels in tumor spreading was already characterized in this model. Indeed, xenograft-induced neovascularization and the resulting dissemination of fluorescent labeled tumor cells were described [ 123 , 124 ]. The zebrafish is still underused as a model for tumor-induced LA [ 125 ]. To our knowledge, there is no zebrafish model yet that has proved its suitability for investigating LA in a tumoral context. Indeed, to date, zebrafish is essentially used for lymphatic vessel development study, the associated factor identification, and anti-lymphangiogenic molecule screening. In this model, the pro-lymphangiogenic activity of actors such as VEGF-C, VEGF-D, and YAP1 in lymphatic system growth and development was characterized [ 126 , 127 , 128 , 129 , 130 ]. The elaboration of therapeutic strategies, including the design of specific inhibitors, indeed represents a huge fraction of the zebrafish usage and essentially targets tumor angiogenesis. The development of anti-vascular drugs in zebrafish is mainly based on the combination between transgenic line availability and high-end imaging techniques [ 122 ]. Several tested compounds displayed an anti-lymphangiogenic effect. The formation of the thoracic duct in zebrafishes was prevented by the kaempferol, leflunomide, cinnarizine, and flunarizine [ 104 ]. As a result of this screening, the use of kaempferol displayed a reduction in tumor-associated lymphatic vessels and LN metastases in a breast cancer xenograft mouse model. In addition, a VEGF-C-VEGFR3-Erk pathway blocking strategy exhibits an anti-lymphangiogenic response [ 131 ]. For several years now, the zebrafish has demonstrated its usefulness in the discovery of new compounds by screening, strengthening the fact that it is a specialized system with unique research possibilities in in vivo cancer research alongside the mouse. Even if the study of tumor-induced LA in zebrafish is not as common as it is in mouse models, the interest in this topic and the availability of imaging techniques might lead to these models becoming important in gaining tumor LA understanding, as well as in future treatment development. 4.3. Imaging Several animal models are now available to mimic the tumoral process and further study the multiple associated features. However, to measure and assess the impact of the tumor and tumor-derived factors on biological structures such as the lymphatic compartment or even the effect of drugs and inhibitors, researchers have to be able to visualize and quantify this network. That is where in vivo imaging techniques are needed. In fundamental research, it has become possible to image lymphatic vessels by exploiting the specificity of some LEC markers (described above, see Section 2 ) thanks to transgenic mouse strains and a better monitoring of proteins with fluorescent properties. Through the years and studies, several constructs implicating Prox1 were designed: Prox1-GFP, Prox1-tdTomato, Prox1-mOrange2, BACTg(Prox1-EGFP), and Prox1-EGFP BAC [ 132 , 133 , 134 , 135 , 136 ]. For LYVE1 and VEGFR-3, the following reporter protein constructs are described: Lyve1CreERT2 tdt , Vegfr3 EGFPLUC and Vegfr3-YFP [ 137 , 138 , 139 ]. A more detailed list of these fluorescent lymphangiogenic reporters was addressed in 2018 in the review of Susan et al. [ 140 ]. Regarding transgenic zebrafish strains, there is no zebrafish model investigating tumor-associated LA, as said above. Transgenic fish lines nevertheless exist to study LA in other contexts [ 104 , 141 , 142 , 143 ]. Combined with tumor models, the latter could enable the zebrafish to become an interesting tool and a reference for LA in the context of tumor progression. Regarding the (pre-)clinical (live) imaging of the lymphatic vasculature, non-invasive and sensitive visualization of the lymphatic system and vasculature is of paramount importance for diagnosis, treatments, and surgery. However, unfortunately, it remains challenging and less studied than the imaging of its blood counterpart [ 144 , 145 , 146 ]. This gap could be explained by the intrinsic properties of lymphatics, such as their size making the insertion of sensors difficult and the filtrating LNs only allowing local and not global contrast. Nevertheless, different methods and technologies enable imaging the lymphatic system and visualizing the process of LA, each with various invasiveness and resolution properties [ 147 ]. For example, with its high affinity for β-lipoproteins, Indocyanine Green possesses the property to accumulate in the lymphatic vessels, considering the high protein content of the lymph. This fluorescent dye is therefore commonly used for imaging the lymphatic system. Indeed, Near-Infrared Fluorescence imaging following Indocyanine Green injection is exploited in real-time during surgery [ 148 ] or for studying lymphatics-related processes, such as the lymphatic drainage in breast-cancer-related lymphedema [ 149 ], but also the LA impact during wound healing, injury repair [ 150 ], and arthritis [ 151 ]. Immuno-positron emission tomography with a radiolabeled lymphatic-specific antibody against LYVE-1 enables the imaging of tumor-induced LN LA [ 152 ]. The notion of the LN pre-metastatic niche is now well established and involves changes of the LN environment in order to be receptive to cancer metastatic cells [ 153 ]. The lymphatic remodeling occurring prior to the arrival of cancer cells, positron emission tomography combined with a lymphatic instead of a cancer marker, is more relevant as a prevention or diagnosis tool. Magnetic Resonance Imaging is another imaging technique used to investigate and visualize LA in various malignancies. For example, Yang et al. combined anti-podoplanin antibodies targeting lymphatic endothelium with GoldMag nanoparticles as a contrast agent and water-soluble polyethylene glycol to increase the stabilization and biocompatibility of the nanoparticles in order to evaluate breast cancer LA [ 154 ]. Other examples of in vivo imaging technologies of the lymphatic system are discussed by Polomska and Proulx [ 155 ] and by Elshikh et al. [ 156 ], the latter focusing on different oncologic imaging techniques of the lymphatic system. 4.4. In Vivo Models—Discussion It is clear that animal models are an important tool in the modeling of cancer and the associated LA and have a place alongside in vitro experiments. Usually, in vitro and in vivo studies require imaging visualization and processing for quantification of the results. Additionally, in silico modeling, an emergent field of science, is about to become unavoidable for a further understanding of such complex biological mechanisms as LA. It should be noticed that imaging is one of the prime ways to connect the in vivo and in silico parts. Through bioinformatics approaches, the multi-omics data from in vitro and in vivo models can be analysed, connecting again biological and engineering techniques. Indeed, the observations of the results of in vivo models can serve as input for the computational methods, which in turn are suitable for providing additional insights over and above what can be imaged. cancers-14-01525-t001_Table 1 Table 1 Advantages and disadvantages of the different cited lymphangiogenesis in vitro and in vivo models. Applications Models Advantages Disadvantages References 2D in vitro LEC physiology LEC/ECM component interactions Adhesion assay - Low cost tests - Inability to model the environment [ 66 , 67 ] Proliferation assay Biological process - Rapid and easy observations Apoptosis assay LEC 2D motility Boyden Chamber - Easy to perform and to quantify - Only 2D Migration Scratch Assay Tubulogenesis - Self-organization - Observation of pseudo-vessel architecture - No distinction between different phenotypes - Limited survival - No flow [ 67 , 69 ] 3D in vitro LEC 3D motility Embryoid bodies - 3D culture - Differentiation between tip and stalk cells - Possibility of lumen formation - No flow - High volumes used for testing - No spatial control of gradients [ 41 , 71 ] Spheroids [ 41 , 71 , 72 ] Lymphatic ring assay [ 71 , 72 ] Lymphatic network Microfluidic chamber - Integration of gradients and flow - Faster lumen formation similar to embryogenesis - Problem of standardization [ 71 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 ] Organ-on-a-chip In vivo Animal models Xenograft - Use of human cells - No impact of immunity in immunosuppressed animals [ 71 , 89 , 90 , 91 , 92 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 ] Syngenic graft - No rejection - Immunocompetent animals - Use of cells with the same genetic background than the host - Inability to use human cells [ 104 , 105 , 106 ] Zebrafish - Pro- and anti-lymphangiogenic factor screening - Developmental studies - Difficult for studying cancer-associated lymphangiogenesis [ 102 , 124 , 128 , 129 ] 4.1. Mouse As a model for human cancer, the mouse has already proven to be an unavoidable research tool thanks to the genetic and physiological homology with human tumors [ 85 ]. As a recipient for tumor cells and/or genetic engineered organisms, the mouse largely contributes to a better understanding of tumorigenesis and its associated hallmarks, including LA. The transplantation of tissue or tumor cells derived from a donor of a different species from the recipient (xenograft) is a predominant approach in cancer modeling. In these model types, human tumor cell lines can be implanted in immunodeficient mice in order to induce tumorigenesis and further study the associated features. Several studies aim to study tumor LA by implanting human tumor cell lines and investigating their impact on the lymphatic network [ 86 , 87 , 88 ]. Developmental studies of the lymphatic vasculature have already contributed to the identification of key lymphangiogenic actors. Indeed, essential agents such as VEGF-C, VEGF-D, or VEGFR-3 were found to be crucial during normal mouse development [ 89 ]. The study of eventual key lymphangiogenic factors can be performed by using the matrigel plug assay, which consists in subcutaneously implanting gel containing compounds to test [ 90 ]. Xenograft-transplanted mouse models have demonstrated their usefulness in confirming the implication of these identified molecules in LA and cancer dissemination. The importance of VEGF-C-induced LA in cancer progression was largely studied in xenograft mouse models. The overexpression of VEGF-C by solid tumors was shown to increase peritumoral and intratumoral lymphatic vessels, as well as metastasis formation [ 91 , 92 , 93 , 94 ]. Moreover, when the RipVEGF-C transgenic mouse strain was crossed with the Rip1Tag2 strain, which is known to generate non-metastatic pancreatic beta cell tumors and VEGF-C-induced LA around the pancreatic beta cells, and promote metastasis in regional lymph nodes [ 92 , 95 ]. In other comparable studies, transplanted VEGF-D-overexpressing tumor cells were shown to promote tumor LA and increase the metastasis rate via dilatation of the collecting lymphatic vessels [ 96 , 97 ]. The ability of xenografts to reproduce the tumoral cascade and to generate a remodeled and extended lymphatic network also opens the way to evaluating anti-lymphangiogenic strategies through inhibitory or blocking compound screening. Such approaches can be used in order to elucidate or confirm the pro-lymphangiogenic role of a protein and even to participate in the emergence of new potential treatments. Several studies have already demonstrated an anti-lymphangiogenic effect by using blocking antibodies [ 96 ]. Because of their predominant role in lymphatic vessel scaffolding, VEGF-C and VEGF-D axis constitute ideal targets. Anti-VEGFR-3 antibodies, as well as soluble VEGFR-3, which competes with the endogenous receptor and traps VEGF-C/-D, showed a deleterious effect on tumor LA and metastasis in transplanted mice [ 86 , 98 , 99 , 100 ]. Antibodies targeting ephrinB2, a ligand of the EphB4 receptor, displayed a lymphatic vessel number decrease in transplanted mice [ 101 ], whereas antibodies against Neuropilin-2 reduced the tumoral LA, in addition to leading to a decrease of the metastasis number in the LN and distant organs [ 102 , 103 ]. The blocking of the ANG2/TIE2 pathway demonstrated an inhibition of lung and LN metastasis via an improved endothelial cell integrity [ 87 ]. Pharmacological compounds have also demonstrated an anti-lymphatic and anti-metastatic activity in a breast cancer mouse model [ 104 ]. More recently, the efficacy of afatinib, an EGFR tyrosine kinase inhibitor, was demonstrated in a lung adenocarcinoma HCC287 xenograft mouse model, where the tumor growth was inhibited and the lymphatic densities as well as the vessel diameter were decreased [ 105 ]. Besides xenografts, syngeneic transplants also present advantages for cancer cascade investigation. In this type of experiment, murine tumor cell lines are implanted to generate a solid tumor in order to further study the mechanisms of cancer. Several murine cell lines derived from spontaneous or chemically induced cancer are described for angiogenesis, metastasis, or LA modeling. For instance, Lewis lung carcinoma, CT26 colon carcinoma, 66cl4 mammary carcinoma, and B16 melanoma cells showed their ability to induce LA in several studies [ 106 ]. These models have the advantage of using immuno-competent and transgenic mice and allow the investigation of the remodeling occurring in draining LNs after tumor cell transplants. The ear sponge assay is an easy and reproductive model. In this system, a gelatin sponge soaked with tumor cells is implanted between the two mouse ear skin layers for 2–4 weeks in order to induce primary tumor growth. This model allows the tumor-associated LA study as well as the mimicking of the metastatic cascade in tumor draining LNs, thus making possible the characterization of the remodeled sentinel LN at the pre-metastatic and metastatic state [ 107 , 108 ]. 4.2. Zebrafish Due to their physiological and genetic similarities with humans, zebrafish constitute another powerful biological tool, which has already contributed to science advancement. Its high fecundity and the low cost of maintenance make it an ideal actor for disease modeling. Indeed, thanks to its ability to grow rapidly and its transparency during the early stages of life, it is ideal for development study [ 109 ], in addition to being largely used in genomics [ 110 , 111 ]. More recently, the use of zebrafish in cancer research became the aim of several studies and reviews [ 112 , 113 ]. Indeed, its properties allow scientists to easily monitor in vivo tumor growth, to perform large drug screening, and to investigate cancer-associated features such as angiogenesis and LA [ 114 , 115 ]. Thanks to the lack of a competent adaptive immune system during the early stages of life and to the apparition of immune-deficient zebrafish strains, xenograft is also an option for cancer study [ 116 ]. Recently, Chen et al. have reported an elegant update of zebrafish xenograft models in cancer research [ 112 ]. It is possible to transplant and to monitor tumor cells both in embryonic and adult animals. Indeed, the native embryonic transparency and the generation of transgenic transparent adult zebrafishes in 2008 open the way to an easier monitoring of in vivo tumor growth and cancer cell dissemination [ 117 , 118 ]. The resulting transparency makes it possible to clearly visualize the transplanted xenograft and to track labelled fluorescent tumor cells as well as extracellular vesicles in vivo [ 119 , 120 ]. Due to constant technical progress, zebrafish also became a powerful tool for in vivo imaging of blood and lymphatic development [ 118 , 121 ]. The generation of transgenic zebrafish lines that express fluorescent labeled vasculature enabled high-resolution real-time imaging of vessels [ 122 ]. In the zebrafish model, tumor neovascularization is so far the most studied. The implication of blood vessels in tumor spreading was already characterized in this model. Indeed, xenograft-induced neovascularization and the resulting dissemination of fluorescent labeled tumor cells were described [ 123 , 124 ]. The zebrafish is still underused as a model for tumor-induced LA [ 125 ]. To our knowledge, there is no zebrafish model yet that has proved its suitability for investigating LA in a tumoral context. Indeed, to date, zebrafish is essentially used for lymphatic vessel development study, the associated factor identification, and anti-lymphangiogenic molecule screening. In this model, the pro-lymphangiogenic activity of actors such as VEGF-C, VEGF-D, and YAP1 in lymphatic system growth and development was characterized [ 126 , 127 , 128 , 129 , 130 ]. The elaboration of therapeutic strategies, including the design of specific inhibitors, indeed represents a huge fraction of the zebrafish usage and essentially targets tumor angiogenesis. The development of anti-vascular drugs in zebrafish is mainly based on the combination between transgenic line availability and high-end imaging techniques [ 122 ]. Several tested compounds displayed an anti-lymphangiogenic effect. The formation of the thoracic duct in zebrafishes was prevented by the kaempferol, leflunomide, cinnarizine, and flunarizine [ 104 ]. As a result of this screening, the use of kaempferol displayed a reduction in tumor-associated lymphatic vessels and LN metastases in a breast cancer xenograft mouse model. In addition, a VEGF-C-VEGFR3-Erk pathway blocking strategy exhibits an anti-lymphangiogenic response [ 131 ]. For several years now, the zebrafish has demonstrated its usefulness in the discovery of new compounds by screening, strengthening the fact that it is a specialized system with unique research possibilities in in vivo cancer research alongside the mouse. Even if the study of tumor-induced LA in zebrafish is not as common as it is in mouse models, the interest in this topic and the availability of imaging techniques might lead to these models becoming important in gaining tumor LA understanding, as well as in future treatment development. 4.3. Imaging Several animal models are now available to mimic the tumoral process and further study the multiple associated features. However, to measure and assess the impact of the tumor and tumor-derived factors on biological structures such as the lymphatic compartment or even the effect of drugs and inhibitors, researchers have to be able to visualize and quantify this network. That is where in vivo imaging techniques are needed. In fundamental research, it has become possible to image lymphatic vessels by exploiting the specificity of some LEC markers (described above, see Section 2 ) thanks to transgenic mouse strains and a better monitoring of proteins with fluorescent properties. Through the years and studies, several constructs implicating Prox1 were designed: Prox1-GFP, Prox1-tdTomato, Prox1-mOrange2, BACTg(Prox1-EGFP), and Prox1-EGFP BAC [ 132 , 133 , 134 , 135 , 136 ]. For LYVE1 and VEGFR-3, the following reporter protein constructs are described: Lyve1CreERT2 tdt , Vegfr3 EGFPLUC and Vegfr3-YFP [ 137 , 138 , 139 ]. A more detailed list of these fluorescent lymphangiogenic reporters was addressed in 2018 in the review of Susan et al. [ 140 ]. Regarding transgenic zebrafish strains, there is no zebrafish model investigating tumor-associated LA, as said above. Transgenic fish lines nevertheless exist to study LA in other contexts [ 104 , 141 , 142 , 143 ]. Combined with tumor models, the latter could enable the zebrafish to become an interesting tool and a reference for LA in the context of tumor progression. Regarding the (pre-)clinical (live) imaging of the lymphatic vasculature, non-invasive and sensitive visualization of the lymphatic system and vasculature is of paramount importance for diagnosis, treatments, and surgery. However, unfortunately, it remains challenging and less studied than the imaging of its blood counterpart [ 144 , 145 , 146 ]. This gap could be explained by the intrinsic properties of lymphatics, such as their size making the insertion of sensors difficult and the filtrating LNs only allowing local and not global contrast. Nevertheless, different methods and technologies enable imaging the lymphatic system and visualizing the process of LA, each with various invasiveness and resolution properties [ 147 ]. For example, with its high affinity for β-lipoproteins, Indocyanine Green possesses the property to accumulate in the lymphatic vessels, considering the high protein content of the lymph. This fluorescent dye is therefore commonly used for imaging the lymphatic system. Indeed, Near-Infrared Fluorescence imaging following Indocyanine Green injection is exploited in real-time during surgery [ 148 ] or for studying lymphatics-related processes, such as the lymphatic drainage in breast-cancer-related lymphedema [ 149 ], but also the LA impact during wound healing, injury repair [ 150 ], and arthritis [ 151 ]. Immuno-positron emission tomography with a radiolabeled lymphatic-specific antibody against LYVE-1 enables the imaging of tumor-induced LN LA [ 152 ]. The notion of the LN pre-metastatic niche is now well established and involves changes of the LN environment in order to be receptive to cancer metastatic cells [ 153 ]. The lymphatic remodeling occurring prior to the arrival of cancer cells, positron emission tomography combined with a lymphatic instead of a cancer marker, is more relevant as a prevention or diagnosis tool. Magnetic Resonance Imaging is another imaging technique used to investigate and visualize LA in various malignancies. For example, Yang et al. combined anti-podoplanin antibodies targeting lymphatic endothelium with GoldMag nanoparticles as a contrast agent and water-soluble polyethylene glycol to increase the stabilization and biocompatibility of the nanoparticles in order to evaluate breast cancer LA [ 154 ]. Other examples of in vivo imaging technologies of the lymphatic system are discussed by Polomska and Proulx [ 155 ] and by Elshikh et al. [ 156 ], the latter focusing on different oncologic imaging techniques of the lymphatic system. 4.4. In Vivo Models—Discussion It is clear that animal models are an important tool in the modeling of cancer and the associated LA and have a place alongside in vitro experiments. Usually, in vitro and in vivo studies require imaging visualization and processing for quantification of the results. Additionally, in silico modeling, an emergent field of science, is about to become unavoidable for a further understanding of such complex biological mechanisms as LA. It should be noticed that imaging is one of the prime ways to connect the in vivo and in silico parts. Through bioinformatics approaches, the multi-omics data from in vitro and in vivo models can be analysed, connecting again biological and engineering techniques. Indeed, the observations of the results of in vivo models can serve as input for the computational methods, which in turn are suitable for providing additional insights over and above what can be imaged. cancers-14-01525-t001_Table 1 Table 1 Advantages and disadvantages of the different cited lymphangiogenesis in vitro and in vivo models. Applications Models Advantages Disadvantages References 2D in vitro LEC physiology LEC/ECM component interactions Adhesion assay - Low cost tests - Inability to model the environment [ 66 , 67 ] Proliferation assay Biological process - Rapid and easy observations Apoptosis assay LEC 2D motility Boyden Chamber - Easy to perform and to quantify - Only 2D Migration Scratch Assay Tubulogenesis - Self-organization - Observation of pseudo-vessel architecture - No distinction between different phenotypes - Limited survival - No flow [ 67 , 69 ] 3D in vitro LEC 3D motility Embryoid bodies - 3D culture - Differentiation between tip and stalk cells - Possibility of lumen formation - No flow - High volumes used for testing - No spatial control of gradients [ 41 , 71 ] Spheroids [ 41 , 71 , 72 ] Lymphatic ring assay [ 71 , 72 ] Lymphatic network Microfluidic chamber - Integration of gradients and flow - Faster lumen formation similar to embryogenesis - Problem of standardization [ 71 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 ] Organ-on-a-chip In vivo Animal models Xenograft - Use of human cells - No impact of immunity in immunosuppressed animals [ 71 , 89 , 90 , 91 , 92 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 ] Syngenic graft - No rejection - Immunocompetent animals - Use of cells with the same genetic background than the host - Inability to use human cells [ 104 , 105 , 106 ] Zebrafish - Pro- and anti-lymphangiogenic factor screening - Developmental studies - Difficult for studying cancer-associated lymphangiogenesis [ 102 , 124 , 128 , 129 ] 5. In Silico Models Defined in analogy to in vitro and in vivo, the term in silico refers to the work performed using mathematical modeling and computer simulations. In silico approaches are increasingly applied in fundamental cancer biology research, focusing on, amongst others, tumor angiogenesis and lymphangiogenesis [ 46 ]. Not all biological features can be studied in in vitro and in vivo models as only a few components can be investigated at the same time, representing one of the limitations of these models. In silico models have a remarkable integration capacity, enabling not only a better understanding of the different actors individually involved in LA but also their interactions. They offer new perspectives and allow the generation of new hypotheses and predictions that are not always straightforward to verify experimentally due to practical, financial, ethical, and timing constraints. Moreover, they can guide scientists towards more informative experiments. Indeed, the in silico techniques coincide with the principle of the 3 Rs: reduction by better planning the experiments, refinement by generating modeling methods to finer extrapolate experimental data, and replacement by enabling a more accurate translation from animal to human experiments [ 48 ]. In silico models are already used during research and development phases, as well as for clinical trial design and optimization [ 49 ]. Regulatory guidelines and standards are becoming available, providing guidance for the verification and validation of in silico methods for clinical and industrial uptake [ 157 , 158 ]. The next paragraphs explain the in silico pipeline and discuss the different existing mathematical and computationnal models related to lymphatics. Although many precise types of modeling techniques are specified hereafter, their subsequent mathematical and computational details will not be addressed, as this is beyond the scope of this review. The diverse ways to classify in silico models and tools in a biological context are reviewed in Appendix A . More information about the specific mathematical formalism (in a biological context) and the in silico pipeline can be found in Bekisz and Geris [ 159 ] and in Lesage et al. [ 160 ]. 5.1. The In Silico Modeling Pipeline The modeling procedure and the development of models, which can be sorted according to different features and mathematical formalism (see Appendix A ), follow a very precise pipeline divided into several distinct steps [ 161 , 162 ]. Specifically, the subject to be addressed and the questions to be asked of the model are first identified and clearly defined. A literature review is then conducted to report all the current data and knowledge about the topic, resulting in the choice of the best modeling formalism for the objectives to be achieved. The model formalism also depends on the biological data availability or at least the possibility to generate these data. A trade-off must be found between an accurate representation of the system under study and the multiple assumptions imposed because of the biological uncertainties and the limited experimental data provision. In this respect, hypotheses are not to be confused with details, whose level has no relation with the model accuracy but rather with the model capacity to answer the stated problem. Let us quote the relevant sentence of Manfred Eigen: "A theory has only the alternative of being right or wrong. A model has a third possibility: it may be right, but irrelevant". The next modeling steps are the mathematical formulation, followed by the computational implementation, because only a few models can be solved purely analytically. The models are often composed with variables and parameters, most of which are not known and must be estimated. Parameter estimation is therefore requested to calibrate the model and consists in determining the optimal parameter values to enter into the model so that the generated output fits as best as possible with the biological data, generated previously or for the purpose. Once the model has been calibrated, it is simulated with efficient algorithms and the in silico outputs are provided. Wrong results are not necessarily related to a wrong mathematical formalism but can also be linked to false starting hypotheses or issues with the data used for calibration (garbage in, garbage out). Coherent model predictions can be validated with new experiments or with previously generated biological observations. Calibration and validation usually do not use the same datasets. Depending on the divergence between the predicted and the observed data, the model might be subjected to modifications and undergo several improvement and optimization cycles for refinement. In addition to reproducing data from biology, the last optimized version of the mathematical model is therefore considered as a tool that can be used to test new experimental conditions. It is important to realize that in silico models (like all other model systems) are not self-sufficient but are constructed, validated, and iteratively refined through in vitro and in vivo experiments before being sufficiently precise and ready to provide insightful outputs. The credibility of in silico models can be established for a precisely defined context of use, by applying the so-called VVUQ (Verification, Validation, and Uncertainty Quantification) [ 163 ]. Figure 3 gathers all the steps outlined above regarding the modeling pipeline and highlights the symbiotic approach, mixing the in vitro, in vivo, and in silico methods. 5.2. In Silico Models of LA For several years, the impact of the vascularization on tumor progression has been intensively studied through mathematical modeling, especially the influence of angiogenesis. Good reviews of in silico models for angiogenesis and its role in tumor progression were written by Peirce [ 164 ], Heck et al. [ 165 ], Chaplain [ 166 ], Scianna et al. [ 47 ], Mantzaris et al. [ 167 ], Levine et al. [ 168 , 169 ], Qutub et al. [ 170 ], Suzuki et al. [ 171 ], and Lowengrub et al. [ 172 ]. Even if some previously reported models of angiogenesis could be easily adapted for LA, this process presents particular features requiring specific dedicated models. Though considerably less widespread than for the process of angiogenesis, several mathematical and computational models have been implemented in the context of the lymphatic system and LA. Currently, very few in silico models specifically focused on tumor LA have been developed. However, the models and tools elaborated in 'simpler' lymphatic contexts will nevertheless be discussed in this review article, sorted by application domain, and can serve as a starting point for more focused tumor LA models. 5.2.1. Lymphatic Flow The principal initiator of the in silico lymphatic system modeling is Reddy, who computationally investigated, among others, the flow through terminal lymphatics but also the lymph circulation biomechanics and the valve physics [ 173 , 174 , 175 , 176 ]. Elhay and Casley-Smith were also pioneers and used normal laws of physics related to flow and diffusion to study the initial lymphatics [ 177 ]. MacDonald adapted one of Reddy's models to study the lymph flow in the collecting lymphatic vessels [ 178 ]. Different modeling techniques of drainage in primary and collecting lymphatics, as well as lymphatic valves and nodes, are reviewed in the paper of Roose and Tabor [ 179 ]. Mozokhina and Savinkov highlighted the different mathematical models implemented to represent and study the lymph flow in the lymphatic system and its subunits (lymphangions, valves, and LNs) [ 180 ]. In this context, the majority of the developed models are built with zero-, quasi-one-, or one-dimensional approaches. Zero-dimensional methods, so-called lumped models, refer to the electrical circuit theory, comparing the pressure, lymph flux, and mass with voltage, current, and charge, respectively. Lymph flow is usually described in one-dimensional approaches with Navier–Stokes equations or with the law of mass and momentum conservation. Comparison between 0D and 1D formulation in the context of modeling lymph flow in the lymphatic system has been explained in the article of Tretyakova [ 181 ]. Cooper et al. used a finite element image-based model to investigate the fluid flow through the LNs [ 182 ]. The effect of a permeable interstitium on a network of initial lymphatics and pre-collectors was investigated by Ikhimwin et al. with a lumped-parameter model composed of differential–algebraic equations [ 183 ]. 5.2.2. Tumor Lymphangiogenesis The pioneer in the field of mathematical tumor LA is Lolas, who developed in collaboration with Friedman a mathematical model of tumor LA through a system of partial differential parabolic equations [ 184 ]. By following the mathematical time and space evolutions of different key variables, such as the LEC and tumor cell densities, the effect of potential anti-cancer drugs has been studied through this model, also considering the effect of the ECM and its components [ 185 ]. This model has been improved by adding the proteolysis effect, enabling the highlighting of the influence of the proteolytically and un-proteolytically processed growth factors on tumor dissemination and LA [ 186 ]. 5.2.3. Cellular Interactions in Lymph Nodes Novkonic et al. investigated LNs and reviewed the different computational models of LNs, especially investigating the interactions between stromal and immune cells [ 187 ]. Indeed, because of their strategic position, their filtration capacity and their link with the immune system, LNs are also widely represented mathematically. Some key regulators of the chemokine gradient formation in LNs could have been predicted by the in silico model developed by Jafarnejad et al. [ 188 ]. Differential equations representing biochemical reactions were combined with well-known fluid mechanics rules describing lymph flow. Benchaid et al. developed two mathematical models for studying the interactions between cancer and immune cells in the LN [ 189 ]. The first model, focusing more on the interactions between cancer and immune cells, represents the spatiotemporal evolutions of proliferating tumor cells, dormant tumor cells, immune cells, and growth factors. The second model, complementary to the first, employs a hybrid multiscale approach, combining continuous and discrete modeling, representing the secondary tumor growth in the LN particularly. In addition to returning the three well-known regimes of tumor growth in the LN (tumor elimination, cancer-immune equilibrium, and tumor evasion) with specific parameters, the numerical simulations suggested that anti-PD-1/PD-L1 therapies could be more effective in the presence of high EGF concentrations in LNs. 5.2.4. Blood and Lymphatic Vessel Interactions Wu et al. integrated blood and lymphatic vascular systems in a hybrid continuous-discrete mathematical model of tumor growth to better elucidate the influence of interstitial fluid pressure and flow [ 190 ]. Their in silico model fitted with already proven biological experiments and generated new suggestions on the influence of the lymphatics in a tumoral environment. Fluid exchanges between lymph and blood vessels in LNs were also investigated by Jafarnejad et al. with a finite volume-based model, confirming that changes in the inflow/outflow conditions seriously impact the LN microenvironment components and therefore modulate the immune response [ 191 ]. 5.2.5. Lymphatic Biomechanics Galie and Spiker used a finite element method to computationally model the transendothelial lymph transport in primary lymphatics, including relevant biomechanics [ 192 ]. Other in silico models of the lymphatic system focusing on its biomechanical behavior are reviewed in the article of Margaris and Black [ 193 ] or in Nipper and Dixon [ 194 ]. 5.2.6. Lymphatic Electrophysiology Ion fluxes in LECs were modeled by Behringer et al. on the basis of the well-known equations of Hodgkin–Huxley [ 195 ]. This model enables a better understanding of the underlying ionic mechanisms of lymphatic endothelial function compared to blood vessels. Remaining in the field of electrophysiology, Contarino and Toro studied the lymphatic dynamical contractions with one-dimensional modeling for collecting lymphatics combined with an electro-fluid-mechanical contraction model for dynamical contractions, which is based on the FitzHugh–Nagumo theory [ 196 ]. 5.2.7. Bioinformatics In the context of increasing computational capabilities going together with the big data era, bioinformatics tools are essential for the treatment and analysis of these data. Transcriptomic and single-cell data from (lymph node) lymphatic vasculature [ 197 , 198 , 199 , 200 , 201 ], as well as metabolomic [ 202 ] and proteomic [ 203 , 204 ] data, have already been produced from experimental lymphatic set-ups. In combination with imaging techniques, Williams et al. also used bioinformatics approaches to study the impact of particular siRNAs on the 2D in vitro migration of endothelial cells [ 68 ]. Regulatory gene and protein networks can be inferred from these large-scale data libraries to study intracellular dynamics governing cellular behavior and identify druggable targets. 5.2.8. Others Even if out of the scope, we mention here the studies by Wertheim and Roose, who proposed a mathematical model of LA in zebrafish embryos [ 205 ], and by Bianchi et al. who investigated LA in the context of wound healing with ordinary differential equations, highlighting the importance of the relative proportion between TGF-β and VEGF-C, rather than their absolute values [ 206 ]. By means of their in silico models, the different suggested biological hypotheses behind the latter process could be sorted. Furthermore, Tretyakova et al. used computational geometry and network graph models to investigate the structure and topology of the lymphatic system [ 207 ]. In a purely schematic and visualization perspective, an interesting library of interactive 3D models representing the lymphatic system and its associated diseases was designed by the Leukemia and Lymphoma Society [ 208 ]. Figure 4 gathers together and illustrates the previously cited in silico models developed in the context of the lymphatic system and the LA process. 5.3. In Silico Models—Discussion It is clear from the variety of in silico models cited above that there is a wide application area that can be covered by said models, in all phases of the R&D pipeline. In addition to the mostly knowledge-driven models cited above, bioinformatics tools are exploited for treatment and analysis of increasingly prevalent high throughput data. Finally, in complement to these in silico models acting as a digital twin of the lymphatic system or one of its features, computational analysis of biological images (often using machine learning or artificial intelligence) is another domain in which computer and mathematical methods are helpful in the context of lymphatic biology and clinical practice. The latter are also indispensable to the interpretation of the results of the models discussed in Section 4 . 5.1. The In Silico Modeling Pipeline The modeling procedure and the development of models, which can be sorted according to different features and mathematical formalism (see Appendix A ), follow a very precise pipeline divided into several distinct steps [ 161 , 162 ]. Specifically, the subject to be addressed and the questions to be asked of the model are first identified and clearly defined. A literature review is then conducted to report all the current data and knowledge about the topic, resulting in the choice of the best modeling formalism for the objectives to be achieved. The model formalism also depends on the biological data availability or at least the possibility to generate these data. A trade-off must be found between an accurate representation of the system under study and the multiple assumptions imposed because of the biological uncertainties and the limited experimental data provision. In this respect, hypotheses are not to be confused with details, whose level has no relation with the model accuracy but rather with the model capacity to answer the stated problem. Let us quote the relevant sentence of Manfred Eigen: "A theory has only the alternative of being right or wrong. A model has a third possibility: it may be right, but irrelevant". The next modeling steps are the mathematical formulation, followed by the computational implementation, because only a few models can be solved purely analytically. The models are often composed with variables and parameters, most of which are not known and must be estimated. Parameter estimation is therefore requested to calibrate the model and consists in determining the optimal parameter values to enter into the model so that the generated output fits as best as possible with the biological data, generated previously or for the purpose. Once the model has been calibrated, it is simulated with efficient algorithms and the in silico outputs are provided. Wrong results are not necessarily related to a wrong mathematical formalism but can also be linked to false starting hypotheses or issues with the data used for calibration (garbage in, garbage out). Coherent model predictions can be validated with new experiments or with previously generated biological observations. Calibration and validation usually do not use the same datasets. Depending on the divergence between the predicted and the observed data, the model might be subjected to modifications and undergo several improvement and optimization cycles for refinement. In addition to reproducing data from biology, the last optimized version of the mathematical model is therefore considered as a tool that can be used to test new experimental conditions. It is important to realize that in silico models (like all other model systems) are not self-sufficient but are constructed, validated, and iteratively refined through in vitro and in vivo experiments before being sufficiently precise and ready to provide insightful outputs. The credibility of in silico models can be established for a precisely defined context of use, by applying the so-called VVUQ (Verification, Validation, and Uncertainty Quantification) [ 163 ]. Figure 3 gathers all the steps outlined above regarding the modeling pipeline and highlights the symbiotic approach, mixing the in vitro, in vivo, and in silico methods. 5.2. In Silico Models of LA For several years, the impact of the vascularization on tumor progression has been intensively studied through mathematical modeling, especially the influence of angiogenesis. Good reviews of in silico models for angiogenesis and its role in tumor progression were written by Peirce [ 164 ], Heck et al. [ 165 ], Chaplain [ 166 ], Scianna et al. [ 47 ], Mantzaris et al. [ 167 ], Levine et al. [ 168 , 169 ], Qutub et al. [ 170 ], Suzuki et al. [ 171 ], and Lowengrub et al. [ 172 ]. Even if some previously reported models of angiogenesis could be easily adapted for LA, this process presents particular features requiring specific dedicated models. Though considerably less widespread than for the process of angiogenesis, several mathematical and computational models have been implemented in the context of the lymphatic system and LA. Currently, very few in silico models specifically focused on tumor LA have been developed. However, the models and tools elaborated in 'simpler' lymphatic contexts will nevertheless be discussed in this review article, sorted by application domain, and can serve as a starting point for more focused tumor LA models. 5.2.1. Lymphatic Flow The principal initiator of the in silico lymphatic system modeling is Reddy, who computationally investigated, among others, the flow through terminal lymphatics but also the lymph circulation biomechanics and the valve physics [ 173 , 174 , 175 , 176 ]. Elhay and Casley-Smith were also pioneers and used normal laws of physics related to flow and diffusion to study the initial lymphatics [ 177 ]. MacDonald adapted one of Reddy's models to study the lymph flow in the collecting lymphatic vessels [ 178 ]. Different modeling techniques of drainage in primary and collecting lymphatics, as well as lymphatic valves and nodes, are reviewed in the paper of Roose and Tabor [ 179 ]. Mozokhina and Savinkov highlighted the different mathematical models implemented to represent and study the lymph flow in the lymphatic system and its subunits (lymphangions, valves, and LNs) [ 180 ]. In this context, the majority of the developed models are built with zero-, quasi-one-, or one-dimensional approaches. Zero-dimensional methods, so-called lumped models, refer to the electrical circuit theory, comparing the pressure, lymph flux, and mass with voltage, current, and charge, respectively. Lymph flow is usually described in one-dimensional approaches with Navier–Stokes equations or with the law of mass and momentum conservation. Comparison between 0D and 1D formulation in the context of modeling lymph flow in the lymphatic system has been explained in the article of Tretyakova [ 181 ]. Cooper et al. used a finite element image-based model to investigate the fluid flow through the LNs [ 182 ]. The effect of a permeable interstitium on a network of initial lymphatics and pre-collectors was investigated by Ikhimwin et al. with a lumped-parameter model composed of differential–algebraic equations [ 183 ]. 5.2.2. Tumor Lymphangiogenesis The pioneer in the field of mathematical tumor LA is Lolas, who developed in collaboration with Friedman a mathematical model of tumor LA through a system of partial differential parabolic equations [ 184 ]. By following the mathematical time and space evolutions of different key variables, such as the LEC and tumor cell densities, the effect of potential anti-cancer drugs has been studied through this model, also considering the effect of the ECM and its components [ 185 ]. This model has been improved by adding the proteolysis effect, enabling the highlighting of the influence of the proteolytically and un-proteolytically processed growth factors on tumor dissemination and LA [ 186 ]. 5.2.3. Cellular Interactions in Lymph Nodes Novkonic et al. investigated LNs and reviewed the different computational models of LNs, especially investigating the interactions between stromal and immune cells [ 187 ]. Indeed, because of their strategic position, their filtration capacity and their link with the immune system, LNs are also widely represented mathematically. Some key regulators of the chemokine gradient formation in LNs could have been predicted by the in silico model developed by Jafarnejad et al. [ 188 ]. Differential equations representing biochemical reactions were combined with well-known fluid mechanics rules describing lymph flow. Benchaid et al. developed two mathematical models for studying the interactions between cancer and immune cells in the LN [ 189 ]. The first model, focusing more on the interactions between cancer and immune cells, represents the spatiotemporal evolutions of proliferating tumor cells, dormant tumor cells, immune cells, and growth factors. The second model, complementary to the first, employs a hybrid multiscale approach, combining continuous and discrete modeling, representing the secondary tumor growth in the LN particularly. In addition to returning the three well-known regimes of tumor growth in the LN (tumor elimination, cancer-immune equilibrium, and tumor evasion) with specific parameters, the numerical simulations suggested that anti-PD-1/PD-L1 therapies could be more effective in the presence of high EGF concentrations in LNs. 5.2.4. Blood and Lymphatic Vessel Interactions Wu et al. integrated blood and lymphatic vascular systems in a hybrid continuous-discrete mathematical model of tumor growth to better elucidate the influence of interstitial fluid pressure and flow [ 190 ]. Their in silico model fitted with already proven biological experiments and generated new suggestions on the influence of the lymphatics in a tumoral environment. Fluid exchanges between lymph and blood vessels in LNs were also investigated by Jafarnejad et al. with a finite volume-based model, confirming that changes in the inflow/outflow conditions seriously impact the LN microenvironment components and therefore modulate the immune response [ 191 ]. 5.2.5. Lymphatic Biomechanics Galie and Spiker used a finite element method to computationally model the transendothelial lymph transport in primary lymphatics, including relevant biomechanics [ 192 ]. Other in silico models of the lymphatic system focusing on its biomechanical behavior are reviewed in the article of Margaris and Black [ 193 ] or in Nipper and Dixon [ 194 ]. 5.2.6. Lymphatic Electrophysiology Ion fluxes in LECs were modeled by Behringer et al. on the basis of the well-known equations of Hodgkin–Huxley [ 195 ]. This model enables a better understanding of the underlying ionic mechanisms of lymphatic endothelial function compared to blood vessels. Remaining in the field of electrophysiology, Contarino and Toro studied the lymphatic dynamical contractions with one-dimensional modeling for collecting lymphatics combined with an electro-fluid-mechanical contraction model for dynamical contractions, which is based on the FitzHugh–Nagumo theory [ 196 ]. 5.2.7. Bioinformatics In the context of increasing computational capabilities going together with the big data era, bioinformatics tools are essential for the treatment and analysis of these data. Transcriptomic and single-cell data from (lymph node) lymphatic vasculature [ 197 , 198 , 199 , 200 , 201 ], as well as metabolomic [ 202 ] and proteomic [ 203 , 204 ] data, have already been produced from experimental lymphatic set-ups. In combination with imaging techniques, Williams et al. also used bioinformatics approaches to study the impact of particular siRNAs on the 2D in vitro migration of endothelial cells [ 68 ]. Regulatory gene and protein networks can be inferred from these large-scale data libraries to study intracellular dynamics governing cellular behavior and identify druggable targets. 5.2.8. Others Even if out of the scope, we mention here the studies by Wertheim and Roose, who proposed a mathematical model of LA in zebrafish embryos [ 205 ], and by Bianchi et al. who investigated LA in the context of wound healing with ordinary differential equations, highlighting the importance of the relative proportion between TGF-β and VEGF-C, rather than their absolute values [ 206 ]. By means of their in silico models, the different suggested biological hypotheses behind the latter process could be sorted. Furthermore, Tretyakova et al. used computational geometry and network graph models to investigate the structure and topology of the lymphatic system [ 207 ]. In a purely schematic and visualization perspective, an interesting library of interactive 3D models representing the lymphatic system and its associated diseases was designed by the Leukemia and Lymphoma Society [ 208 ]. Figure 4 gathers together and illustrates the previously cited in silico models developed in the context of the lymphatic system and the LA process. 5.2.1. Lymphatic Flow The principal initiator of the in silico lymphatic system modeling is Reddy, who computationally investigated, among others, the flow through terminal lymphatics but also the lymph circulation biomechanics and the valve physics [ 173 , 174 , 175 , 176 ]. Elhay and Casley-Smith were also pioneers and used normal laws of physics related to flow and diffusion to study the initial lymphatics [ 177 ]. MacDonald adapted one of Reddy's models to study the lymph flow in the collecting lymphatic vessels [ 178 ]. Different modeling techniques of drainage in primary and collecting lymphatics, as well as lymphatic valves and nodes, are reviewed in the paper of Roose and Tabor [ 179 ]. Mozokhina and Savinkov highlighted the different mathematical models implemented to represent and study the lymph flow in the lymphatic system and its subunits (lymphangions, valves, and LNs) [ 180 ]. In this context, the majority of the developed models are built with zero-, quasi-one-, or one-dimensional approaches. Zero-dimensional methods, so-called lumped models, refer to the electrical circuit theory, comparing the pressure, lymph flux, and mass with voltage, current, and charge, respectively. Lymph flow is usually described in one-dimensional approaches with Navier–Stokes equations or with the law of mass and momentum conservation. Comparison between 0D and 1D formulation in the context of modeling lymph flow in the lymphatic system has been explained in the article of Tretyakova [ 181 ]. Cooper et al. used a finite element image-based model to investigate the fluid flow through the LNs [ 182 ]. The effect of a permeable interstitium on a network of initial lymphatics and pre-collectors was investigated by Ikhimwin et al. with a lumped-parameter model composed of differential–algebraic equations [ 183 ]. 5.2.2. Tumor Lymphangiogenesis The pioneer in the field of mathematical tumor LA is Lolas, who developed in collaboration with Friedman a mathematical model of tumor LA through a system of partial differential parabolic equations [ 184 ]. By following the mathematical time and space evolutions of different key variables, such as the LEC and tumor cell densities, the effect of potential anti-cancer drugs has been studied through this model, also considering the effect of the ECM and its components [ 185 ]. This model has been improved by adding the proteolysis effect, enabling the highlighting of the influence of the proteolytically and un-proteolytically processed growth factors on tumor dissemination and LA [ 186 ]. 5.2.3. Cellular Interactions in Lymph Nodes Novkonic et al. investigated LNs and reviewed the different computational models of LNs, especially investigating the interactions between stromal and immune cells [ 187 ]. Indeed, because of their strategic position, their filtration capacity and their link with the immune system, LNs are also widely represented mathematically. Some key regulators of the chemokine gradient formation in LNs could have been predicted by the in silico model developed by Jafarnejad et al. [ 188 ]. Differential equations representing biochemical reactions were combined with well-known fluid mechanics rules describing lymph flow. Benchaid et al. developed two mathematical models for studying the interactions between cancer and immune cells in the LN [ 189 ]. The first model, focusing more on the interactions between cancer and immune cells, represents the spatiotemporal evolutions of proliferating tumor cells, dormant tumor cells, immune cells, and growth factors. The second model, complementary to the first, employs a hybrid multiscale approach, combining continuous and discrete modeling, representing the secondary tumor growth in the LN particularly. In addition to returning the three well-known regimes of tumor growth in the LN (tumor elimination, cancer-immune equilibrium, and tumor evasion) with specific parameters, the numerical simulations suggested that anti-PD-1/PD-L1 therapies could be more effective in the presence of high EGF concentrations in LNs. 5.2.4. Blood and Lymphatic Vessel Interactions Wu et al. integrated blood and lymphatic vascular systems in a hybrid continuous-discrete mathematical model of tumor growth to better elucidate the influence of interstitial fluid pressure and flow [ 190 ]. Their in silico model fitted with already proven biological experiments and generated new suggestions on the influence of the lymphatics in a tumoral environment. Fluid exchanges between lymph and blood vessels in LNs were also investigated by Jafarnejad et al. with a finite volume-based model, confirming that changes in the inflow/outflow conditions seriously impact the LN microenvironment components and therefore modulate the immune response [ 191 ]. 5.2.5. Lymphatic Biomechanics Galie and Spiker used a finite element method to computationally model the transendothelial lymph transport in primary lymphatics, including relevant biomechanics [ 192 ]. Other in silico models of the lymphatic system focusing on its biomechanical behavior are reviewed in the article of Margaris and Black [ 193 ] or in Nipper and Dixon [ 194 ]. 5.2.6. Lymphatic Electrophysiology Ion fluxes in LECs were modeled by Behringer et al. on the basis of the well-known equations of Hodgkin–Huxley [ 195 ]. This model enables a better understanding of the underlying ionic mechanisms of lymphatic endothelial function compared to blood vessels. Remaining in the field of electrophysiology, Contarino and Toro studied the lymphatic dynamical contractions with one-dimensional modeling for collecting lymphatics combined with an electro-fluid-mechanical contraction model for dynamical contractions, which is based on the FitzHugh–Nagumo theory [ 196 ]. 5.2.7. Bioinformatics In the context of increasing computational capabilities going together with the big data era, bioinformatics tools are essential for the treatment and analysis of these data. Transcriptomic and single-cell data from (lymph node) lymphatic vasculature [ 197 , 198 , 199 , 200 , 201 ], as well as metabolomic [ 202 ] and proteomic [ 203 , 204 ] data, have already been produced from experimental lymphatic set-ups. In combination with imaging techniques, Williams et al. also used bioinformatics approaches to study the impact of particular siRNAs on the 2D in vitro migration of endothelial cells [ 68 ]. Regulatory gene and protein networks can be inferred from these large-scale data libraries to study intracellular dynamics governing cellular behavior and identify druggable targets. 5.2.8. Others Even if out of the scope, we mention here the studies by Wertheim and Roose, who proposed a mathematical model of LA in zebrafish embryos [ 205 ], and by Bianchi et al. who investigated LA in the context of wound healing with ordinary differential equations, highlighting the importance of the relative proportion between TGF-β and VEGF-C, rather than their absolute values [ 206 ]. By means of their in silico models, the different suggested biological hypotheses behind the latter process could be sorted. Furthermore, Tretyakova et al. used computational geometry and network graph models to investigate the structure and topology of the lymphatic system [ 207 ]. In a purely schematic and visualization perspective, an interesting library of interactive 3D models representing the lymphatic system and its associated diseases was designed by the Leukemia and Lymphoma Society [ 208 ]. Figure 4 gathers together and illustrates the previously cited in silico models developed in the context of the lymphatic system and the LA process. 5.3. In Silico Models—Discussion It is clear from the variety of in silico models cited above that there is a wide application area that can be covered by said models, in all phases of the R&D pipeline. In addition to the mostly knowledge-driven models cited above, bioinformatics tools are exploited for treatment and analysis of increasingly prevalent high throughput data. Finally, in complement to these in silico models acting as a digital twin of the lymphatic system or one of its features, computational analysis of biological images (often using machine learning or artificial intelligence) is another domain in which computer and mathematical methods are helpful in the context of lymphatic biology and clinical practice. The latter are also indispensable to the interpretation of the results of the models discussed in Section 4 . 6. Perspectives, Limitations, and Conclusions In a society where cancer is the second leading cause of death and where everyone has at least one relative affected by this disease, the improved understanding of its various aspects is of utmost importance [ 1 ]. Cancer cells notably disseminate and form metastases through tumor-induced vasculature, both blood and lymphatic vessels [ 3 , 4 , 5 , 6 , 7 , 8 ]. The process of lymphangiogenesis has nevertheless often been neglected in favor of the much-studied angiogenesis. However, its relevance in cancer and other pathologies is now well established. The LA study and the discovery of pro-lymphangiogenic drugs have been delayed as compared to angiogenesis, mainly due to the lack of adapted tools, and are now gradually increasing. This review provides a non-exhaustive up-to-date overview of the in vitro, in vivo, and in silico models of LA. These three types of modeling, each presenting advantages and limitations, are required to work all together in an effort to respect the 3R principle, including the reduction in animal experiments. Low cost, tight control of the environmental conditions, and no ethical issues often make in vitro models very attractive, especially now that 3D cultures with flow and organotypic ex vivo approaches have been developed [ 69 , 76 , 77 , 78 , 79 , 81 , 82 , 83 ]. Unfortunately, most in vitro models insufficiently integrate different cell types in a relevant ECM, although that is known to play an essential role in both cancer and LA. In this context, cell-cell and cell-matrix communications are becoming of particular interest as they are at the heart of cell adhesion, migration, and differentiation. Fibroblasts would exert traction forces on the ECM, inducing signals potentially detected by cells sharing the same environment [ 209 ]. Changes in the alignment of collagen fibers in the ECM have been demonstrated in various pathologies, including cancer. The traction of collagen fibers by fibroblasts would impact the migration and invasion of cancer cells. The LEC–fibroblast interactions are therefore of paramount importance and worth considering when studying tumor-induced LA. Still in this context, it could be useful to investigate in depth the heterogeneity/plasticity of fibroblasts and LECs and also how they interact with the surrounding interstitial ECM. In vivo approaches enable the better modeling of complex and multifactorial diseases such as cancer with a systemic integration of the tumoral environment and the inflammation. In the context of tumoral LA, transgenic mouse strains remain the favored animal model, both for their genetic and physiological homologies with humans and also for their ability to be monitored through proteins with fluorescent properties. Anti-lymphangiogenic drugs have even been investigated by means of xenografts reproducing the tumoral cascade with a remodeled and extended lymphatic network [ 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 ]. Because xenografts unfortunately often require immunodeficient mice to avoid graft rejection, approaches with syngeneic transplants have been developed, as in the ear sponge assay investigating the metastatic cascade in LNs [ 107 , 108 ]. Given a facilitated reproduction and decreased ethical constraints, zebrafish could putatively become a model of choice for exploring tumor-associated LA. However, the use of live animals for experimental purposes is increasingly questioned at an ethical level, and the development and uptake of alternative methods, including in silico approaches, are required. The computational and mathematical contributions to biological fundamental research are no longer to be proven. Although not numerous, in silico models of tumor LA, including the ECM, have been developed and enabled to examine the effect of potential anti-cancer drugs [ 187 ]. These models should now be combined with the ones on LNs to have a more integrated and systemic approach to the metastatic cascade. As digital evidence, i.e., the results of computer-based methods, is increasingly included in regulatory filings, a regulation framework is (being) established. A standard is already detailing how to build credibility for computer models used in the context of medical devices (V&V40) [ 158 ]. Regulatory guidelines regarding the introduction of in silico approaches in the pipeline for drug development (beyond the classical pharmacometric models) are also being established [ 210 ]. Additionally, in analogy to good clinical and manufacturing practices, a good simulation practice is being developed by the community as a way to guide proper model development from bench to bedside [ 211 ]. All these activities will allow the building of necessary trust in in silico approaches and allow them to take their place as a third source of data generation in biomedical sciences, next to in vitro and in vivo. The symbiotic approach, mixing in vitro, in vivo, and in silico methods, is of great interest for modeling tumor-associated LA. Each kind of model brings different but complementary information. Whether it is because they are not often conclusive in clinical trials or because of ethical questioning, the animal experiments will be reduced and possibly even eliminated in the future. Alternative and advanced techniques require the combination of in vitro models, prospectively a suite of organ-on-a-chips, with in silico models, prospectively digital twins. In reference to the article of Ingber: "is it time for reviewer 3 to request human organ-on-a-chip experiments instead of animal validation studies?", we aimed to demonstrate in this review that this also holds true also for in silico experiments or a combination of both [ 212 ]. Appendix A.1. Addressed Subject Mathematical modeling extends to many areas of daily life, such as chemistry, engineering, social science, physics, and economics, as well as biology. In biology, in silico modeling is exploited in (amongst others) immunology, drug toxicity, genomics, epidemiology, metabolomics, neurology, and, of course, cancer [ 159 ]. In each of these fields, different levels of details can be addressed. This list is far from exhaustive. Appendix A.2. Length and Time Scales In biology, and in cancer particularly, different length and time scales can be considered and modeled, from the gene and protein levels to the population level [ 157 ]. Regarding the gene and protein levels, high throughput technologies helped to provide access to large amounts of genomic, transcriptomic, proteomic, and metabolomic data. Increasing the computational capabilities going together with the big data era, new bioinformatics analysis tools were developed to infer differential expression patterns and regulatory networks to highlight key actors of this disease frequently connected to genome changes. Single-cell mechanistic behavior, cellular interactions, and the influence of different external clues on individual cells can also be investigated through models, often with a mathematical approach called agent-based modeling. The tissue level focuses more on the influence of growth factors, the ECM, and the vasculature on a group of cells (cancerous here). This tissue context involves many multiscale actors with their own identity and size, making it common to use hybrid modeling, which mixes a discrete formalism to represent the cells of interest with a continuous one to depict the environment and the other molecular species and cells. The influence of endogenous and applied external mechanobiological forces in cells or a set of cells can also be explored in these two previously mentioned levels. The organ level refers to the studies highlighting the structure–function aspect and exploring, for example, the impact of a tumor on a specific organ or biological system (and vice versa). Patient and population levels grow in complexity and are two of the highest (relevant) spatiotemporal scales that can be considered. By integrating several of the aforementioned levels (depending on the question at hand), virtual patients with specific parameters and features can be generated and participate in the reduction challenge of the 3R principle in clinical trials [ 213 ]. Appendix A.3. Mathematical Classification Different mathematical formalisms can be used for modeling a system of interest, focusing on the inputs used as well as the representation of the system. First, models can be distinguished according to the way they represent the system, either in a continuous or discrete manner. Second, besides the assumptions and simplifications, in silico models can be fed with two distinct sorts of information: qualitative knowledge and quantitative data. When only physical principles and physiological knowledge about the studied system are used to implement the model, it is referred to as mechanistic, white-box, physics-based, or knowledge-driven modeling. Contrastingly, models built solely on experimental data are black-box or data-driven empirical models, in which links between the outputs and inputs are established without using prior knowledge. Good illustrations of data-driven model technologies are machine learning, deep learning, and artificial intelligence. However, in practice, many white box models include several empirical elements and black box models are increasingly considering existing knowledge, giving rise to the category of grey-box models which are derived from both previous knowledge and experimental records in various proportions. Models can also be distinguished according to the way they represent the system, either in a continuous or a discrete manner. In continuous modeling, for which partial or ordinary differential equations are often used, the values of the involved variables belong to a continuum, whereas the variable values in discrete models are imposed from a discrete set and implemented through agent-based modeling. Hybrid approaches combine discrete modeling to represent cells of interest and continuous modeling for describing environmental factors and clues.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135112/

Fever in the Returning Traveler

Millions of children travel annually, whether they are refugees, international adoptees, visitors, or vacationers. Although most young travelers do well, many develop a febrile illness during or shortly after their trips. Approaching a fever in the returning traveler requires an appropriate index of suspicion to diagnose and treat in a timely manner. As many as 34% of patients with recent travel history are diagnosed with routine infections, but serious infections such as malaria, enteric fever, and dengue fever should be on the differential diagnosis due the high morbidity and mortality in children. Key points • The initial workup of a febrile child without a clear source will be based on the history, physical examination, and potential risk factors but commonly includes laboratory testing. • Malaria, enteric fever, and dengue fever are some of the most common and serious tropical infections in pediatric travelers. • Clinicians need to remain up-to-date on potential etiologic factors for febrile illnesses to develop a focused plan best suited to the patient's clinical picture. Introduction Millions of children travel annually, whether they are refugees, international adoptees, visitors, or vacationers. 1 , 2 , 3 , 4 In 2015, the International Tourism Organization reported 1.2 billion overseas trips. 5 , 6 Although most young travelers do well, many develop febrile illnesses during or shortly after their journeys. 7 In a study of European children, 53% of all pediatric patients with travel-related infections were visiting friends and relatives (VFRs), 43.4% were tourists, and 2.4% were immigrants. 8 Most illnesses are self-limited childhood infections that do not require subspecialist consultation. However, 28% of 24,920 ill American travelers sought care at travel clinics after returning home. 9 Additionally, young children with fevers can present a diagnostic dilemma because they may not report symptoms and can be at risk for severe disease, such as malaria. As awareness of tropical illnesses rise in parents, such as the increase in multidrug-resistant bacteria worldwide or the emergence of epidemics with Zika virus in South America, families may be more anxious about serious infections as an etiologic factor of fevers. Approaching fevers in the returning traveler requires an appropriate index of suspicion to diagnose and treat the child in a timely manner. This article offers a framework on how to address these issues by discussing diseases based on geography, incubation period, and affected organ systems, as well as risk factors, diagnostic techniques, and resources. General approach A thorough history is an important initial step when evaluating a pediatric traveler with a fever ( Table 1 ). Discussing a detailed travel itinerary develops a timeline of exposures that can be unique to an urban or rural setting ( Table 2 ). Table 1 Patient history for the returning traveler with fever History Implications Travel itinerary Offers information on potential diseases based on geography and other exposures Diet history (improperly cooked meats, unpasteurized dairy products, seafood, or contaminated water and produce) Brucellosis, Campylobacter infection, giardiasis, hepatitis A and E, listeriosis, traveler's diarrhea, enteric fever, trichinosis, viral gastroenteritis (ie, norovirus) Sick contacts (both abroad and since returning to the US) Routine viral or bacterial illnesses, Ebola infection, influenza, meningococcemia, tuberculosis Fresh water exposure Bacterial soft tissue infection ( Aeromonas spp, atypical Mycobacterium ), leptospirosis, schistosomiasis Sexual encounters Acute human immunodeficiency virus (HIV) infection; gonorrhea; hepatitis A, B, or C infection; primary herpesvirus 1 or 2 infection; syphilis; Zika virus infection Insect bites • Fleas: plague, murine typhus, rickettsioses • Flies: African sleeping sickness, leishmaniasis, sandfly fever • Lice: relapsing fever, rickettsioses • Reduviid bugs: Chagas disease • Mosquitoes: Chikungunya virus infection, dengue fever, filiarisis, Japanese encephalitis, West Nile virus infection, Zika virus infection • Ticks: African tick bite fever, babesiosis, Lyme disease, Q fever rickettsioses, tularemia Animal bites Cat-scratch disease, rat bite fever, rabies, simian herpesvirus B infection Animal exposure (including exposure to urine, stool, or animal products; eg, infected carcasses or wool) Anthrax, avian influenza, hantavirus infection, Hendra virus infection, infections from ectoparasites or endoparasites, Nipah virus infection, plague, psittacosis, toxoplasmosis Body fluid exposures (tattoos, piercings, or medical procedures) Acute HIV infection, babesiosis, cytomegalovirus infection, hepatitis B and C, malaria, multidrug-resistant bacteria, trypanosomiasis Medical history (diseases associated with immunosuppression; eg, malignancy, asplenia, or immunodeficiency) Cytomegalovirus infection, Epstein-Barr virus infection, fungal infection, mycobacterial infections Vaccinations and prophylaxis (note: these interventions do not preclude infection with the pathogen prophylaxed against) Malaria prophylaxis, travel-appropriate vaccines Adapted from Refs. 50 , 51 , 52 Table 2 Tropical causes of fever based on geography Location Infection Caribbean Acute histoplasmosis, chikungunya, cholera, dengue fever, leptospirosis, malaria (Haiti, primarily Plasmodium falciparum ) Central America Acute histoplasmosis, coccidioidomycosis, dengue fever, hepatitis A and B, malaria (primarily P vivax ), tuberculosis South America Bartonellosis, dengue fever, malaria (primarily P vivax ), enteric fever, leptospirosis, yellow fever South Central Asia Dengue fever, enteric fever, hepatitis B, Japanese encephalitis, malaria (primarily non-falciparum Plasmodium spp), tuberculosis Southeast Asia Chikungunya, cholera, dengue fever, hepatitis A, Japanese encephalitis, malaria (primarily non-falciparum Plasmodium spp), yellow fever Sub-Saharan Africa Acute schistosomiasis, enteric fever, filariasis, malaria (primarily P falciparum) , meningococcus, rickettsioses, yellow fever Adapted from Centers for Disease Control and Prevention. The yellow book: health information for international travel 2018. Philadelphia: Oxford University Press; 2017. p. 704. Available at: https://wwwnc.cdc.gov/travel/page/yellowbook-home . Accessed July 25, 2017; with permission. Many children receive vaccinations and/or antimicrobial prophylaxis, but reported adherence does not preclude an illness with a particular pathogen. Up to 75% of travelers do not adhere to the recommended malaria prophylaxis. 10 Many travel vaccines, including typhoid vaccine, provide only partial protection despite proper administration of these immunizations. 11 A medically complex individual may have sought care outside of the United States due to necessity or medical tourism, which can increase the risk of infection through body fluid exposures. Multidrug-resistant pathogens can also be associated with health care exposure. Up to half of hospitalized children in Zimbabwe are colonized with extended spectrum beta lactamase producing Enterobacteriaceae on admission to the hospital, 12 a problem that is increasingly seen worldwide. Underlying medical conditions, such as asplenia or immunosuppression from chemotherapy, may predispose children to overwhelming infections and sepsis. Refugee children from countries such as Syria are susceptible to vaccine-preventable diseases such as polio due to infrastructure breakdown. 13 Clinical findings, diagnosis, and management Fever is a common and anxiety-provoking sign for parents that can be exacerbated by overseas travel. Up to 34% of patients with recent travel history are diagnosed with routine infections. 3 Of the 82,825 cases of infection in travelers from 1996 to 2011 reported to GeoSentinel, a worldwide data collection network on travel-related diseases, 4% of cases were considered to be life-threatening. 14 A study in Swiss children showed that 0.45% of emergency room visits were due to travel-related morbidities with fever and gastrointestinal symptoms being the most common complaints in 63% and 50% of patients, respectively. 8 The temporality of travel to the onset of fever can offer important clues to the etiologic factors of fevers ( Table 3 ). Because the causes and clinical outcomes associated with fevers in pediatric travelers vary from self-limited to deadly, a systems-based approach can lead to prompt diagnosis and treatment that evaluates for the most likely and serious diseases early in the illness course. Table 3 Incubation period for common tropical diseases causing Disease Incubation Period Incubation of 10 cm, antimicrobial therapy (albendazole, praziquantel) Traveler's diarrhea Enterotoxigenic Escherichia coli (ETEC) Worldwide Fecal-oral, contaminated food or water 9 h–3 d Abdominal pain, watery diarrhea Clinical diagnosis, NAAT Supportive care, antimicrobial therapy (ciprofloxacin, azithromycin) Fascioliasis Fasciola hepatica and F gigantica South America, Middle East, Southeast Asia Watercress or other aquatic plants, freshwater 6–12 wk Intermittent, fever eosinophilia, abdominal pain, weight loss, urticaria, biliary colic, liver failure Microscopic evaluation of stool, serologies, liver imaging Antimicrobial therapy (triclabendazole) Giardiasis Giardia intestinalis Worldwide Fecal-oral, sexual contact, contaminated water 1–2 wk Abdominal pain, anorexia, foul-smelling diarrhea, flatulence, nausea, reactive arthritis Microscopic evaluation of stool, DFA Antimicrobial therapy (metronidazole, tinidazole, nitazoxanide) Peptic ulcer disease Helicobacter pylori Worldwide Fecal-oral, oral-oral Unknown Epigastric pain, nausea and vomiting, anorexia, gastric cancer Fecal antigen assay, urea breath test Antimicrobial therapy (proton pump inhibitor + clarithromycin + amoxicillin) Pinworm Enterobius vermicularis Worldwide Fecal-oral, contaminated objects 1–2 mo Perianal pruritus Scotch tape test, microscopic evaluation of fingernails Antimicrobial therapy (albendazole, pyrantel pamoate) Sarcocystosis Sarcocystis species Worldwide, especially Southeast Asia Undercooked beef or pork 2 wk Fever, malaise, myalgia, headache, cough, arthralgia, nausea and vomiting, diarrhea, palpitations Microscopic evaluation of stool, PCR, muscle biopsy Antimicrobial therapy (trimethoprim-sulfamethoxazole) Soil-transmitted helminths Ascaris lumbricoides (roundworm), Ancylostoma duodenale (hookworm), Necator americanus (hookworm), Trichuris trichiura (whipworm) Worldwide Fecal-oral, skin penetration with contaminated soil (hookworms) Variable Abdominal pain, malnutrition, bowel obstruction, anemia, cough, chest pain Microscopic evaluation of stool Antimicrobial therapy (albendazole, mebendazole) Strongyloidiasis Strongyloides stercoralis Worldwide Auto-inoculation, skin penetration Variable Pruritic rash at penetration site, serpiginous rashes (larva currens), respiratory symptoms (Löffler-like pneumonitis), abdominal pain, diarrhea, severe disease if immuno-compromised Microscopic evaluation of stool other body fluids if disseminated (eg, sputum, CSF) Antimicrobial therapy (ivermectin, albendazole) Taeniasis Taenia solium (pork) and T saginata or T asiatica (beef) Central and South America, Africa, South and Southeast Asia Undercooked contaminated pork or beef 8–10 wk for T solium , 10–14 wk for T saginata Abdominal discomfort, weight loss, anorexia, perianal pruritus, insomnia, weakness Microscopic evaluation of stool for eggs Antimicrobial therapy (praziquantel, niclosamide unless symptomatic neurocysticercosis) Visceral leishmaniasis Leishmania donovani and L infantum-chagasi South America, Central and Southwest Asia, East Africa Phlebotomine sand fly, blood transfusions Weeks–months Fever, weight loss, hepatosplenomegaly, pancytopenia Light-microscopic evaluation of specimens, culture, molecular methods Antimicrobial therapy (amphotericin B, miltefosine) Yersiniosis Yersinia enterocolitica Japan, Northern Europe Undercooked contaminated pork, contaminated water, unpasteurized dairy 4–6 d Fever, abdominal pain (pseudoappendicitis), bloody diarrhea, necrotizing enterocolitis in infants, reactive arthritis, erythema nodosum Stool culture (or other body sits; eg, CSF, blood) Supportive care, antimicrobial therapy if severe (trimethoprim-sulfamethoxazole, fluoroquinolones, aminoglycosides) Abbreviations: CSF, cerebrospinal fluid; DFA, direct fluorescent antibody. Adapted from Centers for Disease Control and Prevention. The yellow book: health information for international travel 2018. Philadelphia: Oxford University Press; 2017. p. 704. Available at: https://wwwnc.cdc.gov/travel/page/yellowbook-home . Accessed July 25, 2017; with permission. Respiratory Symptoms In the pediatric population, common respiratory infections may be seen on return from international trips including pharyngitis, sinusitis, otitis, and pneumonia from pathogens commonly seen in the United States, such as Streptococcus pneumoniae and rhinovirus. 4 , 43 Local epidemiology of infections can be helpful in diagnosis and management and is available through the CDC. In some tropical regions, influenza may occur throughout the year and should hence remain on the differential for patients who warrant treatment with oseltamivir. 44 Mycobacterium tuberculosis is an important etiologic factor of lower respiratory tract disease worldwide and should be considered in children with risk factors or who do not recover with antimicrobials for bacterial pneumonia. 26 Of note, children younger than 3 years of age are more likely to present with miliary tuberculosis or neurologic involvement than adult patients. There are also many other less common causes of febrile respiratory tract infections ( Table 6 ). Table 6 Tropical diseases associated with respiratory symptoms Disease Etiologic Pathogen Geographic Regions Vector or Exposure Incubation Period Presentation Diagnosis Management Avian bird flu H5N1 and H7N9 influenza A virus East and Southeast Asia Poultry 2–8 d Fever, malaise, myalgia, headache, nasal congestion, cough, acute respiratory distress syndrome (ARDS) RT-PCR Supportive care Diphtheria Corynebacterium diphtheriae Asia, South Pacific, Middle East, Eastern Europe, Caribbean Person-to-person (oral or respiratory droplets), fomites 2–5 d Fever, dysphagia, malaise, anorexia, pseudomembranes Bacterial culture Supportive care, equine diphtheria antitoxin (DAT), antimicrobial therapy (erythromycin, penicillin) Coccidioidomycosis Coccidioides immitis and Coccidioides posadasii Central and South America Inhalation of spores from soil 7–21 d Fever, malaise, cough, headache, night sweats, myalgias, arthritis, rash Culture, IgM and IgG ELISA, immunodiffusion and complement fixation Supportive care, antimicrobial therapy if ill or at high risk of dissemination (amphotericin B, azoles) Histoplasmosis Histoplasma capsulatum Worldwide, especially river valleys Inhalation of spores from soil, bird droppings, bat guano 3–17 d Fever, headache, cough, pleuritic chest pain, malaise Culture, microscopic examination, PCR, EIA on serum or other samples, immunodiffusion complement fixation Supportive care, antimicrobial therapy (azole for mild to moderate disease, amphotericin B for severe) Legionellosis (Legionnaire's disease and Pontiac fever) Legionella species Worldwide Inhalation of freshwater aerosol 2–10 d Fever, headache, myalgias, pneumonia, respiratory distress Urine antigen assay, paired serologies, PCR Antimicrobial therapy (fluoroquinolones, macrolides) Melioidosis Burkholderia pseudomallei Central and Southeast Asia, northern Australia, South America Subcutaneous inoculation, inhalation, ingestion; body fluids 1–21 d Fever, cough, weight loss, pneumonia Culture, indirect hemagglutination assay Antimicrobial therapy (ceftazidime, meropenem) Middle Eastern Respiratory Syndrome (MERS) MERS coronavirus North Africa, Middle East Dromedary camel, person-to-person 2–14 d Fever, cough, arthralgia, diarrhea, myalgia, acute respiratory failure, multiple organ dysfunction RT-PCR Supportive care Pertussis (whooping cough) Bordetella pertussis Worldwide Person-to-person (aerosolized respiratory droplets, respiratory secretions) 7–10 d Paroxysmal cough, post-tussive vomiting, apnea in infants Culture, serologies, PCR Antimicrobial therapy (macrolides) Adapted from Centers for Disease Control and Prevention. The yellow book: health information for international travel 2018. Philadelphia: Oxford University Press; 2017. p. 704. Available at: https://wwwnc.cdc.gov/travel/page/yellowbook-home . Accessed July 25, 2017; with permission Urinary Symptoms Children who present with dysuria, hematuria, and fevers may require urinalysis and culture to evaluate for urinary tract infection and/or pyelonephritis. Gross hematuria with the passage of clots in an afebrile child with exposure to freshwater in Africa, the Middle East, China, and Southeast Asia should be tested for the helminth parasite from the genus Schistosoma via serologies or microscopic identification of eggs in stool. 45 Praziquantel is the treatment of choice and may improve anemia and nutrition in some children. 46 Patients who may have early disease or a high parasite burden may require a repeat treatment. 45 Children who are at risk for sexual abuse and adolescents should undergo testing for sexually transmitted infections such as Chlamydia trachomatis and Neisseria gonorrheae . Dermatologic Symptoms Rashes are a source of concern for parents without the context of travel and may be even more worrisome after going abroad. The differential diagnosis includes typical childhood illnesses, such as roseola or staphylococcal cellulitis, in addition to tropical infections. A study of Canadian travelers from 2009 to 2012 found that cutaneous larva migrans (13%) and skin and soft tissue infections (12.2%) were some of the most common infectious dermatologic complaints among tourists. 47 In countries where vaccination rates are low, varicella zoster virus or rubella may cause disease, especially in young children who have not completed their immunization series. Measles remains an important risk, with tourists comprising 44% of the 94 cases reported to GeoSentinel from 2000 to 2014, and 13% of patients being younger than 18 years of age, although this may represent underreporting due to the surveillance system's primarily adult focus. 48 Petechiae on the extremities in an ill-appearing child may indicate a serious systemic process such as meningococcal or rickettsial infection. There are many other infections with primarily dermatologic manifestations that may not cause fevers ( Table 7 ). 49 Table 7 Tropical diseases associated with dermatologic symptoms Disease Etiologic Pathogen Geographic Regions Vector or Exposure Incubation Period Presentation Diagnosis Management B virus Macacine herpesvirus I or B virus Worldwide Bites, scratches, body fluids of infected macaque 3–30 d Fever, headache, myalgias, vesicular lesions near exposure site with neuropathic pain, ascending encephalomyelitis PCR, virus-specific antibodies Supportive care, postexposure prophylaxis (valacyclovir), antimicrobial therapy (acyclovir, ganciclovir) Cutaneous leishmaniasis Leishmania species Middle East, Southwest and Central Asia, North Africa, Southern Europe, Central and South America Phlebotomine sand fly Weeks–months Papules that progress to ulcerated plaques, regional lymphadenopathy, and nodular lymphangitis Light-microscopy evaluation of specimens, cultures, molecular methods Antimicrobial therapy (miltefosine, amphotericin B) Cutaneous larva migrans Ancylostoma species (hookworms) Caribbean, Africa, Asia, South America Skin contact with contaminated sand 1–5 d Serpiginous track on skin with pruritus and edema Clinical Supportive care, antimicrobial therapy if desired (albendazole, ivermectin) Loiasis (African eye worm) Loa loa Central and West Africa Genus Chrysops (deerflies) 7–12 d Localized edema of extremities and joints (Calabar swelling), diffuse pruritus, eye pruritus and pain, and photophobia Microscopic evaluation of adult worm from eye, microscopic evaluation of microfilariae on blood smear, serologies Surgical excision of adult worms, antimicrobial therapy (diethylcarbamazine, albendazole) Lymphatic filariasis Wuchereria bancrofti , Brugia malayi , and Brugia timori Sub-Saharan Africa, Southern Asia, Pacific Islands, South America, Caribbean Aedes , Culex , Anopheles , Mansonia mosquitoes Years Lymphatic dysfunction with affected limb edema and pain Microscopic evaluation of peripheral blood smear, serologies Antimicrobial therapy (diethylcarbamazine, doxycycline) Myiasis Maggots of Dermatobia hominis (human bot fly), Cochliomyia hominivorax (screw worm), and others Central and South America, Africa, Caribbean Bites of infected flies or egg laying on open wounds 1–2 wk Localized skin nodule, pruritus, discharge from punctum Clinical, serologies Surgical excision of larvae Rat-bite fever Streptobacillus moniliformis and Streptobacillus minus Worldwide Bites, scratches, oral secretions of infected rats; unpasteurized milk or contaminated food or water 7–21 d Relapsing fever, maculopapular or purpuric rash, migratory polyarthritis, lymphadenopathy Culture, darkfield microscopy, stained peripheral blood smear Antimicrobial therapy (penicillin G) River blindness (onchocerciasis) Onchocerca volvulus Sub-Saharan Africa, Middle East, South America Genus Simulium (blackflies) Weeks –years Pruritic, popular rash with subcutaneous nodules, lymphadenitis, ocular lesions, vision loss Microscopic evaluation of skin shavings with microfilariae, histologic evaluation, serologies Antimicrobial therapy (ivermectin + doxycycline) Scabies Sarcoptes scabiei var. Hominis Worldwide Prolonged skin-to-skin contact, fomites if crusted scabies 2–6 wk Nocturnal pruritus, papulovesicular rash, crusts and scales if crusted scabies Microscopic evaluation of skin scraping Antimicrobial therapy (permethrin, ivermectin creams) Strongyloidiasis Strongyloides stercoralis (roundworm) Worldwide Skin penetration with contaminated soil Unknown Localized, pruritic, erythematous popular rash, pulmonary symptoms (Löffler-like pneumonitis), diarrhea, abdominal pain, eosinophilia, serpiginous urticarial rash (larva currens) Microscopic evaluation of stool, peripheral blood eosinophilia if disseminated, serologies Antimicrobial therapy (ivermectin, albendazole) Tungiasis Tunga penetrans (chigoe flea, jigger, sand flea) Africa, South America Skin penetration (especially walking barefoot) 1–2 d Localized pruritus and pain with lesions and ulcerations with central black dot Clinical Extraction of flea using sterile needle Adapted from Beeching N, Beadsworth M. Fever on return from abroad. In: Acute medicine-A practical guide to the management of medical emergencies. 5th edition. 2017. p. 207–14; and Centers for Disease Control and Prevention. The yellow book: health information for international travel 2018. Philadelphia: Oxford University Press; 2017. p. 704. Available at: https://wwwnc.cdc.gov/travel/page/yellowbook-home . Accessed July 25, 2017; with permission. Fever According to GeoSentinel, 91% of patients with an acute, life-threatening illness will present with fever. 14 There are a broad range of potential tropical infections, including malaria, dengue fever, and enteric fever. The incidence of emerging infections such as Zika virus and chikungunya are not yet known. In both adults and children, pneumonia, sepsis, meningococcemia, and urinary tract infections that were acquired at home or overseas should be on the differential diagnosis. The initial workup of a febrile child without a clear source will be based on the history, physical examination, and risk factors but commonly includes a complete blood count, liver function tests, creatinine, urinalysis, and blood cultures. 1 , 3 Malaria smears are also frequently helpful. Other tests to consider include serologies for dengue fever or other potential etiologic agents, polymerase chain reaction for Zika virus or other pathogens, chest radiographs, and cultures of the urine and stool. Patients with altered mental status may require head imaging and lumbar puncture. The most common and concerning causes of fever in a returning pediatric traveler are highlighted next. Malaria Plasmodium falciparum malaria is one of the most common tropical infections. Approximately 15% to 20% of all imported malaria cases are diagnosed in the pediatric population in industrialized countries each year. 3 Malaria is transmitted via the nocturnal-feeding Anopheles genus of mosquito. Children who are VFRs are more likely to become infected with malaria than traditional tourists. 3 Nonimmune children are also susceptible to severe malaria from other malaria strains such as Plasmodium vivax 15 and many young patients can present with atypical symptoms such as abdominal pain and vomiting. 16 Older children may present with paroxysmal fever, fatigue, myalgias, headache, abdominal pain, back pain, hepatosplenomegaly, and hemolytic anemia. Additionally, severe malaria is more common in children after the first month of travel due to the incubation period of P falciparum (7–90 days), especially in those who visited sub-Saharan Africa. 17 , 18 Overall, sub-Saharan Africa is one of the most common geographic regions for acquisition, comprising 71.5% of cases according to a GeoSentinel study of travelers migrating or returning to Canada from 2004 to 2014. 19 Malaria should remain on the differential diagnosis for up to a year in an acutely ill, febrile child after travel to an endemic area where P vivax and P ovale strains are present. 17 Interestingly, 20% of malaria cases can be acquired during trips as short as 2 weeks with less utilization of pretravel services being a contributing factor. 19 A minimum of 3 thick and thin blood smears must be performed before malaria can be excluded, preferably collected during febrile episodes. The specificity of blood smears is high but the sensitivity can be low depending on the experience of the individual interpreting the slides. 17 Rapid diagnostic tests that detect specific proteins or lactate dehydrogenase are alternatives for diagnosis at medical centers with limited experience in microbiologic evaluation for malaria. 20 The result should be confirmed, however, through the state public health department. In general, a febrile child without a localizing source or splenomegaly, thrombocytopenia, or indirect hyperbilirubinemia, in addition to exposure to an endemic area, should be presumptively approached as having malaria until an alternative diagnosis can be made. 21 Treatment of malaria is well-established by the Centers for Disease Control and Prevention (CDC) guidelines. Children with acidosis, hypoglycemia, hyperparasitemia, end-organ dysfunction, and severe anemia meet the criteria for severe malaria and require prompt administration of parenteral medication. There is a growing body of evidence that artesunate may reduce mortality compared with quinidine and is becoming more common as first-line therapy in pediatric patients. 22 , 23 Artesunate must be obtained through the CDC Malaria Hotline (1–770–488–7788) because it is not routinely available in the United States. 24 Quinidine may be initiated until the medication arrives. Completion of therapy with an oral regimen for uncomplicated chloroquine-resistant P falciparum , such as atovaquone-proguanil, can be offered when the child is able to tolerate the medications and the parasite burden has decreased to less than 1%. Severe disease is less common in P vivax and P ovale and infection can be treated with chloroquine or hydroxychloroquine in most areas outside of Indonesia and Papua New Guinea. Enteric fever (typhoid and paratyphoid) Enteric fever accounts for 18% of the 3655 cases with life-threatening tropical diseases reported to GeoSentinel. Most recorded cases were from the Indian subcontinent and in VFRs. 1 Infection with Salmonella typhi and Salmonella paratyphi are clinically indistinguishable with fever, abdominal pain, nausea, vomiting, myalgias, and arthralgias. Diarrhea is greater than 2.5 times more common in infants than older children or adults, 25 although constipation can also be seen. Patients can exhibit a typhoid mask with dull features and confusion, as well as a stepladder fever progression with rising temperatures over time in untreated individuals. Relative bradycardia and rose spots are also classic signs. 25 Complications such as gastrointestinal bleeding are more common in young children who have been ill for 2 weeks or more. 1 Transmission is fecal-oral, and humans, especially adults, may be chronic carriers. Diagnosis of enteric fever is confirmed through cultures. The most sensitive sterile site is bone marrow (80%–95%). Blood culture has the highest yield during the first week of illness (70%), and stool cultures are more sensitive as the duration of illness increases. 26 Stool studies should be performed on all fellow travelers, and they must be monitored for signs of illness. Other abnormal laboratory findings include transaminitis and a normal or decreased white blood cell count. The antimicrobial of choice for treatment varies based on the area in which the infection was acquired because multidrug resistance is increasing. Empiric treatment with ceftriaxone or fluoroquinolones is typically recommended. Strains in Latin America and the Caribbean can be susceptible to ampicillin and trimethoprim-sulfamethoxazole. South and Southeast Asian serovars more frequently require azithromycin or cefixime. 27 , 28 Children with multidrug-resistant strains have more complications such as myocarditis and shock than children infected with susceptible strains but case fatality is similar (1.0% vs 1.3%, respectively). 29 Relapse of infection can occur despite appropriate therapy, with the highest mortality in young children (6%). 29 Dengue fever Dengue remains an important cause of fever in travelers returning from all tropical regions except Africa. 30 The prevalence is rising, even in the United States, with 50 to 100 million global cases reported yearly and 22,000 deaths, primarily in children. 31 Risk factors are dissimilar from those for malaria because transmission occurs in urban areas during the daytime due to the vector Aedes aegypti , whereas malaria transmission is more common in rural areas from dusk to dawn with the Anopheles species mosquito. 32 Some patients may be asymptomatic, whereas others have hemorrhagic fever and shock. The illness presents as 3 distinct phases: (1) febrile phase over 3 to 7 days characterized by myalgias, headache, retroorbital pain, and rash; (2) critical phase of 24 to 48 days with plasma leakage; and (3) convalescent phase. 32 A rising hemoglobin and gallbladder wall thickening due to increased vascular permeability suggests the development of severe dengue in children. Repeat infections with a different strain may lead to more severe disease. 31 Serologies are most commonly used for diagnosis, although some rapid diagnostic tests are available. In cases in which infection is unclear, it may be helpful to repeat serologies 2 weeks after initial testing to monitor for an increase in titers. Other common laboratory findings include leukopenia and thrombocytopenia. 33 Treatment consists of hydration and avoidance of salicylate-containing products to decrease the risk for bleeding. 32 Children who develop severe dengue with hemorrhage and shock may require blood products. No antivirals or vaccines are currently available. Other causes of fever In recent years, arboviral illnesses transmitted via infected Aedes aegypti mosquitos have caused epidemics of Zika virus and chikungunya in South America. A European study of travelers returning from Brazil in 2013 to 2016 reported that of the 29% of patients with travel-related complaints, 6% had dengue fever, 3% had chikungunya, and 3% had Zika virus infection. 34 The prevalence of yellow fever, which is seen throughout low-resource settings and shares the same vector, has remained stable. 35 These infections are difficult to distinguish clinically with fever, retroorbital pain, conjunctivitis, and myalgias. Knowledge on perinatal infection with Zika and the neurodevelopmental sequelae of affected infants is rapidly evolving. 36 A Canadian study found that 5% of travelers developed neurologic complications such as Guillain-Barre syndrome with Zika, suggesting there is much to learn with this disease in nonperinatally acquired infections. 37 At this time, treatment is primarily supportive. Additional tropical diseases associated with fevers are outlined in Table 4 . Table 4 Tropical diseases associated with fever Disease Etiologic Pathogen Geographic Regions Vector or Exposure Incubation Period Presentation Diagnosis Management Acute retroviral syndrome HIV Worldwide, highly prevalent in sub-Saharan Africa Anal or vaginal sex, perinatal, needle stick, blood transfusion 1–3 wk Arthralgia, fever, rash, lymphadenopathy, pharyngitis HIV-1 RNA, p24 antigen, immunoassay for HIV-1 and HIV-2 antibodies (preferred) Antiretroviral therapy, consider trimethoprim-sulfamethoxazole prophylaxis Anthrax Bacillus anthracis Central and South America, sub-Saharan Africa, Central and Southwestern Asia, Eastern Europe Ingestion or handling of contaminated meat, playing drums from contaminated hides, contaminated heroin in drug users Cutaneous: 1–17 d Gastrointestinal: 1–7 d Injection: 1–4 d Inhalation: 7–60 d Varies with infection type; black eschar, cough, fever, nausea and vomiting, meningeal signs, severe soft tissue infection, shock Bacterial culture, RT-PCR Combination antimicrobial therapy Brucellosis Brucella species Central and South America, Africa, Middle East, Mediterranean basin, Eastern Europe Unpasteurized dairy products, undercooked contaminated meat 2–4 wk Fever, headache, malaise, myalgias, night sweats, Culture of sterile site (blood or bone marrow), PCR Combination antimicrobial therapy Carrión's disease (Oroya fever) Bartonella bacilliformis , B rochalimae , and B ancashensis South America, especially Peru Genus Lutzomyia (sandflies) 10–210 d Fever, headache, myalgias, abdominal pain, anemia followed by nodular skin lesions Bacterial culture Antimicrobial therapy (aminoglycosides, tetracyclines, fluoroquinolones) Cat-scratch disease B henselae Worldwide Scratches from infected cats or kittens 1–3 wk Fever, lymphadenitis, follicular conjunctivitis, encephalitis Culture, serologies, PCR Usually self-limited, antimicrobials (macrolides) Chikungunya 33 Chikungunya virus Africa, Asia, Central and South America, Pacific Islands Aedes aegypti and Aedes albopictus mosquito 3–7 d Fever, arthritis, headache, conjunctivitis, maculopapular rash, myalgias Virus-specific IgM, PCR Supportive care, nonsteroidal antiinflammatory drugs for joint pain Ebola & Marburg virus diseases 40 , 41 Ebola virus & Marburg virus Africa Body fluids Rousettus aegyptiacus (fruit bat), nonhuman primate contact, sex 2–21 d Prodrome of fever, arthralgias, headache, myalgias followed by conjunctivitis, coagulopathy, profuse diarrhea, shock Antigen detection, RT-PCR, serologies Experimental immune therapies & antivirals, supportive care Endemic typhus Rickettsia typhi Worldwide, especially Southeast Asia Rodent fleas (eg, Xenopsylla cheopis ) 7–14 d Fever, headache, malaise, nausea and vomiting, rash IgM and IgG ELISA, PCR Antimicrobial therapy (chloramphenicol, doxycycline) Epidemic typhus R prowazekii Central Africa, Asia, Central and South America Pediculus humanus (human body louse) 7–14 d Fever, headache, malaise, nausea and vomiting, rash IgM and IgG ELISA, PCR Antimicrobial therapy (doxycycline) Japanese encephalitis Japanese encephalitis virus Asia, Western Pacific Culex species mosquito 5–15 d Febrile illness, aseptic meningitis, acute encephalitis IgM ELISA Supportive care Lassa fever and other arenaviral infections Argentine hemorrhagic fever, Lassa virus, Lujo virus, LCMV Africa, Asia, Europe, North America, and South America Rodent urine and feces 2–21 d Fever, myalgia, arthralgia, headache, meningeal signs, retrosternal pain, coagulopathy, birth defects (Lassa and LCMV) Cell culture, IgM ELISA, RT-PCR Antimicrobial therapy (ribavirin for Lassa fever), supportive care Leptospirosis Leptospira species Caribbean, sub-Saharan Africa, South America, Southeast Asia Infected animal body fluid or urine, contaminated water, food, or soil 2–30 d Fever, conjunctival suffusion, back pain, rash, diarrhea, vomiting, renal and liver failure IgM and IgG ELISA, PCR Antimicrobial therapy (penicillins, doxycycline) Lyme disease Borrelia burgdorferi Europe, Northern to Central Asia Ixodes ticks 3–30 d Fever, cranial nerve palsy, erythema migrans, headache, malaise, myalgia, myocarditis, meningitis 2-tiered serologic testing (ELISA or IFA & Western blot) Antimicrobial therapy (beta-lactams, doxycycline) Murray Valley encephalitis Murray Valley encephalitis virus New Guinea, Northwestern or southeastern Australia Culex mosquito 7–28 d Fever, meningeal signs, seizures IgM ELISA, neutralizing antibodies, RT-PCR Supportive care Plague Yersinia pestis Central and Southern Africa, Central Asia, Northeastern South America X cheopis flea 1–6 d Varies with infection type; fever, lymphadenitis, overwhelming pneumonia, sepsis with gangrene Culture, serologies Antimicrobial therapy (aminoglycoside, fluoroquinolone, tetracyclines) Poliomyelitis Enterovirus types 1,2,3 Sub-Saharan Africa, Middle East, South and Southeast Asia Fecal-oral 7–21 d Flaccid paralysis, respiratory failure Cell culture, NAAT, PCR Supportive care Q fever Coxiella burnetii Africa, Middle East, Europe Aerosolized birth fluids or feces from infected livestock 2–3 wk Self-limiting respiratory illness, pneumonia, hepatitis, cardiac disease Serial IgG IFA, PCR Antimicrobial therapy (doxycycline, trimethoprim-sulfamethoxazole, fluoroquinolones) Rabies Rabies virus Africa, Asia, Central and South America Saliva from infected animal bite (especially bats) Weeks–months Prodrome of fever, pain, paresthesias followed by hydrophobia, delirium, seizures, death Neutralizing antibodies, RT-PCR, IFA Supportive care, experimental Milwaukee protocol Rat lungworm Angiostrongylus cantonensis Caribbean, Asia, Pacific islands Ingestion of infected snails & slugs or contaminated produce 1–3 wk Fever, meningeal signs, paresthesias Serum antibodies, PCR Supportive care Relapsing fever Borrelia recurrentis Sub-Saharan Africa Pediculus humanus (human body louse) 4–14 d Fever, headache, myalgia, arthralgia, rash Microscopic evaluation of blood smear, IgM and IgG ELISA, PCR Antimicrobial therapy (doxycycline) Rickettsioses Genera Rickettsia , Orientia , Ehrlichia , Neorickettsia , Neoehrlichia , Anaplasma Africa, Europe, India, and Middle East Ectoparasites (fleas, lice, mites and ticks) 7–14 d Fever, headache, eschar ( R conorii ) at bite site, malaise, nausea and vomiting, rash maculopapular or petechial) Clinical diagnosis, PCR, serologies, biopsy of eschar Antimicrobial therapy (doxycycline) RVF and other bunyaviral infections RVF virus, CCHF, hantavirus Africa, Eurasia, Middle East, North and South America Aedes species mosquito, Hyalomma ticks, infected animal carcasses, rodent urine and feces 2–21 d Fever, myalgia, arthralgia, headache, meningeal signs, vision loss (RVF), coagulopathy, renal failure (hantavirus), ecchymoses (CCHF) Cell culture, IgM ELISA, RT-PCR Antimicrobial therapy (ribavirin for CCHF), supportive care Rubella Rubella virus Africa, Middle East, South and Southeast Asia Person-to-person and droplet 14 d Fever, conjunctivitis, lymphadenopathy, rash; congenital defects Serologies, RT-PCR Supportive care Scrub typhus Orientia tsutsugamushi Asia, Pacific regions Larval mite (chigger) 6–20 d Fever, headache, malaise, nausea and vomiting, rash IgM and IgG ELISA, PCR Antimicrobial therapy (chloramphenicol, doxycycline) Sleeping sickness Trypanosoma brucei Sub-Saharan, Central, and Western Africa Glossina species (tsetse) fly 7–21 d Fever, chancre at bite site, splenomegaly, renal failure, sleep cycle disruption Microscopic examination of sterile sites or chancre-tissue biopsy Antimicrobial therapy (suramin for early stage, eflornithine & nifurtimox for late stage) Tetanus Clostridium tetani Worldwide, most common rurally Contaminated wounds with dirt, excrement; punctures 10 d Cranial nerve palsies, muscle spasms and rigidity, respiratory failure Clinical diagnosis Human tetanus immune globulin, tetanus toxoid, supportive care Tick-borne encephalitis 39 Tick-borne encephalitis virus Central and Eastern Europe and Northern Asia Ixodes species ticks, ingestion of unpasteurized dairy products 4–28 d Prodrome of febrile illness followed by aseptic meningitis, encephalitis, myelitis IgM ELISA, RT-PCR Supportive care Toxoplasmosis Toxoplasma gondii Worldwide Ingestion of undercooked meat or contaminated water, cat feces 5–23 d Fever, lymphadenopathy, chorioretinitis, encephalitis or pneumonitis if immunocompromised; congenital syndrome Serologies, ocular examination, computed tomography or MRI for intracranial lesions Supportive care or antimicrobial therapy (pyrimethamine, sulfadiazine, leucovorin) Yellow fever 39 Yellow fever virus Sub-Saharan Africa, South America Aedes species mosquito 3–6 d Fever, headache, back pain, nausea, vomiting, coagulopathy, shock RT-PCR, IgM ELISA Supportive care Zika 35 , 36 Zika virus Africa, Asia, South and Central America Aedes species mosquito, body fluids, sex 3–12 d Fever, arthralgia, conjunctivitis, headache, rash; congenital syndrome RT-PCR, serologies Supportive care Abbreviations: CCHF, Crimean-Congo hemorrhagic fever; ELISA, enzyme-linked immunoassay; Ig, immunoglobulin; IFA, immunofluorescence assay; LCMV, lymphocytic choriomeningitis; NAAT, nucleic acid amplification test; PCR, polymerase chain reaction; RT-PCR, real-time polymerase chain reaction; RVF; Rift Valley fever. Adapted from Centers for Disease Control and Prevention. The yellow book: health information for international travel 2018. Philadelphia: Oxford University Press; 2017. p. 704. Available at: https://wwwnc.cdc.gov/travel/page/yellowbook-home . Accessed July 25, 2017; with permission. Malaria Plasmodium falciparum malaria is one of the most common tropical infections. Approximately 15% to 20% of all imported malaria cases are diagnosed in the pediatric population in industrialized countries each year. 3 Malaria is transmitted via the nocturnal-feeding Anopheles genus of mosquito. Children who are VFRs are more likely to become infected with malaria than traditional tourists. 3 Nonimmune children are also susceptible to severe malaria from other malaria strains such as Plasmodium vivax 15 and many young patients can present with atypical symptoms such as abdominal pain and vomiting. 16 Older children may present with paroxysmal fever, fatigue, myalgias, headache, abdominal pain, back pain, hepatosplenomegaly, and hemolytic anemia. Additionally, severe malaria is more common in children after the first month of travel due to the incubation period of P falciparum (7–90 days), especially in those who visited sub-Saharan Africa. 17 , 18 Overall, sub-Saharan Africa is one of the most common geographic regions for acquisition, comprising 71.5% of cases according to a GeoSentinel study of travelers migrating or returning to Canada from 2004 to 2014. 19 Malaria should remain on the differential diagnosis for up to a year in an acutely ill, febrile child after travel to an endemic area where P vivax and P ovale strains are present. 17 Interestingly, 20% of malaria cases can be acquired during trips as short as 2 weeks with less utilization of pretravel services being a contributing factor. 19 A minimum of 3 thick and thin blood smears must be performed before malaria can be excluded, preferably collected during febrile episodes. The specificity of blood smears is high but the sensitivity can be low depending on the experience of the individual interpreting the slides. 17 Rapid diagnostic tests that detect specific proteins or lactate dehydrogenase are alternatives for diagnosis at medical centers with limited experience in microbiologic evaluation for malaria. 20 The result should be confirmed, however, through the state public health department. In general, a febrile child without a localizing source or splenomegaly, thrombocytopenia, or indirect hyperbilirubinemia, in addition to exposure to an endemic area, should be presumptively approached as having malaria until an alternative diagnosis can be made. 21 Treatment of malaria is well-established by the Centers for Disease Control and Prevention (CDC) guidelines. Children with acidosis, hypoglycemia, hyperparasitemia, end-organ dysfunction, and severe anemia meet the criteria for severe malaria and require prompt administration of parenteral medication. There is a growing body of evidence that artesunate may reduce mortality compared with quinidine and is becoming more common as first-line therapy in pediatric patients. 22 , 23 Artesunate must be obtained through the CDC Malaria Hotline (1–770–488–7788) because it is not routinely available in the United States. 24 Quinidine may be initiated until the medication arrives. Completion of therapy with an oral regimen for uncomplicated chloroquine-resistant P falciparum , such as atovaquone-proguanil, can be offered when the child is able to tolerate the medications and the parasite burden has decreased to less than 1%. Severe disease is less common in P vivax and P ovale and infection can be treated with chloroquine or hydroxychloroquine in most areas outside of Indonesia and Papua New Guinea. Enteric fever (typhoid and paratyphoid) Enteric fever accounts for 18% of the 3655 cases with life-threatening tropical diseases reported to GeoSentinel. Most recorded cases were from the Indian subcontinent and in VFRs. 1 Infection with Salmonella typhi and Salmonella paratyphi are clinically indistinguishable with fever, abdominal pain, nausea, vomiting, myalgias, and arthralgias. Diarrhea is greater than 2.5 times more common in infants than older children or adults, 25 although constipation can also be seen. Patients can exhibit a typhoid mask with dull features and confusion, as well as a stepladder fever progression with rising temperatures over time in untreated individuals. Relative bradycardia and rose spots are also classic signs. 25 Complications such as gastrointestinal bleeding are more common in young children who have been ill for 2 weeks or more. 1 Transmission is fecal-oral, and humans, especially adults, may be chronic carriers. Diagnosis of enteric fever is confirmed through cultures. The most sensitive sterile site is bone marrow (80%–95%). Blood culture has the highest yield during the first week of illness (70%), and stool cultures are more sensitive as the duration of illness increases. 26 Stool studies should be performed on all fellow travelers, and they must be monitored for signs of illness. Other abnormal laboratory findings include transaminitis and a normal or decreased white blood cell count. The antimicrobial of choice for treatment varies based on the area in which the infection was acquired because multidrug resistance is increasing. Empiric treatment with ceftriaxone or fluoroquinolones is typically recommended. Strains in Latin America and the Caribbean can be susceptible to ampicillin and trimethoprim-sulfamethoxazole. South and Southeast Asian serovars more frequently require azithromycin or cefixime. 27 , 28 Children with multidrug-resistant strains have more complications such as myocarditis and shock than children infected with susceptible strains but case fatality is similar (1.0% vs 1.3%, respectively). 29 Relapse of infection can occur despite appropriate therapy, with the highest mortality in young children (6%). 29 Dengue fever Dengue remains an important cause of fever in travelers returning from all tropical regions except Africa. 30 The prevalence is rising, even in the United States, with 50 to 100 million global cases reported yearly and 22,000 deaths, primarily in children. 31 Risk factors are dissimilar from those for malaria because transmission occurs in urban areas during the daytime due to the vector Aedes aegypti , whereas malaria transmission is more common in rural areas from dusk to dawn with the Anopheles species mosquito. 32 Some patients may be asymptomatic, whereas others have hemorrhagic fever and shock. The illness presents as 3 distinct phases: (1) febrile phase over 3 to 7 days characterized by myalgias, headache, retroorbital pain, and rash; (2) critical phase of 24 to 48 days with plasma leakage; and (3) convalescent phase. 32 A rising hemoglobin and gallbladder wall thickening due to increased vascular permeability suggests the development of severe dengue in children. Repeat infections with a different strain may lead to more severe disease. 31 Serologies are most commonly used for diagnosis, although some rapid diagnostic tests are available. In cases in which infection is unclear, it may be helpful to repeat serologies 2 weeks after initial testing to monitor for an increase in titers. Other common laboratory findings include leukopenia and thrombocytopenia. 33 Treatment consists of hydration and avoidance of salicylate-containing products to decrease the risk for bleeding. 32 Children who develop severe dengue with hemorrhage and shock may require blood products. No antivirals or vaccines are currently available. Other causes of fever In recent years, arboviral illnesses transmitted via infected Aedes aegypti mosquitos have caused epidemics of Zika virus and chikungunya in South America. A European study of travelers returning from Brazil in 2013 to 2016 reported that of the 29% of patients with travel-related complaints, 6% had dengue fever, 3% had chikungunya, and 3% had Zika virus infection. 34 The prevalence of yellow fever, which is seen throughout low-resource settings and shares the same vector, has remained stable. 35 These infections are difficult to distinguish clinically with fever, retroorbital pain, conjunctivitis, and myalgias. Knowledge on perinatal infection with Zika and the neurodevelopmental sequelae of affected infants is rapidly evolving. 36 A Canadian study found that 5% of travelers developed neurologic complications such as Guillain-Barre syndrome with Zika, suggesting there is much to learn with this disease in nonperinatally acquired infections. 37 At this time, treatment is primarily supportive. Additional tropical diseases associated with fevers are outlined in Table 4 . Table 4 Tropical diseases associated with fever Disease Etiologic Pathogen Geographic Regions Vector or Exposure Incubation Period Presentation Diagnosis Management Acute retroviral syndrome HIV Worldwide, highly prevalent in sub-Saharan Africa Anal or vaginal sex, perinatal, needle stick, blood transfusion 1–3 wk Arthralgia, fever, rash, lymphadenopathy, pharyngitis HIV-1 RNA, p24 antigen, immunoassay for HIV-1 and HIV-2 antibodies (preferred) Antiretroviral therapy, consider trimethoprim-sulfamethoxazole prophylaxis Anthrax Bacillus anthracis Central and South America, sub-Saharan Africa, Central and Southwestern Asia, Eastern Europe Ingestion or handling of contaminated meat, playing drums from contaminated hides, contaminated heroin in drug users Cutaneous: 1–17 d Gastrointestinal: 1–7 d Injection: 1–4 d Inhalation: 7–60 d Varies with infection type; black eschar, cough, fever, nausea and vomiting, meningeal signs, severe soft tissue infection, shock Bacterial culture, RT-PCR Combination antimicrobial therapy Brucellosis Brucella species Central and South America, Africa, Middle East, Mediterranean basin, Eastern Europe Unpasteurized dairy products, undercooked contaminated meat 2–4 wk Fever, headache, malaise, myalgias, night sweats, Culture of sterile site (blood or bone marrow), PCR Combination antimicrobial therapy Carrión's disease (Oroya fever) Bartonella bacilliformis , B rochalimae , and B ancashensis South America, especially Peru Genus Lutzomyia (sandflies) 10–210 d Fever, headache, myalgias, abdominal pain, anemia followed by nodular skin lesions Bacterial culture Antimicrobial therapy (aminoglycosides, tetracyclines, fluoroquinolones) Cat-scratch disease B henselae Worldwide Scratches from infected cats or kittens 1–3 wk Fever, lymphadenitis, follicular conjunctivitis, encephalitis Culture, serologies, PCR Usually self-limited, antimicrobials (macrolides) Chikungunya 33 Chikungunya virus Africa, Asia, Central and South America, Pacific Islands Aedes aegypti and Aedes albopictus mosquito 3–7 d Fever, arthritis, headache, conjunctivitis, maculopapular rash, myalgias Virus-specific IgM, PCR Supportive care, nonsteroidal antiinflammatory drugs for joint pain Ebola & Marburg virus diseases 40 , 41 Ebola virus & Marburg virus Africa Body fluids Rousettus aegyptiacus (fruit bat), nonhuman primate contact, sex 2–21 d Prodrome of fever, arthralgias, headache, myalgias followed by conjunctivitis, coagulopathy, profuse diarrhea, shock Antigen detection, RT-PCR, serologies Experimental immune therapies & antivirals, supportive care Endemic typhus Rickettsia typhi Worldwide, especially Southeast Asia Rodent fleas (eg, Xenopsylla cheopis ) 7–14 d Fever, headache, malaise, nausea and vomiting, rash IgM and IgG ELISA, PCR Antimicrobial therapy (chloramphenicol, doxycycline) Epidemic typhus R prowazekii Central Africa, Asia, Central and South America Pediculus humanus (human body louse) 7–14 d Fever, headache, malaise, nausea and vomiting, rash IgM and IgG ELISA, PCR Antimicrobial therapy (doxycycline) Japanese encephalitis Japanese encephalitis virus Asia, Western Pacific Culex species mosquito 5–15 d Febrile illness, aseptic meningitis, acute encephalitis IgM ELISA Supportive care Lassa fever and other arenaviral infections Argentine hemorrhagic fever, Lassa virus, Lujo virus, LCMV Africa, Asia, Europe, North America, and South America Rodent urine and feces 2–21 d Fever, myalgia, arthralgia, headache, meningeal signs, retrosternal pain, coagulopathy, birth defects (Lassa and LCMV) Cell culture, IgM ELISA, RT-PCR Antimicrobial therapy (ribavirin for Lassa fever), supportive care Leptospirosis Leptospira species Caribbean, sub-Saharan Africa, South America, Southeast Asia Infected animal body fluid or urine, contaminated water, food, or soil 2–30 d Fever, conjunctival suffusion, back pain, rash, diarrhea, vomiting, renal and liver failure IgM and IgG ELISA, PCR Antimicrobial therapy (penicillins, doxycycline) Lyme disease Borrelia burgdorferi Europe, Northern to Central Asia Ixodes ticks 3–30 d Fever, cranial nerve palsy, erythema migrans, headache, malaise, myalgia, myocarditis, meningitis 2-tiered serologic testing (ELISA or IFA & Western blot) Antimicrobial therapy (beta-lactams, doxycycline) Murray Valley encephalitis Murray Valley encephalitis virus New Guinea, Northwestern or southeastern Australia Culex mosquito 7–28 d Fever, meningeal signs, seizures IgM ELISA, neutralizing antibodies, RT-PCR Supportive care Plague Yersinia pestis Central and Southern Africa, Central Asia, Northeastern South America X cheopis flea 1–6 d Varies with infection type; fever, lymphadenitis, overwhelming pneumonia, sepsis with gangrene Culture, serologies Antimicrobial therapy (aminoglycoside, fluoroquinolone, tetracyclines) Poliomyelitis Enterovirus types 1,2,3 Sub-Saharan Africa, Middle East, South and Southeast Asia Fecal-oral 7–21 d Flaccid paralysis, respiratory failure Cell culture, NAAT, PCR Supportive care Q fever Coxiella burnetii Africa, Middle East, Europe Aerosolized birth fluids or feces from infected livestock 2–3 wk Self-limiting respiratory illness, pneumonia, hepatitis, cardiac disease Serial IgG IFA, PCR Antimicrobial therapy (doxycycline, trimethoprim-sulfamethoxazole, fluoroquinolones) Rabies Rabies virus Africa, Asia, Central and South America Saliva from infected animal bite (especially bats) Weeks–months Prodrome of fever, pain, paresthesias followed by hydrophobia, delirium, seizures, death Neutralizing antibodies, RT-PCR, IFA Supportive care, experimental Milwaukee protocol Rat lungworm Angiostrongylus cantonensis Caribbean, Asia, Pacific islands Ingestion of infected snails & slugs or contaminated produce 1–3 wk Fever, meningeal signs, paresthesias Serum antibodies, PCR Supportive care Relapsing fever Borrelia recurrentis Sub-Saharan Africa Pediculus humanus (human body louse) 4–14 d Fever, headache, myalgia, arthralgia, rash Microscopic evaluation of blood smear, IgM and IgG ELISA, PCR Antimicrobial therapy (doxycycline) Rickettsioses Genera Rickettsia , Orientia , Ehrlichia , Neorickettsia , Neoehrlichia , Anaplasma Africa, Europe, India, and Middle East Ectoparasites (fleas, lice, mites and ticks) 7–14 d Fever, headache, eschar ( R conorii ) at bite site, malaise, nausea and vomiting, rash maculopapular or petechial) Clinical diagnosis, PCR, serologies, biopsy of eschar Antimicrobial therapy (doxycycline) RVF and other bunyaviral infections RVF virus, CCHF, hantavirus Africa, Eurasia, Middle East, North and South America Aedes species mosquito, Hyalomma ticks, infected animal carcasses, rodent urine and feces 2–21 d Fever, myalgia, arthralgia, headache, meningeal signs, vision loss (RVF), coagulopathy, renal failure (hantavirus), ecchymoses (CCHF) Cell culture, IgM ELISA, RT-PCR Antimicrobial therapy (ribavirin for CCHF), supportive care Rubella Rubella virus Africa, Middle East, South and Southeast Asia Person-to-person and droplet 14 d Fever, conjunctivitis, lymphadenopathy, rash; congenital defects Serologies, RT-PCR Supportive care Scrub typhus Orientia tsutsugamushi Asia, Pacific regions Larval mite (chigger) 6–20 d Fever, headache, malaise, nausea and vomiting, rash IgM and IgG ELISA, PCR Antimicrobial therapy (chloramphenicol, doxycycline) Sleeping sickness Trypanosoma brucei Sub-Saharan, Central, and Western Africa Glossina species (tsetse) fly 7–21 d Fever, chancre at bite site, splenomegaly, renal failure, sleep cycle disruption Microscopic examination of sterile sites or chancre-tissue biopsy Antimicrobial therapy (suramin for early stage, eflornithine & nifurtimox for late stage) Tetanus Clostridium tetani Worldwide, most common rurally Contaminated wounds with dirt, excrement; punctures 10 d Cranial nerve palsies, muscle spasms and rigidity, respiratory failure Clinical diagnosis Human tetanus immune globulin, tetanus toxoid, supportive care Tick-borne encephalitis 39 Tick-borne encephalitis virus Central and Eastern Europe and Northern Asia Ixodes species ticks, ingestion of unpasteurized dairy products 4–28 d Prodrome of febrile illness followed by aseptic meningitis, encephalitis, myelitis IgM ELISA, RT-PCR Supportive care Toxoplasmosis Toxoplasma gondii Worldwide Ingestion of undercooked meat or contaminated water, cat feces 5–23 d Fever, lymphadenopathy, chorioretinitis, encephalitis or pneumonitis if immunocompromised; congenital syndrome Serologies, ocular examination, computed tomography or MRI for intracranial lesions Supportive care or antimicrobial therapy (pyrimethamine, sulfadiazine, leucovorin) Yellow fever 39 Yellow fever virus Sub-Saharan Africa, South America Aedes species mosquito 3–6 d Fever, headache, back pain, nausea, vomiting, coagulopathy, shock RT-PCR, IgM ELISA Supportive care Zika 35 , 36 Zika virus Africa, Asia, South and Central America Aedes species mosquito, body fluids, sex 3–12 d Fever, arthralgia, conjunctivitis, headache, rash; congenital syndrome RT-PCR, serologies Supportive care Abbreviations: CCHF, Crimean-Congo hemorrhagic fever; ELISA, enzyme-linked immunoassay; Ig, immunoglobulin; IFA, immunofluorescence assay; LCMV, lymphocytic choriomeningitis; NAAT, nucleic acid amplification test; PCR, polymerase chain reaction; RT-PCR, real-time polymerase chain reaction; RVF; Rift Valley fever. Adapted from Centers for Disease Control and Prevention. The yellow book: health information for international travel 2018. Philadelphia: Oxford University Press; 2017. p. 704. Available at: https://wwwnc.cdc.gov/travel/page/yellowbook-home . Accessed July 25, 2017; with permission. Gastrointestinal Symptoms Vomiting and diarrhea are common complaints in returning travelers. Up to 40% of children less than 2 years of age may develop diarrhea, with 15% requiring medical services. 38 Fevers, nausea, and vomiting can be seen with norovirus that occurs worldwide and is frequently associated with contaminated food and water on cruise ships. 39 Rotavirus, however, is one of the most frequent causes of diarrheal illnesses worldwide and is a common cause of infant mortality in low-resource settings. 5 The hepatitides present with a broad range of disease from mild abdominal pain and vomiting to fulminant liver failure, although serious complications are uncommon in pediatric travelers. 40 Community-acquired Clostridium difficile is uncommon in children but infection should be considered if the patient received recent antimicrobials. 41 GeoSentinel data reported that 2% of patients diagnosed with Clostridium difficile after travel were 10 to 19 years of age. 42 There are many other causes of both febrile and nonfebrile gastrointestinal illness in children ( Table 5 ). Table 5 Tropical diseases associated with gastrointestinal symptoms Disease Etiologic Pathogen Geographic Regions Vector or Exposure Incubation Period Presentation Diagnosis Management amebiasis Entamoeba histolytica Worldwide Fecal-oral, contaminated food or water Days–weeks Abdominal cramps, watery or bloody diarrhea, weight loss, liver abscess with abdominal pain Microscopic evaluation of stool, serologies Antimicrobial therapy (metronidazole + iodoquinol or puromycin) Campylobacteriosis Campylobacter jejuni , Campylobacter coli Worldwide Contaminated foods (raw poultry) and water, unpasteurized milk, fecal-oral 2–4 d Abdominal pain, fever, bloody diarrhea, nausea and vomiting, pseudoappendicitis, reactive arthritis, Guillain-Barre syndrome Stool culture, darkfield microscopy, NAAT Supportive care, antimicrobial therapies (fluoroquinolone, macrolide) Chagas disease T cruzi Central and South America Reduviid bug, contaminated food or water, blood transfusion 7 d Chagoma (eg, Romaña sign), ventricular arrhythmias, megacolon, megaesophagus Microscopic evaluation of blood smear, IgM ELISA, PCR (acute disease only) Antimicrobial therapy (benznidazole, nifurtimox) Cholera Vibrio cholerae O-group 1 or O-group 139 Africa, Caribbean, Southeast Asia Aquatic plants, brackish water, shellfish 5 d Profuse, watery diarrhea, nausea and vomiting, muscle cramps, hypovolemic shock Stool culture Supportive care, antimicrobial therapy (azithromycin, doxycycline) Cyclosporiasis Cyclospora cayetenensis Worldwide Contaminated produce and water 2–14 d Watery diarrhea, anorexia, weight loss, abdominal cramps, myalgias, vomiting Microscopic evaluation of stool for oocysts Antimicrobial therapy (trimethoprim-sulfamethoxazole) Echinococcosis Echinococcus species Eurasia, Central and South America, Africa Contaminated dog feces, contaminated food or water 5–15 y Hydatid cysts in liver and lungs, abdominal pain, liver failure Imaging (ultrasound, computed tomography scan), serologies Supportive care, surgical excision if cyst >10 cm, antimicrobial therapy (albendazole, praziquantel) Traveler's diarrhea Enterotoxigenic Escherichia coli (ETEC) Worldwide Fecal-oral, contaminated food or water 9 h–3 d Abdominal pain, watery diarrhea Clinical diagnosis, NAAT Supportive care, antimicrobial therapy (ciprofloxacin, azithromycin) Fascioliasis Fasciola hepatica and F gigantica South America, Middle East, Southeast Asia Watercress or other aquatic plants, freshwater 6–12 wk Intermittent, fever eosinophilia, abdominal pain, weight loss, urticaria, biliary colic, liver failure Microscopic evaluation of stool, serologies, liver imaging Antimicrobial therapy (triclabendazole) Giardiasis Giardia intestinalis Worldwide Fecal-oral, sexual contact, contaminated water 1–2 wk Abdominal pain, anorexia, foul-smelling diarrhea, flatulence, nausea, reactive arthritis Microscopic evaluation of stool, DFA Antimicrobial therapy (metronidazole, tinidazole, nitazoxanide) Peptic ulcer disease Helicobacter pylori Worldwide Fecal-oral, oral-oral Unknown Epigastric pain, nausea and vomiting, anorexia, gastric cancer Fecal antigen assay, urea breath test Antimicrobial therapy (proton pump inhibitor + clarithromycin + amoxicillin) Pinworm Enterobius vermicularis Worldwide Fecal-oral, contaminated objects 1–2 mo Perianal pruritus Scotch tape test, microscopic evaluation of fingernails Antimicrobial therapy (albendazole, pyrantel pamoate) Sarcocystosis Sarcocystis species Worldwide, especially Southeast Asia Undercooked beef or pork 2 wk Fever, malaise, myalgia, headache, cough, arthralgia, nausea and vomiting, diarrhea, palpitations Microscopic evaluation of stool, PCR, muscle biopsy Antimicrobial therapy (trimethoprim-sulfamethoxazole) Soil-transmitted helminths Ascaris lumbricoides (roundworm), Ancylostoma duodenale (hookworm), Necator americanus (hookworm), Trichuris trichiura (whipworm) Worldwide Fecal-oral, skin penetration with contaminated soil (hookworms) Variable Abdominal pain, malnutrition, bowel obstruction, anemia, cough, chest pain Microscopic evaluation of stool Antimicrobial therapy (albendazole, mebendazole) Strongyloidiasis Strongyloides stercoralis Worldwide Auto-inoculation, skin penetration Variable Pruritic rash at penetration site, serpiginous rashes (larva currens), respiratory symptoms (Löffler-like pneumonitis), abdominal pain, diarrhea, severe disease if immuno-compromised Microscopic evaluation of stool other body fluids if disseminated (eg, sputum, CSF) Antimicrobial therapy (ivermectin, albendazole) Taeniasis Taenia solium (pork) and T saginata or T asiatica (beef) Central and South America, Africa, South and Southeast Asia Undercooked contaminated pork or beef 8–10 wk for T solium , 10–14 wk for T saginata Abdominal discomfort, weight loss, anorexia, perianal pruritus, insomnia, weakness Microscopic evaluation of stool for eggs Antimicrobial therapy (praziquantel, niclosamide unless symptomatic neurocysticercosis) Visceral leishmaniasis Leishmania donovani and L infantum-chagasi South America, Central and Southwest Asia, East Africa Phlebotomine sand fly, blood transfusions Weeks–months Fever, weight loss, hepatosplenomegaly, pancytopenia Light-microscopic evaluation of specimens, culture, molecular methods Antimicrobial therapy (amphotericin B, miltefosine) Yersiniosis Yersinia enterocolitica Japan, Northern Europe Undercooked contaminated pork, contaminated water, unpasteurized dairy 4–6 d Fever, abdominal pain (pseudoappendicitis), bloody diarrhea, necrotizing enterocolitis in infants, reactive arthritis, erythema nodosum Stool culture (or other body sits; eg, CSF, blood) Supportive care, antimicrobial therapy if severe (trimethoprim-sulfamethoxazole, fluoroquinolones, aminoglycosides) Abbreviations: CSF, cerebrospinal fluid; DFA, direct fluorescent antibody. Adapted from Centers for Disease Control and Prevention. The yellow book: health information for international travel 2018. Philadelphia: Oxford University Press; 2017. p. 704. Available at: https://wwwnc.cdc.gov/travel/page/yellowbook-home . Accessed July 25, 2017; with permission. Respiratory Symptoms In the pediatric population, common respiratory infections may be seen on return from international trips including pharyngitis, sinusitis, otitis, and pneumonia from pathogens commonly seen in the United States, such as Streptococcus pneumoniae and rhinovirus. 4 , 43 Local epidemiology of infections can be helpful in diagnosis and management and is available through the CDC. In some tropical regions, influenza may occur throughout the year and should hence remain on the differential for patients who warrant treatment with oseltamivir. 44 Mycobacterium tuberculosis is an important etiologic factor of lower respiratory tract disease worldwide and should be considered in children with risk factors or who do not recover with antimicrobials for bacterial pneumonia. 26 Of note, children younger than 3 years of age are more likely to present with miliary tuberculosis or neurologic involvement than adult patients. There are also many other less common causes of febrile respiratory tract infections ( Table 6 ). Table 6 Tropical diseases associated with respiratory symptoms Disease Etiologic Pathogen Geographic Regions Vector or Exposure Incubation Period Presentation Diagnosis Management Avian bird flu H5N1 and H7N9 influenza A virus East and Southeast Asia Poultry 2–8 d Fever, malaise, myalgia, headache, nasal congestion, cough, acute respiratory distress syndrome (ARDS) RT-PCR Supportive care Diphtheria Corynebacterium diphtheriae Asia, South Pacific, Middle East, Eastern Europe, Caribbean Person-to-person (oral or respiratory droplets), fomites 2–5 d Fever, dysphagia, malaise, anorexia, pseudomembranes Bacterial culture Supportive care, equine diphtheria antitoxin (DAT), antimicrobial therapy (erythromycin, penicillin) Coccidioidomycosis Coccidioides immitis and Coccidioides posadasii Central and South America Inhalation of spores from soil 7–21 d Fever, malaise, cough, headache, night sweats, myalgias, arthritis, rash Culture, IgM and IgG ELISA, immunodiffusion and complement fixation Supportive care, antimicrobial therapy if ill or at high risk of dissemination (amphotericin B, azoles) Histoplasmosis Histoplasma capsulatum Worldwide, especially river valleys Inhalation of spores from soil, bird droppings, bat guano 3–17 d Fever, headache, cough, pleuritic chest pain, malaise Culture, microscopic examination, PCR, EIA on serum or other samples, immunodiffusion complement fixation Supportive care, antimicrobial therapy (azole for mild to moderate disease, amphotericin B for severe) Legionellosis (Legionnaire's disease and Pontiac fever) Legionella species Worldwide Inhalation of freshwater aerosol 2–10 d Fever, headache, myalgias, pneumonia, respiratory distress Urine antigen assay, paired serologies, PCR Antimicrobial therapy (fluoroquinolones, macrolides) Melioidosis Burkholderia pseudomallei Central and Southeast Asia, northern Australia, South America Subcutaneous inoculation, inhalation, ingestion; body fluids 1–21 d Fever, cough, weight loss, pneumonia Culture, indirect hemagglutination assay Antimicrobial therapy (ceftazidime, meropenem) Middle Eastern Respiratory Syndrome (MERS) MERS coronavirus North Africa, Middle East Dromedary camel, person-to-person 2–14 d Fever, cough, arthralgia, diarrhea, myalgia, acute respiratory failure, multiple organ dysfunction RT-PCR Supportive care Pertussis (whooping cough) Bordetella pertussis Worldwide Person-to-person (aerosolized respiratory droplets, respiratory secretions) 7–10 d Paroxysmal cough, post-tussive vomiting, apnea in infants Culture, serologies, PCR Antimicrobial therapy (macrolides) Adapted from Centers for Disease Control and Prevention. The yellow book: health information for international travel 2018. Philadelphia: Oxford University Press; 2017. p. 704. Available at: https://wwwnc.cdc.gov/travel/page/yellowbook-home . Accessed July 25, 2017; with permission Urinary Symptoms Children who present with dysuria, hematuria, and fevers may require urinalysis and culture to evaluate for urinary tract infection and/or pyelonephritis. Gross hematuria with the passage of clots in an afebrile child with exposure to freshwater in Africa, the Middle East, China, and Southeast Asia should be tested for the helminth parasite from the genus Schistosoma via serologies or microscopic identification of eggs in stool. 45 Praziquantel is the treatment of choice and may improve anemia and nutrition in some children. 46 Patients who may have early disease or a high parasite burden may require a repeat treatment. 45 Children who are at risk for sexual abuse and adolescents should undergo testing for sexually transmitted infections such as Chlamydia trachomatis and Neisseria gonorrheae . Dermatologic Symptoms Rashes are a source of concern for parents without the context of travel and may be even more worrisome after going abroad. The differential diagnosis includes typical childhood illnesses, such as roseola or staphylococcal cellulitis, in addition to tropical infections. A study of Canadian travelers from 2009 to 2012 found that cutaneous larva migrans (13%) and skin and soft tissue infections (12.2%) were some of the most common infectious dermatologic complaints among tourists. 47 In countries where vaccination rates are low, varicella zoster virus or rubella may cause disease, especially in young children who have not completed their immunization series. Measles remains an important risk, with tourists comprising 44% of the 94 cases reported to GeoSentinel from 2000 to 2014, and 13% of patients being younger than 18 years of age, although this may represent underreporting due to the surveillance system's primarily adult focus. 48 Petechiae on the extremities in an ill-appearing child may indicate a serious systemic process such as meningococcal or rickettsial infection. There are many other infections with primarily dermatologic manifestations that may not cause fevers ( Table 7 ). 49 Table 7 Tropical diseases associated with dermatologic symptoms Disease Etiologic Pathogen Geographic Regions Vector or Exposure Incubation Period Presentation Diagnosis Management B virus Macacine herpesvirus I or B virus Worldwide Bites, scratches, body fluids of infected macaque 3–30 d Fever, headache, myalgias, vesicular lesions near exposure site with neuropathic pain, ascending encephalomyelitis PCR, virus-specific antibodies Supportive care, postexposure prophylaxis (valacyclovir), antimicrobial therapy (acyclovir, ganciclovir) Cutaneous leishmaniasis Leishmania species Middle East, Southwest and Central Asia, North Africa, Southern Europe, Central and South America Phlebotomine sand fly Weeks–months Papules that progress to ulcerated plaques, regional lymphadenopathy, and nodular lymphangitis Light-microscopy evaluation of specimens, cultures, molecular methods Antimicrobial therapy (miltefosine, amphotericin B) Cutaneous larva migrans Ancylostoma species (hookworms) Caribbean, Africa, Asia, South America Skin contact with contaminated sand 1–5 d Serpiginous track on skin with pruritus and edema Clinical Supportive care, antimicrobial therapy if desired (albendazole, ivermectin) Loiasis (African eye worm) Loa loa Central and West Africa Genus Chrysops (deerflies) 7–12 d Localized edema of extremities and joints (Calabar swelling), diffuse pruritus, eye pruritus and pain, and photophobia Microscopic evaluation of adult worm from eye, microscopic evaluation of microfilariae on blood smear, serologies Surgical excision of adult worms, antimicrobial therapy (diethylcarbamazine, albendazole) Lymphatic filariasis Wuchereria bancrofti , Brugia malayi , and Brugia timori Sub-Saharan Africa, Southern Asia, Pacific Islands, South America, Caribbean Aedes , Culex , Anopheles , Mansonia mosquitoes Years Lymphatic dysfunction with affected limb edema and pain Microscopic evaluation of peripheral blood smear, serologies Antimicrobial therapy (diethylcarbamazine, doxycycline) Myiasis Maggots of Dermatobia hominis (human bot fly), Cochliomyia hominivorax (screw worm), and others Central and South America, Africa, Caribbean Bites of infected flies or egg laying on open wounds 1–2 wk Localized skin nodule, pruritus, discharge from punctum Clinical, serologies Surgical excision of larvae Rat-bite fever Streptobacillus moniliformis and Streptobacillus minus Worldwide Bites, scratches, oral secretions of infected rats; unpasteurized milk or contaminated food or water 7–21 d Relapsing fever, maculopapular or purpuric rash, migratory polyarthritis, lymphadenopathy Culture, darkfield microscopy, stained peripheral blood smear Antimicrobial therapy (penicillin G) River blindness (onchocerciasis) Onchocerca volvulus Sub-Saharan Africa, Middle East, South America Genus Simulium (blackflies) Weeks –years Pruritic, popular rash with subcutaneous nodules, lymphadenitis, ocular lesions, vision loss Microscopic evaluation of skin shavings with microfilariae, histologic evaluation, serologies Antimicrobial therapy (ivermectin + doxycycline) Scabies Sarcoptes scabiei var. Hominis Worldwide Prolonged skin-to-skin contact, fomites if crusted scabies 2–6 wk Nocturnal pruritus, papulovesicular rash, crusts and scales if crusted scabies Microscopic evaluation of skin scraping Antimicrobial therapy (permethrin, ivermectin creams) Strongyloidiasis Strongyloides stercoralis (roundworm) Worldwide Skin penetration with contaminated soil Unknown Localized, pruritic, erythematous popular rash, pulmonary symptoms (Löffler-like pneumonitis), diarrhea, abdominal pain, eosinophilia, serpiginous urticarial rash (larva currens) Microscopic evaluation of stool, peripheral blood eosinophilia if disseminated, serologies Antimicrobial therapy (ivermectin, albendazole) Tungiasis Tunga penetrans (chigoe flea, jigger, sand flea) Africa, South America Skin penetration (especially walking barefoot) 1–2 d Localized pruritus and pain with lesions and ulcerations with central black dot Clinical Extraction of flea using sterile needle Adapted from Beeching N, Beadsworth M. Fever on return from abroad. In: Acute medicine-A practical guide to the management of medical emergencies. 5th edition. 2017. p. 207–14; and Centers for Disease Control and Prevention. The yellow book: health information for international travel 2018. Philadelphia: Oxford University Press; 2017. p. 704. Available at: https://wwwnc.cdc.gov/travel/page/yellowbook-home . Accessed July 25, 2017; with permission. Summary As the numbers of children who travel abroad continues to increase, clinicians need to remain up-to-date on potential etiologic factors for febrile illnesses on families' return home. After ruling out life-threatening disorders that can be acquired locally or internationally, physicians are able to develop a focused diagnosis and management plan best suited to the patient's clinical picture. There is a growing body of resources to assist clinicians, such as the CDC ( www.cdc.gov/travel/ ) and GeoSentinel ( www.istm.org/geosentinel ) for data on epidemiology, geography, and other risk factors. In the future, physicians will need to be prepared to deal with the global epidemic of antimicrobial drug resistance, evolving epidemics and pandemics caused by emerging pathogens, reemerging infections due to vaccine hesitancy or international conflicts, and medical tourism in both healthy and medically complex children.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930730/

Life in vaccine science – a conversation with Stanley Plotkin at the 4th Conference on Vaccines in Dubrovnik, Croatia, September 2017

Note The Eurosurveillance editors acknowledge that this interview by Sanjin Musa, on the margins of a conference is not the usual form of a meeting report in Eurosurveillance . However, we decided to share it exceptionally as such in conjunction with the European Immunisation Week 2018. The opinions expressed in this interview are those of Dr Stanley Plotkin and they do not necessarily reflect the opinions of the journal or its publisher, the European Centre for Disease Prevention and Control (ECDC) or the editorial team or the institutions with which the authors are affiliated.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6011229/

Inflammasomes in livestock and wildlife: Insights into the intersection of pathogens and natural host species

The inflammasome serves as a mechanism by which the body senses damage or danger. These multiprotein complexes form in the cytosol of myeloid, epithelial and potentially other cell types to drive caspase-1 cleavage and the secretion of the pro-inflammatory cytokines IL-1β and IL-18. Different types of inflammasomes, centered on (and named after) their cytosolic NLRs, respond to signals from bacteria, fungi, and viruses, as well as "sterile inflammatory" triggers. Despite the large body of research accumulated on rodent and human inflammasomes over the past 15 years, only recently have studies expanded to consider the role of inflammasomes in veterinary and wildlife species. Due to the key role of inflammasomes in mediating inflammatory responses observed in humans and rodents, characterization of the similarities and differences between humans/rodents and veterinary species is required to identify genetic and evolutionary influences on disease responses and to develop therapeutic candidates for use in veterinary inflammatory syndromes. Here, we summarize recent findings on inflammasomes in swine, cattle, dogs, bats, small ruminants, and birds. We describe current gaps in our knowledge and highlight promising areas for future research.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404922/

Serum response factor modulates neuron survival during peripheral axon injury

Background The transcription factor SRF (serum response factor) mediates neuronal survival in vitro . However, data available so far suggest that SRF is largely dispensable for neuron survival during physiological brain function. Findings Here, we demonstrate that upon neuronal injury, that is facial nerve transection, constitutively-active SRF-VP16 enhances motorneuron survival. SRF-VP16 suppressed active caspase 3 abundance in vitro and enhanced neuron survival upon camptothecin induced apoptosis. Following nerve fiber injury in vitro , SRF-VP16 improved survival of neurons and re-growth of severed neurites. Further, SRF-VP16 enhanced immune responses (that is microglia and T cell activation) associated with neuronal injury in vivo. Genome-wide transcriptomics identified target genes associated with axonal injury and modulated by SRF-VP16. Conclusion In sum, this is a first report describing a neuronal injury-related survival function for SRF. Background The transcription factor SRF (serum response factor) mediates neuronal survival in vitro . However, data available so far suggest that SRF is largely dispensable for neuron survival during physiological brain function. Findings Here, we demonstrate that upon neuronal injury, that is facial nerve transection, constitutively-active SRF-VP16 enhances motorneuron survival. SRF-VP16 suppressed active caspase 3 abundance in vitro and enhanced neuron survival upon camptothecin induced apoptosis. Following nerve fiber injury in vitro , SRF-VP16 improved survival of neurons and re-growth of severed neurites. Further, SRF-VP16 enhanced immune responses (that is microglia and T cell activation) associated with neuronal injury in vivo. Genome-wide transcriptomics identified target genes associated with axonal injury and modulated by SRF-VP16. Conclusion In sum, this is a first report describing a neuronal injury-related survival function for SRF. Background The gene regulator SRF modulates multiple aspects of neuronal motility. In SRF-deficient mice, cell migration, neurite outgrowth, branching, growth cone shape and axon guidance are impaired. In turn, constitutively-active SRF-VP16, a fusion protein of SRF and the viral VP16 transactivation domain, enhances neuronal motility [ 1 ]. Thus, SRF's impact on physiological neuronal motility might proof beneficial also during axonal regeneration, that is the stimulation of regrowth of severed nerve fibers. In addition to cell differentiation, SRF has been implicated in cell survival of various cell types including hepatocytes [ 2 ], thymocytes [ 3 ], heart cells [ 4 ], and during embryogenesis [ 5 ]. In embryonic stem cells lacking SRF apoptosis was strongly upregulated [ 4 ]. The latter result is in line with downregulation of the antiapoptotic protein Bcl-2 upon SRF-deficiency. Bcl-2 was identified as SRF target gene in the same study [ 5 ]. In primary cortical neurons, SRF overexpression mediates BDNF-dependent cell survival in various paradigms of neuronal injury [ 6 ]. Also, SRF conveys expression of the immediate early gene (IEG) Cyr61 during neuronal cell death [ 7 ]. SRF operates through interaction with co-factors of the MRTF (myocardin-related transcription factors) and TCF (ternary complex factors) family. Through interaction with TCFs SRF can mediate an IEG response of for example c-fos, Egr1 and Arc. IEGs are well-established molecular switches of cell survival vs. cell death [ 8 ]. Further, while interacting with MRTFs SRF directs expression of actin isoforms ( Acta, Actb, Actc ) or actin-binding proteins (for example tropomyosin, calponin and gelsolin) thereby regulating cytoskeletal dynamics [ 1 , 9 ]. Similar to SRF, MRTF-A and the TCF Elk-1 enhance cell survival of primary neurons [ 6 , 10 - 13 ] and non-neuronal cells [ 14 ]. In opposite to primary neurons, cell survival and apoptosis are not overtly altered during physiological nervous system development as revealed by SRF-deficient mice [ 15 - 17 ]. Indeed, apoptosis was only elevated in the subventricular zone of SRF-deficient mice [ 15 ] but not documented in for example cortical, hippocampal, striatal and peripheral neurons [ 15 - 17 ]. This suggests that SRF is not a major neuronal survival regulator during physiological brain development. As mentioned above, SRF and co-factors mediate injury-related neuronal survival in vitro . Thus, an in vivo function of SRF in neuronal survival (which has not been demonstrated so far) might become apparent during application of neuronal injury. Here we applied facial nerve injury in adult mice to investigate a role of SRF-VP16 in survival of facial motorneurons in vivo . In mice, the bilateral facial nerve innervates muscles regulating whisker pad and eyelid movements, for example [ 18 ]. Facial nerve axotomy is a model system for studying motorneuron survival, axonal regeneration as well as neuron and immune cell interactions during neuronal injury. We observed an SRF-VP16 dependent increase in motorneuron survival in vivo . In addition , SRF-VP16 enhanced outgrowth and survival of transected primary neurons in vitro . Mechanistically this SRF-VP16 function involves suppression of active caspase 3 expression in vitro and increased microglia and T cell activation around transected motorneurons in vivo . Finally, using transcriptomics, we provide axonal injury-induced and SRF-VP16 modulated target genes potentially associated with neuronal survival. Methods Facial nerve transection The facial nerve transection was performed as described in [ 19 ]. Adult wild-type mice (>2 month) were anaesthetized, a skin incision was made behind the left ear and the facial nerve was exposed. In experiments with no virus application, the nerve was transected with small microscissors about 2 mm posterior to the foramen stylomastoideum. For viral infection, 1 μl virus was injected into the facial nerve using a 26 G Hamilton syringe. Afterwards, the nerve was transected and another 1 μl of virus was injected into the nerve stump. Of note, this virus injection with a syringe causes already a facial nerve lesion. Therefore it is only possible to delineate SRF-VP16 specific effects on the basis of experiments employing control virus, SRF-∆MADS-VP16. Cesium-chloride purified SRF-VP16 (4.6 × 10 12 PFU/mL) and SRF-∆MADS-VP16 (4.9 × 10 12 PFU/mL) adenoviral particles were purchased from Vector Biolabs. Both viruses drive GFP expression via a second CMV promoter. Absence of eyelid closure and whisker movement ensured successful nerve transection. All experiments are in accordance with institutional regulations by the local animal ethical committee (Regierungspräsidium Tübingen). Histology Brains were fixed in 4% PFA/PBS overnight followed by preparation of 60 μm vibratome slices. Immunohistochemistry was performed using Biotin-conjugated secondary antibodies (1:500; Vector) and peroxidase-based detection systems using the ABC complex (Vector) and DAB as substrate. Primary antibodies included anti-IBA1 (rabbit, 1:500; Wako) and anti-CD3 (mouse, 1:1,000; Dr. G. Jung, Tübingen University). Cell biology Primary neurons were prepared as before [ 20 ]. Hippocampal neurons derived from wild-type or SRF-deficient mice [ 15 ] were electroporated with SRF-VP16 or SRF-ΔMADS-VP16 and cultured for 72 h. Neurons were electroporated with 3 μg of the plasmids using Amaxa nucleofection resulting on average in 30% to 40% transfected cells. Neurons were stimulated for 1 h with myelin (12 μg/ml). Protein lysates were prepared as before [ 21 ]. Rabbit anti-active caspase 3 (Cell Signaling; 1:1,000) and mouse anti-GAPDH (Acris; 1:50,000) antibodies were used. For neuronal injury experiments in vitro , hippocampal neurons were grown on poly-L-lysine and laminin coated video dishes. One neurite/neuron was cut with a micro-scalpel driven by an InjectMan® NI 2 Micromanipulator (Eppendorf). The cell reaction was monitored in a life cell imaging set-up (37°C, 5% CO 2 ; Zeiss, Axiovert 200 M) every 5 min for a total of 6 h. Ten neurons/condition in 13 independent experiments were evaluated. Neurons were infected with 1 × 10 8 PFU/ml adenoviral particles expressing GFP alone, SRF-ΔMADS-VP16:GFP or SRF-VP16:GFP 5 h after plating. The next day, cultures were treated overnight (17 h) with camptothecin at 0.1, 1, or 3 μΜ followed by immunocytochemistry. Immunocytochemistry Cells were fixed for 15 min in 4% PFA/5% Sucrose/PBS, permeabilized for 5 min in 0.1% Triton-X-100/PBS and blocked for 30 min in 2% BSA/PBS. Primary antibodies were incubated for 2 h at room temperature as follows: rabbit anti-active caspase 3 (Cell Signaling; 1:750; #6991), mouse anti-GFP (Roche; 1:1,000). First antibodies were detected with Alexa 488, or 546 conjugated secondary antibodies (1:1,000; Molecular Probes), followed by DAPI-staining. Microarrays The facial nuclei were dissected from 300 μm brainstem sections prepared with a tissue chopper using tungsten needles. Facial nuclei of four mice/ condition were pooled and resulted on average between 0.5 and 1 μg RNA. Total RNA was isolated with the RNeasy kit (Qiagen). RNA of 0.1 μg was processed on Affymetrix GeneChips (Mouse Gene 1.0 ST array) according to protocols of the Microarray Facility Tübingen ( http://www.microarray-facility.com/cms/index.php ). Raw data normalized to the control sample were analyzed in such way that only genes with a fold-change of ≥ 1.5 (up- or down-regulated) were carried forward. Genes were considered SRF-VP16 specific if their fold-change differed two-fold from the respective factor obtained for SRF-ΔMADS-VP16. Quantitative real-time PCR (qPCR) Total RNA derived from facial nuclei of four animals was isolated with the RNeasy kit (Qiagen). Reverse transcription was performed with 0.5 to 1 μg RNA using reverse transcriptase (Promega) and random hexamers. qPCR was performed on ABI PRISM 7700 Sequence Detector with the Power PCR SYBR green PCR master mix (Applied Biosystems). Expression was determined in relation to Gapdh RNA levels. Mouse primers used were as follows: Cnn1 (fwd: GAA GGT CAA TGA GTC AAC TCA GAA; rev: CCA TAC TTG GTA ATG GCT TTG A), Sprr1a (fwd: CCT GCT CTT CTC TGA GTA TTA GGA C; rev: GCT GCT TCA CCT GCT GCT), Atf3 (fwd: GCT GGA GTC AGT TAC CGT CAA; rev: CGC CTC CTT TTC CTC TCA T), Gpr15 1 (fwd: TGA CGT GGA GCA GTT TTG G; rev: GGG TCA TTG TCT TGT GCT GA), Gal (fwd: CAG TTT CTT GCA CCT TAA AGA GG; rev: GGT CTC AGG ACT TCT CTA GGT CTT C), Npy (fwd: AGA AAA CGC CCC CAG AAC; rev: GAT GAG GGT GGA AAC TTG GA), Sox11 (fwd: GAG CTG AGC GAG ATG ATC G; rev: GAA CAC CAG GTC GGA GAA GT), Srf (fwd: TGT GCA GGC CAT TCA TGT G; rev: ACA GAC GAC GTC ATG ATG GTG), Egr1 (fwd: GCC GAG CGA ACA ACC CTA T; rev: TCC ACC ATC GCC TTC TCA TT), Actn3 (fwd: ACCACTTTGACCGGAAGCG; rev: GGAGATGAGACAAGCTCGGAA), Acta2 (fwd: CAG CAA ACA GGA ATA CGA CGA A; rev: TGT GTG CTA GAG GCA GAG CAG). Statistics and quantification Numbers of independent experiments or animals are indicated in figure bars. For all cell culture experiments at least three independent cultures derived from different animals were analyzed. For quantification of neuron numbers in facial nerve injury experiments, all sections (that is 10 to 15 sections/animal) were evaluated. Neurons were scored as non-degenerated, if they protruded at least one neurite and if the cell body showed a typical angled shape. For microglia and T cell numbers 4 to 6 sections/ animal were analyzed. Statistical significance was calculated using two-tailed t test or, where appropriate, a one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. *, **, and *** indicates P ≤ 0.05, 0.01, and 0.001, respectively. Standard deviation is provided if not mentioned otherwise. Facial nerve transection The facial nerve transection was performed as described in [ 19 ]. Adult wild-type mice (>2 month) were anaesthetized, a skin incision was made behind the left ear and the facial nerve was exposed. In experiments with no virus application, the nerve was transected with small microscissors about 2 mm posterior to the foramen stylomastoideum. For viral infection, 1 μl virus was injected into the facial nerve using a 26 G Hamilton syringe. Afterwards, the nerve was transected and another 1 μl of virus was injected into the nerve stump. Of note, this virus injection with a syringe causes already a facial nerve lesion. Therefore it is only possible to delineate SRF-VP16 specific effects on the basis of experiments employing control virus, SRF-∆MADS-VP16. Cesium-chloride purified SRF-VP16 (4.6 × 10 12 PFU/mL) and SRF-∆MADS-VP16 (4.9 × 10 12 PFU/mL) adenoviral particles were purchased from Vector Biolabs. Both viruses drive GFP expression via a second CMV promoter. Absence of eyelid closure and whisker movement ensured successful nerve transection. All experiments are in accordance with institutional regulations by the local animal ethical committee (Regierungspräsidium Tübingen). Histology Brains were fixed in 4% PFA/PBS overnight followed by preparation of 60 μm vibratome slices. Immunohistochemistry was performed using Biotin-conjugated secondary antibodies (1:500; Vector) and peroxidase-based detection systems using the ABC complex (Vector) and DAB as substrate. Primary antibodies included anti-IBA1 (rabbit, 1:500; Wako) and anti-CD3 (mouse, 1:1,000; Dr. G. Jung, Tübingen University). Cell biology Primary neurons were prepared as before [ 20 ]. Hippocampal neurons derived from wild-type or SRF-deficient mice [ 15 ] were electroporated with SRF-VP16 or SRF-ΔMADS-VP16 and cultured for 72 h. Neurons were electroporated with 3 μg of the plasmids using Amaxa nucleofection resulting on average in 30% to 40% transfected cells. Neurons were stimulated for 1 h with myelin (12 μg/ml). Protein lysates were prepared as before [ 21 ]. Rabbit anti-active caspase 3 (Cell Signaling; 1:1,000) and mouse anti-GAPDH (Acris; 1:50,000) antibodies were used. For neuronal injury experiments in vitro , hippocampal neurons were grown on poly-L-lysine and laminin coated video dishes. One neurite/neuron was cut with a micro-scalpel driven by an InjectMan® NI 2 Micromanipulator (Eppendorf). The cell reaction was monitored in a life cell imaging set-up (37°C, 5% CO 2 ; Zeiss, Axiovert 200 M) every 5 min for a total of 6 h. Ten neurons/condition in 13 independent experiments were evaluated. Neurons were infected with 1 × 10 8 PFU/ml adenoviral particles expressing GFP alone, SRF-ΔMADS-VP16:GFP or SRF-VP16:GFP 5 h after plating. The next day, cultures were treated overnight (17 h) with camptothecin at 0.1, 1, or 3 μΜ followed by immunocytochemistry. Immunocytochemistry Cells were fixed for 15 min in 4% PFA/5% Sucrose/PBS, permeabilized for 5 min in 0.1% Triton-X-100/PBS and blocked for 30 min in 2% BSA/PBS. Primary antibodies were incubated for 2 h at room temperature as follows: rabbit anti-active caspase 3 (Cell Signaling; 1:750; #6991), mouse anti-GFP (Roche; 1:1,000). First antibodies were detected with Alexa 488, or 546 conjugated secondary antibodies (1:1,000; Molecular Probes), followed by DAPI-staining. Microarrays The facial nuclei were dissected from 300 μm brainstem sections prepared with a tissue chopper using tungsten needles. Facial nuclei of four mice/ condition were pooled and resulted on average between 0.5 and 1 μg RNA. Total RNA was isolated with the RNeasy kit (Qiagen). RNA of 0.1 μg was processed on Affymetrix GeneChips (Mouse Gene 1.0 ST array) according to protocols of the Microarray Facility Tübingen ( http://www.microarray-facility.com/cms/index.php ). Raw data normalized to the control sample were analyzed in such way that only genes with a fold-change of ≥ 1.5 (up- or down-regulated) were carried forward. Genes were considered SRF-VP16 specific if their fold-change differed two-fold from the respective factor obtained for SRF-ΔMADS-VP16. Quantitative real-time PCR (qPCR) Total RNA derived from facial nuclei of four animals was isolated with the RNeasy kit (Qiagen). Reverse transcription was performed with 0.5 to 1 μg RNA using reverse transcriptase (Promega) and random hexamers. qPCR was performed on ABI PRISM 7700 Sequence Detector with the Power PCR SYBR green PCR master mix (Applied Biosystems). Expression was determined in relation to Gapdh RNA levels. Mouse primers used were as follows: Cnn1 (fwd: GAA GGT CAA TGA GTC AAC TCA GAA; rev: CCA TAC TTG GTA ATG GCT TTG A), Sprr1a (fwd: CCT GCT CTT CTC TGA GTA TTA GGA C; rev: GCT GCT TCA CCT GCT GCT), Atf3 (fwd: GCT GGA GTC AGT TAC CGT CAA; rev: CGC CTC CTT TTC CTC TCA T), Gpr15 1 (fwd: TGA CGT GGA GCA GTT TTG G; rev: GGG TCA TTG TCT TGT GCT GA), Gal (fwd: CAG TTT CTT GCA CCT TAA AGA GG; rev: GGT CTC AGG ACT TCT CTA GGT CTT C), Npy (fwd: AGA AAA CGC CCC CAG AAC; rev: GAT GAG GGT GGA AAC TTG GA), Sox11 (fwd: GAG CTG AGC GAG ATG ATC G; rev: GAA CAC CAG GTC GGA GAA GT), Srf (fwd: TGT GCA GGC CAT TCA TGT G; rev: ACA GAC GAC GTC ATG ATG GTG), Egr1 (fwd: GCC GAG CGA ACA ACC CTA T; rev: TCC ACC ATC GCC TTC TCA TT), Actn3 (fwd: ACCACTTTGACCGGAAGCG; rev: GGAGATGAGACAAGCTCGGAA), Acta2 (fwd: CAG CAA ACA GGA ATA CGA CGA A; rev: TGT GTG CTA GAG GCA GAG CAG). Statistics and quantification Numbers of independent experiments or animals are indicated in figure bars. For all cell culture experiments at least three independent cultures derived from different animals were analyzed. For quantification of neuron numbers in facial nerve injury experiments, all sections (that is 10 to 15 sections/animal) were evaluated. Neurons were scored as non-degenerated, if they protruded at least one neurite and if the cell body showed a typical angled shape. For microglia and T cell numbers 4 to 6 sections/ animal were analyzed. Statistical significance was calculated using two-tailed t test or, where appropriate, a one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. *, **, and *** indicates P ≤ 0.05, 0.01, and 0.001, respectively. Standard deviation is provided if not mentioned otherwise. Results SRF-VP16 enhances motorneuron survival in vivo To investigate a role of SRF in neuron survival, we employed a well-established model system of neuronal injury, that is unilateral de-afferentiation of facial motorneurons in mice (Figure 1A ). The transected facial nerve was infected with viral particles expressing GFP in addition to SRF-ΔMADS-VP16 or SRF-VP16. SRF-VP16 consists of SRF fused to the viral VP16 transactivation domain. To control for VP16 off-target effects, SRF-ΔMADS-VP16, lacking DNA binding activity, was used as control [ 20 ]. SRF expression commenced 1 day after infection. Around the virus injection site of the facial nerve, SRF was also found in fibroblasts and glial cells, whereas in the facial nucleus - after retrograde viral transport - SRF expression was motorneuron restricted (Figure 1 Additional file 1 : Movie S1 and Additional file 2 : Movie S2 and data not shown). Figure 1 SRF-VP16 enhances survival of facial motorneurons. ( A ) (left) The facial nerve is outlined in blue. (right) Virus injection (green) and position of axotomy is depicted (arrow). Pictures in ( B-G ) were taken from the facial nucleus whose position is indicated by the red circle. Facial motorneurons express either SRF-VP16 or SRF-ΔMADS-VP16 along with GFP, whose expression is depicted in (B-G). ( B , C ) Facial nuclei of an SRF-ΔMADS-VP16 (B) or SRF-VP16 (C) expressing animal taken at 5 days post infection (d.p.i.) and lesion. No obvious differences were discernable. ( D -G) The facial nucleus of an SRF-ΔMADS-VP16 (D, F) or SRF-VP16 ( E , G ) infected animal at 25 d.p.i/lesion. In SRF-ΔMADS-VP16 (D, F) compared to SRF-VP16 (E, G) numbers of surviving neurons are reduced. SRF-ΔMADS-VP16 expressing neurons are atrophic and assume a "bleb-like" morphology without innervation (see insert in D ). SRF-VP16 neurons protrude neurites and cell bodies are squared in shape (insert in E). ( H ) Numbers of GFP-positive neurons/section are indicated. ( I ) At 25 d.p.i., but not 5 d.p.i. SRF-ΔMADS-VP16, in contrast to SRF-VP16 expressing neurons were degenerated. Dashed lines depict outlines of the facial nuclei. Scale-bar (B-G) = 100 μm; inserts = 10 μm. Survival was quantified by analyzing number and morphology of GFP-positive motorneurons in the facial nucleus. SRF-VP16 increased the number of surviving motorneurons compared to SRF-ΔMADS-VP16 (Figure 1 , Additional file 1 : Movie S1 and Additional file 2 : Movie S2). At 5 days post viral infection (d.p.i.) and axotomy, the number of GFP-positive neurons expressing SRF-VP16 or SRF-ΔMADS-VP16 was comparable (Figure 1B , 1C , and 1H ). However, at 25 d.p.i., numbers of surviving SRF-VP16 positive neurons after transection exceeded those expressing SRF-ΔMADS-VP16 about three-fold (Figure 1D-H ). We also inspected motorneuron morphology. SRF-VP16 expressing neurons appeared less degenerated as assessed by two parameters: neurite innervation and shrunk atrophic cell bodies. At 5 d.p.i, SRF-VP16 and SRF-ΔMADS-VP16 expressing neurons did not differ with regard to these criteria (Figure 1B , 1C and 1I ). At 25 d.p.i about 60% of SRF-ΔMADS-VP16 expressing neurons lost innervation and acquired a 'bleb-like' rounded-up cell morphology (Figure 1D , 1F and 1I ). In contrast, SRF-VP16 suppressed neuronal degeneration, leaving only 35% of neurons atrophic (Figure 1E , 1G and 1I ). This suggests that SRF plays a role in survival of axotomized facial motorneurons. SRF-VP16 suppressed cell death and enhanced neurite regrowth in vitro As shown above (Figure 1 ), SRF-VP16 protects from motorneuron loss upon facial nerve lesion in vivo . To investigate whether SRF-VP16 has also an impact on neuronal survival of primary neurons we employed an in vitro assay of axonal injury (Figure 2 ). Here, neurites of primary neurons were transected using a micro-scalpel followed by recording the neuronal response with time-lapse video-microscopy (Figure 2A-C ). VP16 expressing neurons were identified via GFP-expression. After lesion, neurites of an SRF-ΔMADS-VP16 expressing neuron did not re-grow and neurons frequently died (Figure 2A ). In contrast, neurites of an SRF-VP16 expressing neuron were capable of re-growth, often protruded dynamic growth cones and survived neurite transection (Figure 2B ; quantification in 2C). Thus, SRF-VP16 enhances neuronal survival and re-growth of severed neurites in vitro . Figure 2 SRF-VP16 modulates cell survival in vitro. ( A ) After lesion, the neurite of an SRF-ΔMADS-VP16 expressing neuron is not re-growing and the neuron eventually dies after 200 min. The neurite was severed at the position indicated by the arrow. ( B ) A neuron expressing SRF-VP16. After transection, neurite growth is observed as well as a dynamic growth cone structure (arrowhead). Eventually at 160 min, the neurite has exceeded the original lesion position. ( C ) SRF-VP16 increased the percentage of neurons surviving nerve fiber transection and revealing re-growth of neurites. ( D , E ) SRF-VP16 suppressed active caspase 3 levels in wild-type and more pronounced in SRF-deficient neurons compared to neurons expressing SRF-ΔMADS-VP16. ( F , G ) SRF-VP16 reduced camptothecin induced neuronal cell death as quantified by counting active caspase 3 (F) or surviving GFP-positive (G) neurons. In a next step, we investigated potential mechanisms of SRF's function in neuronal survival. SRF-VP16 might modulate motorneuron survival via blocking apoptosis. To investigate this further, we employed primary neurons assessing protein levels of the pro-apoptotic protein active caspase-3 upon SRF-VP16 expression (Figure 2D , 2E ). In SRF-ΔMADS-VP16 expressing neurons, active caspase 3 levels were strongly induced. In contrast SRF-VP16 suppressed this myelin-induced activation of active caspase-3 (Figure 2D , 2E ). Notably, this effect was more obvious in neurons lacking SRF compared to wild-type neurons (Figure 2D and quantification in 2E). In a further set of experiments we analyzed whether SRF-VP16 might enhance neuronal survival upon camptothecin induced DNA damage (Figure 2F 2G ). In control infected primary neurons either expressing GFP alone or SRF-ΔMADS-VP16, camptothecin induced apoptosis in a concentration dependent manner. This was quantified by either counting numbers of active caspase 3 positive (Figure 2F ) or numbers of surviving GFP-positive neurons (Figure 2G ). In contrast, SRF-VP16 expression reduced this camptothecin induced neuronal cell death compared to control constructs (Figure 2F 2G ). Thus, in cultures infected with adenoviral particles expressing SRF-VP16 (along with GFP) the number of active caspase 3 positive neurons was reduced (Figure 2F ) whereas more GFP-positive neurons survived camptothecin treatment (Figure 2G ). This finding is in agreement with previous observations made with wild-type SRF in cortical neurons [ 6 ]. Thus, results from primary neurons suggest that SRF-VP16 might down-regulate expression of pro-apoptotic proteins to enhance neuronal survival. SRF-VP16 modulates injury associated immune responses Neuronal injury is accompanied by immune responses, for example astrocyte, microglia, and T cell activation and their subsequent infiltration of lesioned neuronal tissue. Injury-related immune responses might dampen as well as exacerbate neuronal loss [ 18 , 22 ]. Regarding the facial nerve lesion model, peri-neuronal accumulation of microglia cells facilitates axonal regeneration [ 23 ]. Also T cells were assigned important roles for the immune surveillance during facial nerve injury [ 24 ]. Given this important link between an immune response and neuronal injury, we asked whether SRF-VP16 might modulate immune responses associated with axon injury (Figure 3 ). Figure 3 SRF-VP16 increases microglia and T cell activation in axonal injury. ( A-H ) Upon axotomy, microglia were activated at both time-points in the lesioned side expressing SRF-ΔMADS-VP16 compared to the control side (compare C , G with A, E ). SRF-VP16 ( B , F and D , H ) enhanced microglia activation at both time-points. In addition, SRF-VP16 enhanced microglia association along the axons (arrows in H) and the nerve exit point (arrowheads in H) at 25 d.p.i. (see insert in H). ( I-L ) At 25 d.p.i., T cells entered the transected facial nucleus in control infected animals ( K ; the insert shows individual T cells), but not the intact facial nucleus (I). In animals expressing SRF-VP16, T cell infiltration was strongly enhanced in the lesion (L) but not the control side ( J ). T cells were also found along nerves (L). ( M , N ) Numbers of microglia/area are indicated for all conditions in the facial nucleus (M) and along the facial nerve (N). ( O ) Numbers of T cells/area are indicated for all conditions in the facial nucleus. Dashed lines depict outlines of the facial nuclei. Dashed boxes point at positions magnified by inserts. Scale-bar (A-L) = 100 μm; inserts (A-D, K, L) = 20 μm; inserts (E-H) = 100 μm. Firstly, we inspected microglia activation in the de-afferented facial nucleus. Microglia were expectedly elevated at the lesion side compared to the control side at 5 and 25 d.p.i (Figure 3A , 3E and 3C , 3G ; 3M ). SRF-VP16 augmented microglia activation in the lesioned facial nucleus at both time-points compared to SRF-ΔMADS-VP16 (Figure 3B , 3 and 3D , 3H ). Notably, SRF-VP16 also enhanced microglia occupancy along the facial nerve axons and the axon exit point (arrows and arrowheads in Figure 3H , respectively; Figure 3N ). Secondly, we investigated T cells labeled with an anti-CD3 directed antibody. T cells did not enter the facial nucleus 5 d.p.i. regardless of virus type (Figure 3O ). In contrast, at 25 d.p.i., we observed T cell infiltration in lesioned SRF-ΔMADS-VP16 expressing neurons but not on the uninfected control side (Figure 3I , K, and 3O ). Similar to results obtained on microglia (Figure 3A-H ), SRF-VP16 also enhanced T cell infiltration around motorneurons and axons (Figure 3L and 3O ). Taken together, microglia and T cell responses are augmented upon SRF-VP16 expression. Microarray analysis of lesion and SRF-VP16 induced transcripts SRF might enhance neuronal survival through various mechanisms including regulation of survival/apoptosis related (Figure 2 ) and immune regulatory genes (Figure 3 ). To identify genes modulated by facial nerve injury per se and by SRF-VP16, we performed transcriptomics after three days of facial nerve lesion (Figures 4 and 5 , Table 1 and Additional file 3 : Table S1). For this, facial nuclei of four mice were pooled for each condition. Figure 4 Transcriptomics of facial nerve injury and SRF-VP16 associated genes. Three days after facial nerve transection, facial nuclei were subjected to microarray analysis. Genes up- or down-regulated (≥ 4-fold) by facial nerve injury alone are depicted in blue. Genes specifically altered by SRF-VP16 upon nerve injury are highlighted in red. Genes in black color are modulated by SRF-ΔMADS-VP16. Genes depicted in green are modulated by lesion alone and SRF-VP16 or SRF-ΔMADS-VP16. Red colors indicate high, whereas blue colors represent low expression. All expression levels were normalized to the control condition (without lesion). Figure 5 Validation of transcriptomics data. cDNAs derived from unlesioned, lesioned, lesioned and SRF-ΔMADS-VP16-positive and lesioned and SRF-VP16-positive facial nuclei were subjected to qPCR analysis with the indicated primers. Every bar reflects mRNA levels obtained from a cDNA sample in which facial nuclei of four independent animals were pooled. Numbers of independent cDNAs are indicated at bars. Statistical significance was calculated in relation to control (no lesion/no virus). Table 1 Summary of genes most strongly regulated by lesion only or SRF-VP16 Lesion-specific genes SRF-VP16-specific genes Gene name Gene symbol Up Gene name Gene symbol Up 1 Small proline-rich protein 2J Sprr2j 80.1 Actin, alpha, cardiac Actc1 13.2 2 G protein-coupled receptor 151 Gpr151 31.0 Protein phosphatase with EF hand Ppef1 12.2 3 Activating transcription factor 3 Atf3 28.6 Tissue inhibitor of metalloprot. 1 Timp1 12.2 4 Glutamate receptor, metab. 3 Grm3 24.9 Ankyrin repeat domain 1 Ankrd1 10.3 5 Protein phosphatase with EF Ppef1 19.9 Calponin 1 Cnn1 9.5 6 Galanin Gal 19.8 Coagulation factor II receptor-like 2 F2rl2 8.7 7 Tissue inhibitor of metalloprot. 1 Timp1 14.3 Actin, alpha 2, smooth muscle Acta2 7.9 8 Neuropeptide Y Npy 13.2 Transgelin (Sm22) Tagln 6.4 9 Annexin a10 Anxa10 12.7 Apolipoprotein L 7b | L 7e Apol7b/e 4.7 10 Small proline-rich protein 2 J Sprr2j 12.7 Serine (or cysteine) peptidase inh. Serpine1 4.5 11 Tubulin, beta 6 Tubb6 11.4 T-box18 Tbx18 3.8 12 Ankyrin repeat domain 1 Ankrd1 11.4 Interleukin 1 receptor, type II Il1r2 3.8 13 Wingless related 2b Wnt2b 10.6 Actinin alpha 3 Actn3 3.7 14 A disintegrin and metallopept. 8 Adam8 9.5 Angiopoietin-like 2 Angptl2 3.4 15 S100 calcium binding prot. A11 S100a11 9.2 Insulin-like growth fac. bind. prot. 6 Igfbp6 3.2 16 Xanthine dehydrogenase Xdh 8.7 Follistatin Fst 3.2 17 Anthrax toxin receptor 2 Antxr2 8.5 GLI pathogenesis-related 1 Glipr1 3.1 18 SH2 domain protein 1B2 Sh2d1b2 8.4 Solute carrier family 38, member 8 Slc38a8 2.9 19 Gastrin releasing peptide Grp 7.5 Early growth response 1 Egr1 2.7 20 Vasoactive intestinal polypeptide Vip 7.4 Dihydroxyacetone kinase 2 hom. Dak 2.7 21 Protein C receptor, endothelial Procr 6.9 Tuftelin 1 Tuft1 2.6 22 Integrin alpha 7 Itga7 6.8 Serum response factor Srf 2.4 23 Lymphocyte antigen 86 Ly86 6.8 Bone marrow stromal cell antigen 1 Bst1 2.4 24 Nerve growth factor Ngf 6.5 Dopamine receptor 2 Drd2 2.4 25 Integrin alpha M Itgam 6.1 Desmocollin 3 Dsc3 2.3 26 Prokineticin receptor 2 Prokr2 6.0 Blood vessel epicardial substance Bves 2.2 27 Serine (or cysteine) pept. inh. Serpine1 5.9 ALX homeobox 1 Alx1 2.2 28 G protein-coupled receptor 133 Gpr133 5.7 Neuronal pentraxin 2 Nptx2 2.2 29 Cyclin-dep. kinase inh.1A (P21) Cdkn1a 5.6 Glucagon-like peptide 1 receptor Glp1r 1.9 30 GalaninCD180 antigen Cd180 5.4 Regulator of G-protein signaling 4 Rgs4 1.7 Upon lesion only, 1,088 genes (858 up, 230 down) were regulated more than 1.5-fold compared to un-lesioned facial nuclei. Figure 4 represents those genes modulated by facial nerve injury alone regulated by a factor ≥ 4 (colored in blue). These included reported genes induced by facial nerve injury such as Atf3 Gal Tubb6 Avpr1a Vip , and Itga7 [ 18 , 25 ]. In addition, we noted that many genes encoding G-protein coupled receptors (GPCRs), hormones and small neuropeptides were modulated by facial nerve axotomy including Avpr1a Grm3 Prokr2 Npr3 Gpr161 Gpr133 Gpr84 Gal Npy Vip , and Grp (Figure 4 , Table 1 , and Additional file 3 : Table S1). SRF-VP16 specific genes modulated after facial nerve injury are depicted in red (Figure 4 , Table 1 ). SRF-VP16 modulated two well-known gene sets, IEGs (for example Egr1 , Egr2 ) and actin cytoskeletal genes ( Actc1 , Cnn1 , Acta2 , and Actn3 ). Similar to facial nerve injury alone (see above) we noted that several potential SRF target genes encoded components of GPCR signaling ( F2rl2 , Glp1r , Rgs4 , Crhbp , Nms , Galp ; Figure 4 and Table 1 ). Finally we observed genes modulated by SRF-VP16 which might link SRF activity to immune responses investigated above (Figure 1 ). These include tissue inhibitor of metalloproteinase ( Timp1 ), interleukin receptor ( Il1r2) , galanin-like peptide ( Galp ), neuromedin ( Nms ), and interleukin 1 ( Il1f9 ) (Figure 4 , Table 1 and Additional file 3 : Table S1; see also discussion). To corroborate microarray data (Figure 4 ), qPCR analysis of selected genes employing independent cDNA samples was performed. Indeed fold changes obtained in this qPCR analysis were comparable to the microarray data (Figure 5 ). SRF-VP16 enhances motorneuron survival in vivo To investigate a role of SRF in neuron survival, we employed a well-established model system of neuronal injury, that is unilateral de-afferentiation of facial motorneurons in mice (Figure 1A ). The transected facial nerve was infected with viral particles expressing GFP in addition to SRF-ΔMADS-VP16 or SRF-VP16. SRF-VP16 consists of SRF fused to the viral VP16 transactivation domain. To control for VP16 off-target effects, SRF-ΔMADS-VP16, lacking DNA binding activity, was used as control [ 20 ]. SRF expression commenced 1 day after infection. Around the virus injection site of the facial nerve, SRF was also found in fibroblasts and glial cells, whereas in the facial nucleus - after retrograde viral transport - SRF expression was motorneuron restricted (Figure 1 Additional file 1 : Movie S1 and Additional file 2 : Movie S2 and data not shown). Figure 1 SRF-VP16 enhances survival of facial motorneurons. ( A ) (left) The facial nerve is outlined in blue. (right) Virus injection (green) and position of axotomy is depicted (arrow). Pictures in ( B-G ) were taken from the facial nucleus whose position is indicated by the red circle. Facial motorneurons express either SRF-VP16 or SRF-ΔMADS-VP16 along with GFP, whose expression is depicted in (B-G). ( B , C ) Facial nuclei of an SRF-ΔMADS-VP16 (B) or SRF-VP16 (C) expressing animal taken at 5 days post infection (d.p.i.) and lesion. No obvious differences were discernable. ( D -G) The facial nucleus of an SRF-ΔMADS-VP16 (D, F) or SRF-VP16 ( E , G ) infected animal at 25 d.p.i/lesion. In SRF-ΔMADS-VP16 (D, F) compared to SRF-VP16 (E, G) numbers of surviving neurons are reduced. SRF-ΔMADS-VP16 expressing neurons are atrophic and assume a "bleb-like" morphology without innervation (see insert in D ). SRF-VP16 neurons protrude neurites and cell bodies are squared in shape (insert in E). ( H ) Numbers of GFP-positive neurons/section are indicated. ( I ) At 25 d.p.i., but not 5 d.p.i. SRF-ΔMADS-VP16, in contrast to SRF-VP16 expressing neurons were degenerated. Dashed lines depict outlines of the facial nuclei. Scale-bar (B-G) = 100 μm; inserts = 10 μm. Survival was quantified by analyzing number and morphology of GFP-positive motorneurons in the facial nucleus. SRF-VP16 increased the number of surviving motorneurons compared to SRF-ΔMADS-VP16 (Figure 1 , Additional file 1 : Movie S1 and Additional file 2 : Movie S2). At 5 days post viral infection (d.p.i.) and axotomy, the number of GFP-positive neurons expressing SRF-VP16 or SRF-ΔMADS-VP16 was comparable (Figure 1B , 1C , and 1H ). However, at 25 d.p.i., numbers of surviving SRF-VP16 positive neurons after transection exceeded those expressing SRF-ΔMADS-VP16 about three-fold (Figure 1D-H ). We also inspected motorneuron morphology. SRF-VP16 expressing neurons appeared less degenerated as assessed by two parameters: neurite innervation and shrunk atrophic cell bodies. At 5 d.p.i, SRF-VP16 and SRF-ΔMADS-VP16 expressing neurons did not differ with regard to these criteria (Figure 1B , 1C and 1I ). At 25 d.p.i about 60% of SRF-ΔMADS-VP16 expressing neurons lost innervation and acquired a 'bleb-like' rounded-up cell morphology (Figure 1D , 1F and 1I ). In contrast, SRF-VP16 suppressed neuronal degeneration, leaving only 35% of neurons atrophic (Figure 1E , 1G and 1I ). This suggests that SRF plays a role in survival of axotomized facial motorneurons. SRF-VP16 suppressed cell death and enhanced neurite regrowth in vitro As shown above (Figure 1 ), SRF-VP16 protects from motorneuron loss upon facial nerve lesion in vivo . To investigate whether SRF-VP16 has also an impact on neuronal survival of primary neurons we employed an in vitro assay of axonal injury (Figure 2 ). Here, neurites of primary neurons were transected using a micro-scalpel followed by recording the neuronal response with time-lapse video-microscopy (Figure 2A-C ). VP16 expressing neurons were identified via GFP-expression. After lesion, neurites of an SRF-ΔMADS-VP16 expressing neuron did not re-grow and neurons frequently died (Figure 2A ). In contrast, neurites of an SRF-VP16 expressing neuron were capable of re-growth, often protruded dynamic growth cones and survived neurite transection (Figure 2B ; quantification in 2C). Thus, SRF-VP16 enhances neuronal survival and re-growth of severed neurites in vitro . Figure 2 SRF-VP16 modulates cell survival in vitro. ( A ) After lesion, the neurite of an SRF-ΔMADS-VP16 expressing neuron is not re-growing and the neuron eventually dies after 200 min. The neurite was severed at the position indicated by the arrow. ( B ) A neuron expressing SRF-VP16. After transection, neurite growth is observed as well as a dynamic growth cone structure (arrowhead). Eventually at 160 min, the neurite has exceeded the original lesion position. ( C ) SRF-VP16 increased the percentage of neurons surviving nerve fiber transection and revealing re-growth of neurites. ( D , E ) SRF-VP16 suppressed active caspase 3 levels in wild-type and more pronounced in SRF-deficient neurons compared to neurons expressing SRF-ΔMADS-VP16. ( F , G ) SRF-VP16 reduced camptothecin induced neuronal cell death as quantified by counting active caspase 3 (F) or surviving GFP-positive (G) neurons. In a next step, we investigated potential mechanisms of SRF's function in neuronal survival. SRF-VP16 might modulate motorneuron survival via blocking apoptosis. To investigate this further, we employed primary neurons assessing protein levels of the pro-apoptotic protein active caspase-3 upon SRF-VP16 expression (Figure 2D , 2E ). In SRF-ΔMADS-VP16 expressing neurons, active caspase 3 levels were strongly induced. In contrast SRF-VP16 suppressed this myelin-induced activation of active caspase-3 (Figure 2D , 2E ). Notably, this effect was more obvious in neurons lacking SRF compared to wild-type neurons (Figure 2D and quantification in 2E). In a further set of experiments we analyzed whether SRF-VP16 might enhance neuronal survival upon camptothecin induced DNA damage (Figure 2F 2G ). In control infected primary neurons either expressing GFP alone or SRF-ΔMADS-VP16, camptothecin induced apoptosis in a concentration dependent manner. This was quantified by either counting numbers of active caspase 3 positive (Figure 2F ) or numbers of surviving GFP-positive neurons (Figure 2G ). In contrast, SRF-VP16 expression reduced this camptothecin induced neuronal cell death compared to control constructs (Figure 2F 2G ). Thus, in cultures infected with adenoviral particles expressing SRF-VP16 (along with GFP) the number of active caspase 3 positive neurons was reduced (Figure 2F ) whereas more GFP-positive neurons survived camptothecin treatment (Figure 2G ). This finding is in agreement with previous observations made with wild-type SRF in cortical neurons [ 6 ]. Thus, results from primary neurons suggest that SRF-VP16 might down-regulate expression of pro-apoptotic proteins to enhance neuronal survival. SRF-VP16 modulates injury associated immune responses Neuronal injury is accompanied by immune responses, for example astrocyte, microglia, and T cell activation and their subsequent infiltration of lesioned neuronal tissue. Injury-related immune responses might dampen as well as exacerbate neuronal loss [ 18 , 22 ]. Regarding the facial nerve lesion model, peri-neuronal accumulation of microglia cells facilitates axonal regeneration [ 23 ]. Also T cells were assigned important roles for the immune surveillance during facial nerve injury [ 24 ]. Given this important link between an immune response and neuronal injury, we asked whether SRF-VP16 might modulate immune responses associated with axon injury (Figure 3 ). Figure 3 SRF-VP16 increases microglia and T cell activation in axonal injury. ( A-H ) Upon axotomy, microglia were activated at both time-points in the lesioned side expressing SRF-ΔMADS-VP16 compared to the control side (compare C , G with A, E ). SRF-VP16 ( B , F and D , H ) enhanced microglia activation at both time-points. In addition, SRF-VP16 enhanced microglia association along the axons (arrows in H) and the nerve exit point (arrowheads in H) at 25 d.p.i. (see insert in H). ( I-L ) At 25 d.p.i., T cells entered the transected facial nucleus in control infected animals ( K ; the insert shows individual T cells), but not the intact facial nucleus (I). In animals expressing SRF-VP16, T cell infiltration was strongly enhanced in the lesion (L) but not the control side ( J ). T cells were also found along nerves (L). ( M , N ) Numbers of microglia/area are indicated for all conditions in the facial nucleus (M) and along the facial nerve (N). ( O ) Numbers of T cells/area are indicated for all conditions in the facial nucleus. Dashed lines depict outlines of the facial nuclei. Dashed boxes point at positions magnified by inserts. Scale-bar (A-L) = 100 μm; inserts (A-D, K, L) = 20 μm; inserts (E-H) = 100 μm. Firstly, we inspected microglia activation in the de-afferented facial nucleus. Microglia were expectedly elevated at the lesion side compared to the control side at 5 and 25 d.p.i (Figure 3A , 3E and 3C , 3G ; 3M ). SRF-VP16 augmented microglia activation in the lesioned facial nucleus at both time-points compared to SRF-ΔMADS-VP16 (Figure 3B , 3 and 3D , 3H ). Notably, SRF-VP16 also enhanced microglia occupancy along the facial nerve axons and the axon exit point (arrows and arrowheads in Figure 3H , respectively; Figure 3N ). Secondly, we investigated T cells labeled with an anti-CD3 directed antibody. T cells did not enter the facial nucleus 5 d.p.i. regardless of virus type (Figure 3O ). In contrast, at 25 d.p.i., we observed T cell infiltration in lesioned SRF-ΔMADS-VP16 expressing neurons but not on the uninfected control side (Figure 3I , K, and 3O ). Similar to results obtained on microglia (Figure 3A-H ), SRF-VP16 also enhanced T cell infiltration around motorneurons and axons (Figure 3L and 3O ). Taken together, microglia and T cell responses are augmented upon SRF-VP16 expression. Microarray analysis of lesion and SRF-VP16 induced transcripts SRF might enhance neuronal survival through various mechanisms including regulation of survival/apoptosis related (Figure 2 ) and immune regulatory genes (Figure 3 ). To identify genes modulated by facial nerve injury per se and by SRF-VP16, we performed transcriptomics after three days of facial nerve lesion (Figures 4 and 5 , Table 1 and Additional file 3 : Table S1). For this, facial nuclei of four mice were pooled for each condition. Figure 4 Transcriptomics of facial nerve injury and SRF-VP16 associated genes. Three days after facial nerve transection, facial nuclei were subjected to microarray analysis. Genes up- or down-regulated (≥ 4-fold) by facial nerve injury alone are depicted in blue. Genes specifically altered by SRF-VP16 upon nerve injury are highlighted in red. Genes in black color are modulated by SRF-ΔMADS-VP16. Genes depicted in green are modulated by lesion alone and SRF-VP16 or SRF-ΔMADS-VP16. Red colors indicate high, whereas blue colors represent low expression. All expression levels were normalized to the control condition (without lesion). Figure 5 Validation of transcriptomics data. cDNAs derived from unlesioned, lesioned, lesioned and SRF-ΔMADS-VP16-positive and lesioned and SRF-VP16-positive facial nuclei were subjected to qPCR analysis with the indicated primers. Every bar reflects mRNA levels obtained from a cDNA sample in which facial nuclei of four independent animals were pooled. Numbers of independent cDNAs are indicated at bars. Statistical significance was calculated in relation to control (no lesion/no virus). Table 1 Summary of genes most strongly regulated by lesion only or SRF-VP16 Lesion-specific genes SRF-VP16-specific genes Gene name Gene symbol Up Gene name Gene symbol Up 1 Small proline-rich protein 2J Sprr2j 80.1 Actin, alpha, cardiac Actc1 13.2 2 G protein-coupled receptor 151 Gpr151 31.0 Protein phosphatase with EF hand Ppef1 12.2 3 Activating transcription factor 3 Atf3 28.6 Tissue inhibitor of metalloprot. 1 Timp1 12.2 4 Glutamate receptor, metab. 3 Grm3 24.9 Ankyrin repeat domain 1 Ankrd1 10.3 5 Protein phosphatase with EF Ppef1 19.9 Calponin 1 Cnn1 9.5 6 Galanin Gal 19.8 Coagulation factor II receptor-like 2 F2rl2 8.7 7 Tissue inhibitor of metalloprot. 1 Timp1 14.3 Actin, alpha 2, smooth muscle Acta2 7.9 8 Neuropeptide Y Npy 13.2 Transgelin (Sm22) Tagln 6.4 9 Annexin a10 Anxa10 12.7 Apolipoprotein L 7b | L 7e Apol7b/e 4.7 10 Small proline-rich protein 2 J Sprr2j 12.7 Serine (or cysteine) peptidase inh. Serpine1 4.5 11 Tubulin, beta 6 Tubb6 11.4 T-box18 Tbx18 3.8 12 Ankyrin repeat domain 1 Ankrd1 11.4 Interleukin 1 receptor, type II Il1r2 3.8 13 Wingless related 2b Wnt2b 10.6 Actinin alpha 3 Actn3 3.7 14 A disintegrin and metallopept. 8 Adam8 9.5 Angiopoietin-like 2 Angptl2 3.4 15 S100 calcium binding prot. A11 S100a11 9.2 Insulin-like growth fac. bind. prot. 6 Igfbp6 3.2 16 Xanthine dehydrogenase Xdh 8.7 Follistatin Fst 3.2 17 Anthrax toxin receptor 2 Antxr2 8.5 GLI pathogenesis-related 1 Glipr1 3.1 18 SH2 domain protein 1B2 Sh2d1b2 8.4 Solute carrier family 38, member 8 Slc38a8 2.9 19 Gastrin releasing peptide Grp 7.5 Early growth response 1 Egr1 2.7 20 Vasoactive intestinal polypeptide Vip 7.4 Dihydroxyacetone kinase 2 hom. Dak 2.7 21 Protein C receptor, endothelial Procr 6.9 Tuftelin 1 Tuft1 2.6 22 Integrin alpha 7 Itga7 6.8 Serum response factor Srf 2.4 23 Lymphocyte antigen 86 Ly86 6.8 Bone marrow stromal cell antigen 1 Bst1 2.4 24 Nerve growth factor Ngf 6.5 Dopamine receptor 2 Drd2 2.4 25 Integrin alpha M Itgam 6.1 Desmocollin 3 Dsc3 2.3 26 Prokineticin receptor 2 Prokr2 6.0 Blood vessel epicardial substance Bves 2.2 27 Serine (or cysteine) pept. inh. Serpine1 5.9 ALX homeobox 1 Alx1 2.2 28 G protein-coupled receptor 133 Gpr133 5.7 Neuronal pentraxin 2 Nptx2 2.2 29 Cyclin-dep. kinase inh.1A (P21) Cdkn1a 5.6 Glucagon-like peptide 1 receptor Glp1r 1.9 30 GalaninCD180 antigen Cd180 5.4 Regulator of G-protein signaling 4 Rgs4 1.7 Upon lesion only, 1,088 genes (858 up, 230 down) were regulated more than 1.5-fold compared to un-lesioned facial nuclei. Figure 4 represents those genes modulated by facial nerve injury alone regulated by a factor ≥ 4 (colored in blue). These included reported genes induced by facial nerve injury such as Atf3 Gal Tubb6 Avpr1a Vip , and Itga7 [ 18 , 25 ]. In addition, we noted that many genes encoding G-protein coupled receptors (GPCRs), hormones and small neuropeptides were modulated by facial nerve axotomy including Avpr1a Grm3 Prokr2 Npr3 Gpr161 Gpr133 Gpr84 Gal Npy Vip , and Grp (Figure 4 , Table 1 , and Additional file 3 : Table S1). SRF-VP16 specific genes modulated after facial nerve injury are depicted in red (Figure 4 , Table 1 ). SRF-VP16 modulated two well-known gene sets, IEGs (for example Egr1 , Egr2 ) and actin cytoskeletal genes ( Actc1 , Cnn1 , Acta2 , and Actn3 ). Similar to facial nerve injury alone (see above) we noted that several potential SRF target genes encoded components of GPCR signaling ( F2rl2 , Glp1r , Rgs4 , Crhbp , Nms , Galp ; Figure 4 and Table 1 ). Finally we observed genes modulated by SRF-VP16 which might link SRF activity to immune responses investigated above (Figure 1 ). These include tissue inhibitor of metalloproteinase ( Timp1 ), interleukin receptor ( Il1r2) , galanin-like peptide ( Galp ), neuromedin ( Nms ), and interleukin 1 ( Il1f9 ) (Figure 4 , Table 1 and Additional file 3 : Table S1; see also discussion). To corroborate microarray data (Figure 4 ), qPCR analysis of selected genes employing independent cDNA samples was performed. Indeed fold changes obtained in this qPCR analysis were comparable to the microarray data (Figure 5 ). Discussion So far, SRF signaling was not assigned a major role in neuronal survival in vivo [ 15 , 16 ] in contrast to injury-related survival in vitro [ 6 , 10 , 12 , 26 ]. This suggests that SRF regulates neuron survival primarily in an injury related situation rather than in physiological brain development. In accordance, we here demonstrate a neuroprotective SRF-VP16 function in vivo , that is preventing motorneuron degeneration upon facial nucleus deafferentiation (Figure 1 ). Further, SRF-VP16 prevented expression of proapoptotic active caspase 3, enhanced regrowth of severed neurites in vitro and reduced camptothecin induced apoptosis (Figure 2 ). The latter might be directly linked to SRF-VP16 induced cytoskeletal genes such as actin isoforms ( Actc1 Acta2), calponin ( Cnn1 ), and actinin ( Actn3 ; Figure 4 and Figure 5 ). How might SRF-VP16 enhance facial motorneuron survival? SRF-VP16 suppressed active caspase 3 in vitro and reduced camptothecin-induced neuronal cell death (Figure 2 ). This SRF-VP16 mediated reduction of active caspase 3 was stronger in primary neurons lacking SRF compared to wild-type neurons (Figure 2 ). Such a reduction in proapoptotic protein levels by SRF-VP16 might enhance neuronal survival also upon facial nerve injury in vivo. To modulate expression of apoptosis related proteins, SRF-VP16 might recruit IEGs, known regulators of neuronal survival [ 8 ], such as Egr-1 and Egr-2 [ 27 ] which were induced by SRF-VP16 during facial nerve lesion (Figures 4 and 5 ). In contrast to primary neurons (Figure 2 ), we did not observe any major effect of SRF-VP16 compared to SRF-ΔMADS-VP16 on active caspase 3 and BAX expression upon facial nerve lesion of wild-type mice in vivo (data not shown). In addition SRF-VP16 did not alter Ki-67 expression, a proliferation marker. Ki-67 was strongly induced in lesioned facial motorneurons compared to unlesioned neurons at 7 days but notably not anymore at 21 days after lesion (data not shown). Thus, similar to primary neurons (Figure 2 ), in vivo SRF-VP16's potential to enhance neuronal survival might be more pronounced and only become visible in the absence of endogenous wild-type SRF. Indeed it is known that wild-type SRF competes with SRF-VP16 for access to certain SRF target gene promoters such as Bcl-2 [ 8 ]. Here, SRF-VP16 induced Bcl-2 mRNA levels in SRF-deficient embryonic stem cells whereas SRF-VP16 failed to induce Bcl-2 in wild-type cells [ 8 ]. In sum, using SRF-deficient primary neurons we demonstrate that SRF-VP16 modulates apoptosis in vitro . Thus it will be useful to employ SRF-deficient mice to unmask SRF-VP16's impact on apoptosis also in vivo. SRF-VP16 enhanced injury associated immune responses including microglia and T cell activation (Figure 3 ). SRF-VP16 enhanced microglia occupancy at facial nerve axons (Figure 3 ). In axonal injury, immune cells such as microglia remove myelin debris and have neuroprotective potential which might enhance neuronal survival [ 22 ]. Thus, by stimulating microglia and T-cell number and infiltration in the lesioned FMN and thereby increasing motorneuron cell body and facial nerve axon occupancy with these potentially neuroprotective immune cells, SRF-VP16 might enhance neuronal survival. Of note, SRF-VP16 expression in the facial nucleus was confined to motorneurons (Figure 1 and Additional file 1 : Movie S1 and Additional file 2 : Movie S2). Thus, SRF-VP16 expression in neurons might influence immune cells such as microglia and T cells via a paracrine mechanism. Such a paracrine mechanism whereby neuronal SRF affects neighboring cells via regulation of secreted molecules has been described before, for example in oligodendrocytes [ 28 , 29 ]. With regard to immune responses initiated upon facial nerve injury such a paracrine mechanism might involve, for example, cytokine/hormone secretion by neurons. Indeed, the genome-wide search for SRF-VP16 target genes upon nerve axotomy provides candidates (Figure 4 , Table 1 , and Additional file 3 : Table S1). In microarray results presented in this study (Figures 4 and 5 , Table 1 , and Additional file 3 : Table S1), facial nuclei of four animals were collected in a single biological sample. Thus, although we confirmed some results with independent cDNAs in qPCR (Figure 5 ), interpretation of microarray data is limited by a lack of statistical evaluation. Taking this into account, SRF target genes associated with up-regulated immune responses might include Il1r2 Galp Nms Il1f9 , and Timp1 . For instance Timp1, a regulator of matrix metalloproteases activity and thereby modulator of, for example, microglia migration [ 30 ] is more than 12-fold induced by SRF-VP16 (Table 1 ). How might SRF-VP16 enhance facial motorneuron survival? SRF-VP16 suppressed active caspase 3 in vitro and reduced camptothecin-induced neuronal cell death (Figure 2 ). This SRF-VP16 mediated reduction of active caspase 3 was stronger in primary neurons lacking SRF compared to wild-type neurons (Figure 2 ). Such a reduction in proapoptotic protein levels by SRF-VP16 might enhance neuronal survival also upon facial nerve injury in vivo. To modulate expression of apoptosis related proteins, SRF-VP16 might recruit IEGs, known regulators of neuronal survival [ 8 ], such as Egr-1 and Egr-2 [ 27 ] which were induced by SRF-VP16 during facial nerve lesion (Figures 4 and 5 ). In contrast to primary neurons (Figure 2 ), we did not observe any major effect of SRF-VP16 compared to SRF-ΔMADS-VP16 on active caspase 3 and BAX expression upon facial nerve lesion of wild-type mice in vivo (data not shown). In addition SRF-VP16 did not alter Ki-67 expression, a proliferation marker. Ki-67 was strongly induced in lesioned facial motorneurons compared to unlesioned neurons at 7 days but notably not anymore at 21 days after lesion (data not shown). Thus, similar to primary neurons (Figure 2 ), in vivo SRF-VP16's potential to enhance neuronal survival might be more pronounced and only become visible in the absence of endogenous wild-type SRF. Indeed it is known that wild-type SRF competes with SRF-VP16 for access to certain SRF target gene promoters such as Bcl-2 [ 8 ]. Here, SRF-VP16 induced Bcl-2 mRNA levels in SRF-deficient embryonic stem cells whereas SRF-VP16 failed to induce Bcl-2 in wild-type cells [ 8 ]. In sum, using SRF-deficient primary neurons we demonstrate that SRF-VP16 modulates apoptosis in vitro . Thus it will be useful to employ SRF-deficient mice to unmask SRF-VP16's impact on apoptosis also in vivo. SRF-VP16 enhanced injury associated immune responses including microglia and T cell activation (Figure 3 ). SRF-VP16 enhanced microglia occupancy at facial nerve axons (Figure 3 ). In axonal injury, immune cells such as microglia remove myelin debris and have neuroprotective potential which might enhance neuronal survival [ 22 ]. Thus, by stimulating microglia and T-cell number and infiltration in the lesioned FMN and thereby increasing motorneuron cell body and facial nerve axon occupancy with these potentially neuroprotective immune cells, SRF-VP16 might enhance neuronal survival. Of note, SRF-VP16 expression in the facial nucleus was confined to motorneurons (Figure 1 and Additional file 1 : Movie S1 and Additional file 2 : Movie S2). Thus, SRF-VP16 expression in neurons might influence immune cells such as microglia and T cells via a paracrine mechanism. Such a paracrine mechanism whereby neuronal SRF affects neighboring cells via regulation of secreted molecules has been described before, for example in oligodendrocytes [ 28 , 29 ]. With regard to immune responses initiated upon facial nerve injury such a paracrine mechanism might involve, for example, cytokine/hormone secretion by neurons. Indeed, the genome-wide search for SRF-VP16 target genes upon nerve axotomy provides candidates (Figure 4 , Table 1 , and Additional file 3 : Table S1). In microarray results presented in this study (Figures 4 and 5 , Table 1 , and Additional file 3 : Table S1), facial nuclei of four animals were collected in a single biological sample. Thus, although we confirmed some results with independent cDNAs in qPCR (Figure 5 ), interpretation of microarray data is limited by a lack of statistical evaluation. Taking this into account, SRF target genes associated with up-regulated immune responses might include Il1r2 Galp Nms Il1f9 , and Timp1 . For instance Timp1, a regulator of matrix metalloproteases activity and thereby modulator of, for example, microglia migration [ 30 ] is more than 12-fold induced by SRF-VP16 (Table 1 ). Conclusions In sum, this study revealed a first neuroprotective SRF function during nervous system injury in vivo . SRF is involved in development and physiological function of many other organs including liver, skin, muscle, blood vessels, and, for example, the heart [ 9 ]. Thus, SRF might also be involved in survival and cellular regeneration processes of other injured organs besides the nervous system. Abbreviations d.p.i, Days post infection; BDNF, Brain derived neurotrophic factor; CNS, Central nervous system; FMN, Facial motor nucleus; IEG, Immediate early gene; MRTF, Myocardin related transcription factor; PNS, Peripheral nervous system; SRF, Serum response factor; TCF, Ternary complex factor. Competing interest The authors declare that they have no competing interests. Authors' contributions SS and DS performed and evaluated all experiments. BK designed the study and wrote the manuscript. All authors have read and approved the final version of the manuscript. Supplementary Material Additional flie 1 Movie S1. GFP expression in the nucleus facialis infected with Ad-SRF-VP16. Click here for file Additional file 2 Movie S2. GFP expression in the nucleus facialis infected with Ad-SRF-ΔMADS-VP16. Click here for file Additional file 3 Table S1. Raw and processed data of transcriptomics. Click here for file Acknowledgements BK is supported by the DFG (Deutsche Forschungsgemeinschaft) and grants of the Schram, Gottschalk and Gemeinnützige Hertie foundation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4514824/

Immunization with a Recombinant, Pseudomonas fluorescens -Expressed, Mutant Form of Bacillus anthracis -Derived Protective Antigen Protects Rabbits from Anthrax Infection

Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis . Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B . anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg. Introduction The gram-positive bacterium Bacillus anthracis is regarded as one of the most serious of all bioterror threats because of the persistence and ease of dispersion of B . anthracis spores as well as the rapid onset and lethality of disease resulting from spore inhalation [ 1 ]. After uptake of B . anthracis spores into the lungs, the spores are trafficked to lymph nodes where they germinate, enter the bloodstream and produce large quantities of anthrax toxins, which then play critical roles in disease progression, pathology, and lethality [ 2 ]. Anthrax toxins are composed of binary combinations of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF) [ 3 ]. PA, the cell receptor-binding derivative of the toxin, combines with either LF to form lethal toxin (LT) or EF to form edema toxin (ET). Because of the central role that the toxins play in disease progression, most anthrax vaccines under development are based on neutralization of PA, the common, non-toxic component of LT and ET [ 4 ]. PA-based vaccines include Anthrax Vaccine Adsorbed (AVA or Biothrax), which is a cell-free filtrate of an avirulent, nonencapsulated variant of a B . anthracis culture that contains PA as the principal immunogen [ 5 ]. Other anthrax vaccines under development are composed of purified forms of recombinant PA (rPA) formulated with alum [ 6 – 9 ]. Recombinant PA manufacturing and alum-based formulations have been reported to be hampered by stability, potentially due to proteolytic sites on the rPA molecule [ 10 ]. A mutant form of PA (PA SNKE167-ΔFF-315-E308D, or mrPA) has been reported to have equivalent immunogenicity and increased stability vs. native (wtrPA, wild type) rPA [ 9 , 11 ]. Similar mutant isoforms have also shown wtrPA-equivalent preclinical immunological responses vs. wtrPA [ 12 ]. mrPA has two site mutations that remove proteolytically sensitive sites, altering residues RKKR at positions 164 to 167, to SNKE, and deleting residues FF at positions 314 to 315. Removal of the furin sensitive site RKKR prevents the PA from assuming its heptameric form that is responsible for pore formation and toxin action. Additionally, these mutations render the molecule more stable during post-expression purification steps [ 11 ]. The objective of this study was to test the feasibility to utilize this recombinant mrPA as an alternative to wtrPA in a subunit vaccine by comparing immunogenicity, toxin neutralization capacity, and efficacy of prototype alhydrogel-based vaccines of both wtrPA and mrPA proteins expressed and purified from the novel host system, Pseudomonas fluorescens [ 13 ]. The P . fluorescens system has proven to be a high yield expression system and to provide an excellent source (multiple grams of active protein expressed per liter in fermentation) of both the wtrPA and mrPA molecules for the studies reported herein (J. Allen, Pfenex Inc, Personal Communication). Other reports of immunogenicity of this mutant protein have come from studies in which the mrPA was prepared from derivatives of B . anthracis [ 9 , 11 ]. The series of studies reported here shows that mrPA prepared from this productive recombinant source induces a highly immunogenic and protective response in NZW rabbits, a species and strain commonly chosen to represent potential safety, immunogenicy, and efficacy of vaccines and rPA in humans. Materials and Methods Recombinant Production of Native and Mutant Protective Antigens Genes encoding both the native and mutant forms (PA SNKE167-ΔFF-315-E308D) of PA were cloned into expression plasmids and transformed into derivative strains of Pseudomonas fluorescens strain MB101 [ 13 ]. Purified native (or wild type, wtrPA) and mutant PA (mrPA) were prepared by standard methods following fermentation of P . fluorescens expression strains including mircofluidic cell lysis, lysate clarification by centrifugation and filtration, followed sequentially by ion exchange and hydrophobic interaction chromatography and final filtration steps [J. Allen and D. Retallack (Pfenex Inc), personal communication]. Vaccines and Formulation wtrPA and mrPA products were formulated (Ajinomoto/Althea Technologies, San Diego, CA) to contain 1.0 mg/mL aluminum, added as Alhydrogel (InvivoGen, San Diego, CA) in Dulbecco's phosphate buffered saline (DPBS). Dosage forms were prepared as follows: wtrPA formulations with 20 μg/mL, 5 μg/mL, and 1.25 μg/mL rPA protein in Alhydrogel; mrPA formulations with 20 μg/mL,5 μg/mL, and 1.25 μg/mL mrPA protein in Alhydrogel. The dosing solutions were prepared aseptically as 1 mL of total sample in glass vials and stored refrigerated (2–8°C). The positive control article, Anthrax Vaccine Adsorbed (AVA, trade name BioThrax), Lot FAV363, was characterized by its manufacturer (Emergent BioDefense Corporation, Lansing, MI). Negative control formulations were DPBS containing Alhydrogel. Lots of mrPA and wtrPA vaccines used for this study were characterized for activity by a LBERI Good Laboratory Practice (GLP 21CFR) validated potency assay (mouse anti-PA antibody ELISA and Toxin Neutralization Assay [TNA]) utilizing serum from immunized AJ mice [ 14 , 15 ]. Rabbits Forty-five male and 45 female New Zealand white (NZW) rabbits, certified Pasteurella -free (Covance Inc. Princeton, NJ), were placed on study. The rabbits weighed between 2.57–3.33 kg (females) and 2.45–3.29 kg (males) and were approximately 21–23 weeks old on Day 0 (study start). Rabbits were identified by subcutaneously placed Biometric Data Systems telemetry chips (Seaford, DE) and randomized into study groups using a validated computerized data acquisition system (Provantis, Instem LSS Ltd., Staffordshire, England) based on weight. Body weights of individual animals were ±20% of the group mean for each gender and groups were approximately equal mean body weights at study start prior to vaccination. Rabbits were housed individually in stainless steel/plastic cages in temperature and humidity controlled rooms on a 12h light cycle and fed Harlan Teklad Global Diet 2031C (Madison, WI) with free access to water. Prior to pathogen challenge, all test rabbits were conditioned and acclimated to restraint by being placed into rabbit exposure boxes for 10 ± 5, 30 ± 5 and 60 ± 5 minutes on separate days. The last conditioning session occurred within three days of exposure. All animimal procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Lovelace Respiratory Research Institute Animal Care and Use Committee. Animals were observed at least twice per day (morning and afternoon) prior to B . anthracis spore challenge and three times per day after challenge (see below) for signs of morbidity and mortality. Based on individual animal condition, additional observations for morbity and mortality occurred at the discretion of the Study Director in consultation with technical and veterinary staff. Examinations were oriented toward (1) identifying dead or moribund animals, and (2) documenting the onset of any abnormal clinical signs. The Study Director in consultations with the veterinary and technical staff made decisions regarding the euthanasia of moribund animals. Criteria for moribund status included severe respiratory distress, persistent recumbency and weakness, unresponsiveness to touch or external stimuli, extreme weight loss, or a combination of these observations. When morbidity was observed, all feasible actions were taken to limit pain and suffering by euthanizing animals by intravenous (IV) injection of an overdose of a barbiturate based sedative (Euthasol, Virbac, Ft. Worth, TX). Due to the nature of the study investigating the efficacy of a vaccine, no analgesics or anesthetics could be administered. Vaccinations and Study Design The study design and endpoint analyses are described in Tables 1 and 2 . Animal groups were derived from similar sample sizes utilized in previous reports of rPA and alum-based experimental animal vaccinations, immunogenicity, antibody neutralization, and efficacy/ protection endpoint determinations [ 14 – 17 ]. Groups of five male and five female New Zealand white (NZW) rabbits were vaccinated via intramuscular (IM) injection (hind leg) on Days 0 and 28 with 0.5 mL of one of six lots of wtrPA or mrPA as described above. Vaccinations were performed by group on each vaccination day. An AVA positive control group (human dose, 0.5 mL) and a vehicle control group were also included and dosed (0.5 mL, IM) on Days 0 and 28. An untreated control group was also included. Rabbits surviving to study conclusion were observed for 84 days following initial vaccination. All rabbits survived through the 28-day blood draw. Ten weeks after initial vaccination, rabbits were challenged with a target dose of 200 ± 50 LD 50 of B . anthracis Ames spores (1.1 X 10 5 ) [ 16 , 18 ]. Note: Two rabbits (one mrPA-10 and and one wtrPA-10) died or were euthanized just prior or just after B . anthracis challenge from non-infection related causes (n = 9 reported in some endpoints). Blood was collected from the marginal ear vein or central ear artery for all blood-associated endpoints. Serum was retained for the TNA and the ELISA at a time point pre-vaccination (baseline); at Days 14, 28, 42, and 65 following initial vaccination; and at euthanasia. Blood was collected on Days 65, 72, and 74 post-challenge and at euthanasia for hematology and clinical chemistry endpoints. Blood was also collected on Days 65, 71 through 74 and at post-challenge euthanasia for bacteriology. Sera were isolated on Days 65, 71 through 74 and at post-challenge euthanasia to detect PA by the electrochemiluminescence assay (ECL). Following challenge, rabbits that were found dead or euthanized were subject to a limited necropsy and samples were collected for bacteriology and histopathology assessments. 10.1371/journal.pone.0130952.t001 Table 1 Experimental Design—Vaccination a . Test Material Group Test Material (Formulation Concentration, μg/ mL) Target Dosage Route Vaccination Schedule Dose Volume Number of Rabbits b 1 Control None None None None 5 / 5 = 10 2 Vehicle Control (0 μg) IM Day 0 and 28 0.5 mL 5 / 5 = 10 3 wtrPA- 20 High (10 μg) IM Days 0 and 28 0.5 mL 5 / 5 = 10 4 wtrPA- 5 Mid (2.5 μg) IM Days 0 and 28 0.5 mL 5 / 5 = 10 5 wtrPA- 1.25 Low (0.625μg) IM Days 0 and 28 0.5 mL 5 / 5 = 10 6 mrPA-20 High (10 μg) IM Day 0 and 28 0.5 mL 5 / 5 = 10 7 mrPA-5 Mid (2.5 μg) IM Day 0 and 28 0.5 mL 5 / 5 = 10 8 mrPA-1.25 Low (0.625μg) IM Days 0 and 28 0.5 mL 5 / 5 = 10 9 AVA Human dose IM Days 0 and 28 0.5 mL 5 / 5 = 10 Total = 45 / 45 = 90 a Animals vaccinated (0.5 mL per injection) with test or control article on Days 0 and 28. b Equivalent numbers per sex, 5 males and 5 females per group. 10.1371/journal.pone.0130952.t002 Table 2 Experimental Procedures and Schedule. Study Day Procedure Pre 0 7 14 21 28 35 42 Pre 70 71 72 73 74 84 Clinical Observations a X ← X (2x daily) → X (3x daily) X Body Weight b X ← X (weekly) → Vaccination c X X Blood (ELISA, TNA) d X X X X X X Challenge e X Blood (Hematology) f X X X X Blood (Clin.Chem.) g X X X X Blood (Bacteriology) h X X X X X X Blood (ECL) i X X X X X X Target Blood Volume (mL) 5 5 5 5 8 2 4 2 4 9 Necropsy X j X k a Thrice daily observations performed on Days 72–74. Twice daily observations performed other days. b Body weight obtained at randomization, on Day 0 and weekly thereafter. c Animals vaccinated via intramuscular injection on Day 0 or Days 0 and 28 (see Table 1 ). d Blood collected and sera isolated for TNA and ELISA on Days -6, 14, 28, 42 and 65 ± 4, when moribund euthanized or at terminal euthanasia (Day 84). e Rabbits challenged with 200 x ± 50 LD 50 B . anthracis (Ames) spores. The published inhalation LD 50 for NZW rabbits is 1.1 x 10 5 spores. f Blood collected for CBC and differential. g Blood collected and sera isolated for clinical chemistry parameters. h Blood collected for quantitative bacteriology. i Blood collected and sera isolated for electrochemiluminescence assay (ECL). j Moribund euthanized or found dead rabbits received a limited gross necropsy. k Euthanized or found dead rabbits received a gross necropsy and select tissues collected for bacteriology or histopathology. Enzyme-linked Immunosorbent Assay (ELISA) Sera collected pre-vaccination and on Days 14, 28, 42, 65 and 84 were assayed for the presence of anti-PA antibodies by an Lovelace Respiratory Research Institute-developed Good Laboratory Practice (GLP 21 CFR) validated ELISA following general methods [ 16 ]. Sera collected at necropsy were 0.2 μM filter-sterilized prior to analyses. Antibodies to PA were measured in 96-well format plates (NUNC flat-bottomed wells; ThermoFisher, Waltham, MA) coated with 1 μg/mL of rPA diluted in Phosphate Buffered Saline (PBS) in a volume of 100 μL per well, sealed and incubated at 4°C for 12–18 hours. After incubation, plates were washed six times with wash buffer (0.1% Tween 20 in PBS) using a Bio-Tek ELx405 plate washer (Winooski, VT). Serum samples were two-fold serially diluted in assay buffer (5% non-fat dry milk, 0.1% Tween 20; PBS) following an initial 1:250 dilution in assay buffer. Single samples were added to the plate at a volume of 100 μl per well and incubated for one hour at 37°C. Following six washes, 100 μL of alkaline phosphatase-labeled goat anti-rabbit IgG (H+L-specific Catalog # 4751–1516; Kirkegard & Perry Laboratories, Gaithersburg, MD) diluted 1:2000 in assay buffer was added to each of the wells and incubated for one hour at 37°C. After six washes, 100 μl of 4-Nitrophenphenyl phosphate disodium salt hexahydrate in detection buffer (12.1% Tris (hydroxymethl) animomethane in PBS) was added to each of the wells and incubated covered for 30 minutes at 37°C. Plates were read at 405 nm using a Bio-Tek μQuant microplate reader and the data analyzed using Gen5 Data Analysis Software (Bio-Tek). Logistic regression analysis based on a 4-paramater curve was performed to determine the antibody concentration of various dilutions of test samples based on a standard curve constructed from pooled rabbit sera immunized with rPA with a concentration of 480 μg/mL anti-PA IgG [NR-3839; Biodefense and Emerging Infections Research Resources Repository (BEI), Manassas, VA]. At least five of seven data points in a two-fold dilution series were used to construct the standard curve based on the best R2 value. Only samples that fell between the upper and lower asymptotes of the 4-paramater curve were analyzed. If multiple dilutions of unknown samples interpolated to the standard curve, the μg/mL assigned was an average of those returned from each dilution. The lower limit of detection was determined by using the greatest dilution of rabbit reference serum that interpolated accurately (+/- 20%) to the standard curve and was at least three times the O.D. of the blank wells multiplied by the lowest dilution of the rabbit reference sera (1:250). Samples interpolating below the limit of detection were assigned that limit of detection multiplied by the lowest dilution of that sera. Toxin Neutralization Assay (TNA) Sera collected pre-vaccination and on Days 14, 28, 42, 65 and 84 were assayed utilizing a Good Laboratory Practice (GLP 21 CFR) validated method for the presence of functional antibodies capable of neutralizing the toxic activity of B . anthracis lethal toxin based on that described [ 15 ]. Sera collected at necropsy were 0.2 μM filter-sterilized prior to analyses. J774A.1 mouse macrophage cells (American Type Culture Collection, Manassas, VA) were plated at 4 x 10 5 cells per mL of maintenance medium (Dulbecco's Modified Eagles medium with high glucose [4.5 g/L], and L-glutamine) in a 96-well flat bottom cell-culture plate (Costar, Corning, Tewksbury MA). The media was supplemented with 5% Fetal Bovine Serum, 1 mM sodium pyruvate, 100 units/mL penicillin and 100 μg/mL streptomycin sulfate, and 10 mM HEPES buffer, and the plates were incubated for 17–19 hours at 37°C with 5% CO 2 . Anthrax lethal toxin was made by the addition of rPA (BEI: NR-140; 0.1 μg/mL) and recombinant Lethal Factor (rLF; BEI: NR-4367; 0.08 μg/mL) to maintenance medium. Sera from the experimental rabbits was diluted 1:100 in maintenance medium and then two-fold serially diluted in a 96-well round bottom plate (Costar) to a final volume of 75 μL per well. An equal amount of lethal toxin was added to each well, except the reference row which received maintenance medium (75 μL) only. The positive control rabbit reference serum (BEI; NR-3839) was diluted 1:5 in maintenance medium prior to the 1:100 dilution for a working dilution of 1:500. The lethal toxin and rabbit sera were incubated together at 37°C for 30 minutes. The medium was removed from the cells and was replaced with 100 μL of dilutions of the toxin/neutralizing antibody mixtures, after which incubation proceeded as above for 4 hours. To assess cell viability, 25 μL of tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; 5 mg/mL) was added to each of the wells at the end of the 4 hour incubation and the plates were then incubated as above for an additional 2 hours. Afterwards, 100 μL of solubilization buffer (50% dimethyl formamide, Sigma with 200 mg/mL sodium dodecyl sulfate) was added to each of the wells and the final incubation proceeded as above for 16–20 hours. Plates were read at 570 nm using a Bio-Tek μQuant microplate reader and the data were analyzed using Gen5 Data Analysis Software (Bio-Tek). The ED 50 , the reciprocal of the dilution that inhibits 50% of the cell death due to the lethal toxin, was obtained by analysis of the 4-parameter curve. The Neutralization Factor (NF 50 ) was determined as the ratio of the test sample ED 50 to ED 50 of the positive control reference serum. B . anthracis Spore Preparation B . anthracis Ames spores used for rabbit inhalation challenge studies were prepared essentially as described [ 17 ]. Briefly, the B . anthracis Ames strain was propagated at 34 ± 2°C in a shaker incubator for 48 hrs in Modified Schaeffer's Medium (2xSG). Spores were heat-shocked (65°C for 45 minutes), harvested by centrifugation (washed three times/ centrifuged-isolated in sterile water for injection), suspended in sterile water for injection and kept at 4 ± 2°C until use. Spores were heat-shocked a second time prior to use. Spore purity was verified on tryptic soy blood agar, MacConkey's agar and phenylethyl alcohol agar by incubation at 37°C for up to 48 hrs. Spore lot titer was determined from serial dilutions of the spore preparation plated on tryptic soy agar (TSA) by incubation at 37°C for up to 48 hrs. Spore content was microscopically verified as >95% and presence of virulence plasmids PX01 and PX02 were confirmed by PCR. Inhalation Challenge On Day 70 post-vaccination rabbits received a targeted dose of 200 ± 50 LD 50 of B . anthracis Ames by nose-only exposure [ 16 , 18 ]. The spores were nebulized in a Collison nebulizer (MRE-3 jet, BGI, Inc., Waltham, MA). An all glass impinger (AGI) sample of the bioaerosol was obtained to characterize presented inhalation dose. The un-anesthetized rabbit rested in a purpose built plethysmography box juxtaposed for nose only exposure essentially as described [ 16 ]. The rabbit's breathing frequency, tidal volume, and minute volume were each measured during the exposure [ 16 , 17 , 19 ]. The duration of the exposure was based on the total volume of air inhaled by the rabbit. Bacterial aerosol concentrations were confirmed by quantitative bacterial culture of AGI samples using standard dilution plating on TSA plates. Cultures were incubated at 37 ± 2°C for 18–24 hours prior to counting. The target particle size of the aerosol was 1 to 3 μm and was determined using a GRIMM Portable Aerosol Spectrometer Model 1.109 [GRIMM Aerosol Technik GmbH & Co. KG, Ainring, Germany] for 0.5 to 20 μm particles. Aerosol dose was calculated after direct measurement of inhaled volume and aerosol concentration using the following formula: Dose = (C × V), where C is the concentration of viable pathogen in the exposure atmosphere, and V is the volume inhaled. Hematology and Serum Chemistries Blood was collected on Days 65, 72, 74 and 84 post vaccination for complete blood count (CBC) and sera isolated for a standard panel of serum parameters. CBC determinations were made using an Advia 120 (Siemens AG, Erlangen, Germany). Serum chemistries were determined using a Hitachi 911 Chemistry Analyzer or a Hitachi Modular Analytics Clinical Chemistry System (Roche Diagnostics, Indianapolis, IN). Electrochemiluminescence Assay (ECL) Sera collected on Days 65, 71 through 74 and at euthanasia was assayed for the presence of anthrax protective antigen in serum using an in-house, GLP validated, qualitative ECL assay [ 20 ]. This assay measured the PA in serum using Meso-Scale Discovery (MSD, Rockville, MD) ECL technology and the MSD B . anthracis PA assay kit. The PA was detected by the addition of the MSD detector antibody (STAG [sulfonated derivative of Ruthenium (II) tris-bipyridine tag]-labeled anti-PA antibody). The amount of PA present in the sample was determined by the amount of light that is emitted upon electrochemical stimulation of the STAG initiated at the electrode surface of the kit's microplate. Bacteriology Blood was collected on Days 65, 71 through 74 and 84 for determination of qualitative or quantitative (Day 84) bacteriology. No baseline serological analyses were reported. All rabbits that were found dead or euthanized post-challenge were necropsied and had select tissues assayed for quantitative bacteriology. For qualitative assessments, collected blood was plated onto a single, sterile 90 mm tryptic soy agar (TSA) plate and incubated at 37°C for 16–24 hr, after which the presence or absence of B . anthracis colonies was determined. For quantitative assessments, blood and tissue samples were serially diluted in sterile 1% peptone. From each dilution, 100 μL was removed and plated onto sterile 90 mm TSA plates in triplicate and allowed to incubate at 37°C for 16–24 hr. B . anthracis titer (colony forming units [CFU]/mL) was calculated: C F U m L = m e a n C F U c o u n t × T o t a l d i l u t i o n f a c t o r The total dilution factor included the media plate inoculum volume of 100 μL (i.e., an additional 10-fold dilution was included in the final calculations to achieve the titer in terms of CFU/mL). Necropsy and Pathology Rabbits surviving to the scheduled sacrifice or determined to be moribund were euthanized by intravenous (IV) injection of an overdose of a barbiturate based sedative (Euthasol, Virbac, Ft. Worth, TX). Lung, spleen, heart, and tracheal-bronchial lymph nodes (when present) were collected for bacteriology and histopathology. Tissue sections were fixed in 10% neutral-buffered formalin (NBF), cut, mounted on slides, stained with hematoxylin and eosin (H&E) and microscopic findings evaluated for incidence and severity by a board certified veterinary pathologist. Findings were given a score from 1 to 4, based upon a subjective assessment of the overall severity in the tissue present on the slide (1 = minimal; 2 = mild; 3 = moderate; 4 = marked). Where appropriate, an assessment of distribution was also made (F = focal, M = multifocal, D = diffuse, Ws = widespread, Lx = locally extensive). A qualitative assessment of bacterial burden was also made where appropriate. Statistics and Primary Experimental Outcomes Assessed Survival, immunogenicity, antibody nuetralizations were the primary outcomes assessed for this study. The number of rabbits dying and mean time to death and/or mean survival time was determined for each group. Survival analysis was tested by log-rank (Mantel-Cox) test analysis [Prism 5.04 (GraphPad Software, Inc.)]. In addition, survival at study end for each experimental group was analyzed by Chi-square test. The body weight/change data were statistically analyzed for each time point for differences in treatment group by analysis of variance (ANOVA) and, if appropriate, Dunnett's test. A p value of ≤ 0.05 was considered significant. TNA and ELISA data were log-transformed prior to being statistically analyzed by repeated-measures ANOVA to determine whether any differences exist between the experimental groups. A p 95% and presence of virulence plasmids PX01 and PX02 were confirmed by PCR. Inhalation Challenge On Day 70 post-vaccination rabbits received a targeted dose of 200 ± 50 LD 50 of B . anthracis Ames by nose-only exposure [ 16 , 18 ]. The spores were nebulized in a Collison nebulizer (MRE-3 jet, BGI, Inc., Waltham, MA). An all glass impinger (AGI) sample of the bioaerosol was obtained to characterize presented inhalation dose. The un-anesthetized rabbit rested in a purpose built plethysmography box juxtaposed for nose only exposure essentially as described [ 16 ]. The rabbit's breathing frequency, tidal volume, and minute volume were each measured during the exposure [ 16 , 17 , 19 ]. The duration of the exposure was based on the total volume of air inhaled by the rabbit. Bacterial aerosol concentrations were confirmed by quantitative bacterial culture of AGI samples using standard dilution plating on TSA plates. Cultures were incubated at 37 ± 2°C for 18–24 hours prior to counting. The target particle size of the aerosol was 1 to 3 μm and was determined using a GRIMM Portable Aerosol Spectrometer Model 1.109 [GRIMM Aerosol Technik GmbH & Co. KG, Ainring, Germany] for 0.5 to 20 μm particles. Aerosol dose was calculated after direct measurement of inhaled volume and aerosol concentration using the following formula: Dose = (C × V), where C is the concentration of viable pathogen in the exposure atmosphere, and V is the volume inhaled. Hematology and Serum Chemistries Blood was collected on Days 65, 72, 74 and 84 post vaccination for complete blood count (CBC) and sera isolated for a standard panel of serum parameters. CBC determinations were made using an Advia 120 (Siemens AG, Erlangen, Germany). Serum chemistries were determined using a Hitachi 911 Chemistry Analyzer or a Hitachi Modular Analytics Clinical Chemistry System (Roche Diagnostics, Indianapolis, IN). Electrochemiluminescence Assay (ECL) Sera collected on Days 65, 71 through 74 and at euthanasia was assayed for the presence of anthrax protective antigen in serum using an in-house, GLP validated, qualitative ECL assay [ 20 ]. This assay measured the PA in serum using Meso-Scale Discovery (MSD, Rockville, MD) ECL technology and the MSD B . anthracis PA assay kit. The PA was detected by the addition of the MSD detector antibody (STAG [sulfonated derivative of Ruthenium (II) tris-bipyridine tag]-labeled anti-PA antibody). The amount of PA present in the sample was determined by the amount of light that is emitted upon electrochemical stimulation of the STAG initiated at the electrode surface of the kit's microplate. Bacteriology Blood was collected on Days 65, 71 through 74 and 84 for determination of qualitative or quantitative (Day 84) bacteriology. No baseline serological analyses were reported. All rabbits that were found dead or euthanized post-challenge were necropsied and had select tissues assayed for quantitative bacteriology. For qualitative assessments, collected blood was plated onto a single, sterile 90 mm tryptic soy agar (TSA) plate and incubated at 37°C for 16–24 hr, after which the presence or absence of B . anthracis colonies was determined. For quantitative assessments, blood and tissue samples were serially diluted in sterile 1% peptone. From each dilution, 100 μL was removed and plated onto sterile 90 mm TSA plates in triplicate and allowed to incubate at 37°C for 16–24 hr. B . anthracis titer (colony forming units [CFU]/mL) was calculated: C F U m L = m e a n C F U c o u n t × T o t a l d i l u t i o n f a c t o r The total dilution factor included the media plate inoculum volume of 100 μL (i.e., an additional 10-fold dilution was included in the final calculations to achieve the titer in terms of CFU/mL). Necropsy and Pathology Rabbits surviving to the scheduled sacrifice or determined to be moribund were euthanized by intravenous (IV) injection of an overdose of a barbiturate based sedative (Euthasol, Virbac, Ft. Worth, TX). Lung, spleen, heart, and tracheal-bronchial lymph nodes (when present) were collected for bacteriology and histopathology. Tissue sections were fixed in 10% neutral-buffered formalin (NBF), cut, mounted on slides, stained with hematoxylin and eosin (H&E) and microscopic findings evaluated for incidence and severity by a board certified veterinary pathologist. Findings were given a score from 1 to 4, based upon a subjective assessment of the overall severity in the tissue present on the slide (1 = minimal; 2 = mild; 3 = moderate; 4 = marked). Where appropriate, an assessment of distribution was also made (F = focal, M = multifocal, D = diffuse, Ws = widespread, Lx = locally extensive). A qualitative assessment of bacterial burden was also made where appropriate. Statistics and Primary Experimental Outcomes Assessed Survival, immunogenicity, antibody nuetralizations were the primary outcomes assessed for this study. The number of rabbits dying and mean time to death and/or mean survival time was determined for each group. Survival analysis was tested by log-rank (Mantel-Cox) test analysis [Prism 5.04 (GraphPad Software, Inc.)]. In addition, survival at study end for each experimental group was analyzed by Chi-square test. The body weight/change data were statistically analyzed for each time point for differences in treatment group by analysis of variance (ANOVA) and, if appropriate, Dunnett's test. A p value of ≤ 0.05 was considered significant. TNA and ELISA data were log-transformed prior to being statistically analyzed by repeated-measures ANOVA to determine whether any differences exist between the experimental groups. A p < 0.05 was considered to be significant. If significant, a post hoc multiple comparison t-test with Bonferroni's adjustment was performed. A significant p value was dependent on whether it is smaller than 0.05/ (number of tests). Statistical analyses were conducted in Statview 5.0.1 (SAS, Cary NC). Data is presented as a group with a geometric mean of 95% confidence intervals. Results Induction and Measurement of Anti-PA Antibodies A rabbit immunogenicity and B . anthracis spore challenge study was designed (Tables 1 and 2 ) to test the effectiveness of both the wtrPA and mrPA molecules at a broad range of doses (dosed at over a 16-fold range) in a two dose vaccine administration regimen to induce an anti-PA antibody response. The approved anthrax vaccine (Biothrax) administered at the human dose, 0.5 mL, served as the positive control. Results for the anti-PA ELISA analyses performed on serum samples collected as outlined in Table 2 are shown in Fig 1 and Tables 3 and 4 . Multiple vaccine groups' immunogenicity responses over time (curve response) were significantly different from other groups when compared by repeated-measures ANOVA and Bonferroni/Dunn post-hoc test when IgG anti-PA values from Days -1 through 65 were analyzed ( Table 4 ). The highest dose of wtrPA (10 μg) induced a significantly greater immune response than did the two lower doses (2.5 and 0.625 μg) when considering the blood draws Days -1 through 65, Table 4 . The highest dose of mrPA induced a significantly higher total antibody titer than either of the two lower doses. The high dose of the mrPA also induced a significantly higher anti-PA response than the human dose of AVA. The 2.5μg mrPA dose was not statistically different from AVA. The mrPA outperformed the wtrPA at all comparable doses. 10.1371/journal.pone.0130952.g001 Fig 1 Ig anti-PA ELISA values (μg/mL). (A) generated from vaccinated wild type and (B) mutant rPA (geometric mean with error bars representing the 95% confidence interval). 10.1371/journal.pone.0130952.t003 Table 3 Study Summary of Fate and Immune Status Relevant to B. anthracis Challenge on Day 70. IgG anti- PA (μg/mL) Geometric Mean ± C.I. TNA ED50 Geometric Mean ± C.I. Test Group (μg rPA) Survival (Alive/Total) Day 42 Day 65 Day 42 Day 65 Naïve Control 0% (0/10) 15 (15, 15) a 17 (12, 24) 1 (1, 1) b 1 (1, 1) Vehicle Control (0 μg rPA) 0% (0/10) 15 (15, 15) 15 (15, 15) 1 (1, 1) 1 (1, 1) wtrPA 10 100% (9/9) 869(674, 1121) 303(240, 381) 27324(18802, 39709) 6608(4522, 9656) wtrPA 2.5 90% (9/10) 456(325, 640) 190(136, 264) 17670(11320, 27583) 4282(2310, 7937) wtrPA0.625 80% (8/10) 234(182, 301) 78(61, 99) 8992(6394, 12644) 1176(499, 2773) mrPA 10 100% (9/9) 1008(800, 1271) 416(338, 512) 37725(20959, 67903) 13,437(7561, 23882) mrPA 2.5 100% (10/10) 800(553, 1158) 306(217, 432) 22176(14214, 34598) 8695(4706, 16065) mrPA 0.625 100% (10/10) 456(335, 621) 159(114, 224) 13674(9504, 19676) 2947(1366, 6358) AVA 100% (10/10) 974(802, 1183) 363(298, 443) 17523(10933, 28085) 4798(2975, 7737) a Lower limit of quantitation < 30 μg/mL. b Samples which returned an incalculable ED50 (by Gen 5 software) were assigned an ED50 value of 1. 10.1371/journal.pone.0130952.t004 Table 4 p Values Associated with Comparison of Ig anti-PA Values between Vaccine Groups as Analyzed by Repeated Measures ANOVA. Vaccine Group WTrPA10 WTrPA2.5 WTrPA0.625 mrPA10 mrPA2.5 mrPA 0.625 AVA wtrPA 10 μg – wtrPA 2.5 μg < .0011 ↓ – wtrPA 0.625 μg < .0001 ↓ NS – mrPA 10 μg < .0001 ↑ < .0001 ↑ < .0001 ↑ – mrPA 2.5 μg NS < .0001 ↑ < .0001 ↑ < .0001 ↓ – mrPA 0.625 μg NS < .0008 ↑ < .0001 ↑ < .0001 ↓ NS – AVA < .0002 ↑ < .0001 ↑ < .0001 ↑ < .0009 ↓ NS < .0003 ↑ – Ig anti-PA levels from blood draws between day -1 and 65 were analyzed by repeated measures ANOVA. The Ig anti-PA response of non-vaccinated groups (None and Vehicle, not shown above) were significantly lower (p < 0.0001) than every other group except each other. ↓ Indicates the Ig anti-PA response of the vaccine group listed on the left is significantly less than the corresponding vaccine group listed on the top of the table. ↑ Indicates the Ig anti-PA response of the vaccine group listed on the left is significantly greater than the corresponding vaccine group listed on the top of the table. NS, no statistical significance between the two groups. Subsequent to the boost vaccination delivered on Day 28, anti-PAantibodies increased in all dose groups to a similar level prior to challenge. The levels of total anti-PA antibody increased post-aerosol challenge in all vaccine groups, Fig 1 . Often the highest post-challenge responses were measured in those rabbits vaccinated with the lowest dose of rPA. Induction and Measurement of Toxin Neutralizing Antibodies In addition to the characterization of the appearance of total anti-PA antibodies (described above), anthrax holotoxin neutralizing anti-PA-antibodies were also measured by a toxin neutralization assay (Tables 1 and 2 ). The approved anthrax vaccine (Biothrax) administered at the human dose, 0.5 mL, once again served as the positive control. Fig 2 and Tables 3 and 5 illustrate the results of the TNA for each vaccine group. Multiple vaccine groups were significantly different from other groups when time-based ED 50 data were analyzed by repeated-measures ANOVA and Bonferroni/Dunn post-hoc test over Days -1 through 65, Table 5 . The highest dose of wtrPA induced a significantly greater immune response than did the two lower doses of wtrPA when considering the blood draws Days -1 through 65. The highest dose of mrPA was similar to the 2.5μg dose of mrPA, but induced a significantly higher amount of functional antibody than either the lowest dose of mrPA or both the 2.5 μg and 0.625 μg doses of wtrPA. Also, higher ED 50 values were measured in rabbits from the highest mrPA dose group (10 μg, p = 0.0002) as compared to AVA. The mrPA outperformed wtrPA at all comparable doses and the two lower doses of mrPA were equivalent and not statistically different from the highest wtrPA doses or AVA. 10.1371/journal.pone.0130952.g002 Fig 2 rPA antibody-specific Toxin Neutralization Assay (TNA) and ED50 values (μg/mL). (A) generated from vaccinated wild type and (B) mutant rPA (geometric mean with error bars representing the 95% confidence interval). 10.1371/journal.pone.0130952.t005 Table 5 p Values Associated with Comparison of ED50 Values between Vaccine Groups as Analyzed by Repeated Measures ANOVA Vaccine Group WTrPA10 WTrPA2.5 WTrPA0.625 mrPA10 mrPA2.5 mrPA0.625 AVA wtrPA 10 μg – wtrPA 2.5 μg < .0001 ↓ – wtrPA 0.625 μg < .0001 ↓ NS – mrPA 10 μg .0003 ↑ < .0001 ↑ < .0001 ↑ – mrPA 2.5 μg NS < .0001 ↑ < .0001 ↑ NS – mrPA 0.625 μg NS < .0001 ↑ < .0001 ↑ < .0001 ↓ NS – AVA NS < .0001 ↑ < .0001 ↑ .0002 ↓ NS NS – TNA ED 50 values from blood draws between day -1 and 65 were analyzed by repeated measures ANOVA. The ED 50 response of non-vaccinated groups (None and Vehicle, not shown) were significantly lower (p < 0.0001) than every other group except each other. ↓ Indicates the ED 50 response of the vaccine group listed on the left is less than the corresponding vaccine group listed on the top of the table. ↑ Indicates the ED 50 response of the vaccine group listed on the left is greater than the corresponding vaccine group listed on the top of the table. NS, no statistical significance between the reactions of the two groups. In general, an rPA dose response was observed prior to the Day 28 boost, particularly in the wtrPA dosed rabbits. After the Day 28 boost, the differences between doses of rPA, either wild type or mutant, became less apparent and the levels declined between Day 48 and 65 prior to challenge. Neutralizing antibody levels increased post-aerosol challenge in all vaccine groups as seen in Fig 2 . Mortality Following B. anthracis Spore Challenge Mortality data and Kaplan-Meier data are shown in Table 3 and Fig 3 . All rabbits survived until challenge with the exception of single 10ug mrPA female rabbit that died during conditioning to inhalation exposure restraint. One 10 μg wtrPA rabbit was euthanized subsequent to challenge due to partial paralysis unrelated to exposure. All Naïve and Vehicle Control group rabbits died due to anthrax infection two to five days post-challenge (Days 72–75). One 1.25 and two 0.625 μg wtrPA-immunized rabbits died due to anthrax infection on Day 74. All 10 μg wtrPA, all mrPA vaccinated, and AVA rabbits survived challenge. There were statistically significant differences (p < 0.0001, Mantel-Cox; Chi-square) in survival and time to death for the Naïve and Vehicle Control groups compared to all other test groups. A statistically significant difference was not observed for the vaccinated groups [2.5 μg wtrPA and 0.625 μg wtrPA)] that had post-challenge deaths compared to the remaining test groups. 10.1371/journal.pone.0130952.g003 Fig 3 Kaplan-Meier Survival Curve of vaccinated, unvaccinated control, and sham vaccinated control rabbits. Bacteriological Evaluation of Vaccinated Rabbits Challenged with B . anthracis Spores Six of ten naïve control rabbits and eight of ten vehicle control rabbits were positive for bacteremia in the blood (data not shown). Of vaccinated rabbits, there were only three rabbits that demonstrated bacteremia at the initial blood (Day 71) draw following challenge, but this resolved in these rabbits by the next blood collection time point (Day 74) and the rabbits survived to Day 84. Of the three wtrPA-vaccinated rabbits that succumbed to disease, none had bacteremia present in any blood sample. Negative control rabbits (unvaccinated and those vaccinated with vehicle) were shown to have high levels of tissue burden for all tissues tested, Table 6 and Fig 4 . All other rabbits that succumbed to disease presented with marked tissue burden in the majority of tissues tested. In the rabbits that succumbed to disease, lung burden ranged from 7.56 x 10 4 to 2.21 x 10 8 CFU/mL. The rabbits who survived to study termination were found to have tissue burden below the detectable limits or, in a few cases, low tissue burden was seen in the lungs. In the three wtrPA vaccinated rabbits that succumbed to disease prior to study termination, high tissue burden was seen in the lung and low levels in the tracheobronchial lymph nodes (TBLN) and spleen. One of the animals that succumbed had high tissue burden in the lung as well as all other tissues collected. The rabbits in the mrPA vaccine groups all survived to study termination and any B . anthracis present in the tissues was at lower levels (less than 30 CFU/100 μL inocula) than wtrPA vaccine group rabbits. 10.1371/journal.pone.0130952.t006 Table 6 Summary of Recovery of B . anthracis from Select Tissues of Inhalational Spore Challenged New Zealand White Rabbits. Dose a Lung Liver Spleen TBLN Naïve Control Mean b 7.13 6.25 6.00 6.87 SD — 0.600 1.293 0.408 GeoM c 1.34 x 10 7 1.79 x 10 6 9.94 x 10 5 7.38 x 10 6 N 1 10 10 10 Vaccinated Control Mean 8.08 6.33 6.24 6.81 SD — 1.118 1.314 0.563 GeoM 1.20 x 10 8 2.14 x 10 6 1.75 x 10 6 6.41 x 10 6 N 1 10 10 10 wtrPA 10 mg Mean 3.29 BDL BDL d BDL SD — BDL BDL BDL GeoM 1.95 x 10 3 BDL BDL BDL N 1 10 10 10 wtrPA 2.5 mg (2.5 mg) Mean 2.51 BDL 0.22 0.75 SD 1.585 BDL 0.700 1.022 GeoM 3.24 x 10 2 BDL 6.65 x 10 −1 4.57 x 10 0 N 7 10 10 10 wtrPA 0.625 mg Mean 5.56 0.72 1.05 1.38 SD 3.916 1.917 2.393 2.639 GeoM 3.59 x 10 5 4.28 x 10 0 1.02 x 10 1 2.31 x 10 1 N 2 10 10 10 mrPA 10 mg Mean 2.03 0.26 BDL 0.35 SD 1.755 0.835 BDL 0.744 GeoM 1.05 x 10 2 8.37E-01 BDL 1.25 x 10 0 N 3 10 10 10 mrPA 2.5 mg Mean 3.40 BDL BDL 0.40 SD — BDL BDL 0.853 GeoM 2.49 x 10 3 BDL BDL 1.53 x 10 0 N 1 10 10 10 mrPA 0.625 Mean 2.67 BDL BDL 0.23 SD 0.005 BDL BDL 0.736 GeoM 4.71 x 10 2 BDL BDL 7.09 x 10 −1 N 2 10 10 10 AVA (human dose) Mean 3.01 BDL BDL 0.17 SD 0.761 BDL BDL 0.543 GeoM 1.03 x 10 3 BDL BDL 4.85E-01 N 3 10 10 10 a Treatment = Dose delivered intramuscularly (IM) on Days 0 and 28 in 0.5 mL volume b Mean = Log 10 (CFU/g + 1) c Geometric mean of CFU/g d BDL = Below detection limit (< 68–70 CFU/g) 10.1371/journal.pone.0130952.g004 Fig 4 Recovery of B . anthracis from Select Tissues of vaccinated, unvaccinated control, and sham vaccinated control rabbits. Clinical Pathology Most elevated clinical pathology parameters were associated with rabbits that succumbed to challenge in the vehicle control and naïve animal groups (data not shown and no statistical analyses performed). Elevated parameters included blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, triglycerides and gamma glutamyltransferase. Hematology parameters were generally unremarkable prior to challenge (data not shown and no statistical analyses performed). Subsequent to challenge white blood cell (WBC) counts increased in virtually all rabbits. In those rabbits that survived, WBC counts returned to normal by sacrifice. Pathology The microscopic findings in the untreated and vehicle control rabbits were characteristic of anthrax and were pooled for baseline data, Table 7 [ 18 ]. Lungs retained bacteria in the septal capillaries and larger arteries and veins. Most rabbits had some degree of heterophilic inflammation and necrosis/fibrosis within the septal capillaries, along with more variable hemorrhage and edema. Of the tracheobronchial lymph nodes collected (11 of 20) and presumably not destroyed by anthrax infection, the majority exhibited necrosis, depletion of lymphocytes, and general inflammation. Bacteria were present in the lymph node sinuses and lymphatics as well as the blood vessels. Spleens were characterized by necrosis, depletion of the white pulp lymphocytes, and fibrosis and inflammation with necrosis of the red pulp. Bacteria were identified in the red pulp of all unvaccinated rabbits. Liver inflammation, fibrosis and necrosis along with associated cell loss occurred to some extent in unvaccinated-challenged rabbits. Bacteria were present in the blood vessels of the brain meninges in these animals. 10.1371/journal.pone.0130952.t007 Table 7 Incidence of Pulmonary Microscopic Findings in the Lungs of B . anthracis Spore Exposed Rabbits by Group (values represent the number of animals within a study group that possessed the finding). Group (μg) Number of Animals on Study: Naïve Control Vehicle Control * wtrPA 10 wtrPA 2.5 wtrPA 0.615 mrPA 10 mrPA 2.5 mrPA 0.625 AVA 10 10 10 10 10 9 10 10 10 LUNG(S) # Examined 10 10 10 10 10 9 10 10 10 # Within Normal Limits 0 0 0 0 0 1 0 0 0 Congestion 9 10 9 10 10 8 10 10 9 Minimal 1 0 0 3 3 2 1 1 0 1 Mild 2 1 1 3 5 6 4 6 5 4 Moderate 3 8 9 3 1 1 3 3 4 2 Severe 4 0 0 0 0 0 0 0 1 1 Hemorrhage 0 4 0 1 3 1 0 0 1 Minimal 1 0 1 0 0 0 1 0 0 0 Mild 2 0 3 0 0 1 0 0 0 1 Moderate 3 0 0 0 1 2 0 0 0 0 Edema 9 6 0 1 4 0 0 0 0 Minimal 1 2 1 0 0 1 0 0 0 0 Mild 2 4 3 0 0 1 0 0 0 0 Moderate 3 3 2 0 1 2 0 0 0 0 Bacteremia 10 10 0 0 1 0 0 0 0 Mild 2 0 3 0 0 0 0 0 0 0 Moderate 3 4 4 0 0 0 0 0 0 0 Severe 4 6 3 0 0 1 0 0 0 0 nfiltration, Heterophils, Fibrin, ±Necrotic Cell Debri; Interstitium; Capillary 10 9 0 0 1 0 0 0 0 Minimal 1 6 1 0 0 0 0 0 0 0 Mild 2 3 8 0 0 1 0 0 0 0 Moderate 3 1 0 0 0 0 0 0 0 0 Increased; Mucosa Associated Lymphoid Tissue 0 0 9 10 8 8 10 10 10 Minimal 1 0 0 3 2 2 5 3 4 2 Mild 2 0 0 5 8 6 3 7 5 7 Moderate 3 0 0 1 0 0 0 0 1 1 Infiltration, Lymphocytic, Histiocytic; Interstitium 0 1 7 9 10 8 9 8 9 Minimal 1 0 0 6 3 3 4 5 5 4 Mild 2 0 1 1 5 5 4 4 2 5 Moderate 3 0 0 0 1 2 0 0 1 0 Accumulation; Alveolus; Macrophage 0 0 5 8 6 6 6 7 5 Minimal 1 0 0 4 4 1 6 5 5 4 Mild 2 0 0 0 3 4 0 1 1 1 Moderate 3 0 0 1 1 1 0 0 1 0 Inflammation, Heterophils; Peribronchiolar; Lymphatic 0 4 2 5 6 2 2 4 2 Minimal 1 0 0 1 0 0 1 2 3 1 Mild 2 0 3 1 3 3 1 0 1 1 Moderate 3 0 1 0 2 3 0 0 0 0 Inflammation, Granulomatous; Lymphatic; Interstitium 0 0 2 6 4 1 1 1 3 Minimal 1 0 0 2 4 0 1 1 0 1 Mild 2 0 0 0 2 3 0 0 0 2 Moderate 3 0 0 0 0 0 0 0 1 0 Severe 4 0 0 0 0 1 0 0 0 0 Abscess 0 0 0 1 2 0 0 1 0 Mild 2 0 0 0 1 1 0 0 1 0 Moderate 3 0 0 0 0 1 0 0 0 0 Inflammation, Lymphocytic, Histiocytic; Pleura 0 0 0 0 1 0 0 0 0 Mild 2 0 0 0 0 1 0 0 0 0 Inflammation, Histiocytic, Heterophilic; Alveolus 0 0 0 1 1 0 0 0 0 Moderate 3 0 0 0 0 1 0 0 0 0 Severe 4 0 0 0 1 0 0 0 0 0 Bacteria; Lymphatic; Alveolus 0 0 0 1 1 0 0 0 0 Moderate 3 0 0 0 0 1 0 0 0 0 Severe 4 0 0 0 1 0 0 0 0 0 *Rabbit included that was euthanized post exposure (9h) due to causes unrelated to treatment. All pathology findings were within normal ranges. Microscopic findings in AVA-vaccinated rabbits were consistent with a response to a high dose of inhaled spores in immunized rabbits. Inflammatory cells were scattered throughout the pulmonary interstitium in most rabbits, and there was an increase in the number of alveolar macrophages, many of which contained cytoplasmic particles approximately 1–2 microns in diameter (possible spores). A few rabbits had foci of granulomatous inflammation associated with lymphatic vessels and/or heterophilic (rabbit neutrophil equivalent) inflammation associated with the conducting airways and lymphatics. All rabbits had an increase in the mucosal-associated lymphoid tissues of the airways and follicles with germinal centers in the tracheobronchial lymph nodes, and 8/10 had an increase in the paracortical zone lymphocytes and/or medullary plasma cells. Spleens and livers were generally unremarkable. The microscopic findings in rabbits from all 10 μg wtrPA, all 2.5 and 10 μg mrPA, eight of ten 2.5 μg wtrPA, six of ten 0.625 μg wtrPA, and nine of ten 0.625 μg mrPA treated rabbits were generally equivalent to the AVA group and survived to Day 84 necropsy. In addition to an AVA-like inflammatory response, a single 0.625 μg mrPA, one 2.5 μg wtrPA and two 0.625 μg wtrPA treated rabbits additionally retained small inflammatory abscesses in a single lung lobe at Day 84. Of the 2.5 μg wtrPA and two 0.625 μg wtrPA treated rabbits that succumbed to B . anthracis spore exposure, the 2.5 μg wtrPA treated rabbit and one of the 0.625 μg wtrPA treated rabbits retained bacteria in the pulmonary lymphatics and alveolar spaces and generalized heterophilic inflammation. The remaining 0.625 μg wtrPA treated rabbit death had findings similar to unvaccinated rabbits. Electrochemiluminescence Assay (ECL) for Residual Circulating Protective Antigen All animals lacked detectable circulating PA in the serum prior to challenge and almost 19/20 control rabbits were PA positive by Study Day 72. Untreated and vehicle-treated B . anthracis spore-exposed control animals remained PA positive until death. Subjects in vaccinated groups had sporadic, transient PA positive test samples; the 10μg wtrPA (3/10) and 2.5 μg wtrPA (4/10) had the highest number of subjects with a PA positive test sample, but all PA positive test subjects in vaccinated animals returned to a PA negative state by the end of the study. Induction and Measurement of Anti-PA Antibodies A rabbit immunogenicity and B . anthracis spore challenge study was designed (Tables 1 and 2 ) to test the effectiveness of both the wtrPA and mrPA molecules at a broad range of doses (dosed at over a 16-fold range) in a two dose vaccine administration regimen to induce an anti-PA antibody response. The approved anthrax vaccine (Biothrax) administered at the human dose, 0.5 mL, served as the positive control. Results for the anti-PA ELISA analyses performed on serum samples collected as outlined in Table 2 are shown in Fig 1 and Tables 3 and 4 . Multiple vaccine groups' immunogenicity responses over time (curve response) were significantly different from other groups when compared by repeated-measures ANOVA and Bonferroni/Dunn post-hoc test when IgG anti-PA values from Days -1 through 65 were analyzed ( Table 4 ). The highest dose of wtrPA (10 μg) induced a significantly greater immune response than did the two lower doses (2.5 and 0.625 μg) when considering the blood draws Days -1 through 65, Table 4 . The highest dose of mrPA induced a significantly higher total antibody titer than either of the two lower doses. The high dose of the mrPA also induced a significantly higher anti-PA response than the human dose of AVA. The 2.5μg mrPA dose was not statistically different from AVA. The mrPA outperformed the wtrPA at all comparable doses. 10.1371/journal.pone.0130952.g001 Fig 1 Ig anti-PA ELISA values (μg/mL). (A) generated from vaccinated wild type and (B) mutant rPA (geometric mean with error bars representing the 95% confidence interval). 10.1371/journal.pone.0130952.t003 Table 3 Study Summary of Fate and Immune Status Relevant to B. anthracis Challenge on Day 70. IgG anti- PA (μg/mL) Geometric Mean ± C.I. TNA ED50 Geometric Mean ± C.I. Test Group (μg rPA) Survival (Alive/Total) Day 42 Day 65 Day 42 Day 65 Naïve Control 0% (0/10) 15 (15, 15) a 17 (12, 24) 1 (1, 1) b 1 (1, 1) Vehicle Control (0 μg rPA) 0% (0/10) 15 (15, 15) 15 (15, 15) 1 (1, 1) 1 (1, 1) wtrPA 10 100% (9/9) 869(674, 1121) 303(240, 381) 27324(18802, 39709) 6608(4522, 9656) wtrPA 2.5 90% (9/10) 456(325, 640) 190(136, 264) 17670(11320, 27583) 4282(2310, 7937) wtrPA0.625 80% (8/10) 234(182, 301) 78(61, 99) 8992(6394, 12644) 1176(499, 2773) mrPA 10 100% (9/9) 1008(800, 1271) 416(338, 512) 37725(20959, 67903) 13,437(7561, 23882) mrPA 2.5 100% (10/10) 800(553, 1158) 306(217, 432) 22176(14214, 34598) 8695(4706, 16065) mrPA 0.625 100% (10/10) 456(335, 621) 159(114, 224) 13674(9504, 19676) 2947(1366, 6358) AVA 100% (10/10) 974(802, 1183) 363(298, 443) 17523(10933, 28085) 4798(2975, 7737) a Lower limit of quantitation < 30 μg/mL. b Samples which returned an incalculable ED50 (by Gen 5 software) were assigned an ED50 value of 1. 10.1371/journal.pone.0130952.t004 Table 4 p Values Associated with Comparison of Ig anti-PA Values between Vaccine Groups as Analyzed by Repeated Measures ANOVA. Vaccine Group WTrPA10 WTrPA2.5 WTrPA0.625 mrPA10 mrPA2.5 mrPA 0.625 AVA wtrPA 10 μg – wtrPA 2.5 μg < .0011 ↓ – wtrPA 0.625 μg < .0001 ↓ NS – mrPA 10 μg < .0001 ↑ < .0001 ↑ < .0001 ↑ – mrPA 2.5 μg NS < .0001 ↑ < .0001 ↑ < .0001 ↓ – mrPA 0.625 μg NS < .0008 ↑ < .0001 ↑ < .0001 ↓ NS – AVA < .0002 ↑ < .0001 ↑ < .0001 ↑ < .0009 ↓ NS < .0003 ↑ – Ig anti-PA levels from blood draws between day -1 and 65 were analyzed by repeated measures ANOVA. The Ig anti-PA response of non-vaccinated groups (None and Vehicle, not shown above) were significantly lower (p < 0.0001) than every other group except each other. ↓ Indicates the Ig anti-PA response of the vaccine group listed on the left is significantly less than the corresponding vaccine group listed on the top of the table. ↑ Indicates the Ig anti-PA response of the vaccine group listed on the left is significantly greater than the corresponding vaccine group listed on the top of the table. NS, no statistical significance between the two groups. Subsequent to the boost vaccination delivered on Day 28, anti-PAantibodies increased in all dose groups to a similar level prior to challenge. The levels of total anti-PA antibody increased post-aerosol challenge in all vaccine groups, Fig 1 . Often the highest post-challenge responses were measured in those rabbits vaccinated with the lowest dose of rPA. Induction and Measurement of Toxin Neutralizing Antibodies In addition to the characterization of the appearance of total anti-PA antibodies (described above), anthrax holotoxin neutralizing anti-PA-antibodies were also measured by a toxin neutralization assay (Tables 1 and 2 ). The approved anthrax vaccine (Biothrax) administered at the human dose, 0.5 mL, once again served as the positive control. Fig 2 and Tables 3 and 5 illustrate the results of the TNA for each vaccine group. Multiple vaccine groups were significantly different from other groups when time-based ED 50 data were analyzed by repeated-measures ANOVA and Bonferroni/Dunn post-hoc test over Days -1 through 65, Table 5 . The highest dose of wtrPA induced a significantly greater immune response than did the two lower doses of wtrPA when considering the blood draws Days -1 through 65. The highest dose of mrPA was similar to the 2.5μg dose of mrPA, but induced a significantly higher amount of functional antibody than either the lowest dose of mrPA or both the 2.5 μg and 0.625 μg doses of wtrPA. Also, higher ED 50 values were measured in rabbits from the highest mrPA dose group (10 μg, p = 0.0002) as compared to AVA. The mrPA outperformed wtrPA at all comparable doses and the two lower doses of mrPA were equivalent and not statistically different from the highest wtrPA doses or AVA. 10.1371/journal.pone.0130952.g002 Fig 2 rPA antibody-specific Toxin Neutralization Assay (TNA) and ED50 values (μg/mL). (A) generated from vaccinated wild type and (B) mutant rPA (geometric mean with error bars representing the 95% confidence interval). 10.1371/journal.pone.0130952.t005 Table 5 p Values Associated with Comparison of ED50 Values between Vaccine Groups as Analyzed by Repeated Measures ANOVA Vaccine Group WTrPA10 WTrPA2.5 WTrPA0.625 mrPA10 mrPA2.5 mrPA0.625 AVA wtrPA 10 μg – wtrPA 2.5 μg < .0001 ↓ – wtrPA 0.625 μg < .0001 ↓ NS – mrPA 10 μg .0003 ↑ < .0001 ↑ < .0001 ↑ – mrPA 2.5 μg NS < .0001 ↑ < .0001 ↑ NS – mrPA 0.625 μg NS < .0001 ↑ < .0001 ↑ < .0001 ↓ NS – AVA NS < .0001 ↑ < .0001 ↑ .0002 ↓ NS NS – TNA ED 50 values from blood draws between day -1 and 65 were analyzed by repeated measures ANOVA. The ED 50 response of non-vaccinated groups (None and Vehicle, not shown) were significantly lower (p < 0.0001) than every other group except each other. ↓ Indicates the ED 50 response of the vaccine group listed on the left is less than the corresponding vaccine group listed on the top of the table. ↑ Indicates the ED 50 response of the vaccine group listed on the left is greater than the corresponding vaccine group listed on the top of the table. NS, no statistical significance between the reactions of the two groups. In general, an rPA dose response was observed prior to the Day 28 boost, particularly in the wtrPA dosed rabbits. After the Day 28 boost, the differences between doses of rPA, either wild type or mutant, became less apparent and the levels declined between Day 48 and 65 prior to challenge. Neutralizing antibody levels increased post-aerosol challenge in all vaccine groups as seen in Fig 2 . Mortality Following B. anthracis Spore Challenge Mortality data and Kaplan-Meier data are shown in Table 3 and Fig 3 . All rabbits survived until challenge with the exception of single 10ug mrPA female rabbit that died during conditioning to inhalation exposure restraint. One 10 μg wtrPA rabbit was euthanized subsequent to challenge due to partial paralysis unrelated to exposure. All Naïve and Vehicle Control group rabbits died due to anthrax infection two to five days post-challenge (Days 72–75). One 1.25 and two 0.625 μg wtrPA-immunized rabbits died due to anthrax infection on Day 74. All 10 μg wtrPA, all mrPA vaccinated, and AVA rabbits survived challenge. There were statistically significant differences (p < 0.0001, Mantel-Cox; Chi-square) in survival and time to death for the Naïve and Vehicle Control groups compared to all other test groups. A statistically significant difference was not observed for the vaccinated groups [2.5 μg wtrPA and 0.625 μg wtrPA)] that had post-challenge deaths compared to the remaining test groups. 10.1371/journal.pone.0130952.g003 Fig 3 Kaplan-Meier Survival Curve of vaccinated, unvaccinated control, and sham vaccinated control rabbits. Bacteriological Evaluation of Vaccinated Rabbits Challenged with B . anthracis Spores Six of ten naïve control rabbits and eight of ten vehicle control rabbits were positive for bacteremia in the blood (data not shown). Of vaccinated rabbits, there were only three rabbits that demonstrated bacteremia at the initial blood (Day 71) draw following challenge, but this resolved in these rabbits by the next blood collection time point (Day 74) and the rabbits survived to Day 84. Of the three wtrPA-vaccinated rabbits that succumbed to disease, none had bacteremia present in any blood sample. Negative control rabbits (unvaccinated and those vaccinated with vehicle) were shown to have high levels of tissue burden for all tissues tested, Table 6 and Fig 4 . All other rabbits that succumbed to disease presented with marked tissue burden in the majority of tissues tested. In the rabbits that succumbed to disease, lung burden ranged from 7.56 x 10 4 to 2.21 x 10 8 CFU/mL. The rabbits who survived to study termination were found to have tissue burden below the detectable limits or, in a few cases, low tissue burden was seen in the lungs. In the three wtrPA vaccinated rabbits that succumbed to disease prior to study termination, high tissue burden was seen in the lung and low levels in the tracheobronchial lymph nodes (TBLN) and spleen. One of the animals that succumbed had high tissue burden in the lung as well as all other tissues collected. The rabbits in the mrPA vaccine groups all survived to study termination and any B . anthracis present in the tissues was at lower levels (less than 30 CFU/100 μL inocula) than wtrPA vaccine group rabbits. 10.1371/journal.pone.0130952.t006 Table 6 Summary of Recovery of B . anthracis from Select Tissues of Inhalational Spore Challenged New Zealand White Rabbits. Dose a Lung Liver Spleen TBLN Naïve Control Mean b 7.13 6.25 6.00 6.87 SD — 0.600 1.293 0.408 GeoM c 1.34 x 10 7 1.79 x 10 6 9.94 x 10 5 7.38 x 10 6 N 1 10 10 10 Vaccinated Control Mean 8.08 6.33 6.24 6.81 SD — 1.118 1.314 0.563 GeoM 1.20 x 10 8 2.14 x 10 6 1.75 x 10 6 6.41 x 10 6 N 1 10 10 10 wtrPA 10 mg Mean 3.29 BDL BDL d BDL SD — BDL BDL BDL GeoM 1.95 x 10 3 BDL BDL BDL N 1 10 10 10 wtrPA 2.5 mg (2.5 mg) Mean 2.51 BDL 0.22 0.75 SD 1.585 BDL 0.700 1.022 GeoM 3.24 x 10 2 BDL 6.65 x 10 −1 4.57 x 10 0 N 7 10 10 10 wtrPA 0.625 mg Mean 5.56 0.72 1.05 1.38 SD 3.916 1.917 2.393 2.639 GeoM 3.59 x 10 5 4.28 x 10 0 1.02 x 10 1 2.31 x 10 1 N 2 10 10 10 mrPA 10 mg Mean 2.03 0.26 BDL 0.35 SD 1.755 0.835 BDL 0.744 GeoM 1.05 x 10 2 8.37E-01 BDL 1.25 x 10 0 N 3 10 10 10 mrPA 2.5 mg Mean 3.40 BDL BDL 0.40 SD — BDL BDL 0.853 GeoM 2.49 x 10 3 BDL BDL 1.53 x 10 0 N 1 10 10 10 mrPA 0.625 Mean 2.67 BDL BDL 0.23 SD 0.005 BDL BDL 0.736 GeoM 4.71 x 10 2 BDL BDL 7.09 x 10 −1 N 2 10 10 10 AVA (human dose) Mean 3.01 BDL BDL 0.17 SD 0.761 BDL BDL 0.543 GeoM 1.03 x 10 3 BDL BDL 4.85E-01 N 3 10 10 10 a Treatment = Dose delivered intramuscularly (IM) on Days 0 and 28 in 0.5 mL volume b Mean = Log 10 (CFU/g + 1) c Geometric mean of CFU/g d BDL = Below detection limit (< 68–70 CFU/g) 10.1371/journal.pone.0130952.g004 Fig 4 Recovery of B . anthracis from Select Tissues of vaccinated, unvaccinated control, and sham vaccinated control rabbits. Clinical Pathology Most elevated clinical pathology parameters were associated with rabbits that succumbed to challenge in the vehicle control and naïve animal groups (data not shown and no statistical analyses performed). Elevated parameters included blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, triglycerides and gamma glutamyltransferase. Hematology parameters were generally unremarkable prior to challenge (data not shown and no statistical analyses performed). Subsequent to challenge white blood cell (WBC) counts increased in virtually all rabbits. In those rabbits that survived, WBC counts returned to normal by sacrifice. Pathology The microscopic findings in the untreated and vehicle control rabbits were characteristic of anthrax and were pooled for baseline data, Table 7 [ 18 ]. Lungs retained bacteria in the septal capillaries and larger arteries and veins. Most rabbits had some degree of heterophilic inflammation and necrosis/fibrosis within the septal capillaries, along with more variable hemorrhage and edema. Of the tracheobronchial lymph nodes collected (11 of 20) and presumably not destroyed by anthrax infection, the majority exhibited necrosis, depletion of lymphocytes, and general inflammation. Bacteria were present in the lymph node sinuses and lymphatics as well as the blood vessels. Spleens were characterized by necrosis, depletion of the white pulp lymphocytes, and fibrosis and inflammation with necrosis of the red pulp. Bacteria were identified in the red pulp of all unvaccinated rabbits. Liver inflammation, fibrosis and necrosis along with associated cell loss occurred to some extent in unvaccinated-challenged rabbits. Bacteria were present in the blood vessels of the brain meninges in these animals. 10.1371/journal.pone.0130952.t007 Table 7 Incidence of Pulmonary Microscopic Findings in the Lungs of B . anthracis Spore Exposed Rabbits by Group (values represent the number of animals within a study group that possessed the finding). Group (μg) Number of Animals on Study: Naïve Control Vehicle Control * wtrPA 10 wtrPA 2.5 wtrPA 0.615 mrPA 10 mrPA 2.5 mrPA 0.625 AVA 10 10 10 10 10 9 10 10 10 LUNG(S) # Examined 10 10 10 10 10 9 10 10 10 # Within Normal Limits 0 0 0 0 0 1 0 0 0 Congestion 9 10 9 10 10 8 10 10 9 Minimal 1 0 0 3 3 2 1 1 0 1 Mild 2 1 1 3 5 6 4 6 5 4 Moderate 3 8 9 3 1 1 3 3 4 2 Severe 4 0 0 0 0 0 0 0 1 1 Hemorrhage 0 4 0 1 3 1 0 0 1 Minimal 1 0 1 0 0 0 1 0 0 0 Mild 2 0 3 0 0 1 0 0 0 1 Moderate 3 0 0 0 1 2 0 0 0 0 Edema 9 6 0 1 4 0 0 0 0 Minimal 1 2 1 0 0 1 0 0 0 0 Mild 2 4 3 0 0 1 0 0 0 0 Moderate 3 3 2 0 1 2 0 0 0 0 Bacteremia 10 10 0 0 1 0 0 0 0 Mild 2 0 3 0 0 0 0 0 0 0 Moderate 3 4 4 0 0 0 0 0 0 0 Severe 4 6 3 0 0 1 0 0 0 0 nfiltration, Heterophils, Fibrin, ±Necrotic Cell Debri; Interstitium; Capillary 10 9 0 0 1 0 0 0 0 Minimal 1 6 1 0 0 0 0 0 0 0 Mild 2 3 8 0 0 1 0 0 0 0 Moderate 3 1 0 0 0 0 0 0 0 0 Increased; Mucosa Associated Lymphoid Tissue 0 0 9 10 8 8 10 10 10 Minimal 1 0 0 3 2 2 5 3 4 2 Mild 2 0 0 5 8 6 3 7 5 7 Moderate 3 0 0 1 0 0 0 0 1 1 Infiltration, Lymphocytic, Histiocytic; Interstitium 0 1 7 9 10 8 9 8 9 Minimal 1 0 0 6 3 3 4 5 5 4 Mild 2 0 1 1 5 5 4 4 2 5 Moderate 3 0 0 0 1 2 0 0 1 0 Accumulation; Alveolus; Macrophage 0 0 5 8 6 6 6 7 5 Minimal 1 0 0 4 4 1 6 5 5 4 Mild 2 0 0 0 3 4 0 1 1 1 Moderate 3 0 0 1 1 1 0 0 1 0 Inflammation, Heterophils; Peribronchiolar; Lymphatic 0 4 2 5 6 2 2 4 2 Minimal 1 0 0 1 0 0 1 2 3 1 Mild 2 0 3 1 3 3 1 0 1 1 Moderate 3 0 1 0 2 3 0 0 0 0 Inflammation, Granulomatous; Lymphatic; Interstitium 0 0 2 6 4 1 1 1 3 Minimal 1 0 0 2 4 0 1 1 0 1 Mild 2 0 0 0 2 3 0 0 0 2 Moderate 3 0 0 0 0 0 0 0 1 0 Severe 4 0 0 0 0 1 0 0 0 0 Abscess 0 0 0 1 2 0 0 1 0 Mild 2 0 0 0 1 1 0 0 1 0 Moderate 3 0 0 0 0 1 0 0 0 0 Inflammation, Lymphocytic, Histiocytic; Pleura 0 0 0 0 1 0 0 0 0 Mild 2 0 0 0 0 1 0 0 0 0 Inflammation, Histiocytic, Heterophilic; Alveolus 0 0 0 1 1 0 0 0 0 Moderate 3 0 0 0 0 1 0 0 0 0 Severe 4 0 0 0 1 0 0 0 0 0 Bacteria; Lymphatic; Alveolus 0 0 0 1 1 0 0 0 0 Moderate 3 0 0 0 0 1 0 0 0 0 Severe 4 0 0 0 1 0 0 0 0 0 *Rabbit included that was euthanized post exposure (9h) due to causes unrelated to treatment. All pathology findings were within normal ranges. Microscopic findings in AVA-vaccinated rabbits were consistent with a response to a high dose of inhaled spores in immunized rabbits. Inflammatory cells were scattered throughout the pulmonary interstitium in most rabbits, and there was an increase in the number of alveolar macrophages, many of which contained cytoplasmic particles approximately 1–2 microns in diameter (possible spores). A few rabbits had foci of granulomatous inflammation associated with lymphatic vessels and/or heterophilic (rabbit neutrophil equivalent) inflammation associated with the conducting airways and lymphatics. All rabbits had an increase in the mucosal-associated lymphoid tissues of the airways and follicles with germinal centers in the tracheobronchial lymph nodes, and 8/10 had an increase in the paracortical zone lymphocytes and/or medullary plasma cells. Spleens and livers were generally unremarkable. The microscopic findings in rabbits from all 10 μg wtrPA, all 2.5 and 10 μg mrPA, eight of ten 2.5 μg wtrPA, six of ten 0.625 μg wtrPA, and nine of ten 0.625 μg mrPA treated rabbits were generally equivalent to the AVA group and survived to Day 84 necropsy. In addition to an AVA-like inflammatory response, a single 0.625 μg mrPA, one 2.5 μg wtrPA and two 0.625 μg wtrPA treated rabbits additionally retained small inflammatory abscesses in a single lung lobe at Day 84. Of the 2.5 μg wtrPA and two 0.625 μg wtrPA treated rabbits that succumbed to B . anthracis spore exposure, the 2.5 μg wtrPA treated rabbit and one of the 0.625 μg wtrPA treated rabbits retained bacteria in the pulmonary lymphatics and alveolar spaces and generalized heterophilic inflammation. The remaining 0.625 μg wtrPA treated rabbit death had findings similar to unvaccinated rabbits. Electrochemiluminescence Assay (ECL) for Residual Circulating Protective Antigen All animals lacked detectable circulating PA in the serum prior to challenge and almost 19/20 control rabbits were PA positive by Study Day 72. Untreated and vehicle-treated B . anthracis spore-exposed control animals remained PA positive until death. Subjects in vaccinated groups had sporadic, transient PA positive test samples; the 10μg wtrPA (3/10) and 2.5 μg wtrPA (4/10) had the highest number of subjects with a PA positive test sample, but all PA positive test subjects in vaccinated animals returned to a PA negative state by the end of the study. Discussion In order to test the feasibility of developing a recombinantly-derived mutant form of B . anthracis protective antigen as a vaccine, the current study was undertaken to compare the efficacy of native, wtrPA and mutant, mrPA vaccines to protect rabbits from an aerosol challenge with B . anthracis spores. Animal numbers were statistically sufficient to answer this question and were consistent with previous literature-based investigations of rPA vaccines [ 21 ]. The mutant form of the rPA studied here may present production and functional advantages as a vaccine antigen. The mutant has had two proteolytically sensitive sites removed. These mutations may serve to protect the intact antigen product to proteolytic loss over long periods of storage, making it a better candidate for a stock-piled vaccine. Ramirez et al . (2002) have also found an increased yield of this protein compared to the native molecule when prepared from a recombinant B . anthracis host [ 11 ], presumably due to the mutations. Also, because the wtrPA must be cleaved to assume its active configuration, a proteolytically resistant form may have advantages for a post exposure prophylactic use. In such cases, the vaccine will not introduce any further component of the anthrax toxin to exposed individuals [ 22 ]. A 24-month stability study utilizing a GLP validated mouse potency assay is currently underway assessing mrPA and wtrPA proteins expressed and isolated from P . fluorescens and formulated in alum (data not shown). The data that will be derived from this study will reveal any differentiation of the stability of wtrPA vs. mrPA formulated with Alhydrogel. Immunogenicity results from the initial preclinical work performed [ 11 ] as well as a Phase I clinical study [ 9 ] investigating the mrPA vs. wtrPA mirror those of the current study. The mrPA performed at least as well as the wtrPA and was comparable to AVA. ELISA and neutralizing antibody responses are very similar to those produced by the native protein. In the current study, ELISA and TNA results from both rPA molecules were consistent with previously reported alum-based rPA anthrax vaccinations and challenge studies [ 21 ]. However, this study is the first to show a detailed comparative assessment of the immunogenicity, TNA response, and survival of rabbits vaccinated with alum formulations of mrPA vs. wtrPA derived from a novel recombinant source, P . fluorescens , and challenged with B . anthracis spores. mrPA generated circulating and neutralizing antibodies that were superior to all comparable doses of wtrPA and superior to the human dose of AVA when administered at the 10 μg/dose level. The mrPA and wtrPA comparative antibody response was reflected in trends observed in survival, general bacteremia and pathology, suggesting that the mrPA conferred superior protection compared to the wtrPA protein. Such mutants that cannot heptamerize have been shown to be more effective antigens. Perhaps this is because they disrupt normal cellular trafficking, making them more susceptible targets for the host's immune system [ 23 ]. Bacteriology, pathology and survival also suggest that the antibodies produced by mrPA are at least as protective as the human dose of AVA. It is interesting to consider how these results could potentially be compared to long term rabbit vaccination and challenge studies, as well as human anti-PA IgG and TNA responses generated from AVA and other rPA-based vaccines under development. However, the 14-day post challenge sacrifice date used in the current study design was not long enough in duration to determine whether or not full B . anthracis lung clearance could be achieved at an associated TNA or anti-PA value. In summary, these data demonstrate that recombinant mrPA and wtrPA produced in a novel expression system can be formulated with alum to achieve protection that is equivalent to the clinical administration of AVA. Under the conditions of this study, mrPA outperformed the wtrPA formulation and was equivalent to AVA at dose levels as low as 0.625 μg in a two dose regimen. These data combined with ongoing stability studies currently suggest that both the alum-formulated wtrPA and mrPA proteins expressed in P . fluorescens are viable vaccine candidates suitable for further development [ 24 ].

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588557/

Validated Methods for Removing Select Agent Samples from Biosafety Level 3 Laboratories

The Federal Select Agent Program dictates that all research entities in the United States must rigorously assess laboratory protocols to sterilize samples being removed from containment areas. We validated procedures using sterile filtration and methanol to remove the following select agents: Francisella tularensis , Burkholderia pseudomallei , B. mallei , Yersinia pestis , and Bacillus anthracis . We validated methanol treatment for B. pseudomallei . These validations reaffirm safety protocols that enable researchers to keep samples sufficiently intact when samples are transferred between laboratories. Materials and Methods Biosafety We tested all protocols in a BSL-3 laboratory at the University of Florida, which is registered and licensed with the Centers for Disease Control and Prevention and the Animal and Plant Health Inspection Service, US Department of Agriculture, to conduct select agent research. The containment laboratory uses a high-efficiency particulate air filter to decontaminate discharged air. All staff must don facility-dedicated scrubs, Tyvek suits (Dupont, https://www.dupont.com ), respiratory protection, double gloves, and shoe covers. All bacterial work is performed in a class II Biosafety cabinet, and all waste is removed using pass-through autoclaves. Bacterial Strains and Growth Conditions We used the following strains from the Biodefense and Emerging Infections Resource Repository: B. anthracis (Ames), Y. pestis (CO92), F. tularensis (SchuS4), B. pseudomallei (1026b), and B. mallei (China 7). We isolated B. anthracis spores according to Leighton and Doi ( 4 ) and maintained the spores in refrigerated sterile water at »1 × 10 10 CFU/mL. We verified this concentration by serial dilution in sterile water onto sheep blood agar plates as previously stated ( 5 ). We cultured Y. pestis CO92 from frozen stock on sheep blood agar (Becton Dickinson, https://www.bd.com ) and incubated it for 48 h at 28°C. We then removed colonies from the stock plate and suspended them in 1 mL heart infusion broth (Becton Dickinson). We added this suspension to 100 mL heart infusion broth containing 2 mL 10% xylose (Indofine, https://indofinechemical.com ). We incubated this mixture in a 500 mL flask with agitation for 18–24 h. We then cultured B. mallei China 7 and B. pseudomallei 1026b from frozen stock vials on tryptic soy agar and incubated them at 35°C for 24–48 h to generate a stock plate of each strain. We selected 2–3 colonies from each incubated stock plate and inoculated them in brain heart infusion (BHI) broth (Becton Dickinson) overnight culture. We then incubated the cultures at 35°C with agitation for 16–20 h. We also cultured F. tularensis SchuS4 from frozen stock onto chocolate agar (Becton Dickinson) and incubated it at 35°C for 48 h. We selected colonies from the agar plate and used them to inoculate a BHI culture containing 2% Isovitalex (Becton Dickinson). We incubated this culture for 18–20 h at 35°C with agitation. Matrices We tested the filtration protocol with murine lung BAL fluid, serum, plasma, and the listed culture mediums ( Table 2 ). For the spore preparation, we used BHI as the culture media. We purchased the murine serum, plasma, and BAL from BioreclamationIVT ( https://bioivt.com ). We used mouse plasma from Balb/c mice collected in sodium citrate–containing tubes and pooled across sex. We also used mouse BAL and serum from Balb/c mice and pooled across sex. Table 2 Preparation of select agents in different matrices* Agent CFU/mL (matrix) BAL fluid Serum and plasma, µL Culture BAL cell pellet Bacillus anthracis 10 10 (spore prep†) 20 μL 20§ 20 µL NT Yersinia pestis 10 9 (overnight culture) Resuspend pellet¶ 20 Resuspend pellet¶ NT Burkholderia mallei 10 9 (overnight culture) Resuspend pellet# 20** Resuspend pellet††NT Burkholderia pseudomallei 10 9 (overnight culture) 200 μL + 1.8 mL BAL 20‡‡ Resuspend pellet§§ 2 × 10 6 CFU Francisella tularensis 10 9 (overnight culture‡) 20 μL 20¶¶ Resuspend pellet## NT *BAL, bronchoalveolar lavage; NT, not tested. †Spores for aerosol challenge were maintained in sterile water and diluted to the nebulizer-challenge concentration of »1 × 10 10 CFU/mL. ‡All broth cultures will require a 2% supplement with Isovitalex (Becton Dickinson, https://www.bd.com ) to obtain growth of F . tularensis . §Dilute spore prep 1:1000; transfer 20 μL to serum and plasma. ¶Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL BAL fluid or culture media. #Centrifuge 2 mL of overnight culture, resuspend in 2 mL BAL fluid. **Dilute overnight culture 1:100; transfer 20 μL to BAL fluid. ††Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL culture media. ‡‡Dilute overnight culture 1:10 transfer 20 μL to serum or plasma. §§Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL culture media. ¶¶Dilute overnight culture 1:10 transfer 20 μL to BAL fluid. ##Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL culture media. Test Sample Preparation All matrices had a final volume of 2 mL. We selected test sample starting concentrations that exceeded the maximum published bacterial concentrations ( Table 1 ). We established a conversion factor for each species on the basis of serial dilution plate counts and optical density (OD) measurements at 600 nm (H. Heine, unpub. data). We used these conversion factors to determine the concentrations of overnight cultures and spore preparations. Y. pestis had a conversion factor of 5.34 × 10 8 CFU/OD, B. mallei and B. pseudomallei 1.57 × 10 9 CFU/OD, and F. tularensis 3.89 × 10 10 CFU/OD. Table 1 Maximum bacterial concentrations of select agents in tissues of infected mice* Agent (reference) Source of samples, bacterial load Lung, per g Cell pellet, per mL BAL Blood, per mL Overnight culture, per mL Bacillus anthracis ( 5 , 6 ) <10 8 Not tested <10 4 10 8 Yersinia pestis ( 7 ) <10 10 Not tested <10 6 10 9 Burkholderia mallei ( 8 – 11 ) <10 9 †Not tested <10 4 10 9 Burkholderia pesudomallei ( 11 , 12 ) <10 8 10 5 ‡ <10 5 10 9 Francisella tularensis ( 13 ) 10 7 Not tested <10 5 10 9 *BAL, bronchoalveolar lavage. †References ( 7 ) and ( 8 ) use a different strain of B. mallei ‡Value determined through in-house testing of lung samples. For B. anthracis Ames strain, we prepared spores and spiked the different matrices. We used 20 μL of the spore preparation for BAL and culture medium samples. We diluted the spore preparation 1:1000 and used 20 μL of the diluted solution to spike each serum and plasma sample ( Table 2 ). We prepared test samples for Y. pestis from the incubated 100 mL broth culture. We took an OD reading from serially diluted broth culture and conversion factors to determine the culture concentration. We centrifuged 20 mL of this culture at 3,500 × g for 15 min. We then resuspended this pellet in 2 mL of BAL fluid ( Table 2 ). We repeated the process for the culture medium. We inoculated serum and plasma samples with a uncentrifuged overnight culture ( Table 2 ). We prepared B. mallei test samples from the overnight broth cultures incubated previously. We prepared BAL fluid test samples by centrifuging 2 mL overnight broth culture at 3,500 rpm for 15 min and then resuspending the pellet in 2 mL BAL fluid. We inoculated serum and plasma with an overnight culture that had been diluted 1:100, then added 20 μL to each matrix ( Table 2 ). We inoculated culture medium by centrifuging 20 mL of the overnight culture then suspending the pellet in 2 mL of culture media ( Table 2 ). We prepared B. pseudomallei test samples for culture medium as stated for B. mallei and Y. pestis using the conversion factor. We prepared BAL fluid samples by adding 200 μL overnight culture to 1.8 mL BAL fluid ( Table 2 ). We inoculated serum and plasma with 20 μL of overnight culture that was first diluted 1:10 ( Table 2 ). We prepared F. tularensis samples for culture medium with a final concentration of 2% Isovitalex. We took an OD reading and used the conversion factor to concentrate samples appropriately. We centrifuged 20 mL of an overnight culture and resuspended it in culture medium with 2% Isovitalex. We spiked serum and plasma samples with 20 μL of an overnight culture that was first diluted 1:10 and inoculated BAL fluid with 20 μL of an overnight culture ( Table 2 ). Methanol Test Sample Preparation Test samples, positive controls, and the negative control of BAL fluid for the methanol treatment procedure all had a final volume of 500 μL. We used stock plates to grow bacteria, then selected colonies and suspended them in 3 mL of sterile water for injection (GE Healthcare, https://www.gehealthcare.com ). We took an OD reading at 600 nm on a spectrophotometer (ThermoFisher Scientific, https://www.thermofisher.com ) using a 1 cm 2 cuvette (ThermoFisher Scientific). We converted this value to an approximate CFU per milliliter value using a conversion factor as stated in test sample preparation. We calculated the total volume needed to spike each sample so that each sample would have 2 × 10 6 CFU ( Table 2 ). Filtration Procedure We conducted all filtration test procedures in triplicate for each matrix type. For negative controls, we used uninoculated matrix samples. For positive controls, we used 100 μL of unfiltered inoculated test samples suspended in broth culture medium. We then placed 450 μL of each test sample into a clean 0.2 μm PALL Nanosep Bio-Inert centrifuge filter (Pall Corporation, https://www.pall.com ) with a sterile microcentrifuge tube. In accordance with the manufacturer's recommendations, we centrifuged the filters for 3 min at 14,000 × g . We then transferred the filtrate to a clean tube and sealed it to prevent secondary contamination. We emphasize that the filtrate collection tubes should not be sealed with the same cap used to close the centrifuge filter before spinning because this cap could be contaminated with residual unfiltered sample and thus might yield false positive outcomes. We then suspended the filtrate in 4.5 mL BHI and incubated it at 35°C for 2 d. We incubated the positive controls in the same manner. After 48 h, we checked the tubes for turbidity and plated 5 × 200 μL samples onto the appropriate media. We incubated these samples at 35°C for an additional 7 d to ensure complete sterility. We considered this method to be validated only if all 3 replicates of all matrices were sterile in both broth and agar medium. Any failure, defined here as positive growth on agar or in broth media, prompted a review of the procedures. Once we determined the cause of the failure, we made the appropriate adjustments and reconducted the procedure in 3 replicates. Methanol Procedure We centrifuged BAL fluid for 5 min at 5,000 × g . We removed the supernatant and decontaminated it using the filtration procedure detailed in the previous section. We suspended the pellet in 500 μL of 80% methanol (ThermoFisher Scientific) and incubated it for 10 min. We placed 10% of this sample into 9.5 mL Dey-Engley neutralization broth (D/E media) (Becton Dickinson) and incubated it at 35°C for 5 d. After 5 d, we plated 200 μL of the D/E media onto 5 agar plates specific to each bacterial species and incubated them at 35°C for an additional 2 d. For positive controls, we used D/E media inoculated with bacteria and D/E media with 80% methanol added to the same volume as the test sample (50 μL of 80% methanol into 9.5 mL D/E media). We incubated this tube for 10 min and then inoculated it with bacteria. We also used growth media specific to each bacterial species as positive controls. For negative controls, we used uninoculated D/E media and D/E media inoculated with methanol treated bacteria. Biosafety We tested all protocols in a BSL-3 laboratory at the University of Florida, which is registered and licensed with the Centers for Disease Control and Prevention and the Animal and Plant Health Inspection Service, US Department of Agriculture, to conduct select agent research. The containment laboratory uses a high-efficiency particulate air filter to decontaminate discharged air. All staff must don facility-dedicated scrubs, Tyvek suits (Dupont, https://www.dupont.com ), respiratory protection, double gloves, and shoe covers. All bacterial work is performed in a class II Biosafety cabinet, and all waste is removed using pass-through autoclaves. Bacterial Strains and Growth Conditions We used the following strains from the Biodefense and Emerging Infections Resource Repository: B. anthracis (Ames), Y. pestis (CO92), F. tularensis (SchuS4), B. pseudomallei (1026b), and B. mallei (China 7). We isolated B. anthracis spores according to Leighton and Doi ( 4 ) and maintained the spores in refrigerated sterile water at »1 × 10 10 CFU/mL. We verified this concentration by serial dilution in sterile water onto sheep blood agar plates as previously stated ( 5 ). We cultured Y. pestis CO92 from frozen stock on sheep blood agar (Becton Dickinson, https://www.bd.com ) and incubated it for 48 h at 28°C. We then removed colonies from the stock plate and suspended them in 1 mL heart infusion broth (Becton Dickinson). We added this suspension to 100 mL heart infusion broth containing 2 mL 10% xylose (Indofine, https://indofinechemical.com ). We incubated this mixture in a 500 mL flask with agitation for 18–24 h. We then cultured B. mallei China 7 and B. pseudomallei 1026b from frozen stock vials on tryptic soy agar and incubated them at 35°C for 24–48 h to generate a stock plate of each strain. We selected 2–3 colonies from each incubated stock plate and inoculated them in brain heart infusion (BHI) broth (Becton Dickinson) overnight culture. We then incubated the cultures at 35°C with agitation for 16–20 h. We also cultured F. tularensis SchuS4 from frozen stock onto chocolate agar (Becton Dickinson) and incubated it at 35°C for 48 h. We selected colonies from the agar plate and used them to inoculate a BHI culture containing 2% Isovitalex (Becton Dickinson). We incubated this culture for 18–20 h at 35°C with agitation. Matrices We tested the filtration protocol with murine lung BAL fluid, serum, plasma, and the listed culture mediums ( Table 2 ). For the spore preparation, we used BHI as the culture media. We purchased the murine serum, plasma, and BAL from BioreclamationIVT ( https://bioivt.com ). We used mouse plasma from Balb/c mice collected in sodium citrate–containing tubes and pooled across sex. We also used mouse BAL and serum from Balb/c mice and pooled across sex. Table 2 Preparation of select agents in different matrices* Agent CFU/mL (matrix) BAL fluid Serum and plasma, µL Culture BAL cell pellet Bacillus anthracis 10 10 (spore prep†) 20 μL 20§ 20 µL NT Yersinia pestis 10 9 (overnight culture) Resuspend pellet¶ 20 Resuspend pellet¶ NT Burkholderia mallei 10 9 (overnight culture) Resuspend pellet# 20** Resuspend pellet††NT Burkholderia pseudomallei 10 9 (overnight culture) 200 μL + 1.8 mL BAL 20‡‡ Resuspend pellet§§ 2 × 10 6 CFU Francisella tularensis 10 9 (overnight culture‡) 20 μL 20¶¶ Resuspend pellet## NT *BAL, bronchoalveolar lavage; NT, not tested. †Spores for aerosol challenge were maintained in sterile water and diluted to the nebulizer-challenge concentration of »1 × 10 10 CFU/mL. ‡All broth cultures will require a 2% supplement with Isovitalex (Becton Dickinson, https://www.bd.com ) to obtain growth of F . tularensis . §Dilute spore prep 1:1000; transfer 20 μL to serum and plasma. ¶Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL BAL fluid or culture media. #Centrifuge 2 mL of overnight culture, resuspend in 2 mL BAL fluid. **Dilute overnight culture 1:100; transfer 20 μL to BAL fluid. ††Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL culture media. ‡‡Dilute overnight culture 1:10 transfer 20 μL to serum or plasma. §§Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL culture media. ¶¶Dilute overnight culture 1:10 transfer 20 μL to BAL fluid. ##Centrifuge 20 mL of overnight culture, resuspend pellet in 2 mL culture media. Test Sample Preparation All matrices had a final volume of 2 mL. We selected test sample starting concentrations that exceeded the maximum published bacterial concentrations ( Table 1 ). We established a conversion factor for each species on the basis of serial dilution plate counts and optical density (OD) measurements at 600 nm (H. Heine, unpub. data). We used these conversion factors to determine the concentrations of overnight cultures and spore preparations. Y. pestis had a conversion factor of 5.34 × 10 8 CFU/OD, B. mallei and B. pseudomallei 1.57 × 10 9 CFU/OD, and F. tularensis 3.89 × 10 10 CFU/OD. Table 1 Maximum bacterial concentrations of select agents in tissues of infected mice* Agent (reference) Source of samples, bacterial load Lung, per g Cell pellet, per mL BAL Blood, per mL Overnight culture, per mL Bacillus anthracis ( 5 , 6 ) <10 8 Not tested <10 4 10 8 Yersinia pestis ( 7 ) <10 10 Not tested <10 6 10 9 Burkholderia mallei ( 8 – 11 ) <10 9 †Not tested <10 4 10 9 Burkholderia pesudomallei ( 11 , 12 ) <10 8 10 5 ‡ <10 5 10 9 Francisella tularensis ( 13 ) 10 7 Not tested <10 5 10 9 *BAL, bronchoalveolar lavage. †References ( 7 ) and ( 8 ) use a different strain of B. mallei ‡Value determined through in-house testing of lung samples. For B. anthracis Ames strain, we prepared spores and spiked the different matrices. We used 20 μL of the spore preparation for BAL and culture medium samples. We diluted the spore preparation 1:1000 and used 20 μL of the diluted solution to spike each serum and plasma sample ( Table 2 ). We prepared test samples for Y. pestis from the incubated 100 mL broth culture. We took an OD reading from serially diluted broth culture and conversion factors to determine the culture concentration. We centrifuged 20 mL of this culture at 3,500 × g for 15 min. We then resuspended this pellet in 2 mL of BAL fluid ( Table 2 ). We repeated the process for the culture medium. We inoculated serum and plasma samples with a uncentrifuged overnight culture ( Table 2 ). We prepared B. mallei test samples from the overnight broth cultures incubated previously. We prepared BAL fluid test samples by centrifuging 2 mL overnight broth culture at 3,500 rpm for 15 min and then resuspending the pellet in 2 mL BAL fluid. We inoculated serum and plasma with an overnight culture that had been diluted 1:100, then added 20 μL to each matrix ( Table 2 ). We inoculated culture medium by centrifuging 20 mL of the overnight culture then suspending the pellet in 2 mL of culture media ( Table 2 ). We prepared B. pseudomallei test samples for culture medium as stated for B. mallei and Y. pestis using the conversion factor. We prepared BAL fluid samples by adding 200 μL overnight culture to 1.8 mL BAL fluid ( Table 2 ). We inoculated serum and plasma with 20 μL of overnight culture that was first diluted 1:10 ( Table 2 ). We prepared F. tularensis samples for culture medium with a final concentration of 2% Isovitalex. We took an OD reading and used the conversion factor to concentrate samples appropriately. We centrifuged 20 mL of an overnight culture and resuspended it in culture medium with 2% Isovitalex. We spiked serum and plasma samples with 20 μL of an overnight culture that was first diluted 1:10 and inoculated BAL fluid with 20 μL of an overnight culture ( Table 2 ). Methanol Test Sample Preparation Test samples, positive controls, and the negative control of BAL fluid for the methanol treatment procedure all had a final volume of 500 μL. We used stock plates to grow bacteria, then selected colonies and suspended them in 3 mL of sterile water for injection (GE Healthcare, https://www.gehealthcare.com ). We took an OD reading at 600 nm on a spectrophotometer (ThermoFisher Scientific, https://www.thermofisher.com ) using a 1 cm 2 cuvette (ThermoFisher Scientific). We converted this value to an approximate CFU per milliliter value using a conversion factor as stated in test sample preparation. We calculated the total volume needed to spike each sample so that each sample would have 2 × 10 6 CFU ( Table 2 ). Filtration Procedure We conducted all filtration test procedures in triplicate for each matrix type. For negative controls, we used uninoculated matrix samples. For positive controls, we used 100 μL of unfiltered inoculated test samples suspended in broth culture medium. We then placed 450 μL of each test sample into a clean 0.2 μm PALL Nanosep Bio-Inert centrifuge filter (Pall Corporation, https://www.pall.com ) with a sterile microcentrifuge tube. In accordance with the manufacturer's recommendations, we centrifuged the filters for 3 min at 14,000 × g . We then transferred the filtrate to a clean tube and sealed it to prevent secondary contamination. We emphasize that the filtrate collection tubes should not be sealed with the same cap used to close the centrifuge filter before spinning because this cap could be contaminated with residual unfiltered sample and thus might yield false positive outcomes. We then suspended the filtrate in 4.5 mL BHI and incubated it at 35°C for 2 d. We incubated the positive controls in the same manner. After 48 h, we checked the tubes for turbidity and plated 5 × 200 μL samples onto the appropriate media. We incubated these samples at 35°C for an additional 7 d to ensure complete sterility. We considered this method to be validated only if all 3 replicates of all matrices were sterile in both broth and agar medium. Any failure, defined here as positive growth on agar or in broth media, prompted a review of the procedures. Once we determined the cause of the failure, we made the appropriate adjustments and reconducted the procedure in 3 replicates. Methanol Procedure We centrifuged BAL fluid for 5 min at 5,000 × g . We removed the supernatant and decontaminated it using the filtration procedure detailed in the previous section. We suspended the pellet in 500 μL of 80% methanol (ThermoFisher Scientific) and incubated it for 10 min. We placed 10% of this sample into 9.5 mL Dey-Engley neutralization broth (D/E media) (Becton Dickinson) and incubated it at 35°C for 5 d. After 5 d, we plated 200 μL of the D/E media onto 5 agar plates specific to each bacterial species and incubated them at 35°C for an additional 2 d. For positive controls, we used D/E media inoculated with bacteria and D/E media with 80% methanol added to the same volume as the test sample (50 μL of 80% methanol into 9.5 mL D/E media). We incubated this tube for 10 min and then inoculated it with bacteria. We also used growth media specific to each bacterial species as positive controls. For negative controls, we used uninoculated D/E media and D/E media inoculated with methanol treated bacteria. Results After following the described procedures, we observed that all samples (except 1) were sterilized in broth culture after 48 h incubation. The samples remained sterile after plating on agar medium incubated for 7 d ( Table 3 ). We determined that the test sample that had not been sterilized had sustained secondary contamination from the centrifuge filter unit cap. The PALL centrifuge filters are supplied as a filter and tube unit; they do not come with sterile secondary caps. To avoid secondary contamination, we transferred the filtrate to a clean tube immediately after spinning. We also observed that all samples were sterilized after treatment with 80% methanol and after incubation in broth culture for 5 d. The samples remained sterile on agar after an additional 2 d incubation. Table 3 Sterility of select agent samples after sterile filtration and methanol procedure*‡ Agent (reference) Positive serum Positive plasma Positive BAL Positive overnight culture Positive BAL cell pellet Bacillus anthracis 0/3 0/3 0/3 0/3 NT Yersinia pestis 0/3 0/3 0/3 0/3 NT Burkholderia mallei 0/3 0/3 0/3 0/3 NT Burkholderia pesudomallei 0/3 0/3 0/3 0/3 0/3 Francisella tularensis ( 14 ) 0/6 0/6 1/6†1/6†NT *BAL, bronchoalveolar lavage; NT, not tested. †Negative result caused by contaminated tube cap. ‡Total success rate for filtration: 97% Discussion Validating sterility procedures is a time-intensive and costly necessity for removing select agent samples from BSL-3 laboratories. Researchers can streamline this process by publishing validated methods in peer-reviewed journals. We described and validated reproducible procedures for select agent sample removal. However, researchers should ascertain that none of their sample is lost because of binding to the filter material. In this study, we checked 100% of the sample as a proof of concept, although we recognize the impossibility of incubating 100% of the sample to ensure sterility during actual experiments. Our laboratory now samples 10% of the filtrate to verify successful disinfection. We have found that these filters have an approximate failure rate of 0.1%; however, other researchers such as Dauphin et al. have found a failure rate closer to 3% ( 14 ). The differences in failure rates, variety of available filter membranes, and new methods of sterilization showcase the need for clear, detailed, and reproducible published methods.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272745/

cAMP signaling primes lung endothelial cells to activate caspase-1 during Pseudomonas aeruginosa infection

Activation of the inflammasome-caspase-1 axis in lung endothelial cells is emerging as a novel arm of the innate immune response to pneumonia and sepsis caused by Pseudomonas aeruginosa . Increased levels of circulating autacoids are hallmarks of pneumonia and sepsis and induce physiological responses via cAMP signaling in targeted cells. However, it is unknown whether cAMP affects other functions, such as P. aeruginosa- induced caspase-1 activation. Herein, we describe the effects of cAMP signaling on caspase-1 activation using a single cell flow cytometry-based assay. P. aeruginosa infection of cultured lung endothelial cells caused caspase-1 activation in a distinct population of cells. Unexpectedly, pharmacological cAMP elevation increased the total number of lung endothelial cells with activated caspase-1. Interestingly, addition of cAMP agonists augmented P. aeruginosa infection of lung endothelial cells as a partial explanation underlying cAMP priming of caspase-1 activation. The cAMP effect(s) appeared to function as a priming signal because addition of cAMP agonists was required either before or early during the onset of infection. However, absolute cAMP levels measured by ELISA were not predictive of cAMP-priming effects. Importantly, inhibition of de novo cAMP synthesis decreased the number of lung endothelial cells with activated caspase-1 during infection. Collectively, our data suggest that lung endothelial cells rely on cAMP signaling to prime caspase-1 activation during P. aeruginosa infection.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1619899/

Mining Microarray Data at NCBI’s Gene Expression Omnibus (GEO) *

Summary The Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) has emerged as the leading fully public repository for gene expression data. This chapter describes how to use Web-based interfaces, applications, and graphics to effectively explore, visualize, and interpret the hundreds of microarray studies and millions of gene expression patterns stored in GEO. Data can be examined from both experiment-centric and gene-centric perspectives using user-friendly tools that do not require specialized expertise in microarray analysis or time-consuming download of massive data sets. The GEO database is publicly accessible through the World Wide Web at http://www.ncbi.nlm.nih.gov/geo .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8789007/

Photophysical Details and O 2 -Sensing Analysis of a Eu(III) Complex in Polymer Composite Nanofibers Prepared by Electrospinning

An as-synthesized Eu(III) complex, denoted as Eu(N-DPNQ)(TTD) 3 , was prepared and characterized, and the antenna mechanism between these ligands and central metal emitter was studied. Here DPNQ means 10-ethyl-10H-indolo [2′,3':5,6]pyrazino[2,3-f][1,10]phenanthroline and TTD is 4,4,4-trifluoro-1-(thiophen-2-yl)butane-1,3-dione. We find that Eu(N-DPNQ)(TTD) 3 emission intensity dependents on oxygen concentration, and O 2 -sensing skill of Eu(N-DPNQ)(TTD) 3 in polymer composite nanofibers of poly (vinylpyrrolidone) (PVP) prepared by electrospinning is investigated. Results reveal that the emission quenching of Eu(N-DPNQ)(TTD) 3 is caused by the ground state (triplet) oxygen quenching on antenna ligands triplet state. The Eu(N-DPNQ)(TTD) 3 doped composite nanofiber with a loading level of 6 wt% exhibits the best result with sensitivity of 2.43 and response time of 10 s, along with linear response. Introduction Rare earth metal compounds are attractive ones, showing wide applications in laser and luminescence and serving as probes in porous and bio-active materials. Their advantages include efficient emission, long-lived fluorescence and sharp emission peaks ( Sun et al., 2002 ; Xu et al., 2002 ; Gunnlaugsson et al., 2003 ; Sun et al., 2003 ; Gunnlaugsson et al., 2004 ; Nakamura et al., 2007 ; Ai et al., 2009 ; Zhang et al., 2009 ; Park et al., 2010 ; Zhang et al., 2010 ). Very recently, the emission features of Eu(III) emitters grafted on a solid host have been systematically investigated ( Xu et al., 2002 ; Li et al., 2006 ; Li and Yan, 2008 ; Yan and Wang, 2008 ; Li Kong et al., 2009 ). Some Eu(III) complexes have been demonstrated as optical O 2 -sensing probes ( Amao et al., 2000a ; Amao et al., 2000b ; Zuo et al., 2010 ). Long nanofibers have found their potential applications such as reinforcement, filters, textiles, catalysis and medicine. Electrospinning is a fascinating method for preparation of fibers at nanometer level, and has been already applied in many technological areas ( Huang et al., 2003 ). As a fibers drawing technique, electrospinning is finished via the drawing of polymer solutions and melts ( Zhang et al., 2007a ). The fiber shape and morphology are controlled by many parameters, such as polymer nature (molecular weight and its distribution, glass temperature and compatibility), physical characters of the precursor solution (doping level, conductivity, tension and so on), solvent partial pressure, field strength, and environmental humidity. It is particularly fascinating that the polymers can be decorated and adjusted by various dopants such as luminescent phosphors and dyes by electrospinning ( Greiner and Wendorff, 2007 ). Therefore, several functionalized composite nanofibers have been proposed, along with their practical application ( Wang et al., 2002 ; Baldé et al., 2008 ; Dodiuk-kenig et al., 2008 ; Liu et al., 2008 ; Tan et al., 2008 ; Takahashi et al., 2009 ; Wang F. et al., 2009 ; Wang W. et al., 2009 ; Wang Y. et al., 2009 ). One of the most important application of composite nanofibers is chemical sensing or biosensing profiting from their vast surface area, which may lead to super-sensitive and instant response during sensing process. In this article, a novel oxygen-sensing Eu(III) probe of Eu(N-DPNQ)(TTD) 3 is synthesized, and incorporated into polymer composite nanofibers using electrospinning method. Here, a polymer PVP is selected as the supporting host for this oxygen-sensing probe, owing to its virtues of good mechanical strength, stable physical property and good compatibility with various dopants ( Wang et al., 2002 ; Wang W. et al., 2009 ; Wang Y. et al., 2009 ). The vast surface area and the uniform dispersal of the resulting composite samples accelerate oxygen diffusion, leading to sensitivity improvement and response time decrease of oxygen sensor. O 2 -sensing parameters of the composite nanofibers upon three loading contents are compared and investigated, as well as luminescence quenching mechanism. Experimental Materials and Apparatus PVP ( molecular weight ≈ 60000) was purchased from Tanggu Chemicals Corporation (China). Eu 2 O 3 and 1, 10-Phenanthroline were bought via Shanghai Chemical Ltd. (China). Isatin and Pd/C were ordered from Aldrich Chemical Ltd. N, N-dimethylformamide (DMF), bromoethane, EtOH, 1, 2-dichloroethane and concentrated HCl were supplied by Tianjin Chemicals Corporation. Elemental analysis was obtained using a Vario Element Analyzer. 1 H NMR experiment was deployed on a Bruker-DPX-300 spectrometer. IR experiment was finished by a Magna560 spectrometer. Thermogravimetric experiment was performed on a Perkin-Elmer thermal analyzer. Phosphorescence spectrum was determined at liquid N 2 temperature by FLS 920 spectrometer. The fiber size and morphology were obtained using a Hitachi S-4800 microscopy. Absorption experiment was done on a Cary 500 spectrometer. Emission experiment was done by a Hitachi F-4500 spectrometer. For Stern-Volmer plots experiment, O 2 and N 2 were controlled by gas flowmeters and mixed in a quartz chamber. Synthesis of N-DPNQ and its Complex Related synthetic schemes for N-DPNQ and its europium complex are summarized as Scheme 1 . SCHEME 1 Synthetic routes for N-DPNQ and Eu (N-DPNQ) (TTD) 3 . N-DPNQ: the related two starting compounds were obtained following reported methods ( Bolger et al., 1996 ; Bian et al., 2002 ). N-DPNQ was synthesized by modification of the literature method ( Bodige and MacDonnell, 1997 ). The detailed synthetic routes were described as follows: (0.756 g, 3.6 mmol) 1, 10-phenanthroline-5, 6-diamine was added into 80 ml of methanol under, and (0.525g, 3 mmol) 1-ethylindoline-2,3-dione was added to the solution. This solution was refluxed for 8 h. After cooling the mixture, solid product was filtered off and washed by EtOH. The solid powder wasrecrystallized from methanol. 1 H NMR(CDCl 3 , 500 MHz ) δ [ppm]: 9.22 (d, 1H), 9.13 (d, 1H), 9.05 (d, 1H), 8.52 (d, 1H), 7.66–7.72 (m, 2H), 7.57 (d, 1H), 7.34 (d, 1H), 7.28 (s, 2H), 4.34 (t, 2H), and 1.34 (t, 3H). 13 C NMR δ [ppm]: 150.2, 147.7, 145.8, 139.9, 138.8, 137.7, 137.0, 134.9, 127.8, 124.2, 123.0, 121.5, 120.2, 109.8, 39.4, 14.2. Calculated for C 22 H 15 N 5 : 349.1, MS Found: 349.0 [M] + . Eu(N-DPNQ)(TTD) 3 . This compound was prepared in accordance with a published method ( Bauer et al., 1964 ) (0.3 mmol) TTD and (0.11 mmol) N-DPNQ were mixed. Then EtOH (5 ml) was added. The mixture pH was modified as 7.0 with NaOH. Finally, (0.1 mmol) EuCl 3 ·6H 2 O and H 2 O (2 ml) were poured into above EtOH solution. After reacting at 59°C (60 min), solid sample was filtered off and purified in EtOH. Elemental analysis, Found: C, 47.59; H, 2.32; N, 5.98. IR (KBr, cm −1 ): 1598 (C=O), 464 (Eu-O). 1 H NMR(CDCl 3 , 500 MHz ) δ [ppm]: 8.55–8.59 (m, 2H), 8.45–8.49 (m, 2H), 8.21–8.24 (m, 2H), 7.82 (s, 1H), 7.59–7.56 (m, 3H), 7.42–7.45 (m, 6H), 7.12 (d, 3H), 6.28 (s, 3H), 4.44 (t, 2H), and 1.39 (t, 3H). 13 C NMR δ [ppm]: 172.5, 162.3, 147.6, 141.7, 140.2, 138.8, 136.9, 132.5, 128.1, 127.8, 126.5, 123.1, 121.5, 120.4, 116.6, 109.8, 89.4, 39.3, 14.1. Calculated for C 46 H 27 N 5 EuF 9 O 6 S 3 : 1164.88, MS Found: 1165.0 [M] + . Synthesis of Gd Reference Compounds Gd(N-DPNQ) 2 Cl 3 : (0.07 g, 0.2 mmol) N-DPNQ was mixed with 5 ml of EtOH. Later, (0.037 g, 0.1 mmol) GdCl 3 ·6H 2 O and ten drops of water were incorporated under stirring. The suspension was refluxed for 2 h at 80°C, and solid powder was resulted by filtration. Calculated for C 22 H 23 Cl 3 N 5 GdO 4 : 686.0, MS Found: 686.1 [M] + . Gd(TTD) 3 ·(H 2 O) 2 : (0.067 g, 0.3 mmol) HTTD was added into 5 ml ethanol, and 1.0 mol/L 0.3 ml of NaOH was slowly incorporated. After vigorous stirring of 20 min, (0.037 g, 0.1 mmol) GdCl 3 ·6H 2 O and ten drops of water were incorporated under stirring. The suspension was refluxed 2 h at 80°C. Solid powder was obtained by precipitating. Calculated for C 24 H 16 F 9 GdO 8 S 3 : 856.91, MS Found: 856.9 [M] + . Preparation of Electrospinning Solutions A mixed solvent 1, 2-dichloroethane/ethanol (v:v = 1:1) containing 1 g of PVP was prepared. After the solvent mixtures was stirred, a controlled amount of Eu(N-DPNQ)(TTD) 3 (0.4, 0.6, and 0.8%) relative to PVP weight was added to PVP solutions. Electrospinning Process When preparing the electrospinning nanofibers, the precursor solution was poured into a glass syring. Its plastic needle was wired to the anode of a high voltage generator. A plate of Al foil was placed under the plastic needle, serving as collector plat. The voltage was set as 18 kV with collecting distance between needle and collector plate of 20 cm. The current was less than 0.01 mA. The composite fibrous samples (0.4, 0.6, and 0.8%) are denoted as Eu 1 , Eu 2 , and Eu 3 , respectively. Materials and Apparatus PVP ( molecular weight ≈ 60000) was purchased from Tanggu Chemicals Corporation (China). Eu 2 O 3 and 1, 10-Phenanthroline were bought via Shanghai Chemical Ltd. (China). Isatin and Pd/C were ordered from Aldrich Chemical Ltd. N, N-dimethylformamide (DMF), bromoethane, EtOH, 1, 2-dichloroethane and concentrated HCl were supplied by Tianjin Chemicals Corporation. Elemental analysis was obtained using a Vario Element Analyzer. 1 H NMR experiment was deployed on a Bruker-DPX-300 spectrometer. IR experiment was finished by a Magna560 spectrometer. Thermogravimetric experiment was performed on a Perkin-Elmer thermal analyzer. Phosphorescence spectrum was determined at liquid N 2 temperature by FLS 920 spectrometer. The fiber size and morphology were obtained using a Hitachi S-4800 microscopy. Absorption experiment was done on a Cary 500 spectrometer. Emission experiment was done by a Hitachi F-4500 spectrometer. For Stern-Volmer plots experiment, O 2 and N 2 were controlled by gas flowmeters and mixed in a quartz chamber. Synthesis of N-DPNQ and its Complex Related synthetic schemes for N-DPNQ and its europium complex are summarized as Scheme 1 . SCHEME 1 Synthetic routes for N-DPNQ and Eu (N-DPNQ) (TTD) 3 . N-DPNQ: the related two starting compounds were obtained following reported methods ( Bolger et al., 1996 ; Bian et al., 2002 ). N-DPNQ was synthesized by modification of the literature method ( Bodige and MacDonnell, 1997 ). The detailed synthetic routes were described as follows: (0.756 g, 3.6 mmol) 1, 10-phenanthroline-5, 6-diamine was added into 80 ml of methanol under, and (0.525g, 3 mmol) 1-ethylindoline-2,3-dione was added to the solution. This solution was refluxed for 8 h. After cooling the mixture, solid product was filtered off and washed by EtOH. The solid powder wasrecrystallized from methanol. 1 H NMR(CDCl 3 , 500 MHz ) δ [ppm]: 9.22 (d, 1H), 9.13 (d, 1H), 9.05 (d, 1H), 8.52 (d, 1H), 7.66–7.72 (m, 2H), 7.57 (d, 1H), 7.34 (d, 1H), 7.28 (s, 2H), 4.34 (t, 2H), and 1.34 (t, 3H). 13 C NMR δ [ppm]: 150.2, 147.7, 145.8, 139.9, 138.8, 137.7, 137.0, 134.9, 127.8, 124.2, 123.0, 121.5, 120.2, 109.8, 39.4, 14.2. Calculated for C 22 H 15 N 5 : 349.1, MS Found: 349.0 [M] + . Eu(N-DPNQ)(TTD) 3 . This compound was prepared in accordance with a published method ( Bauer et al., 1964 ) (0.3 mmol) TTD and (0.11 mmol) N-DPNQ were mixed. Then EtOH (5 ml) was added. The mixture pH was modified as 7.0 with NaOH. Finally, (0.1 mmol) EuCl 3 ·6H 2 O and H 2 O (2 ml) were poured into above EtOH solution. After reacting at 59°C (60 min), solid sample was filtered off and purified in EtOH. Elemental analysis, Found: C, 47.59; H, 2.32; N, 5.98. IR (KBr, cm −1 ): 1598 (C=O), 464 (Eu-O). 1 H NMR(CDCl 3 , 500 MHz ) δ [ppm]: 8.55–8.59 (m, 2H), 8.45–8.49 (m, 2H), 8.21–8.24 (m, 2H), 7.82 (s, 1H), 7.59–7.56 (m, 3H), 7.42–7.45 (m, 6H), 7.12 (d, 3H), 6.28 (s, 3H), 4.44 (t, 2H), and 1.39 (t, 3H). 13 C NMR δ [ppm]: 172.5, 162.3, 147.6, 141.7, 140.2, 138.8, 136.9, 132.5, 128.1, 127.8, 126.5, 123.1, 121.5, 120.4, 116.6, 109.8, 89.4, 39.3, 14.1. Calculated for C 46 H 27 N 5 EuF 9 O 6 S 3 : 1164.88, MS Found: 1165.0 [M] + . Synthesis of Gd Reference Compounds Gd(N-DPNQ) 2 Cl 3 : (0.07 g, 0.2 mmol) N-DPNQ was mixed with 5 ml of EtOH. Later, (0.037 g, 0.1 mmol) GdCl 3 ·6H 2 O and ten drops of water were incorporated under stirring. The suspension was refluxed for 2 h at 80°C, and solid powder was resulted by filtration. Calculated for C 22 H 23 Cl 3 N 5 GdO 4 : 686.0, MS Found: 686.1 [M] + . Gd(TTD) 3 ·(H 2 O) 2 : (0.067 g, 0.3 mmol) HTTD was added into 5 ml ethanol, and 1.0 mol/L 0.3 ml of NaOH was slowly incorporated. After vigorous stirring of 20 min, (0.037 g, 0.1 mmol) GdCl 3 ·6H 2 O and ten drops of water were incorporated under stirring. The suspension was refluxed 2 h at 80°C. Solid powder was obtained by precipitating. Calculated for C 24 H 16 F 9 GdO 8 S 3 : 856.91, MS Found: 856.9 [M] + . Preparation of Electrospinning Solutions A mixed solvent 1, 2-dichloroethane/ethanol (v:v = 1:1) containing 1 g of PVP was prepared. After the solvent mixtures was stirred, a controlled amount of Eu(N-DPNQ)(TTD) 3 (0.4, 0.6, and 0.8%) relative to PVP weight was added to PVP solutions. Electrospinning Process When preparing the electrospinning nanofibers, the precursor solution was poured into a glass syring. Its plastic needle was wired to the anode of a high voltage generator. A plate of Al foil was placed under the plastic needle, serving as collector plat. The voltage was set as 18 kV with collecting distance between needle and collector plate of 20 cm. The current was less than 0.01 mA. The composite fibrous samples (0.4, 0.6, and 0.8%) are denoted as Eu 1 , Eu 2 , and Eu 3 , respectively. Results and Discussion Thermal Property To discuss the thermal stability of Eu(N-DPNQ)(TTD) 3 , its decomposition temperature is determined from TGA (thermal gravimetric analysis) curve shown in Figure 1 . Corresponding DTG (differential thermogravimetric analysis) curve is shown for comparison as well. It is clear that Eu(N-DPNQ)(TTD) 3 is thermally stable below 300°C, and the 10% weight reduction temperature of Eu(N-DPNQ)(TTD) 3 is calculated to be 302°C. There are two regions of weight loss in the TGA curve of Eu(N-DPNQ)(TTD) 3 . The first decomposition region from 300 to 358°C is attributed to release of three TTD ligands (calculated 57.3%, found 61%). Upon higher temperature of 380°C, the leaving of ligand N-DPNQ leads the gradual weight loss of Eu(N-DPNQ)(TTD) 3 . There is still residual weight (19.4%) at temperature higher than 500°C. This residual weight is attributed to the remaining Eu element (13.0%) and O element (8.2%). It is assumed that Eu oxides are formed and finally preserved at the end of thermal decomposition. FIGURE 1 TGA and DTG curves of Eu (N-DPNQ) (TTD) 3 . Photophysical Properties Eu(N-DPNQ)(TTD) 3 absorption, excitation, and luminescence spectra of in dichloromethane (10 μM) solutions are observed in Figure 2 . The UV-vis absorption spectra of free N-DPNQ and free HTTD are shown in Figure 2 as well. The absorption bands for Eu(N-DPNQ)(TTD) 3 locating at around 227 and 275 nm, which well matches that of N-DPNQ, are assigned as introligand π-π* electron transitions of N-DPNQ. The absorption of 339 nm corresponds to the π-π* electron transition of HTTD ligand. Eu(N-DPNQ)(TTD) 3 excitation bands shown in Figure 2 are similar to its corresponding absorption spectrum. On the other hand, spectral shift and narrowed band are observed for the excitation spectra. This result suggests an indirect energy transfer dynamic from ligands to central metal ion since ligands have to experience a series of energy-wasting procedures, such as geometric relaxation, intersystem crossing and potential surface crossing, before transferring their energy to central metal ion. This statement is consistent with the antenna energy transfer procedure in rare earth complexes. The emission spectrum of Eu(N-DPNQ)(TTD) 3 in dichloromethane is also given in Figure 2 . Eu(N-DPNQ)(TTD) 3 showed typical photoluminescence (PL) peaks of Eu(III) with five bands peaking at 578, 590, 610, 649, and 699 nm, which correspond to 5 D 0 → 7 F n ones ( n = 0–4), respectively. FIGURE 2 Absorption, excitation, and PL spectra of Eu (N-DPNQ) (TTD) 3 , N-DPNQ and HTTD in dichloromethane. The PL quantum yield (Φ) of Eu(N-DPNQ)(TTD) 3 is determined with the help of a reference sample whose PL quantum yield is well determined, according to below formula. Φ unk = Φ std Ι unk / A unk A std / I std η unk 2 / η std 2 (1) Here Ф unk means Φ of unknown target. Ф std =0.546 means the Φ of standard sample ( Ye et al., 2005 ). I unk and I std denote the emission intensity (integrated areas) of unknown target and standard sample, respectively. A unk and A std indicate the absorbance of unknown target and standard sample with specific excitation position. η unk and η std denote solvent refractive index values of unknown target and standard sample solutions. The Ф of Eu(N-DPNQ)(TTD) 3 is calculated to be 0.12. The emissive dynamic decay of Eu(N-DPNQ)(TTD) 3 is also discussed. In Figure 3 , Eu(N-DPNQ)(TTD) 3 shows a biexponential decay pattern with a mean lifetime of 268.3 μs. Corresponding two decay components are τ1 = 0.00004 s and τ2 = 0.00027 s. These two lifetime components are rather different from each other, indicating their different decay paths. Generally, the observation of strong absorption in UV-Vis region and a short-lived emissive center indicate a potential surface crossing procedure ( Wang et al., 2002 ). In this case, the long-lived emissive center is attributed to the decay of Eu(III) f-f transitions, while the short-lived emissive center is assigned as the decay of ligand energy transfer to metal center. This assignment is consistent with its small proportion to the emissive center. FIGURE 3 Emission dynamics of 5 D 0 - 7 F 2 transition of Eu (N-DPNQ) (TTD) 3 upon air, pure N 2 and pure O 2 conditions. As shown in Figure 3 , the lifetimes of Eu(N-DPNQ)(TTD) 3 in solid state are determined as 367.5 μs (τ1 = 0.00009 s and τ2 = 0.0004 s) upon 100% N 2 and 137.2 μs (τ1 = 0.00005 s and τ2 = 0.00015 s) upon 100% O 2 , respectively, indicating an obvious oxygen quenching effect. The PL spectra of Eu(N-DPNQ)(TTD) 3 in solid state upon air, 100% N 2 and 100% O 2 are also measured. It is clearly observed from Figure 4 that the PL intensity of Eu(N-DPNQ)(TTD) 3 is significantly influenced by oxygen concentration. The Ф values of Eu(N-DPNQ)(TTD) 3 in solid state are determined as 0.18 upon 100% N 2 and 0.06 upon 100% O 2 , respectively, compared to that upon air condition of 0.12. The emission intensity of 5 D 0 → 7 F 2 transitions has the most obvious change among the Eu 3+ emission lines upon 100% O 2 . This observation suggests that the emission of Eu(N-DPNQ)(TTD) 3 complex is probably oxygen sensitive and could be applied for O 2 -sensing. FIGURE 4 PL spectra of Eu (N-DPNQ) (TTD) 3 upon various conditions. Excitation = 365 nm. Micromorphology and Structure of Eu(N-DPNQ)(TTD) 3 /PVP To further realize the practical application and optimize oxygen-sensing properties, Eu(N-DPNQ)(TTD) 3 is incorporated in one-dimensional nanofibers of PVP. The SEM photos of all three fibrous samples are shown in Figures 5A–D , respectively. As shown in Figure 5 , the uniform nanofibers have been formed through electrospinning process. The average diameters for Eu 1 , Eu 2 , and Eu 3 are 400, 600, and 900 nm, respectively. FIGURE 5 SEM photos of Eu (N-DPNQ) (TTD)3/PVP: (A) Eu 1 , (B) Eu 2 , (C) Eu 3 and (D) a large scale view of Eu 2 . Figure 6 exhibits the IR peaks of Eu(N-DPNQ)(TTD) 3 , PVP, Eu 1 , Eu 2 , and Eu 3 . For PVP nanofibers, the band around 1674 cm −1 is related with the stretching vibration of C=O. This is a characteristic band of PVP. However, this C=O band is shifted to 1665 cm −1 for Eu 1 , Eu 2 , and Eu 3 . The decreased wavenumber of this C=O band is attributed to the electron-accepting effect of dopant Eu(N-DPNQ)(TTD) 3 on the O atom of PVP chain. This result confirms a close and direct contact between Eu(N-DPNQ)(TTD) 3 molecules and PVP network. In other words, dopant molecuels have been well captured by PVP host. A similar IR spectral red shift has been reported in composite samples ( Zhang et al., 2008 ). Furthermore, the IR spectra of three fibrous samples are quite similar with that of pure PVP nanofiber, confirming that Eu(N-DPNQ)(TTD) 3 is well capped by PVP matrix ( Zhang et al., 2007b ). FIGURE 6 IR spectra of Eu(N-DPNQ)(TTD) 3 , PVP, Eu 1 , Eu 2 , and Eu 3 . Oxygen-Sensing Properties and Sensing Mechanism To assess the oxygen-sensing ability of the composite nanofibers, the PL spectra of three fibrous samples at various O 2 levels are demonstrated in Figure 7 . The PL spectra of Eu 1 , Eu 2 , and Eu 3 are similar to the PL bands of complex Eu(N-DPNQ)(TTD) 3 , showing characteristic emissions of Eu 3+ ion with 5 D 0 → 7 F n ( n = 0–4) transitions. The emission intensity of 5 D 0 → 7 F 2 transition for Eu 1 , Eu 2 , and Eu 3 is quenched greatly by O 2 . FIGURE 7 PL spectra of Eu 1 , Eu 2 , and Eu 3 at different oxygen concentrations. Excitation = 365 nm. To further investigate the quenching mechanism, energy levels of relevant electronic states of N-DPNQ and HTTD are measured. The singlet level (S 1 ) values of N-DPNQ and HTTD are measured as 3.11 eV (398 nm) and 3.08 eV (402 nm) which correspond to their absorption cutting-off values. The phosphorescence spectra of Gd(N-DPNQ) 2 Cl 3 and Gd (TTD) 3 (H 2 O) 2 at 77 K are given in Figure 8 . Correspondingly, the triplet levels (T 1 ) of Gd(N-DPNQ) 2 Cl 3 and Gd (TTD) 3 are determined as 2.75 eV (450 nm) and 2.32 eV (533 nm). FIGURE 8 Phosphorus spectra of Gd (N-DPNQ) 2 Cl 3 and Gd (TTD) 3 at 77 K. A schematic presentation for energy transfer mechanism is shown as Figure 9 according to above mentioned experimental results. For Eu(N-DPNQ)(TTD) 3 , the positive energy transfer of N-DPNQ S 1 excited state to TTD S 1 excited state is difficult due to the same S 1 level. But partial S 1 energy of N-DPNQ can be transferred to N-DPNQ T 1 excited state. Hence such partial energy could be transferred to the lowest triplet level of TTD and then to 5 D 0 for Eu 3+ . Furthermore, excitation energy could be transferred from TTD singlet level to TTD triplet level, and finally to 5 D 0 for Eu 3+ ( Xin et al., 2004 ; Xu et al., 2006 ). Generally, there are three main steps involved in a sensitized Eu 3+ luminescence process, as shown in Figure 10 ( Parker, 2000 ). Firstly, the antenna ligand absorbs energy and is excited to its singlet level. Then this energy is migrated to the T 1 excited state of antenna ligands by inter-system crossing (ISC). Next, the excitation procedure from the T 1 level of antenna ligands to Eu 3+ ion excited state happens in this process. If the rate of energy transfer is sufficiently slow so as to lead a deactivation of the T 1 excited state of antenna ligands, the quenching by molecular oxygen could occur in this process. Finally, Eu 3+ ion excited state energy could be transferred to the ground state, then Eu 3+ luminescence is generated. Hence, the emission quenching of Eu 3+ probe by molecular oxygen is based on intermolecular collision of S 1 /T 1 state of antenna ligands with O 2 S 0 state. The above procedure can be presented below: * L − Eu + O 2 → L − Eu + O 2 * (2) here L and Eu denote antenna ligands and Eu(III) complex, and * stands for the excited state. FIGURE 9 Energy levels of ligands and Eu center in Eu (N-DPNQ) (TTD) 3 . FIGURE 10 Key photophysical processes of sensitized Eu 3+ luminescence. The quenching of luminescent molecule in a homogeneous medium with non-obvious host afluoence is supposed to be a simple exponential dynamic procedure. The PL intensity variation against O 2 levels should follow Stern-Volmer relationship. I 0 I = τ 0 τ = 1 + K s v [ O 2 ] (3) Here I and τ denote emission intensity and dynamic lifespan, respectively. I 0 is the intrinsic emission intensity with no O 2 . K SV shall be the Stern-Volmer constant. K q stands for a fixed fitting parameter. [O 2 ] means O 2 ratio. The curve of I 0 /I against [O 2 ] shall be a linear plot. Its slope shall be K SV . Typical intensity-formed Stern-Volmer fitting curves for Eu 1 , Eu 2 , and Eu 3 are presented in Figure 11 . These plots for Eu 1 , Eu 2 , and Eu 3 are well fitted by Eq. 3 . The parameters are also found in Table 1 . Eu 2 (with doping level of 0.6wt%) seems the optimal sample by showing the highest sensitivity of 2.43. As observed, Eu 1 and Eu 2 show a good linear relationship, whereas Eu 3 shows a poor linearity with a linearly dependent coefficient R ( Nakamura et al., 2007 ) of 0.969. Since Eu(N-DPNQ)(TTD) 3 could be effectively quenched by molecular O 2 , the increase in amount of Eu(N-DPNQ)(TTD) 3 results in increased molar fractions of oxygen-quenchable dye and hence the sensitivity and linearity of Eu 2 are superior to the corresponding values of Eu 1 . If the distribution of Eu-probe in matrix is changed by increasing amount of Eu(N-DPNQ)(TTD) 3 , this could affect the sensitivity and linearity of the sensor. In fact, the increasing of Eu(III) complex in Eu 3 leads to self-aggregation of Eu-probe molecules in the matrix, indicating that there is a change in the micro-environment of the composite nanofibers ( Lee and Okura, 1997 ; Shi et al., 2009 ). FIGURE 11 Intensity-based Stern-Volmer plots of Eu 1 , Eu 2 , and Eu 3 at different oxygen concentrations. TABLE 1 Key sensing parameters for Eu 1 , Eu 2 , and Eu 3 . Loading levels (wt%) t ↓ (s) t ↑ (s) I 0 / I 100 K SV (O 2 % −1 ) R Nakamura et al. (2007) Eu 1 , 0.4 8 14 1.93 0.00949 ± 0.00007 0.998 Eu 2 , 0.6 10 10 2.43 0.01444 ± 0.00008 0.999 Eu 3 , 0.8 7 12 2.31 0.01191 ± 0.00039 0.969 The response and recovery characteristics are very fundamental parameters for oxygen-sensing materials. Generally, response (t ↓ ) and recovery (t ↑ ) parameters are determined by calculating the time for each sample to lose or restore ninety five percent of maximum original emission intensity when testing atmosphere is changed between 100% N 2 and 100 O 2 . Figure 12 shows the emission variation of Eu 1 , Eu 2 , and Eu 3 upon surrounding atmosphere cycle of 100% N 2 -100% O 2 -100% N 2 . Based on the dynamic variation measurements, the values of t ↓ and t ↑ are measured and summarized in Table 1 . It is clear in Figure 12 that repeatable emission responses are detected with Eu 1 , Eu 2 , and Eu 3 . There is slice drift intensity. Furthermore, we also have monitored the mean sensitivity and response behavior of Eu 1 , Eu 2 , and Eu 3 over 12 weeks (interval = 15 days). The detected aging effect on sensitivity and response behavior is neglectable. The oxygen-sensing properties of Eu 1 , Eu 2 , and Eu 3 at 15 and 30 days also have been measured and the results are very similar, as shown in Table 2 . FIGURE 12 Response time of Eu 1 , Eu 2 , and Eu 3 upon various environmental atmospheres, emission = 610 nm. TABLE 2 Sensing parameters upon different aging days. Sample Sensitivity/response 0 day 15 days 30 days 45 days 60 days 75 days 84 days Eu 1 1.93/8 1.92/8 1.92/8 1.90/9 1.90/9 1.89/9 1.88/9 Eu 2 2.43/10 2.41/10 2.41/10 2.40/10 2.40/12 2.38/12 2.37/12 Eu 3 2.31/7 2.31/7 2.30/8 2.30/9 2.29/9 2.28/9 2.25/10 Considering the sensing mechanism of a dynamic collision between O 2 ground state and excited state probe, it is assumed that these composite nanofibers should have good sensing selectivity towards O 2 since most other gases have closed-shell structures and thus are not open for probe energy transfer. Aiming at a primitive evaluation on the sensing selectivity of these composite nanofibers, five typical interfering gases are selected, including CO 2 , benzene, toluene, CHCl 3 and CH 2 Cl 2 . The emission spectra of a representative sample Eu 2 are recorded and compared in Supplementary Figure S1 (Supporting Information). No obvious spectral shift or intensity variation is observed upon these interfering gases, which shall be attributed to the unique f-f transitions of these Eu(III) probes. As a consequence, it is concluded that these composite nanofibers have good selectivity for O 2 . On the other hand, such f-f transitions needs a complicated energy transfer procedure, which makes the sensitivity far away from satisfactory. For later improvement, the antenna energy transfer from ligand to central metal ion should be simplified, so that the O 2 quenching effect on excited probe shall be efficient and complete, leading to improved sensitivity. Thermal Property To discuss the thermal stability of Eu(N-DPNQ)(TTD) 3 , its decomposition temperature is determined from TGA (thermal gravimetric analysis) curve shown in Figure 1 . Corresponding DTG (differential thermogravimetric analysis) curve is shown for comparison as well. It is clear that Eu(N-DPNQ)(TTD) 3 is thermally stable below 300°C, and the 10% weight reduction temperature of Eu(N-DPNQ)(TTD) 3 is calculated to be 302°C. There are two regions of weight loss in the TGA curve of Eu(N-DPNQ)(TTD) 3 . The first decomposition region from 300 to 358°C is attributed to release of three TTD ligands (calculated 57.3%, found 61%). Upon higher temperature of 380°C, the leaving of ligand N-DPNQ leads the gradual weight loss of Eu(N-DPNQ)(TTD) 3 . There is still residual weight (19.4%) at temperature higher than 500°C. This residual weight is attributed to the remaining Eu element (13.0%) and O element (8.2%). It is assumed that Eu oxides are formed and finally preserved at the end of thermal decomposition. FIGURE 1 TGA and DTG curves of Eu (N-DPNQ) (TTD) 3 . Photophysical Properties Eu(N-DPNQ)(TTD) 3 absorption, excitation, and luminescence spectra of in dichloromethane (10 μM) solutions are observed in Figure 2 . The UV-vis absorption spectra of free N-DPNQ and free HTTD are shown in Figure 2 as well. The absorption bands for Eu(N-DPNQ)(TTD) 3 locating at around 227 and 275 nm, which well matches that of N-DPNQ, are assigned as introligand π-π* electron transitions of N-DPNQ. The absorption of 339 nm corresponds to the π-π* electron transition of HTTD ligand. Eu(N-DPNQ)(TTD) 3 excitation bands shown in Figure 2 are similar to its corresponding absorption spectrum. On the other hand, spectral shift and narrowed band are observed for the excitation spectra. This result suggests an indirect energy transfer dynamic from ligands to central metal ion since ligands have to experience a series of energy-wasting procedures, such as geometric relaxation, intersystem crossing and potential surface crossing, before transferring their energy to central metal ion. This statement is consistent with the antenna energy transfer procedure in rare earth complexes. The emission spectrum of Eu(N-DPNQ)(TTD) 3 in dichloromethane is also given in Figure 2 . Eu(N-DPNQ)(TTD) 3 showed typical photoluminescence (PL) peaks of Eu(III) with five bands peaking at 578, 590, 610, 649, and 699 nm, which correspond to 5 D 0 → 7 F n ones ( n = 0–4), respectively. FIGURE 2 Absorption, excitation, and PL spectra of Eu (N-DPNQ) (TTD) 3 , N-DPNQ and HTTD in dichloromethane. The PL quantum yield (Φ) of Eu(N-DPNQ)(TTD) 3 is determined with the help of a reference sample whose PL quantum yield is well determined, according to below formula. Φ unk = Φ std Ι unk / A unk A std / I std η unk 2 / η std 2 (1) Here Ф unk means Φ of unknown target. Ф std =0.546 means the Φ of standard sample ( Ye et al., 2005 ). I unk and I std denote the emission intensity (integrated areas) of unknown target and standard sample, respectively. A unk and A std indicate the absorbance of unknown target and standard sample with specific excitation position. η unk and η std denote solvent refractive index values of unknown target and standard sample solutions. The Ф of Eu(N-DPNQ)(TTD) 3 is calculated to be 0.12. The emissive dynamic decay of Eu(N-DPNQ)(TTD) 3 is also discussed. In Figure 3 , Eu(N-DPNQ)(TTD) 3 shows a biexponential decay pattern with a mean lifetime of 268.3 μs. Corresponding two decay components are τ1 = 0.00004 s and τ2 = 0.00027 s. These two lifetime components are rather different from each other, indicating their different decay paths. Generally, the observation of strong absorption in UV-Vis region and a short-lived emissive center indicate a potential surface crossing procedure ( Wang et al., 2002 ). In this case, the long-lived emissive center is attributed to the decay of Eu(III) f-f transitions, while the short-lived emissive center is assigned as the decay of ligand energy transfer to metal center. This assignment is consistent with its small proportion to the emissive center. FIGURE 3 Emission dynamics of 5 D 0 - 7 F 2 transition of Eu (N-DPNQ) (TTD) 3 upon air, pure N 2 and pure O 2 conditions. As shown in Figure 3 , the lifetimes of Eu(N-DPNQ)(TTD) 3 in solid state are determined as 367.5 μs (τ1 = 0.00009 s and τ2 = 0.0004 s) upon 100% N 2 and 137.2 μs (τ1 = 0.00005 s and τ2 = 0.00015 s) upon 100% O 2 , respectively, indicating an obvious oxygen quenching effect. The PL spectra of Eu(N-DPNQ)(TTD) 3 in solid state upon air, 100% N 2 and 100% O 2 are also measured. It is clearly observed from Figure 4 that the PL intensity of Eu(N-DPNQ)(TTD) 3 is significantly influenced by oxygen concentration. The Ф values of Eu(N-DPNQ)(TTD) 3 in solid state are determined as 0.18 upon 100% N 2 and 0.06 upon 100% O 2 , respectively, compared to that upon air condition of 0.12. The emission intensity of 5 D 0 → 7 F 2 transitions has the most obvious change among the Eu 3+ emission lines upon 100% O 2 . This observation suggests that the emission of Eu(N-DPNQ)(TTD) 3 complex is probably oxygen sensitive and could be applied for O 2 -sensing. FIGURE 4 PL spectra of Eu (N-DPNQ) (TTD) 3 upon various conditions. Excitation = 365 nm. Micromorphology and Structure of Eu(N-DPNQ)(TTD) 3 /PVP To further realize the practical application and optimize oxygen-sensing properties, Eu(N-DPNQ)(TTD) 3 is incorporated in one-dimensional nanofibers of PVP. The SEM photos of all three fibrous samples are shown in Figures 5A–D , respectively. As shown in Figure 5 , the uniform nanofibers have been formed through electrospinning process. The average diameters for Eu 1 , Eu 2 , and Eu 3 are 400, 600, and 900 nm, respectively. FIGURE 5 SEM photos of Eu (N-DPNQ) (TTD)3/PVP: (A) Eu 1 , (B) Eu 2 , (C) Eu 3 and (D) a large scale view of Eu 2 . Figure 6 exhibits the IR peaks of Eu(N-DPNQ)(TTD) 3 , PVP, Eu 1 , Eu 2 , and Eu 3 . For PVP nanofibers, the band around 1674 cm −1 is related with the stretching vibration of C=O. This is a characteristic band of PVP. However, this C=O band is shifted to 1665 cm −1 for Eu 1 , Eu 2 , and Eu 3 . The decreased wavenumber of this C=O band is attributed to the electron-accepting effect of dopant Eu(N-DPNQ)(TTD) 3 on the O atom of PVP chain. This result confirms a close and direct contact between Eu(N-DPNQ)(TTD) 3 molecules and PVP network. In other words, dopant molecuels have been well captured by PVP host. A similar IR spectral red shift has been reported in composite samples ( Zhang et al., 2008 ). Furthermore, the IR spectra of three fibrous samples are quite similar with that of pure PVP nanofiber, confirming that Eu(N-DPNQ)(TTD) 3 is well capped by PVP matrix ( Zhang et al., 2007b ). FIGURE 6 IR spectra of Eu(N-DPNQ)(TTD) 3 , PVP, Eu 1 , Eu 2 , and Eu 3 . Oxygen-Sensing Properties and Sensing Mechanism To assess the oxygen-sensing ability of the composite nanofibers, the PL spectra of three fibrous samples at various O 2 levels are demonstrated in Figure 7 . The PL spectra of Eu 1 , Eu 2 , and Eu 3 are similar to the PL bands of complex Eu(N-DPNQ)(TTD) 3 , showing characteristic emissions of Eu 3+ ion with 5 D 0 → 7 F n ( n = 0–4) transitions. The emission intensity of 5 D 0 → 7 F 2 transition for Eu 1 , Eu 2 , and Eu 3 is quenched greatly by O 2 . FIGURE 7 PL spectra of Eu 1 , Eu 2 , and Eu 3 at different oxygen concentrations. Excitation = 365 nm. To further investigate the quenching mechanism, energy levels of relevant electronic states of N-DPNQ and HTTD are measured. The singlet level (S 1 ) values of N-DPNQ and HTTD are measured as 3.11 eV (398 nm) and 3.08 eV (402 nm) which correspond to their absorption cutting-off values. The phosphorescence spectra of Gd(N-DPNQ) 2 Cl 3 and Gd (TTD) 3 (H 2 O) 2 at 77 K are given in Figure 8 . Correspondingly, the triplet levels (T 1 ) of Gd(N-DPNQ) 2 Cl 3 and Gd (TTD) 3 are determined as 2.75 eV (450 nm) and 2.32 eV (533 nm). FIGURE 8 Phosphorus spectra of Gd (N-DPNQ) 2 Cl 3 and Gd (TTD) 3 at 77 K. A schematic presentation for energy transfer mechanism is shown as Figure 9 according to above mentioned experimental results. For Eu(N-DPNQ)(TTD) 3 , the positive energy transfer of N-DPNQ S 1 excited state to TTD S 1 excited state is difficult due to the same S 1 level. But partial S 1 energy of N-DPNQ can be transferred to N-DPNQ T 1 excited state. Hence such partial energy could be transferred to the lowest triplet level of TTD and then to 5 D 0 for Eu 3+ . Furthermore, excitation energy could be transferred from TTD singlet level to TTD triplet level, and finally to 5 D 0 for Eu 3+ ( Xin et al., 2004 ; Xu et al., 2006 ). Generally, there are three main steps involved in a sensitized Eu 3+ luminescence process, as shown in Figure 10 ( Parker, 2000 ). Firstly, the antenna ligand absorbs energy and is excited to its singlet level. Then this energy is migrated to the T 1 excited state of antenna ligands by inter-system crossing (ISC). Next, the excitation procedure from the T 1 level of antenna ligands to Eu 3+ ion excited state happens in this process. If the rate of energy transfer is sufficiently slow so as to lead a deactivation of the T 1 excited state of antenna ligands, the quenching by molecular oxygen could occur in this process. Finally, Eu 3+ ion excited state energy could be transferred to the ground state, then Eu 3+ luminescence is generated. Hence, the emission quenching of Eu 3+ probe by molecular oxygen is based on intermolecular collision of S 1 /T 1 state of antenna ligands with O 2 S 0 state. The above procedure can be presented below: * L − Eu + O 2 → L − Eu + O 2 * (2) here L and Eu denote antenna ligands and Eu(III) complex, and * stands for the excited state. FIGURE 9 Energy levels of ligands and Eu center in Eu (N-DPNQ) (TTD) 3 . FIGURE 10 Key photophysical processes of sensitized Eu 3+ luminescence. The quenching of luminescent molecule in a homogeneous medium with non-obvious host afluoence is supposed to be a simple exponential dynamic procedure. The PL intensity variation against O 2 levels should follow Stern-Volmer relationship. I 0 I = τ 0 τ = 1 + K s v [ O 2 ] (3) Here I and τ denote emission intensity and dynamic lifespan, respectively. I 0 is the intrinsic emission intensity with no O 2 . K SV shall be the Stern-Volmer constant. K q stands for a fixed fitting parameter. [O 2 ] means O 2 ratio. The curve of I 0 /I against [O 2 ] shall be a linear plot. Its slope shall be K SV . Typical intensity-formed Stern-Volmer fitting curves for Eu 1 , Eu 2 , and Eu 3 are presented in Figure 11 . These plots for Eu 1 , Eu 2 , and Eu 3 are well fitted by Eq. 3 . The parameters are also found in Table 1 . Eu 2 (with doping level of 0.6wt%) seems the optimal sample by showing the highest sensitivity of 2.43. As observed, Eu 1 and Eu 2 show a good linear relationship, whereas Eu 3 shows a poor linearity with a linearly dependent coefficient R ( Nakamura et al., 2007 ) of 0.969. Since Eu(N-DPNQ)(TTD) 3 could be effectively quenched by molecular O 2 , the increase in amount of Eu(N-DPNQ)(TTD) 3 results in increased molar fractions of oxygen-quenchable dye and hence the sensitivity and linearity of Eu 2 are superior to the corresponding values of Eu 1 . If the distribution of Eu-probe in matrix is changed by increasing amount of Eu(N-DPNQ)(TTD) 3 , this could affect the sensitivity and linearity of the sensor. In fact, the increasing of Eu(III) complex in Eu 3 leads to self-aggregation of Eu-probe molecules in the matrix, indicating that there is a change in the micro-environment of the composite nanofibers ( Lee and Okura, 1997 ; Shi et al., 2009 ). FIGURE 11 Intensity-based Stern-Volmer plots of Eu 1 , Eu 2 , and Eu 3 at different oxygen concentrations. TABLE 1 Key sensing parameters for Eu 1 , Eu 2 , and Eu 3 . Loading levels (wt%) t ↓ (s) t ↑ (s) I 0 / I 100 K SV (O 2 % −1 ) R Nakamura et al. (2007) Eu 1 , 0.4 8 14 1.93 0.00949 ± 0.00007 0.998 Eu 2 , 0.6 10 10 2.43 0.01444 ± 0.00008 0.999 Eu 3 , 0.8 7 12 2.31 0.01191 ± 0.00039 0.969 The response and recovery characteristics are very fundamental parameters for oxygen-sensing materials. Generally, response (t ↓ ) and recovery (t ↑ ) parameters are determined by calculating the time for each sample to lose or restore ninety five percent of maximum original emission intensity when testing atmosphere is changed between 100% N 2 and 100 O 2 . Figure 12 shows the emission variation of Eu 1 , Eu 2 , and Eu 3 upon surrounding atmosphere cycle of 100% N 2 -100% O 2 -100% N 2 . Based on the dynamic variation measurements, the values of t ↓ and t ↑ are measured and summarized in Table 1 . It is clear in Figure 12 that repeatable emission responses are detected with Eu 1 , Eu 2 , and Eu 3 . There is slice drift intensity. Furthermore, we also have monitored the mean sensitivity and response behavior of Eu 1 , Eu 2 , and Eu 3 over 12 weeks (interval = 15 days). The detected aging effect on sensitivity and response behavior is neglectable. The oxygen-sensing properties of Eu 1 , Eu 2 , and Eu 3 at 15 and 30 days also have been measured and the results are very similar, as shown in Table 2 . FIGURE 12 Response time of Eu 1 , Eu 2 , and Eu 3 upon various environmental atmospheres, emission = 610 nm. TABLE 2 Sensing parameters upon different aging days. Sample Sensitivity/response 0 day 15 days 30 days 45 days 60 days 75 days 84 days Eu 1 1.93/8 1.92/8 1.92/8 1.90/9 1.90/9 1.89/9 1.88/9 Eu 2 2.43/10 2.41/10 2.41/10 2.40/10 2.40/12 2.38/12 2.37/12 Eu 3 2.31/7 2.31/7 2.30/8 2.30/9 2.29/9 2.28/9 2.25/10 Considering the sensing mechanism of a dynamic collision between O 2 ground state and excited state probe, it is assumed that these composite nanofibers should have good sensing selectivity towards O 2 since most other gases have closed-shell structures and thus are not open for probe energy transfer. Aiming at a primitive evaluation on the sensing selectivity of these composite nanofibers, five typical interfering gases are selected, including CO 2 , benzene, toluene, CHCl 3 and CH 2 Cl 2 . The emission spectra of a representative sample Eu 2 are recorded and compared in Supplementary Figure S1 (Supporting Information). No obvious spectral shift or intensity variation is observed upon these interfering gases, which shall be attributed to the unique f-f transitions of these Eu(III) probes. As a consequence, it is concluded that these composite nanofibers have good selectivity for O 2 . On the other hand, such f-f transitions needs a complicated energy transfer procedure, which makes the sensitivity far away from satisfactory. For later improvement, the antenna energy transfer from ligand to central metal ion should be simplified, so that the O 2 quenching effect on excited probe shall be efficient and complete, leading to improved sensitivity. Conclusion A Eu(III) compound have been successfully synthesized for the first time, to the best of our knowledge. Based on the quenching of luminescence for Eu(III) complex by molecular oxygen, the oxygen-sensing Eu(N-DIIQ)(TTD) 3 /PVP composite nanofibers are prepared using elctrospinning method. This work shall be extended to obtain oxygen-sensing fibrous composites having good sensitivity and linearity by altering loading levels of oxygen-sensing dye. The oxygen-sensing Eu(N-DIIQ)(TTD) 3 /PVP composite nanofibers possess good operational stability and reproducibility. The optimized fibrous composite shows the best result with sensitivity as high as 2.43, quick response as short as 10 s and linear behavior. Data Availability Statement The original contributions presented in the study are included in the article/ Supplementary Material , further inquiries can be directed to the corresponding author. Author Contributions CC, Writing and reviewing; LS, Data; CL, Data analysis; TL, Data; KS, Supervision. CC and LS contributed equally to this study. Funding This work was financially supported by the Natural science foundation of Jilin Province (No. 20190201048JC) and the Department of Education of Jilin Province (JJKH20201031KJ). Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's Note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary Material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2021.812461/full#supplementary-material Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7129276/

Vaccines, adjuvants and autoimmunity

Graphical abstract 1 Introduction Vaccines have been a preventive treatment option available for over 200 years. They have been proven to be effective in preventing infections that previously had high morbidity and mortality. An example of this is the eradication of small pox, which was mainly attributed to successful vaccination programs. Preventing a high burden disease has since proven to be a cost effective measure and, as such, vaccines have become a part of multiple national health programs. These promising results led to the development of more and more vaccines and to the study of its applicability in other fields such as cancer prevention and treatment. Vaccines are drugs administered to healthy individuals, and much like other drugs, vaccines are associated with adverse events. Usually the described adverse events are transient and acute, but may rarely present with hypersensitivity and induction of autoimmunity that may be severe and fatal. These adverse events play an important role in the life of the vaccinated patients. Immune mediated diseases arise from various different sources; these include environmental, genetic, hormonal and immune defects. The combination of these defects can be described as the mosaic of autoimmunity [1] . Patient background can be used as a clue to determine the response that may be elicited following drug administration. It has been proven that infectious agents may elicit an autoimmune disease in a prone subject through various mechanisms, including, but not limited to, molecular mimicry, epitope spreading and polyclonal activation [2] . Scientific findings suggest that autoimmunity may be triggered by vaccine adjuvants, of which aluminum compounds (aluminum hydroxide and phosphate) have been the most studied and the most widely used. Adjuvants are molecules, which, in combination with antigens, enhance immunological response. This enables an easier and more effective recognition of "non self", which in turn permits the triggering of adaptive and innate immune responses [3] . Recently a new syndrome was described: "Autoimmune/inflammatory syndrome induced by adjuvants" (ASIA). This embodies a spectrum of reactions, which are usually mild but may also be severe. These reactions are attributed to adjuvant stimulation, which can include chronic exposure to silicone, tetramethylpentadecane, pristane, aluminum, infectious components and other adjuvants. All of these environmental factors have been found to induce autoimmunity and inflammatory manifestations by themselves both in animal models and in humans. The mechanisms of this disease will be described in further detail [4] . This review will focus on general mechanism of vaccines, adjuvant-induced autoimmunity, and on vaccines and the specific autoimmune diseases that they may trigger. 2 General mechanisms of vaccines and adjuvant induced autoimmunity 2.1 Adjuvants role in infections and autoimmunity Adjuvants approved to date for human vaccines are: aluminum, MF59 in some viral vaccines, MPL, AS04, AS01B and AS02A against viral and parasitic infections, virosomes for hepatitis B virus (HBV), human papilloma virus (HPV), hepatitis A virus (HAV), and cholera toxin for cholera. Adjuvants may be composed of several different compounds. Currently, oil based adjuvants, virosomes, toll-like receptors (TLRs) related adjuvants, MPL, adjuvants made of unmethylated CpG dinucleotides and tuftsin have all been described. It is of great interest the understanding of the mechanisms related to the adjuvant effect, as well as to aluminum salts. Aluminum acts through multiple pathways, which do not depend solely on TLRs signaling. Each of these pathways leads to an enhanced host immune response [5] . There are many oil based adjuvants. One is incomplete Freund adjuvant (IFA), which contains water in oil emulsion. Another is complete Freund adjuvant (CFA), which is the same as IFA, except that it also contains killed Mycobacteria in addition to water in oil emulsion. Usually, CFA is used for primary vaccination and IFA for boosting. Recent oil based adjuvants that have been developed are MF59 (Novartis ® ), AS03 (GlaxoSmithKline ® ), Advax™ which are based on inulin compounds (Vaxine™ Pty) and Qs-21/ISCOMs, which are immune stimulating complexes composed of cholesterol and phospholipid with or without antigen ( Table 1 ). Table 1 Types of adjuvants in development or use. Type of Adjuvants Name of compound Vaccines in test or use Related to Toll like receptors (TLRs) Aluminum hydroxide and phosphate PCV7, PCV13, MenC, HPV, HAV, Hib; tetanus vaccine IC31 Influenza [14] ASO4 (MPL + QS-21), ASO2A (MPL + Alum), CPG 7907, and GM-CSF Papilloma virus, hepatitis B, malaria [15] RD-529, ISS, Flagellin TLR agonists Oil based emulsions CFA, IFA, MF59 TM montanide, adjuvant 65, lipovant, QS-21 [16] ISCOMs, ADVAX™, algammulin Influenza Xenobiotic adjuvants Unmethylated CpG dinucleotides [17] Hepatitis B, allergens, tumor cells Tuftsin auto adjuvant Tuftsin Influenza, malaria, autoimmune encephalomyelitis, restoration of innate immune system (HIV patients), SLE [18] , [19] , [20] , [21] CFA: complete freund adjuvant; IFA: incomplete freund adjuvant; PCV: pneumococcal conjugated vaccine; MenC: meningitis C; HPV: human papiloma Virus; HAV: hepatitis A virus; Hib: haemophilus influenza type b. Virosomes are adjuvants that contain a membrane-bound hemagglutinin and neuraminidase obtained from the influenza virus. Both components facilitate the uptake into antigen presenting cells (APC) and mimic the natural immune response [6] . Leucocyte membranes have membrane bound pattern recognition receptors (PRRs) called TLRs, which are responsible for detecting most (although certainly not all) antigen-mediated infections. Their activation leads to adaptive immune responses. For this reason, many adjuvants that are used today are directed to PRRs. These adjuvants are called TLRs related adjuvants [7] . MPL is a series of 4'monophosphoryl lipid A obtained from the purification of a modified lipopolysaccharide (LPS) of Salmonella Minnesota. Bacterial deoxyribonucleic acid (DNA) is immunostimulatory due to Unmethylated CpG dinucleotides. Vertebrate DNA has relatively low amounts of unmethylated CpG compared to Bacterial DNA. The adjuvant effect of CpG is enhanced when conjugated to protein antigens. This adjuvant is being tested in vaccines directed at infectious agents, allergens and tumor cells [8] , [9] , [10] . Another type of adjuvant is tuftsin. Tuftsin is an auto adjuvant, which is a natural self-immunostimulating tetrapeptide (Thr–Lys–Pro–Arg). This tetrapeptide is a fraction of the IgG heavy chain molecule produced by enzymatic cleavage in the spleen [11] . Its functions include: binding to receptors on neutrophils and macrophages to stimulate their phagocytic activity, increasing tumor necrosis factor alpha (TNFα) release from human Kupffer cells enhancing secretion of IL1 by activating macrophages, activation of macrophages expressing nitric oxide (NO) synthase to produce NO and enhancement of murine natural cell mediated cytotoxicity in vitro [11] , [12] , [13] . In summary, it is an adjuvant with minor side effects with a promising effect in restoring innate immune mediated response. 2.1.1 Mechanisms of adjuvanticity Adjuvants may exert their immune enhancing effects according to five immune functional activities: 1. Translocation of antigens to the lymph nodes where they can be recognized by T cells. 2. Antigen protection enabling longer exposure. 3. Enhanced local reaction at the injection site. 4. Induction of the release of inflammatory cytokines. 5. Interaction with PRRs, specifically TLRs [22] . a Adjuvant effect The term "adjuvant effect" refers to the co-administration of an antigen with a microbial specific factor to enhance an antigen-specific immune response in vivo. The microbial components of adjuvants activate APCs to produce pro-inflammatory cytokines ("non-specific" signal 2) and to up-regulate molecules essential for antigen presentation. These molecules include major histocompatibility complex (MHC) class II (antigen-specific signal 1) and B7-1/2. These innate immune events allow a more effective presentation to the adaptive immune system, resulting in an augmented activation and clonal expansion of T cells [23] . In accordance to this effect, if self-antigens are used, an autoimmune response can be elicited [24] . It has been shown that auto-reactive T-cells that surpass tolerance mechanisms can be triggered by exogenous adjuvants to become auto-aggressive [25] . Infectious agents are able to naturally generate their adjuvant effect and can induce autoimmunity [26] . An example of this is the causality between viral infection and myocarditis. Half the cases of myocarditis are preceded by an acute viral infection. Infectious myocarditis in humans can be reproduced in experimental murine models of myocarditis [27] . It has also been shown that the autoimmune reaction elicited by an infectious agent can be effective in treating cancer. An example of this is that bladder administration of BCG ( bacille Calmette–Guérin ) has been shown to be effective against superficial bladder cancer development [28] . It can be inferred that the adjuvant effect can be used against specific tumor derived molecules, so that these molecules can be recognized as "non self". 2.1.2 Innate immune pattern recognition of pathogens and adjuvants PRR-PAMP (Pattern Recognition Receptor—Pathogen-Associated Molecular Patterns) interactions activate the APCs to promote antigen-specific lymphocytic responses [29] . The definition of PAMPs has now broadened, in that the recognized structures do not need to be pathogens. Thus the concept of "microbe-associated molecular patterns" (MAMPs) and of "danger/damage-associated molecular patterns" (DAMPs) were defined based on the notion that the endogenous host molecules signal danger or damage to the immune system [30] . 2.1.3 Innate immune response mediates the adjuvant effect TLRs are single-transmembrane PRRs localized on cell surface and endosomal membranes. From all the PRRs, these are the most studied. TLRs play a crucial role in innate immune response to "non self" and are biosensors of tissue damage. The interaction between the four known TLRs adapters: MyD88, TIRAP/Mal, TRAM and TRIF, in TLR signaling, shape the innate immune response. Besides PRRs the innate immune system also detects proteolytic enzymes generated during infection [31] . Merging the response to different PRRs signaling may be the pathway for developing customized responses to different aggressions [32] . b Experimental models of adjuvants Many animals have been used in experimental models of adjuvant-related autoimmune conditions [33] . These include primates, salmons, rabbits and swine; however, the most common are murine models. Murine models include autoimmune prone strains, models of autoimmune disease and autoimmune resistant strains ( Table 2 ). Table 2 Experimental models of adjuvant autoimmunity Experimental models Strain Disease model or related signs and symptoms Adjuvant Murine Rats DA (dark agouti) rats Rheumatoid arthritis Mineral oil (CFA, pristane, squalene,avridine) [34] , [35] Arthritis Collagen [36] Sprague Dawley rats Arthritis CFA [37] MMF Aluminum [38] Mice BALB/c Plasmacytomas Mineral oil, pristane [39] Sclerosing lipogranulomas SC injection of mineral oil [40] SLE-related autoantibodies Pristane, CFA, squalene [41] C57BL/6 Antiphospholipid-like syndrome CFA, IFA [42] NZB/NZWF1 SLE, lupus like GLN CFA, alum [43] Salmons Impaired growth rate, decreased carcass quality, spinal deformities, uveitis, inflammatory reactions in the abdominal cavity, RF, ANA, ANCA, immune-complex GLN and chronic granulomatous inflammation Vaccines with adjuvants such as oils [44] Rabbits Inflammation at injection site Vaccine: CFA, IFA, montanide [45] Swine Granulomatous inflammation Adverse local reactions Mineral oils [46] Primates Rhesus macaque Potential delayed acquisition of neonatal reflexes aluminum contained in pre clinical vaccine testing [47] C57BL/6 (transgenic factor V Leiden-mutated C57/BL6-back-crossed mice); RF: rheumatoid factor; ANA: antinuclear autoantibodies; ANCA: anti-cytoplasmic autoantibodies; GLN: glumerulonephritis; SLE: systemic lupus erythematous; MMF: macrophagic myofasciitis. An interesting model is that described by Lujan et al. The authors described that a commercial sheep, inoculated repetitively with aluminum-containing adjuvants vaccinations, developed an acute neurological episode with low response to external stimuli and acute meningoencephalitis few days after immunization. An excitatory phase, followed by weakness, extreme cachexia, tetraplegia and death appeared. This was suggested to be part of the spectrum of ASIA syndrome. Moreover, the biopsy of the nervous tissue of experimental animals indicated the presence of alum [48] . c Toxicity of aluminum adjuvants Aluminum nanoparticles have both a unique capacity of surpassing the blood brain barrier (BBB) and of eliciting immune inflammatory responses. These are probably the reasons why Aluminums' most sensitive target is the brain, and also why documented side effects are mostly neurologic or neuropsychiatric [49] , [50] . Aluminum is present in nature, not only as a vaccine adjuvant, but also in food, water and cosmetics. It has been described as a neurotoxin because even when a relatively small amount of Aluminium reaches the brain [49] , is can act as a genotoxin [51] , a prooxidant [52] , it can be proinflammatory [51] , act as an immunotoxin [5] and also as an endocrine disruptor [53] . Aluminum interferes with many essential cellular processes. Memory, concentration, speech deficits, impaired psychomotor control, reduced seizure tolerance and altered behaviour are manifestations of aluminium neurotoxicity. Moreover, Alzheimer's [54] , amyotrophic lateral sclerosis, Parkinsonism dementia [55] , multiple sclerosis [56] , and neurological impairments in children have been linked to aluminum neurotoxicity [57] . Brain susceptibility to aluminum compounds is possibly due to the brain's high metabolic requirement, to the fact that it possesses a large area of biological membranes and to the relatively low concentration of antioxidants [54] . Aluminum adjuvants exert their immunostimulatory effect through many different pathways that activate both the innate and adaptive immune systems. One of the most significant is the activation of the NLRP3 inflammasome pathway [58] . NLPR3 activation has been shown to trigger type 2 diabetes. By using NLPR3 knockout mice it has been demonstrated that the absence of inflammasome components leads to a better maintenance of glucose homeostasis and higher insulin sensitivity [59] . On the other hand, activation of the inflammasome and its downstream components: pro-inflammatory cytokines IL-1β and IL-18 are strongly implicated in the development of several central nervous system (CNS) disorders [60] . The vast majority of people are consuming higher amounts of aluminum through dietary and parenteral intake than what expert authorities consider safe. Upper limits set by US food and drug administrations (FDA) for aluminum in vaccines are set at no more than 850 μg/dose. These values were not based on toxicity studies, but on the minimum amount needed for aluminum to exert its effect as an adjuvant [51] . The quantities of aluminum to which infants, in their first year of age are exposed, have been considered safe by the FDA. However the scientific basis for this recommendation does not take into account aluminum persistence in the body. The concern about aluminum in dietary intake has been reinforced by the Food and Agriculture (FAO) WHO Expert Committee, which lowered the provisional tolerable weekly intake of aluminum from 7 mg/kg/bw (490 mg/week, for an average 70 kg human) to 1 mg/kg/bw (70 mg/week) [61] . The amount of dietary intake of aluminum has risen in urban societies to up to 100 mg/day considering the widespread use of processed convenience foods. However, only about 0.25% of dietary aluminum is absorbed into systemic circulation and most of it is thereafter eliminated through the kidneys [54] . Absorption of aluminum by the skin from ointments and cosmetics containing aluminum has been shown. Moreover, the presence of aluminum in breast tissue was associated with breast cancer [62] . Aluminum compounds persist for up to 8–11 years post vaccination in human body. This fact, combined with repeated exposure, may account for a hyper activation of the immune system and subsequent chronic inflammation [63] . The clinical and experimental evidence collected so far identify at least three main risks associated with aluminum in vaccines: 1. It can persist in the body. 2. It can trigger pathological immunological responses. 3. It can pass through the BBB into the CNS where it can trigger immuno-inflammatory processes, resulting in brain inflammation and long-term neural dysfunction. 2.2 Allergy and autoimmunity caused by metals There is a link between allergies and autoimmunity since both are the result of an abnormal immune response [3] , [4] . Metals such as mercury, aluminum, nickel and gold are known to induce immunotoxic effects in humans. The immunologic effects of these metals include immunomodulation, allergies and autoimmunity. They may act either as immunosuppressants or as immune adjuvants. Metals bind firmly to cells and proteins and thus have the ability to modify autologous epitopes (hapetenization). T-cells then recognize the proteins as foreign and trigger an autoimmune response [64] . Hypersensitivity caused by metals may be referred to as Type IV delayed hypersensitivity. The reaction is considered delayed because the first symptoms appear 24–48 h after exposure, because it is mostly T-cell mediated and the gold standard for diagnosis of delayed type hypersensitivity is patch testing [65] . In mercury-sensitized patients, even mercury concentrations within the normal range might provoke neuroallergic reactions in the brain [66] . Identifying metal sensitivity and removal of the sensitizing metals, such as dental amalgam, have been proved successful by showing symptom improvement in patients with previous autoimmune diseases. These diseases included fibromyalgia, autoimmune thyroid diseases and orofacial granulomatosis [67] , [68] , [69] , [70] ( Table 3 ). Table 3 Metals reported side effects. Metal Derivatives Main cause of exposure Side effects Mercury Methyl mercury Skin ointments Dental amalgam fillings Kidney disease [71] ; peripheral neuropathy; multiple sclerosis [72] ; ANA positivity [73] Polluted fish Thimerosal and phenyl mercury Antiseptics/preservatives in eye drops vaccines Flu like symptoms Eyelid eczema and edema Gold Colloidal gold [74] Treatment for RA Nephropathy Nickel [75] , [76] Food Jewelry Tobacco allergic and autoimmune symptoms; scleroderma-related autoantibodies and cutaneous sclerosis Aluminum [4] , [77] Food Vaccines Neurotoxic; delayed type hypersensitivity; ASIA syndrome; chronic fatigue syndrome; macrophagic myofasciitis RA: rheumatoid arthritis. 2.3 Genetics and vaccinology The timeline regarding the field of vaccinology has been divided in two generations, the first regarding the administration of inactivated pathogens in whole or live attenuated forms (e.g., Bacillus Calmette Guerin (BCG), plague, pertussis, polio, rabies, and smallpox) and the second regarding vaccines assembled from purified microbial cell components, also referred as subunit vaccines (e.g., polysaccharides, or protein antigens) [78] . This latter approach relies on recombinant DNA technology and polysaccharide chemistry. There are obstacles to conventional vaccine development methods such as non-cultivable in vitro pathogens (e.g., hepatitis C, papilloma virus types 16 and 18, and Mycobacterium leprae), antigen hypervariability (e.g., serogroup B meningococcus, gonococcus, malaria), opportunistic pathogens (e.g., Staphylococcus aureus) and rapid evolving pathogens such as Human immunodeficiency virus (HIV) [79] . Vaccine research gained a new perspective as the genomics field emerged over the last decades. Bacterial genomes have been sequenced and analyzed making it possible to choose the best candidate vaccine antigens by using the concept of reverse vaccinology [80] . The main known factors influencing the observed heterogeneity for immune responses induced by vaccines are gender, age, ethnicity, co-morbidity, immune system, and genetic background. The interaction between genetic and environmental components will dictate the response to vaccines. Studying the vaccine and the host will enable the development of customized treatment options. The combination of genetics, epidemiology and genomics in vaccine design has been denominated "vaccinomics" [81] . The importance of genetic influence is supported by twins and siblings studies, which show familial aggregation. This suggests that genomics is crucial in inter-individual variations in vaccine immune responses [82] . Both Human leukocyte antigen (HLA) and non-HLA gene markers have been identified as markers for immune response to vaccines. Multiple studies have shown connections between HLA gene polymorphisms and non-responsiveness to the HBV vaccine [83] . HLA region is divided in three sub regions: Class I is associated with the induction and maintenance of cell-mediated immune response, class II is associated with presentation of exogenous antigens to helper T CD4+ cells and class III, where immune non HLA related genes are located. Normal human tissue has at least 12HLA antigens, and although new recombinant haplotypes may occur, it is inherited mostly intact from progenitors [84] . HLA allelic differences are associated with different responses to vaccines, either by hyper or hypo responsiveness. We can infer that a similar response may be associated with different safety in relation to the development of autoimmune reactions to vaccines, particularly in the patients with genetic predisposition to an enhanced response to vaccine inoculation [85] . Furthermore, patients that share the same HLA, for instance siblings, have been diagnosed with ASIA following similar environmental stimuli [86] , [87] . 2.4 Autoantibodies induced by vaccines Autoantibodies help to diagnose certain autoimmune diseases, however, they can also be found in healthy individuals. Thus, autoimmune diseases cannot be diagnosed based solely on antibody detection [88] . Inoculation of vaccines triggers autoimmune responses that result in the development of autoantibodies. Many studies have been carried out in animals, healthy subjects and patients with autoimmune diseases to understand if this development is of clinical significance [89] , [90] , [91] , [92] . A difference in eliciting the production of autoantibodies in healthy humans has been observed between adjuvanted and non-adjuvanted influenza vaccines [93] . The annual influenza vaccine has been the most heavily researched vaccine, along with HPV and Pneumococcal vaccines as far as their relationship with patients who have previously been diagnosed with an autoimmune disease [94] , [95] , [96] . Autoantibody induction after HPV vaccination was also shown in adolescent girls with systemic lupus erythematosus (SLE) [97] . Although induction of autoantibodies was proven following vaccine administration, there have been no proven relation with disease diagnosis in either of the specific groups studied so far [92] , [98] . It has been widely demonstrated that autoantibodies can develop years before the manifestation of a full-blown autoimmune disease [99] . Moreover, the development of a specific autoantibody is also genetically determined, and the link between genetic, autoantibodies and vaccines may become an even more intriguing area of research [100] . 2.5 Siliconosis and autoimmune (auto-inflammatory) syndrome induced by adjuvants (ASIA) Silicones are synthetic polymers that can be used as fluids, emulsions, resins and elastomers making them useful in diverse fields. They were thought to be biologically inert substances and were incorporated in a multitude of medical devices such as joint implants, artificial heart valves, catheters, drains and shunts. Of all the silicone-containing products, the most famous are most likely breast implants. Silicon is one of the substances suspected to induce ASIA [5] . It is currently believed that exposure alone is not enough to trigger the disease but that it requires the presence of additional risk factors (e.g., genetic susceptibility, other environmental factors) [4] . Silicone exerts local tissue reactions. Some of these reactions are considered para-physiological, such as capsular tissue formation around an implant. Other reactions are viewed as abnormal, like when capsular contractures and allergic reactions to silicone or platinum (catalyst used in silicone polymerization found in minute concentrations in implants) occur [101] . Cutaneous exposure to silicone with cosmetics or baby bottles could potentially sensitize patients [102] . There is also a systemic component of silicone exposure related to diffusion of silicone through the elastomer envelope, commonly termed "bleeding". It may arouse systemic effects as it degrades and fragments in tissue, it can also spread throughout the body and lead to the development of cancer or autoimmune phenomena [103] . Patients with ruptured implants complain more frequently of pain and chronic fatigue when compared to patients with intact implants [104] . Anti-silicone antibodies were found to be present in human sera more frequently in patients who have undergone silicone breast implants, however, their pathological significance remains uncertain [105] . The same was seen for other antibodies such as autoantibodies directed against dsDNA, ssDNA, SSB/La, silicone and collagen II, which were found to be present in increased levels in patients after exposure to silicone [106] . It has also been shown that the formation of autoantibodies is directly related to implant duration. Several autoimmune diseases have been linked to silicone exposure including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), polymyositis, systemic sclerosis (SSc) and fibromyalgia. Although ASIA symptoms may arise 24 years after the onset of exposure to silicone implants [107] , most of the follow-up periods are short and concluding evidence is yet to come regarding this causality. 2.6 Vaccines and autoimmune diseases There have been published case reports, epidemiologic and research studies that suggest a connection between several vaccines and certain autoimmune conditions, notwithstanding that, overall the benefits of vaccination outweigh the risks. 2.1 Adjuvants role in infections and autoimmunity Adjuvants approved to date for human vaccines are: aluminum, MF59 in some viral vaccines, MPL, AS04, AS01B and AS02A against viral and parasitic infections, virosomes for hepatitis B virus (HBV), human papilloma virus (HPV), hepatitis A virus (HAV), and cholera toxin for cholera. Adjuvants may be composed of several different compounds. Currently, oil based adjuvants, virosomes, toll-like receptors (TLRs) related adjuvants, MPL, adjuvants made of unmethylated CpG dinucleotides and tuftsin have all been described. It is of great interest the understanding of the mechanisms related to the adjuvant effect, as well as to aluminum salts. Aluminum acts through multiple pathways, which do not depend solely on TLRs signaling. Each of these pathways leads to an enhanced host immune response [5] . There are many oil based adjuvants. One is incomplete Freund adjuvant (IFA), which contains water in oil emulsion. Another is complete Freund adjuvant (CFA), which is the same as IFA, except that it also contains killed Mycobacteria in addition to water in oil emulsion. Usually, CFA is used for primary vaccination and IFA for boosting. Recent oil based adjuvants that have been developed are MF59 (Novartis ® ), AS03 (GlaxoSmithKline ® ), Advax™ which are based on inulin compounds (Vaxine™ Pty) and Qs-21/ISCOMs, which are immune stimulating complexes composed of cholesterol and phospholipid with or without antigen ( Table 1 ). Table 1 Types of adjuvants in development or use. Type of Adjuvants Name of compound Vaccines in test or use Related to Toll like receptors (TLRs) Aluminum hydroxide and phosphate PCV7, PCV13, MenC, HPV, HAV, Hib; tetanus vaccine IC31 Influenza [14] ASO4 (MPL + QS-21), ASO2A (MPL + Alum), CPG 7907, and GM-CSF Papilloma virus, hepatitis B, malaria [15] RD-529, ISS, Flagellin TLR agonists Oil based emulsions CFA, IFA, MF59 TM montanide, adjuvant 65, lipovant, QS-21 [16] ISCOMs, ADVAX™, algammulin Influenza Xenobiotic adjuvants Unmethylated CpG dinucleotides [17] Hepatitis B, allergens, tumor cells Tuftsin auto adjuvant Tuftsin Influenza, malaria, autoimmune encephalomyelitis, restoration of innate immune system (HIV patients), SLE [18] , [19] , [20] , [21] CFA: complete freund adjuvant; IFA: incomplete freund adjuvant; PCV: pneumococcal conjugated vaccine; MenC: meningitis C; HPV: human papiloma Virus; HAV: hepatitis A virus; Hib: haemophilus influenza type b. Virosomes are adjuvants that contain a membrane-bound hemagglutinin and neuraminidase obtained from the influenza virus. Both components facilitate the uptake into antigen presenting cells (APC) and mimic the natural immune response [6] . Leucocyte membranes have membrane bound pattern recognition receptors (PRRs) called TLRs, which are responsible for detecting most (although certainly not all) antigen-mediated infections. Their activation leads to adaptive immune responses. For this reason, many adjuvants that are used today are directed to PRRs. These adjuvants are called TLRs related adjuvants [7] . MPL is a series of 4'monophosphoryl lipid A obtained from the purification of a modified lipopolysaccharide (LPS) of Salmonella Minnesota. Bacterial deoxyribonucleic acid (DNA) is immunostimulatory due to Unmethylated CpG dinucleotides. Vertebrate DNA has relatively low amounts of unmethylated CpG compared to Bacterial DNA. The adjuvant effect of CpG is enhanced when conjugated to protein antigens. This adjuvant is being tested in vaccines directed at infectious agents, allergens and tumor cells [8] , [9] , [10] . Another type of adjuvant is tuftsin. Tuftsin is an auto adjuvant, which is a natural self-immunostimulating tetrapeptide (Thr–Lys–Pro–Arg). This tetrapeptide is a fraction of the IgG heavy chain molecule produced by enzymatic cleavage in the spleen [11] . Its functions include: binding to receptors on neutrophils and macrophages to stimulate their phagocytic activity, increasing tumor necrosis factor alpha (TNFα) release from human Kupffer cells enhancing secretion of IL1 by activating macrophages, activation of macrophages expressing nitric oxide (NO) synthase to produce NO and enhancement of murine natural cell mediated cytotoxicity in vitro [11] , [12] , [13] . In summary, it is an adjuvant with minor side effects with a promising effect in restoring innate immune mediated response. 2.1.1 Mechanisms of adjuvanticity Adjuvants may exert their immune enhancing effects according to five immune functional activities: 1. Translocation of antigens to the lymph nodes where they can be recognized by T cells. 2. Antigen protection enabling longer exposure. 3. Enhanced local reaction at the injection site. 4. Induction of the release of inflammatory cytokines. 5. Interaction with PRRs, specifically TLRs [22] . a Adjuvant effect The term "adjuvant effect" refers to the co-administration of an antigen with a microbial specific factor to enhance an antigen-specific immune response in vivo. The microbial components of adjuvants activate APCs to produce pro-inflammatory cytokines ("non-specific" signal 2) and to up-regulate molecules essential for antigen presentation. These molecules include major histocompatibility complex (MHC) class II (antigen-specific signal 1) and B7-1/2. These innate immune events allow a more effective presentation to the adaptive immune system, resulting in an augmented activation and clonal expansion of T cells [23] . In accordance to this effect, if self-antigens are used, an autoimmune response can be elicited [24] . It has been shown that auto-reactive T-cells that surpass tolerance mechanisms can be triggered by exogenous adjuvants to become auto-aggressive [25] . Infectious agents are able to naturally generate their adjuvant effect and can induce autoimmunity [26] . An example of this is the causality between viral infection and myocarditis. Half the cases of myocarditis are preceded by an acute viral infection. Infectious myocarditis in humans can be reproduced in experimental murine models of myocarditis [27] . It has also been shown that the autoimmune reaction elicited by an infectious agent can be effective in treating cancer. An example of this is that bladder administration of BCG ( bacille Calmette–Guérin ) has been shown to be effective against superficial bladder cancer development [28] . It can be inferred that the adjuvant effect can be used against specific tumor derived molecules, so that these molecules can be recognized as "non self". 2.1.2 Innate immune pattern recognition of pathogens and adjuvants PRR-PAMP (Pattern Recognition Receptor—Pathogen-Associated Molecular Patterns) interactions activate the APCs to promote antigen-specific lymphocytic responses [29] . The definition of PAMPs has now broadened, in that the recognized structures do not need to be pathogens. Thus the concept of "microbe-associated molecular patterns" (MAMPs) and of "danger/damage-associated molecular patterns" (DAMPs) were defined based on the notion that the endogenous host molecules signal danger or damage to the immune system [30] . 2.1.3 Innate immune response mediates the adjuvant effect TLRs are single-transmembrane PRRs localized on cell surface and endosomal membranes. From all the PRRs, these are the most studied. TLRs play a crucial role in innate immune response to "non self" and are biosensors of tissue damage. The interaction between the four known TLRs adapters: MyD88, TIRAP/Mal, TRAM and TRIF, in TLR signaling, shape the innate immune response. Besides PRRs the innate immune system also detects proteolytic enzymes generated during infection [31] . Merging the response to different PRRs signaling may be the pathway for developing customized responses to different aggressions [32] . b Experimental models of adjuvants Many animals have been used in experimental models of adjuvant-related autoimmune conditions [33] . These include primates, salmons, rabbits and swine; however, the most common are murine models. Murine models include autoimmune prone strains, models of autoimmune disease and autoimmune resistant strains ( Table 2 ). Table 2 Experimental models of adjuvant autoimmunity Experimental models Strain Disease model or related signs and symptoms Adjuvant Murine Rats DA (dark agouti) rats Rheumatoid arthritis Mineral oil (CFA, pristane, squalene,avridine) [34] , [35] Arthritis Collagen [36] Sprague Dawley rats Arthritis CFA [37] MMF Aluminum [38] Mice BALB/c Plasmacytomas Mineral oil, pristane [39] Sclerosing lipogranulomas SC injection of mineral oil [40] SLE-related autoantibodies Pristane, CFA, squalene [41] C57BL/6 Antiphospholipid-like syndrome CFA, IFA [42] NZB/NZWF1 SLE, lupus like GLN CFA, alum [43] Salmons Impaired growth rate, decreased carcass quality, spinal deformities, uveitis, inflammatory reactions in the abdominal cavity, RF, ANA, ANCA, immune-complex GLN and chronic granulomatous inflammation Vaccines with adjuvants such as oils [44] Rabbits Inflammation at injection site Vaccine: CFA, IFA, montanide [45] Swine Granulomatous inflammation Adverse local reactions Mineral oils [46] Primates Rhesus macaque Potential delayed acquisition of neonatal reflexes aluminum contained in pre clinical vaccine testing [47] C57BL/6 (transgenic factor V Leiden-mutated C57/BL6-back-crossed mice); RF: rheumatoid factor; ANA: antinuclear autoantibodies; ANCA: anti-cytoplasmic autoantibodies; GLN: glumerulonephritis; SLE: systemic lupus erythematous; MMF: macrophagic myofasciitis. An interesting model is that described by Lujan et al. The authors described that a commercial sheep, inoculated repetitively with aluminum-containing adjuvants vaccinations, developed an acute neurological episode with low response to external stimuli and acute meningoencephalitis few days after immunization. An excitatory phase, followed by weakness, extreme cachexia, tetraplegia and death appeared. This was suggested to be part of the spectrum of ASIA syndrome. Moreover, the biopsy of the nervous tissue of experimental animals indicated the presence of alum [48] . c Toxicity of aluminum adjuvants Aluminum nanoparticles have both a unique capacity of surpassing the blood brain barrier (BBB) and of eliciting immune inflammatory responses. These are probably the reasons why Aluminums' most sensitive target is the brain, and also why documented side effects are mostly neurologic or neuropsychiatric [49] , [50] . Aluminum is present in nature, not only as a vaccine adjuvant, but also in food, water and cosmetics. It has been described as a neurotoxin because even when a relatively small amount of Aluminium reaches the brain [49] , is can act as a genotoxin [51] , a prooxidant [52] , it can be proinflammatory [51] , act as an immunotoxin [5] and also as an endocrine disruptor [53] . Aluminum interferes with many essential cellular processes. Memory, concentration, speech deficits, impaired psychomotor control, reduced seizure tolerance and altered behaviour are manifestations of aluminium neurotoxicity. Moreover, Alzheimer's [54] , amyotrophic lateral sclerosis, Parkinsonism dementia [55] , multiple sclerosis [56] , and neurological impairments in children have been linked to aluminum neurotoxicity [57] . Brain susceptibility to aluminum compounds is possibly due to the brain's high metabolic requirement, to the fact that it possesses a large area of biological membranes and to the relatively low concentration of antioxidants [54] . Aluminum adjuvants exert their immunostimulatory effect through many different pathways that activate both the innate and adaptive immune systems. One of the most significant is the activation of the NLRP3 inflammasome pathway [58] . NLPR3 activation has been shown to trigger type 2 diabetes. By using NLPR3 knockout mice it has been demonstrated that the absence of inflammasome components leads to a better maintenance of glucose homeostasis and higher insulin sensitivity [59] . On the other hand, activation of the inflammasome and its downstream components: pro-inflammatory cytokines IL-1β and IL-18 are strongly implicated in the development of several central nervous system (CNS) disorders [60] . The vast majority of people are consuming higher amounts of aluminum through dietary and parenteral intake than what expert authorities consider safe. Upper limits set by US food and drug administrations (FDA) for aluminum in vaccines are set at no more than 850 μg/dose. These values were not based on toxicity studies, but on the minimum amount needed for aluminum to exert its effect as an adjuvant [51] . The quantities of aluminum to which infants, in their first year of age are exposed, have been considered safe by the FDA. However the scientific basis for this recommendation does not take into account aluminum persistence in the body. The concern about aluminum in dietary intake has been reinforced by the Food and Agriculture (FAO) WHO Expert Committee, which lowered the provisional tolerable weekly intake of aluminum from 7 mg/kg/bw (490 mg/week, for an average 70 kg human) to 1 mg/kg/bw (70 mg/week) [61] . The amount of dietary intake of aluminum has risen in urban societies to up to 100 mg/day considering the widespread use of processed convenience foods. However, only about 0.25% of dietary aluminum is absorbed into systemic circulation and most of it is thereafter eliminated through the kidneys [54] . Absorption of aluminum by the skin from ointments and cosmetics containing aluminum has been shown. Moreover, the presence of aluminum in breast tissue was associated with breast cancer [62] . Aluminum compounds persist for up to 8–11 years post vaccination in human body. This fact, combined with repeated exposure, may account for a hyper activation of the immune system and subsequent chronic inflammation [63] . The clinical and experimental evidence collected so far identify at least three main risks associated with aluminum in vaccines: 1. It can persist in the body. 2. It can trigger pathological immunological responses. 3. It can pass through the BBB into the CNS where it can trigger immuno-inflammatory processes, resulting in brain inflammation and long-term neural dysfunction. 2.1.1 Mechanisms of adjuvanticity Adjuvants may exert their immune enhancing effects according to five immune functional activities: 1. Translocation of antigens to the lymph nodes where they can be recognized by T cells. 2. Antigen protection enabling longer exposure. 3. Enhanced local reaction at the injection site. 4. Induction of the release of inflammatory cytokines. 5. Interaction with PRRs, specifically TLRs [22] . a Adjuvant effect The term "adjuvant effect" refers to the co-administration of an antigen with a microbial specific factor to enhance an antigen-specific immune response in vivo. The microbial components of adjuvants activate APCs to produce pro-inflammatory cytokines ("non-specific" signal 2) and to up-regulate molecules essential for antigen presentation. These molecules include major histocompatibility complex (MHC) class II (antigen-specific signal 1) and B7-1/2. These innate immune events allow a more effective presentation to the adaptive immune system, resulting in an augmented activation and clonal expansion of T cells [23] . In accordance to this effect, if self-antigens are used, an autoimmune response can be elicited [24] . It has been shown that auto-reactive T-cells that surpass tolerance mechanisms can be triggered by exogenous adjuvants to become auto-aggressive [25] . Infectious agents are able to naturally generate their adjuvant effect and can induce autoimmunity [26] . An example of this is the causality between viral infection and myocarditis. Half the cases of myocarditis are preceded by an acute viral infection. Infectious myocarditis in humans can be reproduced in experimental murine models of myocarditis [27] . It has also been shown that the autoimmune reaction elicited by an infectious agent can be effective in treating cancer. An example of this is that bladder administration of BCG ( bacille Calmette–Guérin ) has been shown to be effective against superficial bladder cancer development [28] . It can be inferred that the adjuvant effect can be used against specific tumor derived molecules, so that these molecules can be recognized as "non self". 2.1.2 Innate immune pattern recognition of pathogens and adjuvants PRR-PAMP (Pattern Recognition Receptor—Pathogen-Associated Molecular Patterns) interactions activate the APCs to promote antigen-specific lymphocytic responses [29] . The definition of PAMPs has now broadened, in that the recognized structures do not need to be pathogens. Thus the concept of "microbe-associated molecular patterns" (MAMPs) and of "danger/damage-associated molecular patterns" (DAMPs) were defined based on the notion that the endogenous host molecules signal danger or damage to the immune system [30] . 2.1.3 Innate immune response mediates the adjuvant effect TLRs are single-transmembrane PRRs localized on cell surface and endosomal membranes. From all the PRRs, these are the most studied. TLRs play a crucial role in innate immune response to "non self" and are biosensors of tissue damage. The interaction between the four known TLRs adapters: MyD88, TIRAP/Mal, TRAM and TRIF, in TLR signaling, shape the innate immune response. Besides PRRs the innate immune system also detects proteolytic enzymes generated during infection [31] . Merging the response to different PRRs signaling may be the pathway for developing customized responses to different aggressions [32] . b Experimental models of adjuvants Many animals have been used in experimental models of adjuvant-related autoimmune conditions [33] . These include primates, salmons, rabbits and swine; however, the most common are murine models. Murine models include autoimmune prone strains, models of autoimmune disease and autoimmune resistant strains ( Table 2 ). Table 2 Experimental models of adjuvant autoimmunity Experimental models Strain Disease model or related signs and symptoms Adjuvant Murine Rats DA (dark agouti) rats Rheumatoid arthritis Mineral oil (CFA, pristane, squalene,avridine) [34] , [35] Arthritis Collagen [36] Sprague Dawley rats Arthritis CFA [37] MMF Aluminum [38] Mice BALB/c Plasmacytomas Mineral oil, pristane [39] Sclerosing lipogranulomas SC injection of mineral oil [40] SLE-related autoantibodies Pristane, CFA, squalene [41] C57BL/6 Antiphospholipid-like syndrome CFA, IFA [42] NZB/NZWF1 SLE, lupus like GLN CFA, alum [43] Salmons Impaired growth rate, decreased carcass quality, spinal deformities, uveitis, inflammatory reactions in the abdominal cavity, RF, ANA, ANCA, immune-complex GLN and chronic granulomatous inflammation Vaccines with adjuvants such as oils [44] Rabbits Inflammation at injection site Vaccine: CFA, IFA, montanide [45] Swine Granulomatous inflammation Adverse local reactions Mineral oils [46] Primates Rhesus macaque Potential delayed acquisition of neonatal reflexes aluminum contained in pre clinical vaccine testing [47] C57BL/6 (transgenic factor V Leiden-mutated C57/BL6-back-crossed mice); RF: rheumatoid factor; ANA: antinuclear autoantibodies; ANCA: anti-cytoplasmic autoantibodies; GLN: glumerulonephritis; SLE: systemic lupus erythematous; MMF: macrophagic myofasciitis. An interesting model is that described by Lujan et al. The authors described that a commercial sheep, inoculated repetitively with aluminum-containing adjuvants vaccinations, developed an acute neurological episode with low response to external stimuli and acute meningoencephalitis few days after immunization. An excitatory phase, followed by weakness, extreme cachexia, tetraplegia and death appeared. This was suggested to be part of the spectrum of ASIA syndrome. Moreover, the biopsy of the nervous tissue of experimental animals indicated the presence of alum [48] . c Toxicity of aluminum adjuvants Aluminum nanoparticles have both a unique capacity of surpassing the blood brain barrier (BBB) and of eliciting immune inflammatory responses. These are probably the reasons why Aluminums' most sensitive target is the brain, and also why documented side effects are mostly neurologic or neuropsychiatric [49] , [50] . Aluminum is present in nature, not only as a vaccine adjuvant, but also in food, water and cosmetics. It has been described as a neurotoxin because even when a relatively small amount of Aluminium reaches the brain [49] , is can act as a genotoxin [51] , a prooxidant [52] , it can be proinflammatory [51] , act as an immunotoxin [5] and also as an endocrine disruptor [53] . Aluminum interferes with many essential cellular processes. Memory, concentration, speech deficits, impaired psychomotor control, reduced seizure tolerance and altered behaviour are manifestations of aluminium neurotoxicity. Moreover, Alzheimer's [54] , amyotrophic lateral sclerosis, Parkinsonism dementia [55] , multiple sclerosis [56] , and neurological impairments in children have been linked to aluminum neurotoxicity [57] . Brain susceptibility to aluminum compounds is possibly due to the brain's high metabolic requirement, to the fact that it possesses a large area of biological membranes and to the relatively low concentration of antioxidants [54] . Aluminum adjuvants exert their immunostimulatory effect through many different pathways that activate both the innate and adaptive immune systems. One of the most significant is the activation of the NLRP3 inflammasome pathway [58] . NLPR3 activation has been shown to trigger type 2 diabetes. By using NLPR3 knockout mice it has been demonstrated that the absence of inflammasome components leads to a better maintenance of glucose homeostasis and higher insulin sensitivity [59] . On the other hand, activation of the inflammasome and its downstream components: pro-inflammatory cytokines IL-1β and IL-18 are strongly implicated in the development of several central nervous system (CNS) disorders [60] . The vast majority of people are consuming higher amounts of aluminum through dietary and parenteral intake than what expert authorities consider safe. Upper limits set by US food and drug administrations (FDA) for aluminum in vaccines are set at no more than 850 μg/dose. These values were not based on toxicity studies, but on the minimum amount needed for aluminum to exert its effect as an adjuvant [51] . The quantities of aluminum to which infants, in their first year of age are exposed, have been considered safe by the FDA. However the scientific basis for this recommendation does not take into account aluminum persistence in the body. The concern about aluminum in dietary intake has been reinforced by the Food and Agriculture (FAO) WHO Expert Committee, which lowered the provisional tolerable weekly intake of aluminum from 7 mg/kg/bw (490 mg/week, for an average 70 kg human) to 1 mg/kg/bw (70 mg/week) [61] . The amount of dietary intake of aluminum has risen in urban societies to up to 100 mg/day considering the widespread use of processed convenience foods. However, only about 0.25% of dietary aluminum is absorbed into systemic circulation and most of it is thereafter eliminated through the kidneys [54] . Absorption of aluminum by the skin from ointments and cosmetics containing aluminum has been shown. Moreover, the presence of aluminum in breast tissue was associated with breast cancer [62] . Aluminum compounds persist for up to 8–11 years post vaccination in human body. This fact, combined with repeated exposure, may account for a hyper activation of the immune system and subsequent chronic inflammation [63] . The clinical and experimental evidence collected so far identify at least three main risks associated with aluminum in vaccines: 1. It can persist in the body. 2. It can trigger pathological immunological responses. 3. It can pass through the BBB into the CNS where it can trigger immuno-inflammatory processes, resulting in brain inflammation and long-term neural dysfunction. 2.2 Allergy and autoimmunity caused by metals There is a link between allergies and autoimmunity since both are the result of an abnormal immune response [3] , [4] . Metals such as mercury, aluminum, nickel and gold are known to induce immunotoxic effects in humans. The immunologic effects of these metals include immunomodulation, allergies and autoimmunity. They may act either as immunosuppressants or as immune adjuvants. Metals bind firmly to cells and proteins and thus have the ability to modify autologous epitopes (hapetenization). T-cells then recognize the proteins as foreign and trigger an autoimmune response [64] . Hypersensitivity caused by metals may be referred to as Type IV delayed hypersensitivity. The reaction is considered delayed because the first symptoms appear 24–48 h after exposure, because it is mostly T-cell mediated and the gold standard for diagnosis of delayed type hypersensitivity is patch testing [65] . In mercury-sensitized patients, even mercury concentrations within the normal range might provoke neuroallergic reactions in the brain [66] . Identifying metal sensitivity and removal of the sensitizing metals, such as dental amalgam, have been proved successful by showing symptom improvement in patients with previous autoimmune diseases. These diseases included fibromyalgia, autoimmune thyroid diseases and orofacial granulomatosis [67] , [68] , [69] , [70] ( Table 3 ). Table 3 Metals reported side effects. Metal Derivatives Main cause of exposure Side effects Mercury Methyl mercury Skin ointments Dental amalgam fillings Kidney disease [71] ; peripheral neuropathy; multiple sclerosis [72] ; ANA positivity [73] Polluted fish Thimerosal and phenyl mercury Antiseptics/preservatives in eye drops vaccines Flu like symptoms Eyelid eczema and edema Gold Colloidal gold [74] Treatment for RA Nephropathy Nickel [75] , [76] Food Jewelry Tobacco allergic and autoimmune symptoms; scleroderma-related autoantibodies and cutaneous sclerosis Aluminum [4] , [77] Food Vaccines Neurotoxic; delayed type hypersensitivity; ASIA syndrome; chronic fatigue syndrome; macrophagic myofasciitis RA: rheumatoid arthritis. 2.3 Genetics and vaccinology The timeline regarding the field of vaccinology has been divided in two generations, the first regarding the administration of inactivated pathogens in whole or live attenuated forms (e.g., Bacillus Calmette Guerin (BCG), plague, pertussis, polio, rabies, and smallpox) and the second regarding vaccines assembled from purified microbial cell components, also referred as subunit vaccines (e.g., polysaccharides, or protein antigens) [78] . This latter approach relies on recombinant DNA technology and polysaccharide chemistry. There are obstacles to conventional vaccine development methods such as non-cultivable in vitro pathogens (e.g., hepatitis C, papilloma virus types 16 and 18, and Mycobacterium leprae), antigen hypervariability (e.g., serogroup B meningococcus, gonococcus, malaria), opportunistic pathogens (e.g., Staphylococcus aureus) and rapid evolving pathogens such as Human immunodeficiency virus (HIV) [79] . Vaccine research gained a new perspective as the genomics field emerged over the last decades. Bacterial genomes have been sequenced and analyzed making it possible to choose the best candidate vaccine antigens by using the concept of reverse vaccinology [80] . The main known factors influencing the observed heterogeneity for immune responses induced by vaccines are gender, age, ethnicity, co-morbidity, immune system, and genetic background. The interaction between genetic and environmental components will dictate the response to vaccines. Studying the vaccine and the host will enable the development of customized treatment options. The combination of genetics, epidemiology and genomics in vaccine design has been denominated "vaccinomics" [81] . The importance of genetic influence is supported by twins and siblings studies, which show familial aggregation. This suggests that genomics is crucial in inter-individual variations in vaccine immune responses [82] . Both Human leukocyte antigen (HLA) and non-HLA gene markers have been identified as markers for immune response to vaccines. Multiple studies have shown connections between HLA gene polymorphisms and non-responsiveness to the HBV vaccine [83] . HLA region is divided in three sub regions: Class I is associated with the induction and maintenance of cell-mediated immune response, class II is associated with presentation of exogenous antigens to helper T CD4+ cells and class III, where immune non HLA related genes are located. Normal human tissue has at least 12HLA antigens, and although new recombinant haplotypes may occur, it is inherited mostly intact from progenitors [84] . HLA allelic differences are associated with different responses to vaccines, either by hyper or hypo responsiveness. We can infer that a similar response may be associated with different safety in relation to the development of autoimmune reactions to vaccines, particularly in the patients with genetic predisposition to an enhanced response to vaccine inoculation [85] . Furthermore, patients that share the same HLA, for instance siblings, have been diagnosed with ASIA following similar environmental stimuli [86] , [87] . 2.4 Autoantibodies induced by vaccines Autoantibodies help to diagnose certain autoimmune diseases, however, they can also be found in healthy individuals. Thus, autoimmune diseases cannot be diagnosed based solely on antibody detection [88] . Inoculation of vaccines triggers autoimmune responses that result in the development of autoantibodies. Many studies have been carried out in animals, healthy subjects and patients with autoimmune diseases to understand if this development is of clinical significance [89] , [90] , [91] , [92] . A difference in eliciting the production of autoantibodies in healthy humans has been observed between adjuvanted and non-adjuvanted influenza vaccines [93] . The annual influenza vaccine has been the most heavily researched vaccine, along with HPV and Pneumococcal vaccines as far as their relationship with patients who have previously been diagnosed with an autoimmune disease [94] , [95] , [96] . Autoantibody induction after HPV vaccination was also shown in adolescent girls with systemic lupus erythematosus (SLE) [97] . Although induction of autoantibodies was proven following vaccine administration, there have been no proven relation with disease diagnosis in either of the specific groups studied so far [92] , [98] . It has been widely demonstrated that autoantibodies can develop years before the manifestation of a full-blown autoimmune disease [99] . Moreover, the development of a specific autoantibody is also genetically determined, and the link between genetic, autoantibodies and vaccines may become an even more intriguing area of research [100] . 2.5 Siliconosis and autoimmune (auto-inflammatory) syndrome induced by adjuvants (ASIA) Silicones are synthetic polymers that can be used as fluids, emulsions, resins and elastomers making them useful in diverse fields. They were thought to be biologically inert substances and were incorporated in a multitude of medical devices such as joint implants, artificial heart valves, catheters, drains and shunts. Of all the silicone-containing products, the most famous are most likely breast implants. Silicon is one of the substances suspected to induce ASIA [5] . It is currently believed that exposure alone is not enough to trigger the disease but that it requires the presence of additional risk factors (e.g., genetic susceptibility, other environmental factors) [4] . Silicone exerts local tissue reactions. Some of these reactions are considered para-physiological, such as capsular tissue formation around an implant. Other reactions are viewed as abnormal, like when capsular contractures and allergic reactions to silicone or platinum (catalyst used in silicone polymerization found in minute concentrations in implants) occur [101] . Cutaneous exposure to silicone with cosmetics or baby bottles could potentially sensitize patients [102] . There is also a systemic component of silicone exposure related to diffusion of silicone through the elastomer envelope, commonly termed "bleeding". It may arouse systemic effects as it degrades and fragments in tissue, it can also spread throughout the body and lead to the development of cancer or autoimmune phenomena [103] . Patients with ruptured implants complain more frequently of pain and chronic fatigue when compared to patients with intact implants [104] . Anti-silicone antibodies were found to be present in human sera more frequently in patients who have undergone silicone breast implants, however, their pathological significance remains uncertain [105] . The same was seen for other antibodies such as autoantibodies directed against dsDNA, ssDNA, SSB/La, silicone and collagen II, which were found to be present in increased levels in patients after exposure to silicone [106] . It has also been shown that the formation of autoantibodies is directly related to implant duration. Several autoimmune diseases have been linked to silicone exposure including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), polymyositis, systemic sclerosis (SSc) and fibromyalgia. Although ASIA symptoms may arise 24 years after the onset of exposure to silicone implants [107] , most of the follow-up periods are short and concluding evidence is yet to come regarding this causality. 2.6 Vaccines and autoimmune diseases There have been published case reports, epidemiologic and research studies that suggest a connection between several vaccines and certain autoimmune conditions, notwithstanding that, overall the benefits of vaccination outweigh the risks. 3 The vaccines 3.1 Measles, mumps, rubella (MMR) vaccine Thrombocytopenia has been reported as the main adverse event following MMR vaccine. After MMR vaccine the onset of immune thrombocytopenic purpura (ITP) usually occurred within 6 weeks at a risk rate of 1:22,000–25,000 MMR vaccine doses, while the incidence of ITP following infections is 1:6000 for measles and 1:3000 for rubella [108] . As the risk of thrombocytopenia is higher in patients who experience natural infection with measles, mumps or rubella than in those receiving the vaccine, vaccination is encouraged. Arthralgia complaints have also been reported and they may present as transient arthralgia, acute arthritis and rarely chronic arthritis [109] . Some risk factors have been found to be associated with the development of arthritis in vaccinated patients such as: female gender, older age, prior seronegativity and specific HLA alleles [110] . 3.2 Yellow fever (YF) vaccine YF vaccine is only advisable to people in, or going to endemic areas. The risk of developing YF vaccine-associated neurologic disease (YEL-AND) is inversely proportional to age [111] . This is why children aged 60 years because of possible higher risk of severe adverse effects (SAEs) even though the incidence remains low [113] . 3.3 Bacillus Calmette-Guérin (BCG) vaccine Besides being a vaccine for Mycobacterium tuberculosis (TB), the BCG has proved effective as immunotherapy for bladder cancer. Although the mechanism is yet to be fully understood, it is thought that BCG binds to fibronectin forming complexes that enable the recognition as "non-self" by the innate immune response of Th1 cells. Ultimately the pathways result in the apoptosis of tumor cells [114] . Because of its effect in treating non-muscle-invasive urothelial carcinoma, as well as superficial bladder tumors, it was expected that BCG could play a role in treating other types of cancer, despite data having not corroborated this hypothesis so far. Adverse events vary according to the site and method of administration. Intradermal administration of BCG has been reported to elicit arthritis [115] , dermatomyositis [116] and Takayasu's arteritis (TA) [117] among others. Intravesical treatment for bladder cancer can cause reactive arthritis (ReA) [118] . The risk relies on a systemic reaction composed of an early infective phase (PCR positive and response to anti-TB treatment) and a late hypersensitivity reaction [119] . 3.4 Hepatitis B virus vaccine (HBVacc) HBV is a DNA virus of the Hepadnaviridae family, responsible for acute and chronic liver disease. HBV vaccines are considered the first efficient vaccines against a major human cancer. HBV vaccines have reduced the risk of developing chronic infection and they also have proved to reduce the incidence of liver cancer in children [120] . The vaccine has been associated mainly with autoimmune neuromuscular disorders. They include, but are not limited to: optic neuritis, Guillain-Barre syndrome (GBS), myelitis and multiple sclerosis (MS), systemic lupus erythematosus (SLE), arthritis, vasculitis, antiphospholipid syndrome (APS) and myopathy [121] . HBV vaccine is the most common immunization associated with acute myelitis. There are studies that indicate that the pathogenicity behind such vaccine and autoimmunity might be based on cross-reactivity between HBV antigen (HBsAg) epitopes, yeast antigens, as well as other adjuvants contained in the vaccine itself [122] . 3.5 Human papilloma virus (HPV) vaccine Up to 90% of cervical cancer deaths, occur in developing countries that lack the ability to fully implement the Papanicolau (Pap) screening programs. HPV poses a special challenge in vaccine safety. HPV is necessary for the development of cervical cancer. However, most women infected with HPV will not develop the disease since 70% of infections will resolve within a year and up to 90% within 2 years without specific treatment. Over the course of decades, cancer may result in a small proportion of the remaining infected women. Death rate from cervical cancer in 9–20 year old girls is zero and long-term benefits are yet to be proven. In this specific case, short term risks to healthy subjects can prove to pose a heavier burden than cervical cancer [123] . There are at least 100 types of HPV strains, 15 of which have been pathologically associated with cancer. Two vaccines, Gardasil™ and Cervarix™, are commercially available against HPV. Both contain the L1 capsid proteins of several HPV strains as antigens. Gardasil™ contains serotypes 16, 18, 6, 11. These antigens are combined with aluminum (Al) hydroxyphosphate sulphate as an adjuvant. Cervarix™ contains a combination of the oil-based adjuvant monophosphoryl lipid A (MPL) and Al hydroxide (ASO4) as adjuvant and is directed at strains 16 and 18 [124] . There have been several reports of post-licensure adverse events, some of which have even been fatal [125] . Compared to other vaccines, an unusually high proportion of adverse drug reactions has been reported associated with HPV vaccines [126] . In 2008, Australia reported an annual ADR rate of 7.3/100,000, the highest since 2003. This increase was almost entirely due to ADRs reported following the commencement of the national HPV vaccination program for females aged 12–26 years in April 2007 (705 out of a total of 1538 ADRs records). The numbers only decreased after the cessation of the catch-up schedule. Although the percentage of convulsions attributable to the HPV vaccine decreased, the overall report remained comparable between 2007 and 2009 (51% and 40% respectively). These reports do not prove the association, but show that there is a higher frequency of ADRs related to HPV vaccines reported worldwide, and that they fit a consistent pattern (i.e., nervous system-related disorders rank the highest in frequency) that deserves further investigation [126] , [127] , [128] . Indeed, several autoimmune diseases have been linked to HPV immunization. Examples include GBS, MS, Acute disseminated encephalomyelitis (ADEM), Transverse Myelitis (TM), postural orthostatic tachycardia syndrome (POTS), SLE, primary ovarian failure (POF), pancreatitis, vasculitis, immune thrombocytopenic purpura (ITP) and Autoimmune hepatitis (AH) [123] . 3.6 Influenza Influenza is an acute viral infection that affects the respiratory tract and is caused by influenza type A–C viruses of the Orthomyxoviridae family [129] . H1N1 mortality rates in the 2009 outbreak showed high risk in those aged 70 years and older, presence of chronic diseases and delayed admission. Risk of infection was lower in those who had been vaccinated for seasonal influenza with 2008/9 trivalent inactivated vaccine [130] . Studies have demonstrated that influenza vaccine is safe and immunogenic in patients with SLE or rheumatoid arthritis (RA), diminishing the risk of respiratory infections [129] . It has been shown that adjuvanted vaccine had more local reactions but did not increase systemic adverse reactions [131] . Molecular mimicry has been suggested as a mechanism to explain an autoimmune response following influenza vaccination. However, a causal relationship between influenza vaccines and induction of autoimmune diseases remains unproved [129] . Diseases or symptoms reported after influenza vaccination include mostly neurological syndromes such as GBS [REF]. Nonetheless, influenza vaccines should be recommended for patients with MS, because influenza infection is associated with increased risk of exacerbations. That being said, influenza vaccinations showed increased risk of autoimmune responses suggestive of ASIA [132] , vasculitis [133] and APS [134] among others. 3.7 Meningococcal vaccines Meningococcal disease is caused by Neisseria meningitidis . One of the following five serogroups causes almost every invasive disease: A–C, Y, and W-135. Vaccines available so far for its prevention encompass either pure polysaccharide vaccines that use purified bacterial capsular polysaccharides as antigens, or protein/polysaccharide conjugate vaccines, which use the polysaccharide molecule plus diphtheria or tetanus toxoid as T-cell-stimulating antigens. N. meningitidis serogroup B (MenB) MenB glycoconjugate vaccines are not immunogenic and hence, vaccine design has focused on sub-capsular antigens [135] . MenB capsular polysaccharide is composed of a linear homopolymer of α(2 → 8) N -acetyl-neuroaminic acid (polysialic acid; PSA). MenB PSA and PSA found on neural cell adhesion molecules are structurally identical. As a result of this, it has been proposed that infection with MenB or vaccination with PSA may be associated with subsequent autoimmune or neurological disease [136] . No evidence of increased autoimmunity was found to be associated with meningococcal serogroup B infection [136] . Regarding vaccination, the inoculation does not cause autoimmune diseases but may unmask autoimmune phenomena in genetically predisposed individuals. Local reactions are more frequent in individuals vaccinated with quadrivalent meningococcal conjugate vaccines compared to plain polysaccharide vaccines. The intramuscular administration of the conjugate vaccine (versus subcutaneous for that of polysaccharide) may, in part, explain the higher reactivity [137] . Diseases previously associated with meningococcal vaccines are GBS [138] , Henoch-Schönlein Purpura (HSP) [139] and Bullous pemphigoid (BP) [140] . 3.8 Pneumococcal vaccine Streptococcus pneumoniae (Pneumococcus) is the main cause of bacterial community-acquired pneumonia and meningitis in western countries, as well as the cause of more than 800,000 children deaths in developing countries [141] , [142] . There are three anti-pneumococcal vaccines commercially available. Two of these are conjugated to a protein carrier (PCV7 and PCV13) and one is not conjugated (PPV23). PPV23 was licensed in 1983 and consists of the capsular polysaccharides of twenty-three different Streptococcus pneumoniae serotypes (1–5, 6B, 7F, 8, 9N, 9 V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F). It does not elicit immunological memory because the immune response it triggers is T-cell independent. It is usually administered to the elderly (above 65 years), as it is believed to be less effective in children. PCV7 is composed of the most frequent serotypes 4, 6B, 9 V, 14,18C, 19F, and 23F. PCV13 is directed at serotypes 1, 3–5, 6A, 6B, 7F, 9 V, 14, 18C, 19A, 19F, and 23F. Contrary to PPV23 both PCV7 and PCV13 have an aluminum adjuvant in their composition that elicits a T-cell mediated response [143] . Ever since vaccines were introduced in the healthcare system, prevalence, fatality and admissions for invasive pneumococcal disease have decreased significantly [144] . Vaccine adverse events vary depending on whether the vaccine is adjuvanted or not. In a non adjuvanted vaccine, local reactions are present in 9% of people vaccinated intra muscularly and in 24% of those immunized sub-cutaneously [145] . In conjugated vaccines, this percentage rises to 50% [146] . Systemic reactions such as fever, irritability, decreased appetite and sleep disturbances occur in 80–85% of recipients of PCV or PPV. Symptoms like arthralgia, arthritis, myalgia, paresthesia and fatigue are more frequent in patients post PPV. This may be related to the fact that the vaccines are administered to different age groups. Autoimmune risk following PPV vaccine is very low. Only 14 case reports were found after PPV vaccine. Six of these referred to reactivation of a previous autoimmune disorder. Studies directed to access vaccine safety in subjects with autoimmune diseases showed immunization was safe [147] , [148] . 3.9 Tetanus vaccine Tetanus toxoid (TT) is a potent exotoxin produced by the bacteria Clostridium tetani . The toxin has a predominant effect on inhibitory neurons, inhibiting release of γ-aminobutiric acid (GABA). When spinal inhibitory interneurons are affected the symptoms appear [149] . The vaccine against C. tetani contains deactivated tetanus toxoid plus an adjuvant (usually aluminium hydroxide). The most studied and prevalent disease associated with TT is antiphospholipid syndrome (APS), but CNS complications have also been reported such as optic neuritis, acute myelitis and encephalomyelitis [150] . In mice, the immune response to TT depends on genetic background and to the specific adjuvant used for immunization. Naive BALB/c mice, immunized with TT, developed antibodies directed to TT, dsDNA and β2GPI and were extremely sick [90] . 3.1 Measles, mumps, rubella (MMR) vaccine Thrombocytopenia has been reported as the main adverse event following MMR vaccine. After MMR vaccine the onset of immune thrombocytopenic purpura (ITP) usually occurred within 6 weeks at a risk rate of 1:22,000–25,000 MMR vaccine doses, while the incidence of ITP following infections is 1:6000 for measles and 1:3000 for rubella [108] . As the risk of thrombocytopenia is higher in patients who experience natural infection with measles, mumps or rubella than in those receiving the vaccine, vaccination is encouraged. Arthralgia complaints have also been reported and they may present as transient arthralgia, acute arthritis and rarely chronic arthritis [109] . Some risk factors have been found to be associated with the development of arthritis in vaccinated patients such as: female gender, older age, prior seronegativity and specific HLA alleles [110] . 3.2 Yellow fever (YF) vaccine YF vaccine is only advisable to people in, or going to endemic areas. The risk of developing YF vaccine-associated neurologic disease (YEL-AND) is inversely proportional to age [111] . This is why children aged 60 years because of possible higher risk of severe adverse effects (SAEs) even though the incidence remains low [113] . 3.3 Bacillus Calmette-Guérin (BCG) vaccine Besides being a vaccine for Mycobacterium tuberculosis (TB), the BCG has proved effective as immunotherapy for bladder cancer. Although the mechanism is yet to be fully understood, it is thought that BCG binds to fibronectin forming complexes that enable the recognition as "non-self" by the innate immune response of Th1 cells. Ultimately the pathways result in the apoptosis of tumor cells [114] . Because of its effect in treating non-muscle-invasive urothelial carcinoma, as well as superficial bladder tumors, it was expected that BCG could play a role in treating other types of cancer, despite data having not corroborated this hypothesis so far. Adverse events vary according to the site and method of administration. Intradermal administration of BCG has been reported to elicit arthritis [115] , dermatomyositis [116] and Takayasu's arteritis (TA) [117] among others. Intravesical treatment for bladder cancer can cause reactive arthritis (ReA) [118] . The risk relies on a systemic reaction composed of an early infective phase (PCR positive and response to anti-TB treatment) and a late hypersensitivity reaction [119] . 3.4 Hepatitis B virus vaccine (HBVacc) HBV is a DNA virus of the Hepadnaviridae family, responsible for acute and chronic liver disease. HBV vaccines are considered the first efficient vaccines against a major human cancer. HBV vaccines have reduced the risk of developing chronic infection and they also have proved to reduce the incidence of liver cancer in children [120] . The vaccine has been associated mainly with autoimmune neuromuscular disorders. They include, but are not limited to: optic neuritis, Guillain-Barre syndrome (GBS), myelitis and multiple sclerosis (MS), systemic lupus erythematosus (SLE), arthritis, vasculitis, antiphospholipid syndrome (APS) and myopathy [121] . HBV vaccine is the most common immunization associated with acute myelitis. There are studies that indicate that the pathogenicity behind such vaccine and autoimmunity might be based on cross-reactivity between HBV antigen (HBsAg) epitopes, yeast antigens, as well as other adjuvants contained in the vaccine itself [122] . 3.5 Human papilloma virus (HPV) vaccine Up to 90% of cervical cancer deaths, occur in developing countries that lack the ability to fully implement the Papanicolau (Pap) screening programs. HPV poses a special challenge in vaccine safety. HPV is necessary for the development of cervical cancer. However, most women infected with HPV will not develop the disease since 70% of infections will resolve within a year and up to 90% within 2 years without specific treatment. Over the course of decades, cancer may result in a small proportion of the remaining infected women. Death rate from cervical cancer in 9–20 year old girls is zero and long-term benefits are yet to be proven. In this specific case, short term risks to healthy subjects can prove to pose a heavier burden than cervical cancer [123] . There are at least 100 types of HPV strains, 15 of which have been pathologically associated with cancer. Two vaccines, Gardasil™ and Cervarix™, are commercially available against HPV. Both contain the L1 capsid proteins of several HPV strains as antigens. Gardasil™ contains serotypes 16, 18, 6, 11. These antigens are combined with aluminum (Al) hydroxyphosphate sulphate as an adjuvant. Cervarix™ contains a combination of the oil-based adjuvant monophosphoryl lipid A (MPL) and Al hydroxide (ASO4) as adjuvant and is directed at strains 16 and 18 [124] . There have been several reports of post-licensure adverse events, some of which have even been fatal [125] . Compared to other vaccines, an unusually high proportion of adverse drug reactions has been reported associated with HPV vaccines [126] . In 2008, Australia reported an annual ADR rate of 7.3/100,000, the highest since 2003. This increase was almost entirely due to ADRs reported following the commencement of the national HPV vaccination program for females aged 12–26 years in April 2007 (705 out of a total of 1538 ADRs records). The numbers only decreased after the cessation of the catch-up schedule. Although the percentage of convulsions attributable to the HPV vaccine decreased, the overall report remained comparable between 2007 and 2009 (51% and 40% respectively). These reports do not prove the association, but show that there is a higher frequency of ADRs related to HPV vaccines reported worldwide, and that they fit a consistent pattern (i.e., nervous system-related disorders rank the highest in frequency) that deserves further investigation [126] , [127] , [128] . Indeed, several autoimmune diseases have been linked to HPV immunization. Examples include GBS, MS, Acute disseminated encephalomyelitis (ADEM), Transverse Myelitis (TM), postural orthostatic tachycardia syndrome (POTS), SLE, primary ovarian failure (POF), pancreatitis, vasculitis, immune thrombocytopenic purpura (ITP) and Autoimmune hepatitis (AH) [123] . 3.6 Influenza Influenza is an acute viral infection that affects the respiratory tract and is caused by influenza type A–C viruses of the Orthomyxoviridae family [129] . H1N1 mortality rates in the 2009 outbreak showed high risk in those aged 70 years and older, presence of chronic diseases and delayed admission. Risk of infection was lower in those who had been vaccinated for seasonal influenza with 2008/9 trivalent inactivated vaccine [130] . Studies have demonstrated that influenza vaccine is safe and immunogenic in patients with SLE or rheumatoid arthritis (RA), diminishing the risk of respiratory infections [129] . It has been shown that adjuvanted vaccine had more local reactions but did not increase systemic adverse reactions [131] . Molecular mimicry has been suggested as a mechanism to explain an autoimmune response following influenza vaccination. However, a causal relationship between influenza vaccines and induction of autoimmune diseases remains unproved [129] . Diseases or symptoms reported after influenza vaccination include mostly neurological syndromes such as GBS [REF]. Nonetheless, influenza vaccines should be recommended for patients with MS, because influenza infection is associated with increased risk of exacerbations. That being said, influenza vaccinations showed increased risk of autoimmune responses suggestive of ASIA [132] , vasculitis [133] and APS [134] among others. 3.7 Meningococcal vaccines Meningococcal disease is caused by Neisseria meningitidis . One of the following five serogroups causes almost every invasive disease: A–C, Y, and W-135. Vaccines available so far for its prevention encompass either pure polysaccharide vaccines that use purified bacterial capsular polysaccharides as antigens, or protein/polysaccharide conjugate vaccines, which use the polysaccharide molecule plus diphtheria or tetanus toxoid as T-cell-stimulating antigens. N. meningitidis serogroup B (MenB) MenB glycoconjugate vaccines are not immunogenic and hence, vaccine design has focused on sub-capsular antigens [135] . MenB capsular polysaccharide is composed of a linear homopolymer of α(2 → 8) N -acetyl-neuroaminic acid (polysialic acid; PSA). MenB PSA and PSA found on neural cell adhesion molecules are structurally identical. As a result of this, it has been proposed that infection with MenB or vaccination with PSA may be associated with subsequent autoimmune or neurological disease [136] . No evidence of increased autoimmunity was found to be associated with meningococcal serogroup B infection [136] . Regarding vaccination, the inoculation does not cause autoimmune diseases but may unmask autoimmune phenomena in genetically predisposed individuals. Local reactions are more frequent in individuals vaccinated with quadrivalent meningococcal conjugate vaccines compared to plain polysaccharide vaccines. The intramuscular administration of the conjugate vaccine (versus subcutaneous for that of polysaccharide) may, in part, explain the higher reactivity [137] . Diseases previously associated with meningococcal vaccines are GBS [138] , Henoch-Schönlein Purpura (HSP) [139] and Bullous pemphigoid (BP) [140] . 3.8 Pneumococcal vaccine Streptococcus pneumoniae (Pneumococcus) is the main cause of bacterial community-acquired pneumonia and meningitis in western countries, as well as the cause of more than 800,000 children deaths in developing countries [141] , [142] . There are three anti-pneumococcal vaccines commercially available. Two of these are conjugated to a protein carrier (PCV7 and PCV13) and one is not conjugated (PPV23). PPV23 was licensed in 1983 and consists of the capsular polysaccharides of twenty-three different Streptococcus pneumoniae serotypes (1–5, 6B, 7F, 8, 9N, 9 V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F). It does not elicit immunological memory because the immune response it triggers is T-cell independent. It is usually administered to the elderly (above 65 years), as it is believed to be less effective in children. PCV7 is composed of the most frequent serotypes 4, 6B, 9 V, 14,18C, 19F, and 23F. PCV13 is directed at serotypes 1, 3–5, 6A, 6B, 7F, 9 V, 14, 18C, 19A, 19F, and 23F. Contrary to PPV23 both PCV7 and PCV13 have an aluminum adjuvant in their composition that elicits a T-cell mediated response [143] . Ever since vaccines were introduced in the healthcare system, prevalence, fatality and admissions for invasive pneumococcal disease have decreased significantly [144] . Vaccine adverse events vary depending on whether the vaccine is adjuvanted or not. In a non adjuvanted vaccine, local reactions are present in 9% of people vaccinated intra muscularly and in 24% of those immunized sub-cutaneously [145] . In conjugated vaccines, this percentage rises to 50% [146] . Systemic reactions such as fever, irritability, decreased appetite and sleep disturbances occur in 80–85% of recipients of PCV or PPV. Symptoms like arthralgia, arthritis, myalgia, paresthesia and fatigue are more frequent in patients post PPV. This may be related to the fact that the vaccines are administered to different age groups. Autoimmune risk following PPV vaccine is very low. Only 14 case reports were found after PPV vaccine. Six of these referred to reactivation of a previous autoimmune disorder. Studies directed to access vaccine safety in subjects with autoimmune diseases showed immunization was safe [147] , [148] . 3.9 Tetanus vaccine Tetanus toxoid (TT) is a potent exotoxin produced by the bacteria Clostridium tetani . The toxin has a predominant effect on inhibitory neurons, inhibiting release of γ-aminobutiric acid (GABA). When spinal inhibitory interneurons are affected the symptoms appear [149] . The vaccine against C. tetani contains deactivated tetanus toxoid plus an adjuvant (usually aluminium hydroxide). The most studied and prevalent disease associated with TT is antiphospholipid syndrome (APS), but CNS complications have also been reported such as optic neuritis, acute myelitis and encephalomyelitis [150] . In mice, the immune response to TT depends on genetic background and to the specific adjuvant used for immunization. Naive BALB/c mice, immunized with TT, developed antibodies directed to TT, dsDNA and β2GPI and were extremely sick [90] . 4 The diseases 4.1 Anti-phospholipid syndrome (APS) APS is an autoimmune disease characterized by the occurrence of thrombotic events. Patients suffering from this condition have recurrent fetal loss, thromboembolic phenomena, thrombocytopenia as well as neurological, cardiac and dermatological involvement [151] . The serological marker of APS is the presence of anti-phospholipid antibodies (aPL), which bind negatively charged phospholipids, platelets and endothelial cells mainly through the plasma protein beta-2-glycoprotein-I (b2GPI). The presence of IgG and IgM anti-cardiolipin antibodies (aCL) and lupus anticoagulant is associated with thrombosis in patients with APS [151] . β2GPI was identified as the most important antigen in APS. β2GPI has several properties in vitro which define it as an anticoagulant (e.g., inhibition of prothrombinase activity, adenosine diphosphate-induced platelet aggregation, platelet factor IX production) [152] . Passive transfer of anti-β2GPI antibodies induce experimental APS in naïve mice and thrombus formation in ex vivo model [153] . Evidence suggests that the molecular mimicry mechanism between β2GPI and TT is one of the possible causes for APS. Besides TT, APS has also been reported following HBV and influenza virus vaccination, although data are scarce [154] , [155] . 4.2 Systemic lupus erythematosus (SLE) SLE is a multisystem autoimmune disease characterized by the production of a variety of autoantibodies. IgG isotype antibodies to double-stranded DNA (dsDNA) are thought to be diagnostic markers and their presence correlates with disease pathogenesis. Several factors including genetic, hormonal, environmental and immune defects are involved in the induction of autoantibodies in this disease [156] . Post vaccination manifestations of SLE or lupus like syndrome have been reported and range from autoantibody induction to full blown clinical disease. Reports have been published associating SLE to HBV, MMR, dTP, HPV, influenza, BCG, pneumococcal and small pox vaccinations [157] . Vaccination in SLE diagnosed patients is associated with disease exacerbation and decreased antibody response, which may be due to the underlying disease and the frequent use of immunosuppressive drugs [158] . A temporal link between SLE and HBV vaccination is the only relation that has been demonstrated [159] . Several studies have demonstrated an increased prevalence of HPV in individuals with lupus compared to the general population, which has increased awareness for the need to vaccinate this high-risk population [160] . To do so, the association between immunization with HPV vaccines and SLE like symptoms, as well as the higher incidence of flares in known Lupus patients must be taken into account. 4.3 Vasculitis Vasculitis is the name given to a group of autoimmune mediated diseases, which involve blood vessels of different types and sizes. They can be categorized according to several disease features indluding: the type of vessel affected, organ distribution, genetic predisposition and clinical manifestation [161] . 4.3.1 Large vessels vasculitis So far, 18 cases of large vessel vasculitis have been detected. This includes 15 cases of giant cell arteritis (GCA) following influenza vaccination, 2 cases of Takayasu disease (TD), and one case of large cell arteritis involving subclavian and renal arteries following HBV vaccines. Two of these patients had previous received the diagnosis of ankylosing spondylitis and polymyalgia rheumatica (PMR)-like illness [162] . 4.3.2 Medium vessels vasculitis One case of polyarteritis nodosa (PAN) following the administration of Tetanus and BCG vaccine is described. All other cases of PAN in adults follow the administration of HBV vaccine [163] , [164] , [165] . Case reports of medium vessels vasculitis – both polyarteritis nodosa and Kawasaki disease (KD) – have also been published in pediatric patients. KD has been described one day after the second dose of HBV vaccine and following yellow fever vaccine [166] , [167] . Two cases of pediatric patients with PAN have been reported two months after receiving the HBV vaccine [164] , [165] . 4.3.3 Small vessels vasculitis: ANCA-associated vasculitis Eosinophilic granulomatosis with polyangiitis (EGPA) after tetanus vaccination [163] and following HBV vaccine [168] have been reported. There are also 3 cases of microscopic polyangiitis (MPA) and 6 cases of granulomatosis with polyangiitis (GPA) following influenza vaccines in the literature [169] , [170] . 4.3.4 Immune complex small vessels vasculitis Henoch Schönlein purpura (HSP) is the most common vasculitis of childhood. It is generally benign and self-limited. It is mediated by IgA immune complex deposition in various tissues as well as in small-sized blood vessels. Genetic risk factors play an important role in the pathogenesis of the disease: it is associated with HLA-DRB*01, 07 and 11. HSP was associated with seasonal influenza, influenza A (H1N1), pneumococcal and meningococcal disease, hepatitis A virus (HAV), HBV, anti-human papilloma virus (HPV) vaccines, and following multiple combinations of vaccines, such as typhoid, cholera and yellow fever [139] , [171] , [172] , [173] . Leukocytoclastic vasculitis has been associated with several vaccines, including influenza vaccine [174] , HAV vaccine [175] , HBV vaccine [176] , pneumococcal vaccine [177] , varicella [178] , rubella, smallpox [179] and the anthrax vaccine [180] . Dermal vasculitis with pan uveitis has also been described following MMR vaccine [181] . 4.4 Rheumatoid arthritis (RA) RA is the most prevalent chronic inflammatory arthritis affecting the synovial membrane of multiple diarthrodial joints. Although its etiology has not been completely clarified, deregulation of the immune system is evident with a preponderance of inflammatory cytokines and immune cells within the joints. RA has an estimated heritability of 60%, leaving a substantial proportion of risk to environmental factors. Immunizations have previously been proposed as potential environmental triggers for RA. In the Norfolk Arthritis Register database, 19 of the first 588 patients reported receiving a tetanus vaccination within 6 weeks prior to the onset of arthritis. Similarly, a transient rise in RF titer was recorded in 10 out of 245 military recruits 2–3 weeks after receiving concomitant immunization against tetanus, typhoid, paratyphoid, mumps, diphtheria, polio and smallpox. However, only 2 showed a persistent elevation in titer and none developed arthritis [182] . Several mechanisms have been proposed to explain the putative association between vaccination and the initiation of RA, the most prominent of which are molecular mimicry and non-specific immune system activation [182] . Vaccines who have been associated with RA include rubella vaccine in which reactive arthritis occurs in 5% of recipients. Controlled studies failed to show persistent arthritis or arthralgia in these patients [110] . Patients following HBV vaccine showed an increase of arthritis in a VAERS study, but this was not seen in a large retrospective epidemiological study [183] . Data so far suggest that vaccines carry an insignificant role in the pathogenesis of RA. 4.4.1 Vaccines in the therapy of RA Several mechanisms are being studied to produce vaccines mainly targeting inflammatory cytokines as "antigens" such as TNF, aiming to induce high titers of endogenous neutralizing anti-cytokine antibodies with the goal of breaking natural Th tolerance to auto antigens. Other cytokines, namely IL-1 IL-6, MIF, RANTES, IL-18, MCP-1 are also being tested [184] . Another vaccine related therapy uses autologous T cell lines to induce a specific immune response by the host's T cells directed against the autoimmune (vaccine) T cells [185] . This strategy has been successful in mouse models and has shown encouraging results in a small pilot study of 15 RA patients, where 10 patients showed a clinical response, defined by ACR 50 improvement criteria [186] . 4.5 Undifferentiated connective tissue disease (UCTD) UCTD is a clinical condition characterized by signs, symptoms and laboratory tests suggestive of a systemic autoimmune disease but that does not fulfill the criteria for any defined connective tissue disease (CTD). Such patients with clinical manifestations suggestive of systemic connective tissue disease but not fulfilling any existing criteria are quite frequent: 12-20% of the patients initially asking for a rheumatologic evaluation may at least temporarily be diagnosed as affected by 'undefined' or 'undifferentiated' connective tissue disease. Comparing studies on these diseases is unfeasible because of the inexistence of defined criteria for diagnosis [187] . Within 5 years of follow-up, patients usually evolve to defined CTDs, which include SLE, systemic sclerosis (SSc), primary Sjögren's syndrome (pSS), mixed connective tissue disease (MCTD), systemic vasculitis, poly-dermatomyositis (PM/DM) and RA. Maintaining an undefined profile for 5 years makes evolving into CTDs less probable and the diagnosis of "stable UCTD" reliable [188] . Disease etiology is a concern and it has been associated with Vitamin D deficiency and silicone implants, both of which lead to an imbalance in proinflammatory and anti-inflammatory cytokines [189] . Vaccines have also been associated with this disease, namely the HBV vaccine [190] . Etiopathogenesis of UCTD is unknown and it has been suggested it might fall on ASIA spectrum since symptomatic similarities are striking and UCTD etiopathogenesis has been associated with adjuvants [122] . 4.6 Alopecia areata (AA) AA is an autoimmune disease, characterized by one or more well demarcated oval and round non-cicatricial patches of hair loss. The disease may affect any hair bearing part of the body and has a great impact on a patient's self-esteem and quality of life. Depending on ethnicity and location, AA is the most prevalent skin disease. AA prevalence varies and is estimated to be between 0.1–0.2% in the United States and 3.8% in Singapore [191] , [192] . As with any other autoimmune disease, the development of AA encompasses genetic and environmental factors. Environmental factors associated with AA development are emotional and/or physical stress, infections and vaccines [193] . Secondary syphilis is one of the most well studied examples, however Epstein Barr Virus [194] and Herpes Zoster [195] infections have also been related to the development of the disease. As far as vaccines go, HBV vaccine has been associated with AA development. In one study of 60 patients, 48 developed AA after vaccination with HBV vaccine. Of those 48 patients, 16 were re-challenged, and the reappearance of disease was witnessed [196] . In mice this association failed to be established [197] . One case of AA was witnessed following Tetanus Toxoid, as well as two case reports following HPV and MMR vaccine [198] , [199] , [200] . 4.7 Immune thrombocytopenic purpura (ITP) ITP is an autoimmune disease defined by a platelet count of less than 105 platelets/μL without overlapping diseases. It can present with or without anti-platelet–antibodies. Thrombocytopenia is relatively common and the overall probability of developing ITP was 6,9% in a cohort of 260 patients. It was also found that 12% of patients developed an overlapping AID other than ITP [201] . The etiology of the disease is yet to be fully understood but it has been detected following infectious diseases, such as Helicobacter pylori, hepatitis C virus (HCV), novel influenza A infection, rotavirus infection and human immunodeficiency virus (HIV) [202] . ITP onset has also been reported, although rarely, as a severe adverse event following vaccine administration. This was more often observed after measles–mumps–rubella (MMR), hepatitis A and B, diphtheria–tetanus–acellular pertussis (DTaP), and varicella vaccinations [203] . Molecular mimicry has been suggested as a possible mechanism for the development of ITP, namely following Helicobacter Pylori infection. Its eradication has been shown to increase platelet count and diminish the levels of anti-CagA antibody in a subset of H. Pylori infected subjects with ITP [204] . These data point towards a beneficial role of H. pylori eradication in chronic ITP. Two cases of ITP following anti-rabies vaccine have been reported and one after HPV vaccine. Reactivation of ITP was reported two weeks after a tick-borne encephalitis vaccination [202] . The most consistent association with ITP is with the MMR vaccine [205] . However, it should be emphasized that the number of cases are fewer than expected without vaccination. 4.8 Type 1 diabetes (T1D) T1D is due to antigen specific reactions against insulin producing beta cells of the pancreas. Much like other autoimmune diseases, T1D results from a combination of genetic, environmental, hormonal and immunological factors. Environmental factors such as pathogens, diet, toxins, stress and vaccines are believed to be involved in the beginning of the autoimmune process [206] . Although the mechanisms by which viral infections cause autoimmune diabetes have not been fully clarified, there is some evidence to suggest a role for natural infections in the pathogenesis of T1D mellitus in susceptible individuals [207] . It has been hypothesized that vaccination could trigger T1D in susceptible individuals. Although post-vaccination T1D may be biologically plausible, cumulative evidence has not supported an increased risk of T1D following any vaccine [208] . Several experimental data have suggested that, depending on the timing, vaccination might exert a protecting or aggravating effect on the occurrence of diabetes [209] . A study suggests that Haemophillus influenza type b vaccine might be a risk factor in the induction of islet cell and anti-GAD antibodies measured at one year of age [210] but there are previous studies that show no association between Hib and T1D [211] . In a cohort of American military officers diagnosed with T1D, there was no association found between vaccination and T1D diagnosis [212] . Available data about a relation between the mumps vaccine and T1D are still incomplete and their interpretation is difficult because of miscellaneous confounding factors associated with the development of T1D [213] . Association between Hemagglutinin 1 Neuraminidase 1 (H1N1) vaccines and T1D is so far unproven [214] . In humans, it has been hypothesized that early-age BCG vaccination is associated with the risk of T1D. The few studies conducted to date provided no consistent evidence of an association. There are, however, studies showing a possible temporary boost of the immune function after vaccination [215] . Studies also show that among BCG-vaccinated children who test positive for islet autoantibodies, there is a higher cumulative risk of T1D [216] . In animal experiments it has been observed that BCG seems to have a protective effect against diabetes, however researchers have yet to translate this benefit to humans [217] . In all, studies results do not support any strong association between vaccination and T1D. 4.9 Narcolepsy Narcolepsy is a sleep disorder described as excessive sleepiness with abnormal sleep pattern characterized by uncontrollable rapid eye movement (REM) events which occur at any time during the day. These event and may or may not be accompanied by a loss of muscle tone (cataplexy) [218] . A plethora of data indicates that narcolepsy is caused by the lack of orexin (also known as hypocretin), an important neurotransmitter, which is involved in the regulation of the sleep cycle. In Narcolepsy patients, a loss of orexin producing neurons in the hypothalamus and low levels of orexin in the cerebrospinal fluid (CSF) has been reported [218] . Narcolepsy has been shown to have an autoimmune background. Antibodies against Tribbles 2 (Trib2) have been found in these patients, which may be related to the pathogenesis of disease. An experimental model of narcolepsy in mice has been made by passive transfer of total IgG from narcolepsy patients into the animal's brains through intra ventricular injection [219] . Environmental factors like Influenza A virus and streptococcal infections have been associated with disease onset. Interestingly, fever by itself without the diagnosis of an infectious etiology was found to be a risk factor for narcolepsy [220] . Several groups have studied and found an increase in the incidence of narcolepsy diagnosis following the introduction of influenza vaccination, specifically, ASO3-adjuvanted Pandemrix™ vaccine. This association was shown in Finland especially in 4–19 year-olds, but also in case reports from other countries [221] . Other studies failed to find an association. The actual infection with H1N1 has been associated with disease development in China, however no such relationship has been noted in Europe [220] . The above-mentioned associations are specifically related to the ASO3-adjuvanted Pandemrix™ vaccine. The same association has not been reported for other H1N1 adjuvanted or non-adjuvanted vaccines. The major difference between the ASO3 and the MF59 adjuvants is the presence of the α-tocopherol. α-tocopherol is unique in that it can achieve the highest and longest antibody response by producing an enhanced antigen-specific adaptive immune response. In vitro it was shown that α-tocopherol could increase the production of orexin as well as increase the proteosome activity. This increased production of orexin fragments may facilitate antigen presentation to MHC class II, thus triggering an autoimmune process [220] . All these data together support the relationship between the H1N1 Pandemrix™ vaccine and the development of narcolepsy. 4.10 Celiac disease Gluten induced enteropathy, gluten sensitive enteropathy, or more commonly called celiac disease (CD) is a life-long autoimmune condition mainly of the gastrointestinal tract, specifically affecting the small intestine. The abnormal immune response crates autoantigens which are directed towards Tissue transglutaminase (tTG). The two main autoantibodies and the most widespread serological markers to screen for the disease are anti tTG and anti endomysium. Two additional auto-antibodies, namely: anti deaminated gliadin peptide and anti-neoepitope tTG were found recently to be reliable for CD screening as well [222] . CD is an autoimmune disease induced by well-known nutritional environmental factors. The non-dietary ones are less studied and established. Several infectious disease have been linked to its development, the so-called infectome [193] . A clear cause-effect relation is yet to be established for most of the pathogens associated with CD. What has been shown, however, is that in countries with low economic status, inferior hygiene conditions and higher infectious load, CD prevalence is lower [223] . An epidemiologic relationship was established in 2006 between rotavirus infection and CD. Data showed that in genetically predisposed individuals, rotavirus infection was related to childhood CD development [224] . In subsequent research studies, a celiac peptide was recognized and proved to share homology with rotavirus major neutralizing protein VP7 and with the CD autoantigen tTG. The antibodies directed against the viral protein VP7 were shown to predict the onset of CD and induce typical features of CD in the intestinal epithelial cell-line T84 [225] . It has also been suggested that rotavirus vaccine alters B and T behavior, as the percentage of B-cells was higher in the vaccinated infants [226] . Rotavirus vaccine as an inducer of CD is still in discussion and warrants further study. 4.11 Polymyalgia rheumatica (PMR) PMR is an autoimmune inflammatory rheumatic disease characterized by raised inflammatory markers with pain and morning stiffness of shoulders and pelvic girdles and synovitis of proximal joints and extra-articular synovial structures. Its diagnosis is clinical and it is typically a disease of the elderly occurring mainly in subjects above 70. Etiopathogenesis of PMR remains unknown, but genetic and environmental factors play a role [227] . A close temporal relationship has been ascertained concerning epidemics of Mycoplasma pneumoniae , Chlamydia pneumonia , Parvovirus B19 and peaks of cases of PMR and giant cell arteritis, however this is not clearly proven [228] . Cases of PMR following vaccination have rarely been reported. However, it is believed that post vaccination PMR may be underreported due to its symptomatic similarities with the transient effects of vaccines, namely: arthralgia, myalgia and low-grade fever. This leads to failure in establishing a chronological relationship when the disease is diagnosed. Most of the reported cases are associated with seasonal influenza vaccine (Inf-V). Often, the time interval between vaccine administration and symptoms onset varies from one day, to three months. Three cases were reported with associated Giant Cell arthritis. A case report of relapsing PMR after four years of remission following tetanus vaccination has also been reported [229] , [230] . 4.12 Acute disseminated encephalomyelitis (ADEM) Acute disseminated encephalomyelitis (ADEM) is an inflammatory demyelinating disease of the central nervous system (CNS). ADEM is usually poly-symptomatic with encephalopathy (i.e., behavioral change or altered level of consciousness). It affects mostly children and young adults and has higher prevalence in males. Its incidence is 0.6–0.8 per 100 000 per year [231] . Although there is no concrete evidence of a clear pathogenic association, ADEM has been associated with immunization or previous viral infection. Post-vaccination ADEM accounts for only 5-10 percent of all cases, while post-infectious ADEM accounts for 66 percent of all cases of ADEM [232] . The hypothesis that better describes these associations is molecular mimicry. T-cells targeting human herpesvirus-6 (HHV-6), coronavirus, influenza virus and Epstein-Barr virus (EBV) have been shown to cross-react with myelin basic protein (MBP) antigens. Anti-MBP T-cells were detected in patients following vaccination with simple rabies vaccine [233] , [234] , [235] . In a post experimental therapy for Alzheimer's disease with a vaccine that contained aggregates of synthetic Aβ42 fragments of amyloid precursor protein, ADEM was shown to develop in mice [236] . The experimental model of MS, EAE mice, may be induced with injection of Aβ42, but only when the latter is administered together with the complete Freund's adjuvant [237] . This observation points to the importance and central role of the adjuvants in induction of ADEM and autoimmunity in general [238] . The overall incidence of post vaccination ADEM is estimated to be 0.1–0.2 per 100 000 and a higher risk has been reported following immunization against measles. Other vaccines accountable for post-vaccination ADEM include vaccines against the varicella zoster, the rubella, the smallpox and the influenza viruses [239] . Surprisingly, certain vaccines such as anti-tetanus vaccine were shown to have a negative correlation with ADEM (statistically significant decreased risk) [240] . HBV immunization has been studied as a possible cause for ADEM but was later associated with clinically isolated syndrome (CIS) (a first time occurring demyelinating episode that may, or not develop to MS) and complete conversion to MS [241] . As far as case reports are concerned, ADEM was associated with vaccination with influenza, hepatitis A and B, MMR, HPV and tetanus [121] , [242] , [243] . 4.13 Bullous dermatoses Bullous dermatoses are characterized by the presence of blisters and autoantibodies against structural components of the skin: desmosomal proteins (in pemphigus), adhesion molecules of the dermal-epidermal junction (in pemphigoid diseases), and epidermal/ tissue transglutaminase (in dermatitis herpetiformis). The most frequent autoimmune bullous diseases are bullous pemphigoid (BP) and pemphigus vulgaris (PV). BP is more frequently observed in the elderly, while the age of onset of PV is between 40 and 60 years. Neither of the diseases have any gender preference [244] . BP and PV etiology is, so far, poorly understood. Both diseases have been associated with various environmental factors, which include emotional and/or physical stress, infections and vaccinations [244] . Genetic predisposition has also been studied with overexpression of certain HLA class II alleles. These include HLA-DQB1*0301, DRB1*04, DRB1*1101, and DQB1*0302. These alleles have been found to be more prevalent in BP patients than in the general population [245] . PV is associated with certain HLA class II loci such as HLA-DR4 and HLADR14 alleles (DRB1*0401 and DRB1*0402, which is prevalent in Ashkenazi Jews, Iranian and Sardinian patients). Other loci include DRB1*1401 (common among Japanese and Italian patients) and two DQB1 alleles (DQB1*0302 and DQB1*0503), which are strongly associated with PV. BP and PV patients' sera were found to have significantly higher prevalence of antibodies to hepatitis B virus, hepatitis C virus, helicobacter pylori, toxoplasma gondii and cytomegalovirus [244] . As far as vaccination is concerned, BP developed in patients following influenza, diphtheria, tetanus, pertussis, hepatitis B, BCG, polio and herpes zoster vaccines [140] , [246] , [247] Furthermore, reactivation of BP following influenza vaccination was reported in one case report [248] . New onset PV was associated with: influenza vaccine, hepatitis B vaccine, anthrax vaccine, typhoid booster and rabies vaccination. In addition, exacerbation of PV after vaccination was also reported following influenza vaccine and tetanus vaccine [121] . 4.14 Idiopathic inflammatory myopathies (IIM) IIM compose a group of skeletal muscles diseases in which myositis without a recognized cause occurs. IIM is usually subdivided in 4 entities: dermatomyositis (DM), polymyositis (PM), inclusion body myositis (sIBM) non-specific myositis (NSM) and immune mediated necrotizing myopathy (IAM) [249] . IIM prevalence is around 1.1 × 10 −6 cases, with a bimodal age of distribution that peaks in childhood and again between 45 and 55 years. DM is the most common inflammatory myopathy while PM is the least frequent. Despite exhibiting similar clinical symptoms, the subsets of IIM exhibit significant immunopathological variation. DM begins with the activation of the complement and formation of membrane attack complexes (MAC). In PM and sIBM the fundamental process is related to CD8+ T cells mediated cytotoxicity [249] . It is unclear what breaks the tolerance and drives the immune response to induce IIM. So far, DM, PM and sIBM have been linked to vaccination. Several cases have been reported in the literature associating different vaccines with the development of idiopathic inflammatory myopathies. 119 cases of IIM had been reported to VAERS database up to June 2013. Out of these 119 cases, 33 were classified as PM, 85 as DM and an only one as a sIBM. DM has been reported after almost any vaccine, however only a few studies have attempted to clarify the possible relationship between DM and vaccination. PM is a frequent misdiagnosed disorder. Some reports have associated previous immunization, especially hepatitis B vaccine with PM [250] . Despite being recently differentiated from other IIM, sIBM has already been related to HBV vaccine [250] . Some vaccines associated with myositis are MMR vaccine, smallpox vaccine, Poliomyelitis (IPV), diphtheria and tetanus toxoid, influenza, HPV and BCG [250] . 4.15 Fibromyalgia syndrome (FMS) FMS is an entity that is related to the inability of the CNS to modulate pain. The conditioned pain modulation process in the CNS appears to be compromised among many FMS patients, which might explain the enhanced pain sensation experienced by these patients [251] . The etiology of FMS is yet to be unveiled. Genetic predisposition, physical trauma (particularly to the cervical spine), emotional stress (to various stressors) as well as a variety of infections have been linked with FMS. Vaccines have been associated with the triggering of FMS namely rubella and Lyme disease vaccines [252] . There are several reports of fibromyalgia-like disease after vaccination, specifically HPV (Martinez-Lavin Journal of Clinical rheumatology 2014). The medical community and regulatory agencies should be aware of these possible adverse effects aiming at defining their magnitude. 4.16 Chronic fatigue syndrome (CFS)/myalgic encephalomyelitis (ME)/systemic exertion intolerance disease (SEID) Chronic fatigue syndrome (CFS) is a disease characterized by disabling fatigue, headaches, concentration difficulties and memory deficits (90%). Other symptoms such as sore throat (85%), tender lymph nodes (80%), skeletal muscle pain and feverishness (75%), sleep disruption (70%), psychiatric problems (65%) and rapid pulse (10%) are often observed. It more frequently affects women and has a prevalence of 0.2-2.6% [253] . Although disease etiology is still unknown, there are several pathogens, such as Epstein–Barr virus (EBV), which have been associated with CFS. Patients often have higher titers of IgM to the EBV viral capsid antigen. Cytomegalovirus and human herpes virus 6 antibodies were also detected more often in CFS patients, although other reports failed to replicate these results. Parvovirus B19 infection has also been suggested as a trigger to CFS [253] , [254] , [255] . Vaccine inoculation has also been appointed as a probable cause. Vaccinations against rubella, Q fever and hepatitis B were found to be associated with higher risk of developing CFS while meningococcal vaccine, poliovirus and influenza vaccine were not. Surprisingly, staphylococcus toxoid vaccine appeared to have a protective effect [121] , [256] , [257] . 4.17 ASIA syndrome Defined in 2011 by Shoenfeld and Agmon-Levin ASIA syndrome is characterized by hyperactive immune response to adjuvants [4] . As previously stated, ASIA incorporates four known medical conditions: Siliconosis, GWS, MMF, and post-vaccination phenomena [4] . Recently, the sick building syndrome (SBS) was proposed as a candidate for the ASIA spectrum [258] . All of these diseases satisfy several criteria for FMS and SEID [252] . a Macrophagic myofasciitis (MMF) MMF has been described as an emerging condition of unknown cause characterized by a pathognomonic lesion in muscle biopsy mixing large macrophages with submicron to micron-sized agglomerates of nanocrystals in their cytoplasm and lymphocytic infiltrates. These lesions were related to aluminum deposits in muscle following immunization with aluminum containing vaccines [63] . MMF lesion is now universally recognized as indicative of a long-lasting persistence of aluminum adjuvant at the site of prior intramuscular immunization. The long-lasting MMF lesion should be considered as a biomarker of aluminum bio persistence in a given individual. Patients with MMF have higher reported myalgia with incidence being up to 90%. Its etiology is not clear but genuine muscle weakness is rare and the diagnosis of fibromyalgia is also rare. Higher prevalence of chronic fatigue syndrome (CFS) in patients with MMF has been reported as well. Cognitive impairment has been associated with MMF: in one series of 105 MMF patients, up to 97% had attention and memory complaints and neuropsychological tests were abnormal in 89% [259] . b Gulf War syndrome (GWS) GWS is a clinical entity specifically related to a certain time and place in history. It was described among veterans of the military conflict occurring in 1990–1991 in the Persian Gulf. The syndrome is characterized by chronic fatigue, musculoskeletal symptoms, malaise and cognitive impairment. It clinically overlaps with Post Traumatic Stress Disorder (PTSD), FMS, CFS and other functional disorders [260] . The unique conditions that have been associated so far with disease development are the exposure to extreme climate in the Persian Gulf, exposure to various chemicals (pesticides, depleted uranium), stress provoked by prolonged waiting without actual combat and the intense exposure to vaccinations of the soldiers for fear of biological weaponry [260] . Comparing Gulf War veterans and veterans of the Bosnian conflict, multiple vaccinations administered to servicemen in the Gulf War was identified as a unique exposure [261] . The mechanism through which vaccination exposure may lead to the development of functional symptoms is not completely understood. The possibility that a shift from Th1 to Th2 type reactions could be of pathogenic significance was raised and is supported by an increased frequency of allergic reactions, low natural killer cell activity and low levels of interferon γ and IL-2 in these patients [262] . One study with GWS patients showed a connection between anti-squalene antibodies and symptoms development. This was refuted by a larger study that found no association between anti-squalene antibodies and chronic multi-symptom illness [263] . c ASIA registry A registry is a collection of data related to patients with the same specific characteristic. It is often the first approach in the study of an area of inquiry. In rare diseases, registries are often the way to get a sufficiently sized sample of patients which can be used either for epidemiological or research purposes. ASIA syndrome may be underreported because of unawareness and failure to connect the syndrome with the exposure. This registry was created to fully understand the clinical aspects of disease and compare patients from all over the world in order to have fully validated criteria for disease diagnosis and also to define demographic and environmental history of disease. The ASIA Syndrome registry website can be found on the following link: https://ontocrf.costisa.com/en/web/asia . Only cases reported by physicians are accepted. 4.1 Anti-phospholipid syndrome (APS) APS is an autoimmune disease characterized by the occurrence of thrombotic events. Patients suffering from this condition have recurrent fetal loss, thromboembolic phenomena, thrombocytopenia as well as neurological, cardiac and dermatological involvement [151] . The serological marker of APS is the presence of anti-phospholipid antibodies (aPL), which bind negatively charged phospholipids, platelets and endothelial cells mainly through the plasma protein beta-2-glycoprotein-I (b2GPI). The presence of IgG and IgM anti-cardiolipin antibodies (aCL) and lupus anticoagulant is associated with thrombosis in patients with APS [151] . β2GPI was identified as the most important antigen in APS. β2GPI has several properties in vitro which define it as an anticoagulant (e.g., inhibition of prothrombinase activity, adenosine diphosphate-induced platelet aggregation, platelet factor IX production) [152] . Passive transfer of anti-β2GPI antibodies induce experimental APS in naïve mice and thrombus formation in ex vivo model [153] . Evidence suggests that the molecular mimicry mechanism between β2GPI and TT is one of the possible causes for APS. Besides TT, APS has also been reported following HBV and influenza virus vaccination, although data are scarce [154] , [155] . 4.2 Systemic lupus erythematosus (SLE) SLE is a multisystem autoimmune disease characterized by the production of a variety of autoantibodies. IgG isotype antibodies to double-stranded DNA (dsDNA) are thought to be diagnostic markers and their presence correlates with disease pathogenesis. Several factors including genetic, hormonal, environmental and immune defects are involved in the induction of autoantibodies in this disease [156] . Post vaccination manifestations of SLE or lupus like syndrome have been reported and range from autoantibody induction to full blown clinical disease. Reports have been published associating SLE to HBV, MMR, dTP, HPV, influenza, BCG, pneumococcal and small pox vaccinations [157] . Vaccination in SLE diagnosed patients is associated with disease exacerbation and decreased antibody response, which may be due to the underlying disease and the frequent use of immunosuppressive drugs [158] . A temporal link between SLE and HBV vaccination is the only relation that has been demonstrated [159] . Several studies have demonstrated an increased prevalence of HPV in individuals with lupus compared to the general population, which has increased awareness for the need to vaccinate this high-risk population [160] . To do so, the association between immunization with HPV vaccines and SLE like symptoms, as well as the higher incidence of flares in known Lupus patients must be taken into account. 4.3 Vasculitis Vasculitis is the name given to a group of autoimmune mediated diseases, which involve blood vessels of different types and sizes. They can be categorized according to several disease features indluding: the type of vessel affected, organ distribution, genetic predisposition and clinical manifestation [161] . 4.3.1 Large vessels vasculitis So far, 18 cases of large vessel vasculitis have been detected. This includes 15 cases of giant cell arteritis (GCA) following influenza vaccination, 2 cases of Takayasu disease (TD), and one case of large cell arteritis involving subclavian and renal arteries following HBV vaccines. Two of these patients had previous received the diagnosis of ankylosing spondylitis and polymyalgia rheumatica (PMR)-like illness [162] . 4.3.2 Medium vessels vasculitis One case of polyarteritis nodosa (PAN) following the administration of Tetanus and BCG vaccine is described. All other cases of PAN in adults follow the administration of HBV vaccine [163] , [164] , [165] . Case reports of medium vessels vasculitis – both polyarteritis nodosa and Kawasaki disease (KD) – have also been published in pediatric patients. KD has been described one day after the second dose of HBV vaccine and following yellow fever vaccine [166] , [167] . Two cases of pediatric patients with PAN have been reported two months after receiving the HBV vaccine [164] , [165] . 4.3.3 Small vessels vasculitis: ANCA-associated vasculitis Eosinophilic granulomatosis with polyangiitis (EGPA) after tetanus vaccination [163] and following HBV vaccine [168] have been reported. There are also 3 cases of microscopic polyangiitis (MPA) and 6 cases of granulomatosis with polyangiitis (GPA) following influenza vaccines in the literature [169] , [170] . 4.3.4 Immune complex small vessels vasculitis Henoch Schönlein purpura (HSP) is the most common vasculitis of childhood. It is generally benign and self-limited. It is mediated by IgA immune complex deposition in various tissues as well as in small-sized blood vessels. Genetic risk factors play an important role in the pathogenesis of the disease: it is associated with HLA-DRB*01, 07 and 11. HSP was associated with seasonal influenza, influenza A (H1N1), pneumococcal and meningococcal disease, hepatitis A virus (HAV), HBV, anti-human papilloma virus (HPV) vaccines, and following multiple combinations of vaccines, such as typhoid, cholera and yellow fever [139] , [171] , [172] , [173] . Leukocytoclastic vasculitis has been associated with several vaccines, including influenza vaccine [174] , HAV vaccine [175] , HBV vaccine [176] , pneumococcal vaccine [177] , varicella [178] , rubella, smallpox [179] and the anthrax vaccine [180] . Dermal vasculitis with pan uveitis has also been described following MMR vaccine [181] . 4.3.1 Large vessels vasculitis So far, 18 cases of large vessel vasculitis have been detected. This includes 15 cases of giant cell arteritis (GCA) following influenza vaccination, 2 cases of Takayasu disease (TD), and one case of large cell arteritis involving subclavian and renal arteries following HBV vaccines. Two of these patients had previous received the diagnosis of ankylosing spondylitis and polymyalgia rheumatica (PMR)-like illness [162] . 4.3.2 Medium vessels vasculitis One case of polyarteritis nodosa (PAN) following the administration of Tetanus and BCG vaccine is described. All other cases of PAN in adults follow the administration of HBV vaccine [163] , [164] , [165] . Case reports of medium vessels vasculitis – both polyarteritis nodosa and Kawasaki disease (KD) – have also been published in pediatric patients. KD has been described one day after the second dose of HBV vaccine and following yellow fever vaccine [166] , [167] . Two cases of pediatric patients with PAN have been reported two months after receiving the HBV vaccine [164] , [165] . 4.3.3 Small vessels vasculitis: ANCA-associated vasculitis Eosinophilic granulomatosis with polyangiitis (EGPA) after tetanus vaccination [163] and following HBV vaccine [168] have been reported. There are also 3 cases of microscopic polyangiitis (MPA) and 6 cases of granulomatosis with polyangiitis (GPA) following influenza vaccines in the literature [169] , [170] . 4.3.4 Immune complex small vessels vasculitis Henoch Schönlein purpura (HSP) is the most common vasculitis of childhood. It is generally benign and self-limited. It is mediated by IgA immune complex deposition in various tissues as well as in small-sized blood vessels. Genetic risk factors play an important role in the pathogenesis of the disease: it is associated with HLA-DRB*01, 07 and 11. HSP was associated with seasonal influenza, influenza A (H1N1), pneumococcal and meningococcal disease, hepatitis A virus (HAV), HBV, anti-human papilloma virus (HPV) vaccines, and following multiple combinations of vaccines, such as typhoid, cholera and yellow fever [139] , [171] , [172] , [173] . Leukocytoclastic vasculitis has been associated with several vaccines, including influenza vaccine [174] , HAV vaccine [175] , HBV vaccine [176] , pneumococcal vaccine [177] , varicella [178] , rubella, smallpox [179] and the anthrax vaccine [180] . Dermal vasculitis with pan uveitis has also been described following MMR vaccine [181] . 4.4 Rheumatoid arthritis (RA) RA is the most prevalent chronic inflammatory arthritis affecting the synovial membrane of multiple diarthrodial joints. Although its etiology has not been completely clarified, deregulation of the immune system is evident with a preponderance of inflammatory cytokines and immune cells within the joints. RA has an estimated heritability of 60%, leaving a substantial proportion of risk to environmental factors. Immunizations have previously been proposed as potential environmental triggers for RA. In the Norfolk Arthritis Register database, 19 of the first 588 patients reported receiving a tetanus vaccination within 6 weeks prior to the onset of arthritis. Similarly, a transient rise in RF titer was recorded in 10 out of 245 military recruits 2–3 weeks after receiving concomitant immunization against tetanus, typhoid, paratyphoid, mumps, diphtheria, polio and smallpox. However, only 2 showed a persistent elevation in titer and none developed arthritis [182] . Several mechanisms have been proposed to explain the putative association between vaccination and the initiation of RA, the most prominent of which are molecular mimicry and non-specific immune system activation [182] . Vaccines who have been associated with RA include rubella vaccine in which reactive arthritis occurs in 5% of recipients. Controlled studies failed to show persistent arthritis or arthralgia in these patients [110] . Patients following HBV vaccine showed an increase of arthritis in a VAERS study, but this was not seen in a large retrospective epidemiological study [183] . Data so far suggest that vaccines carry an insignificant role in the pathogenesis of RA. 4.4.1 Vaccines in the therapy of RA Several mechanisms are being studied to produce vaccines mainly targeting inflammatory cytokines as "antigens" such as TNF, aiming to induce high titers of endogenous neutralizing anti-cytokine antibodies with the goal of breaking natural Th tolerance to auto antigens. Other cytokines, namely IL-1 IL-6, MIF, RANTES, IL-18, MCP-1 are also being tested [184] . Another vaccine related therapy uses autologous T cell lines to induce a specific immune response by the host's T cells directed against the autoimmune (vaccine) T cells [185] . This strategy has been successful in mouse models and has shown encouraging results in a small pilot study of 15 RA patients, where 10 patients showed a clinical response, defined by ACR 50 improvement criteria [186] . 4.4.1 Vaccines in the therapy of RA Several mechanisms are being studied to produce vaccines mainly targeting inflammatory cytokines as "antigens" such as TNF, aiming to induce high titers of endogenous neutralizing anti-cytokine antibodies with the goal of breaking natural Th tolerance to auto antigens. Other cytokines, namely IL-1 IL-6, MIF, RANTES, IL-18, MCP-1 are also being tested [184] . Another vaccine related therapy uses autologous T cell lines to induce a specific immune response by the host's T cells directed against the autoimmune (vaccine) T cells [185] . This strategy has been successful in mouse models and has shown encouraging results in a small pilot study of 15 RA patients, where 10 patients showed a clinical response, defined by ACR 50 improvement criteria [186] . 4.5 Undifferentiated connective tissue disease (UCTD) UCTD is a clinical condition characterized by signs, symptoms and laboratory tests suggestive of a systemic autoimmune disease but that does not fulfill the criteria for any defined connective tissue disease (CTD). Such patients with clinical manifestations suggestive of systemic connective tissue disease but not fulfilling any existing criteria are quite frequent: 12-20% of the patients initially asking for a rheumatologic evaluation may at least temporarily be diagnosed as affected by 'undefined' or 'undifferentiated' connective tissue disease. Comparing studies on these diseases is unfeasible because of the inexistence of defined criteria for diagnosis [187] . Within 5 years of follow-up, patients usually evolve to defined CTDs, which include SLE, systemic sclerosis (SSc), primary Sjögren's syndrome (pSS), mixed connective tissue disease (MCTD), systemic vasculitis, poly-dermatomyositis (PM/DM) and RA. Maintaining an undefined profile for 5 years makes evolving into CTDs less probable and the diagnosis of "stable UCTD" reliable [188] . Disease etiology is a concern and it has been associated with Vitamin D deficiency and silicone implants, both of which lead to an imbalance in proinflammatory and anti-inflammatory cytokines [189] . Vaccines have also been associated with this disease, namely the HBV vaccine [190] . Etiopathogenesis of UCTD is unknown and it has been suggested it might fall on ASIA spectrum since symptomatic similarities are striking and UCTD etiopathogenesis has been associated with adjuvants [122] . 4.6 Alopecia areata (AA) AA is an autoimmune disease, characterized by one or more well demarcated oval and round non-cicatricial patches of hair loss. The disease may affect any hair bearing part of the body and has a great impact on a patient's self-esteem and quality of life. Depending on ethnicity and location, AA is the most prevalent skin disease. AA prevalence varies and is estimated to be between 0.1–0.2% in the United States and 3.8% in Singapore [191] , [192] . As with any other autoimmune disease, the development of AA encompasses genetic and environmental factors. Environmental factors associated with AA development are emotional and/or physical stress, infections and vaccines [193] . Secondary syphilis is one of the most well studied examples, however Epstein Barr Virus [194] and Herpes Zoster [195] infections have also been related to the development of the disease. As far as vaccines go, HBV vaccine has been associated with AA development. In one study of 60 patients, 48 developed AA after vaccination with HBV vaccine. Of those 48 patients, 16 were re-challenged, and the reappearance of disease was witnessed [196] . In mice this association failed to be established [197] . One case of AA was witnessed following Tetanus Toxoid, as well as two case reports following HPV and MMR vaccine [198] , [199] , [200] . 4.7 Immune thrombocytopenic purpura (ITP) ITP is an autoimmune disease defined by a platelet count of less than 105 platelets/μL without overlapping diseases. It can present with or without anti-platelet–antibodies. Thrombocytopenia is relatively common and the overall probability of developing ITP was 6,9% in a cohort of 260 patients. It was also found that 12% of patients developed an overlapping AID other than ITP [201] . The etiology of the disease is yet to be fully understood but it has been detected following infectious diseases, such as Helicobacter pylori, hepatitis C virus (HCV), novel influenza A infection, rotavirus infection and human immunodeficiency virus (HIV) [202] . ITP onset has also been reported, although rarely, as a severe adverse event following vaccine administration. This was more often observed after measles–mumps–rubella (MMR), hepatitis A and B, diphtheria–tetanus–acellular pertussis (DTaP), and varicella vaccinations [203] . Molecular mimicry has been suggested as a possible mechanism for the development of ITP, namely following Helicobacter Pylori infection. Its eradication has been shown to increase platelet count and diminish the levels of anti-CagA antibody in a subset of H. Pylori infected subjects with ITP [204] . These data point towards a beneficial role of H. pylori eradication in chronic ITP. Two cases of ITP following anti-rabies vaccine have been reported and one after HPV vaccine. Reactivation of ITP was reported two weeks after a tick-borne encephalitis vaccination [202] . The most consistent association with ITP is with the MMR vaccine [205] . However, it should be emphasized that the number of cases are fewer than expected without vaccination. 4.8 Type 1 diabetes (T1D) T1D is due to antigen specific reactions against insulin producing beta cells of the pancreas. Much like other autoimmune diseases, T1D results from a combination of genetic, environmental, hormonal and immunological factors. Environmental factors such as pathogens, diet, toxins, stress and vaccines are believed to be involved in the beginning of the autoimmune process [206] . Although the mechanisms by which viral infections cause autoimmune diabetes have not been fully clarified, there is some evidence to suggest a role for natural infections in the pathogenesis of T1D mellitus in susceptible individuals [207] . It has been hypothesized that vaccination could trigger T1D in susceptible individuals. Although post-vaccination T1D may be biologically plausible, cumulative evidence has not supported an increased risk of T1D following any vaccine [208] . Several experimental data have suggested that, depending on the timing, vaccination might exert a protecting or aggravating effect on the occurrence of diabetes [209] . A study suggests that Haemophillus influenza type b vaccine might be a risk factor in the induction of islet cell and anti-GAD antibodies measured at one year of age [210] but there are previous studies that show no association between Hib and T1D [211] . In a cohort of American military officers diagnosed with T1D, there was no association found between vaccination and T1D diagnosis [212] . Available data about a relation between the mumps vaccine and T1D are still incomplete and their interpretation is difficult because of miscellaneous confounding factors associated with the development of T1D [213] . Association between Hemagglutinin 1 Neuraminidase 1 (H1N1) vaccines and T1D is so far unproven [214] . In humans, it has been hypothesized that early-age BCG vaccination is associated with the risk of T1D. The few studies conducted to date provided no consistent evidence of an association. There are, however, studies showing a possible temporary boost of the immune function after vaccination [215] . Studies also show that among BCG-vaccinated children who test positive for islet autoantibodies, there is a higher cumulative risk of T1D [216] . In animal experiments it has been observed that BCG seems to have a protective effect against diabetes, however researchers have yet to translate this benefit to humans [217] . In all, studies results do not support any strong association between vaccination and T1D. 4.9 Narcolepsy Narcolepsy is a sleep disorder described as excessive sleepiness with abnormal sleep pattern characterized by uncontrollable rapid eye movement (REM) events which occur at any time during the day. These event and may or may not be accompanied by a loss of muscle tone (cataplexy) [218] . A plethora of data indicates that narcolepsy is caused by the lack of orexin (also known as hypocretin), an important neurotransmitter, which is involved in the regulation of the sleep cycle. In Narcolepsy patients, a loss of orexin producing neurons in the hypothalamus and low levels of orexin in the cerebrospinal fluid (CSF) has been reported [218] . Narcolepsy has been shown to have an autoimmune background. Antibodies against Tribbles 2 (Trib2) have been found in these patients, which may be related to the pathogenesis of disease. An experimental model of narcolepsy in mice has been made by passive transfer of total IgG from narcolepsy patients into the animal's brains through intra ventricular injection [219] . Environmental factors like Influenza A virus and streptococcal infections have been associated with disease onset. Interestingly, fever by itself without the diagnosis of an infectious etiology was found to be a risk factor for narcolepsy [220] . Several groups have studied and found an increase in the incidence of narcolepsy diagnosis following the introduction of influenza vaccination, specifically, ASO3-adjuvanted Pandemrix™ vaccine. This association was shown in Finland especially in 4–19 year-olds, but also in case reports from other countries [221] . Other studies failed to find an association. The actual infection with H1N1 has been associated with disease development in China, however no such relationship has been noted in Europe [220] . The above-mentioned associations are specifically related to the ASO3-adjuvanted Pandemrix™ vaccine. The same association has not been reported for other H1N1 adjuvanted or non-adjuvanted vaccines. The major difference between the ASO3 and the MF59 adjuvants is the presence of the α-tocopherol. α-tocopherol is unique in that it can achieve the highest and longest antibody response by producing an enhanced antigen-specific adaptive immune response. In vitro it was shown that α-tocopherol could increase the production of orexin as well as increase the proteosome activity. This increased production of orexin fragments may facilitate antigen presentation to MHC class II, thus triggering an autoimmune process [220] . All these data together support the relationship between the H1N1 Pandemrix™ vaccine and the development of narcolepsy. 4.10 Celiac disease Gluten induced enteropathy, gluten sensitive enteropathy, or more commonly called celiac disease (CD) is a life-long autoimmune condition mainly of the gastrointestinal tract, specifically affecting the small intestine. The abnormal immune response crates autoantigens which are directed towards Tissue transglutaminase (tTG). The two main autoantibodies and the most widespread serological markers to screen for the disease are anti tTG and anti endomysium. Two additional auto-antibodies, namely: anti deaminated gliadin peptide and anti-neoepitope tTG were found recently to be reliable for CD screening as well [222] . CD is an autoimmune disease induced by well-known nutritional environmental factors. The non-dietary ones are less studied and established. Several infectious disease have been linked to its development, the so-called infectome [193] . A clear cause-effect relation is yet to be established for most of the pathogens associated with CD. What has been shown, however, is that in countries with low economic status, inferior hygiene conditions and higher infectious load, CD prevalence is lower [223] . An epidemiologic relationship was established in 2006 between rotavirus infection and CD. Data showed that in genetically predisposed individuals, rotavirus infection was related to childhood CD development [224] . In subsequent research studies, a celiac peptide was recognized and proved to share homology with rotavirus major neutralizing protein VP7 and with the CD autoantigen tTG. The antibodies directed against the viral protein VP7 were shown to predict the onset of CD and induce typical features of CD in the intestinal epithelial cell-line T84 [225] . It has also been suggested that rotavirus vaccine alters B and T behavior, as the percentage of B-cells was higher in the vaccinated infants [226] . Rotavirus vaccine as an inducer of CD is still in discussion and warrants further study. 4.11 Polymyalgia rheumatica (PMR) PMR is an autoimmune inflammatory rheumatic disease characterized by raised inflammatory markers with pain and morning stiffness of shoulders and pelvic girdles and synovitis of proximal joints and extra-articular synovial structures. Its diagnosis is clinical and it is typically a disease of the elderly occurring mainly in subjects above 70. Etiopathogenesis of PMR remains unknown, but genetic and environmental factors play a role [227] . A close temporal relationship has been ascertained concerning epidemics of Mycoplasma pneumoniae , Chlamydia pneumonia , Parvovirus B19 and peaks of cases of PMR and giant cell arteritis, however this is not clearly proven [228] . Cases of PMR following vaccination have rarely been reported. However, it is believed that post vaccination PMR may be underreported due to its symptomatic similarities with the transient effects of vaccines, namely: arthralgia, myalgia and low-grade fever. This leads to failure in establishing a chronological relationship when the disease is diagnosed. Most of the reported cases are associated with seasonal influenza vaccine (Inf-V). Often, the time interval between vaccine administration and symptoms onset varies from one day, to three months. Three cases were reported with associated Giant Cell arthritis. A case report of relapsing PMR after four years of remission following tetanus vaccination has also been reported [229] , [230] . 4.12 Acute disseminated encephalomyelitis (ADEM) Acute disseminated encephalomyelitis (ADEM) is an inflammatory demyelinating disease of the central nervous system (CNS). ADEM is usually poly-symptomatic with encephalopathy (i.e., behavioral change or altered level of consciousness). It affects mostly children and young adults and has higher prevalence in males. Its incidence is 0.6–0.8 per 100 000 per year [231] . Although there is no concrete evidence of a clear pathogenic association, ADEM has been associated with immunization or previous viral infection. Post-vaccination ADEM accounts for only 5-10 percent of all cases, while post-infectious ADEM accounts for 66 percent of all cases of ADEM [232] . The hypothesis that better describes these associations is molecular mimicry. T-cells targeting human herpesvirus-6 (HHV-6), coronavirus, influenza virus and Epstein-Barr virus (EBV) have been shown to cross-react with myelin basic protein (MBP) antigens. Anti-MBP T-cells were detected in patients following vaccination with simple rabies vaccine [233] , [234] , [235] . In a post experimental therapy for Alzheimer's disease with a vaccine that contained aggregates of synthetic Aβ42 fragments of amyloid precursor protein, ADEM was shown to develop in mice [236] . The experimental model of MS, EAE mice, may be induced with injection of Aβ42, but only when the latter is administered together with the complete Freund's adjuvant [237] . This observation points to the importance and central role of the adjuvants in induction of ADEM and autoimmunity in general [238] . The overall incidence of post vaccination ADEM is estimated to be 0.1–0.2 per 100 000 and a higher risk has been reported following immunization against measles. Other vaccines accountable for post-vaccination ADEM include vaccines against the varicella zoster, the rubella, the smallpox and the influenza viruses [239] . Surprisingly, certain vaccines such as anti-tetanus vaccine were shown to have a negative correlation with ADEM (statistically significant decreased risk) [240] . HBV immunization has been studied as a possible cause for ADEM but was later associated with clinically isolated syndrome (CIS) (a first time occurring demyelinating episode that may, or not develop to MS) and complete conversion to MS [241] . As far as case reports are concerned, ADEM was associated with vaccination with influenza, hepatitis A and B, MMR, HPV and tetanus [121] , [242] , [243] . 4.13 Bullous dermatoses Bullous dermatoses are characterized by the presence of blisters and autoantibodies against structural components of the skin: desmosomal proteins (in pemphigus), adhesion molecules of the dermal-epidermal junction (in pemphigoid diseases), and epidermal/ tissue transglutaminase (in dermatitis herpetiformis). The most frequent autoimmune bullous diseases are bullous pemphigoid (BP) and pemphigus vulgaris (PV). BP is more frequently observed in the elderly, while the age of onset of PV is between 40 and 60 years. Neither of the diseases have any gender preference [244] . BP and PV etiology is, so far, poorly understood. Both diseases have been associated with various environmental factors, which include emotional and/or physical stress, infections and vaccinations [244] . Genetic predisposition has also been studied with overexpression of certain HLA class II alleles. These include HLA-DQB1*0301, DRB1*04, DRB1*1101, and DQB1*0302. These alleles have been found to be more prevalent in BP patients than in the general population [245] . PV is associated with certain HLA class II loci such as HLA-DR4 and HLADR14 alleles (DRB1*0401 and DRB1*0402, which is prevalent in Ashkenazi Jews, Iranian and Sardinian patients). Other loci include DRB1*1401 (common among Japanese and Italian patients) and two DQB1 alleles (DQB1*0302 and DQB1*0503), which are strongly associated with PV. BP and PV patients' sera were found to have significantly higher prevalence of antibodies to hepatitis B virus, hepatitis C virus, helicobacter pylori, toxoplasma gondii and cytomegalovirus [244] . As far as vaccination is concerned, BP developed in patients following influenza, diphtheria, tetanus, pertussis, hepatitis B, BCG, polio and herpes zoster vaccines [140] , [246] , [247] Furthermore, reactivation of BP following influenza vaccination was reported in one case report [248] . New onset PV was associated with: influenza vaccine, hepatitis B vaccine, anthrax vaccine, typhoid booster and rabies vaccination. In addition, exacerbation of PV after vaccination was also reported following influenza vaccine and tetanus vaccine [121] . 4.14 Idiopathic inflammatory myopathies (IIM) IIM compose a group of skeletal muscles diseases in which myositis without a recognized cause occurs. IIM is usually subdivided in 4 entities: dermatomyositis (DM), polymyositis (PM), inclusion body myositis (sIBM) non-specific myositis (NSM) and immune mediated necrotizing myopathy (IAM) [249] . IIM prevalence is around 1.1 × 10 −6 cases, with a bimodal age of distribution that peaks in childhood and again between 45 and 55 years. DM is the most common inflammatory myopathy while PM is the least frequent. Despite exhibiting similar clinical symptoms, the subsets of IIM exhibit significant immunopathological variation. DM begins with the activation of the complement and formation of membrane attack complexes (MAC). In PM and sIBM the fundamental process is related to CD8+ T cells mediated cytotoxicity [249] . It is unclear what breaks the tolerance and drives the immune response to induce IIM. So far, DM, PM and sIBM have been linked to vaccination. Several cases have been reported in the literature associating different vaccines with the development of idiopathic inflammatory myopathies. 119 cases of IIM had been reported to VAERS database up to June 2013. Out of these 119 cases, 33 were classified as PM, 85 as DM and an only one as a sIBM. DM has been reported after almost any vaccine, however only a few studies have attempted to clarify the possible relationship between DM and vaccination. PM is a frequent misdiagnosed disorder. Some reports have associated previous immunization, especially hepatitis B vaccine with PM [250] . Despite being recently differentiated from other IIM, sIBM has already been related to HBV vaccine [250] . Some vaccines associated with myositis are MMR vaccine, smallpox vaccine, Poliomyelitis (IPV), diphtheria and tetanus toxoid, influenza, HPV and BCG [250] . 4.15 Fibromyalgia syndrome (FMS) FMS is an entity that is related to the inability of the CNS to modulate pain. The conditioned pain modulation process in the CNS appears to be compromised among many FMS patients, which might explain the enhanced pain sensation experienced by these patients [251] . The etiology of FMS is yet to be unveiled. Genetic predisposition, physical trauma (particularly to the cervical spine), emotional stress (to various stressors) as well as a variety of infections have been linked with FMS. Vaccines have been associated with the triggering of FMS namely rubella and Lyme disease vaccines [252] . There are several reports of fibromyalgia-like disease after vaccination, specifically HPV (Martinez-Lavin Journal of Clinical rheumatology 2014). The medical community and regulatory agencies should be aware of these possible adverse effects aiming at defining their magnitude. 4.16 Chronic fatigue syndrome (CFS)/myalgic encephalomyelitis (ME)/systemic exertion intolerance disease (SEID) Chronic fatigue syndrome (CFS) is a disease characterized by disabling fatigue, headaches, concentration difficulties and memory deficits (90%). Other symptoms such as sore throat (85%), tender lymph nodes (80%), skeletal muscle pain and feverishness (75%), sleep disruption (70%), psychiatric problems (65%) and rapid pulse (10%) are often observed. It more frequently affects women and has a prevalence of 0.2-2.6% [253] . Although disease etiology is still unknown, there are several pathogens, such as Epstein–Barr virus (EBV), which have been associated with CFS. Patients often have higher titers of IgM to the EBV viral capsid antigen. Cytomegalovirus and human herpes virus 6 antibodies were also detected more often in CFS patients, although other reports failed to replicate these results. Parvovirus B19 infection has also been suggested as a trigger to CFS [253] , [254] , [255] . Vaccine inoculation has also been appointed as a probable cause. Vaccinations against rubella, Q fever and hepatitis B were found to be associated with higher risk of developing CFS while meningococcal vaccine, poliovirus and influenza vaccine were not. Surprisingly, staphylococcus toxoid vaccine appeared to have a protective effect [121] , [256] , [257] . 4.17 ASIA syndrome Defined in 2011 by Shoenfeld and Agmon-Levin ASIA syndrome is characterized by hyperactive immune response to adjuvants [4] . As previously stated, ASIA incorporates four known medical conditions: Siliconosis, GWS, MMF, and post-vaccination phenomena [4] . Recently, the sick building syndrome (SBS) was proposed as a candidate for the ASIA spectrum [258] . All of these diseases satisfy several criteria for FMS and SEID [252] . a Macrophagic myofasciitis (MMF) MMF has been described as an emerging condition of unknown cause characterized by a pathognomonic lesion in muscle biopsy mixing large macrophages with submicron to micron-sized agglomerates of nanocrystals in their cytoplasm and lymphocytic infiltrates. These lesions were related to aluminum deposits in muscle following immunization with aluminum containing vaccines [63] . MMF lesion is now universally recognized as indicative of a long-lasting persistence of aluminum adjuvant at the site of prior intramuscular immunization. The long-lasting MMF lesion should be considered as a biomarker of aluminum bio persistence in a given individual. Patients with MMF have higher reported myalgia with incidence being up to 90%. Its etiology is not clear but genuine muscle weakness is rare and the diagnosis of fibromyalgia is also rare. Higher prevalence of chronic fatigue syndrome (CFS) in patients with MMF has been reported as well. Cognitive impairment has been associated with MMF: in one series of 105 MMF patients, up to 97% had attention and memory complaints and neuropsychological tests were abnormal in 89% [259] . b Gulf War syndrome (GWS) GWS is a clinical entity specifically related to a certain time and place in history. It was described among veterans of the military conflict occurring in 1990–1991 in the Persian Gulf. The syndrome is characterized by chronic fatigue, musculoskeletal symptoms, malaise and cognitive impairment. It clinically overlaps with Post Traumatic Stress Disorder (PTSD), FMS, CFS and other functional disorders [260] . The unique conditions that have been associated so far with disease development are the exposure to extreme climate in the Persian Gulf, exposure to various chemicals (pesticides, depleted uranium), stress provoked by prolonged waiting without actual combat and the intense exposure to vaccinations of the soldiers for fear of biological weaponry [260] . Comparing Gulf War veterans and veterans of the Bosnian conflict, multiple vaccinations administered to servicemen in the Gulf War was identified as a unique exposure [261] . The mechanism through which vaccination exposure may lead to the development of functional symptoms is not completely understood. The possibility that a shift from Th1 to Th2 type reactions could be of pathogenic significance was raised and is supported by an increased frequency of allergic reactions, low natural killer cell activity and low levels of interferon γ and IL-2 in these patients [262] . One study with GWS patients showed a connection between anti-squalene antibodies and symptoms development. This was refuted by a larger study that found no association between anti-squalene antibodies and chronic multi-symptom illness [263] . c ASIA registry A registry is a collection of data related to patients with the same specific characteristic. It is often the first approach in the study of an area of inquiry. In rare diseases, registries are often the way to get a sufficiently sized sample of patients which can be used either for epidemiological or research purposes. ASIA syndrome may be underreported because of unawareness and failure to connect the syndrome with the exposure. This registry was created to fully understand the clinical aspects of disease and compare patients from all over the world in order to have fully validated criteria for disease diagnosis and also to define demographic and environmental history of disease. The ASIA Syndrome registry website can be found on the following link: https://ontocrf.costisa.com/en/web/asia . Only cases reported by physicians are accepted. 5 Vaccination in autoimmune diseases. 5.1 Autoimmune rheumatic diseases ( Table 4 ) To make an informed decision in medicine, there is always a need to weigh the pros and cons. ARDs may play an important role in deciding whether vaccination is or is not appropriate to a patient. In these cases, patients are immunosuppressed on account of their diagnosis and even more so if they are under specific immunomodelatory medication [4] . If the efficacy of vaccination is reduced, there is a potential for development of disease flares following vaccination. In the case of live vaccines, its inoculation may even be enough to trigger disease in the host. For these specific reasons, live vaccines are generally contraindicated in patients receiving immunosuppressant medication. There is a need for screening and treatment of Latent Tuberculosis Infection (LTBI) before starting anti-TNF-alpha therapy. The same is true for vaccination. Preferably, even recommended vaccination (see Table 5 ) should be administered before the initiation of Disease-Modifying Anti-rheumatic Drugs (DMARDs) because these may reduce vaccine efficacy [264] . Table 4 Most common autoimmune inflammatory rheumatic diseases (AIIRDs) and non-inflammatory autoimmune rheumatic diseases (ARDs). AIIRDs ARDs Rheumatoid arthritis Ankylosing spondylitis Reactive arthritis Connective tissue diseases Polymyalgia rheumatica Degenerative spine diseases Osteoarthritis Osteoporosis Fibromyalgia Table 5 Vaccination recommendation in ARDs [265] . Vaccines Recommended Not recommended Special 5emarks Live BCG X Herpes zoster Previous contact with varicella (vaccine/infection) Highly immunosuppressed patients a Single dose >50 y Yellow fever Endemic areas [266] Routine immunization not recommended MMR X Non-live Influenza X Allergy to egg or the vaccine itself; GBS up to 6 weeks after vaccination Annual Rituximab: before starting/6 mts after 1st infusion/4 wks before next dose [148] , [267] Pneumococcal X 1 Initial dose + 1 booster (5 y later) DTaP and DT X DTaP every 10 y Tetanus Igb if exp Meningococcal X Low data support [268] Hep A X Hep B Neg HBsAg in serum HPV Adolescents and young women Preferably before initiating sexual activity Hib X X – for all ARDs patients; MMR: measles, mumps and rubella; Hib: haemophilus Influenza type B; DTaP: diphteria, tetanus and pertussis; DT: diphteria and tetanus; Hep: hepatitis; Igb: immunoglobulin; y: years; mts: months; wks: weeks; HPV: human papilloma virus; GBS: guillain barré syndrome; exp: exposure; Neg HBsAG: negative hepatitis B antigen. a Highly immunosuppressed patients: high doses of corticosteroids (>20 mg of prednisone per day or equivalent) for 2 weeks or longer, pulse therapy, cytotoxic or alkylating agents, synthetic DMARDs at doses above those recommended, or immunobiological therapy [264] . 5.2 Autoimmune inflammatory rheumatic diseases (AIIRD) Immunosuppression equals high risk of infection and lower vaccine efficacy. Taking into account safety concerns and efficacy, the EULAR recommendations for immunizations in AIIRD patients are: • Assess vaccination status in initial investigation. • Administer vaccines in a stable disease phase. • Live attenuated vaccines are to be avoided especially if immunosuppressive agents are being administered. BCG is not recommended. • Administer vaccines ideally before starting DMARDs and anti-TNFα agents. • Influenza and 23-valent polysaccharide pneumococcal vaccination is recommended. • Tetanus toxoid vaccination is recommended following recommendations of general population, in case of major and/or contaminated wounds in patients receiving rituximab in the previous 24 weeks Tetanus Ig is indicated. • HPV and Herpes Zoster should be considered. • In hyposplenic/asplenic patients, influenza, pneumococcal, Haemophilus Influenza b and Meningococcal C are advisable. • Hepatitis A and B is recommended in patients at risk. • Travel patients should be immunized according to general population guidelines except for live attenuated vaccines, which are to be avoided [148] . 5.1 Autoimmune rheumatic diseases ( Table 4 ) To make an informed decision in medicine, there is always a need to weigh the pros and cons. ARDs may play an important role in deciding whether vaccination is or is not appropriate to a patient. In these cases, patients are immunosuppressed on account of their diagnosis and even more so if they are under specific immunomodelatory medication [4] . If the efficacy of vaccination is reduced, there is a potential for development of disease flares following vaccination. In the case of live vaccines, its inoculation may even be enough to trigger disease in the host. For these specific reasons, live vaccines are generally contraindicated in patients receiving immunosuppressant medication. There is a need for screening and treatment of Latent Tuberculosis Infection (LTBI) before starting anti-TNF-alpha therapy. The same is true for vaccination. Preferably, even recommended vaccination (see Table 5 ) should be administered before the initiation of Disease-Modifying Anti-rheumatic Drugs (DMARDs) because these may reduce vaccine efficacy [264] . Table 4 Most common autoimmune inflammatory rheumatic diseases (AIIRDs) and non-inflammatory autoimmune rheumatic diseases (ARDs). AIIRDs ARDs Rheumatoid arthritis Ankylosing spondylitis Reactive arthritis Connective tissue diseases Polymyalgia rheumatica Degenerative spine diseases Osteoarthritis Osteoporosis Fibromyalgia Table 5 Vaccination recommendation in ARDs [265] . Vaccines Recommended Not recommended Special 5emarks Live BCG X Herpes zoster Previous contact with varicella (vaccine/infection) Highly immunosuppressed patients a Single dose >50 y Yellow fever Endemic areas [266] Routine immunization not recommended MMR X Non-live Influenza X Allergy to egg or the vaccine itself; GBS up to 6 weeks after vaccination Annual Rituximab: before starting/6 mts after 1st infusion/4 wks before next dose [148] , [267] Pneumococcal X 1 Initial dose + 1 booster (5 y later) DTaP and DT X DTaP every 10 y Tetanus Igb if exp Meningococcal X Low data support [268] Hep A X Hep B Neg HBsAg in serum HPV Adolescents and young women Preferably before initiating sexual activity Hib X X – for all ARDs patients; MMR: measles, mumps and rubella; Hib: haemophilus Influenza type B; DTaP: diphteria, tetanus and pertussis; DT: diphteria and tetanus; Hep: hepatitis; Igb: immunoglobulin; y: years; mts: months; wks: weeks; HPV: human papilloma virus; GBS: guillain barré syndrome; exp: exposure; Neg HBsAG: negative hepatitis B antigen. a Highly immunosuppressed patients: high doses of corticosteroids (>20 mg of prednisone per day or equivalent) for 2 weeks or longer, pulse therapy, cytotoxic or alkylating agents, synthetic DMARDs at doses above those recommended, or immunobiological therapy [264] . 5.2 Autoimmune inflammatory rheumatic diseases (AIIRD) Immunosuppression equals high risk of infection and lower vaccine efficacy. Taking into account safety concerns and efficacy, the EULAR recommendations for immunizations in AIIRD patients are: • Assess vaccination status in initial investigation. • Administer vaccines in a stable disease phase. • Live attenuated vaccines are to be avoided especially if immunosuppressive agents are being administered. BCG is not recommended. • Administer vaccines ideally before starting DMARDs and anti-TNFα agents. • Influenza and 23-valent polysaccharide pneumococcal vaccination is recommended. • Tetanus toxoid vaccination is recommended following recommendations of general population, in case of major and/or contaminated wounds in patients receiving rituximab in the previous 24 weeks Tetanus Ig is indicated. • HPV and Herpes Zoster should be considered. • In hyposplenic/asplenic patients, influenza, pneumococcal, Haemophilus Influenza b and Meningococcal C are advisable. • Hepatitis A and B is recommended in patients at risk. • Travel patients should be immunized according to general population guidelines except for live attenuated vaccines, which are to be avoided [148] . 6 Conclusions and future perspectives Vaccines have many beneficial effects in combating infectious diseases and preventing mortality and morbidity. They have also proved to be effective cancer treatments by immunomodulation, as demonstrated by the intravesical administration of BCG to treat superficial bladder cancer [28] . Vaccines are however, linked to autoimmunity. Beneficial outcomes, like the adjuvant effect are based on immunity triggering and enhanced immunity mechanisms. These same responses account for autoimmunity exertion. Vaccines induce the production of autoantibodies, but their pathologic effect is yet to be unveiled. Although vaccines are widely considered safe, there are subjects with predispositions to whom vaccines pose a bigger threat. An example is the fact that animal models with autoimmune predispositions develop autoimmune disease following adjuvant exposure. As many as 1% of recipients of aluminum containing adjuvants may be sensitized to future exposure [269] . Silicon-induced inflammatory fibro proliferative response is irrefutable and well documented. The presence of anti-silicone antibodies and silicone-associated autoimmune phenomena seems very plausible. ASIA syndrome and aluminum safety studies show that the use of aluminum containing "placebo" in control groups in vaccine safety studies should be carefully evaluated. New studies must be performed using a proper placebo to adequately test vaccine safety. Another evident failure in vaccine safety studies are the short-term periods which are evaluated. Continued immune system activation has been observed to be a potential mechanism of disease. A disease which is poorly understood so far. Vaccine recommendations should be reassessed frequently in different subsets of the population. This does not invalidate the need for vaccines, however, the lower the possibility of exerting adverse events, the easier it will be for the potential benefits to outweigh the risks. Vaccinomics represents a major breakthrough in vaccine development and can lead to the development of targeted vaccines to peptides most likely to be immunogenic [81] . A predictable response to vaccine can be achieved by differentiating the host variability. This can be achieved namely in genetics and pathogen variability. Developing a vaccine accordingly will lead to increased specificity in treatment and leave less room for adverse events. By using immunomodulation, vaccinomics can also give rise to novel therapies for autoimmune diseases. There are several reports of cases of autoimmunity diseases following vaccines but despite in vitro positive results and due to both the limited number of cases and the long latency period of the diseases, every attempt for an epidemiological study has failed to deliver a connection. Classification as ASIA syndrome, in detriment of classic specific autoimmune diseases, could be the key to finding effective preventative therapeutic strategies. It will enable the study of bigger patient clusters with earlier diagnoses. Future studies that could help clarify the association between vaccinations, adjuvants and autoimmunity should ideally have a different design, more long-term data and should include autoimmune phenomena as well as large-scale epidemiological studies of autoimmune diseases.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131351/

ITGA2, LAMB3, and LAMC2 may be the potential therapeutic targets in pancreatic ductal adenocarcinoma: an integrated bioinformatics analysis

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer with an abysmal prognosis rate over the last few decades. Early diagnosis and prevention could effectively combat this malignancy. Therefore, it is crucial to discover potential biomarkers to identify asymptomatic premalignant or early malignant tumors of PDAC. Gene expression analysis is a powerful technique to identify candidate biomarkers involved in disease progression. In the present study, five independent gene expression datasets, including 321 PDAC tissues and 208 adjacent non-cancerous tissue samples, were subjected to statistical and bioinformatics analysis. A total of 20 differentially expressed genes (DEGs) were identified in PDAC tissues compared to non-cancerous tissue samples. Gene ontology and pathway enrichment analysis showed that DEGs were mainly enriched in extracellular matrix (ECM), cell adhesion, ECM–receptor interaction, and focal adhesion signaling. The protein–protein interaction network was constructed, and the hub genes were evaluated. Collagen type XII alpha 1 chain (COL12A1), fibronectin 1 (FN1), integrin subunit alpha 2 (ITGA2), laminin subunit beta 3 (LAMB3), laminin subunit gamma 2 (LAMC2), thrombospondin 2 (THBS2), and versican (VCAN) were identified as hub genes. The correlation analysis revealed that identified hub genes were significantly interconnected. Wherein COL12A1, FN1, ITGA2, LAMB3, LAMC2, and THBS2 were significantly associated with PDAC pathological stages. The Kaplan–Meier survival plots revealed that ITGA2, LAMB3, and LAMC2 expression were inversely correlated with a prolonged patient survival period. Furthermore, the Human Protein Atlas database was used to validate the expression and cellular origins of hub genes encoded proteins. The protein expression of hub genes was higher in pancreatic cancer tissue than in normal pancreatic tissue samples, wherein ITGA2, LAMB3, and LAMC2 were exclusively expressed in pancreatic cancer cells. Pancreatic cancer cell-specific expression of these three proteins may play pleiotropic roles in cancer progression. Our results collectively suggest that ITGA2, LAMB3, and LAMC2 could provide deep insights into pancreatic carcinogenesis molecular mechanisms and provide attractive therapeutic targets. Introduction Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive and common form of pancreatic cancer, accounting for 95% of all pancreatic malignant neoplasms 1 . The 5-year overall survival rate for patients with PDAC is less than 8% despite advances in medical oncology 2 . The poor prognosis of PDAC may be due to the lack of precise molecular biomarkers for early diagnosis and prognosis 3 . Therefore, there is an urgent need for more effective targeted therapies to improve the survival rate of patients with PDAC 4 . Gene expression microarrays and gene chips are extensively applied to reveal genetic aspects of diseases. These techniques are routinely used to monitor genome-wide expression levels of genes and are particularly suitable for screening differentially expressed genes (DEGs) between two samples 5 . The identification of DEGs may elucidate cancer pathogenesis, provide early diagnosis, and improve treatment. Hence, gene expression microarray analysis could be a promising approach to identify candidate biomarkers involved in disease progression. The gene expression profiles from diverse microarray platforms are submitted to several public databases, including Gene Expression Omnibus (GEO: https://www.ncbi.nlm.nih.gov/gds/ ). Several previous studies used gene expression microarray technology to underpinning the DEGs of PDAC in recent years 6 – 8 . However, the results were inconsistent, and various aspects remain unclear due to sample heterogeneity. Moreover, those studies have not considered ethnic differences, and many studies have proven that ethnic differences may have relevance for disease gene expression profiles 9 , 10 . The present study aimed to improve DEGs accuracy and reliability in PDAC compared to adjacent non-cancerous tissue samples using several datasets from different ethnicities. In the current study, gene expression datasets from PDAC were analyzed to identify DEGs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed using an online toolset. Then, the protein interaction networks were constructed and the hub genes were identified and further verified. The identified hub genes may serve as potential diagnostic and prognostic biomarkers and could be a promising approach for the treatment of PDAC. To the best of our knowledge, this analysis is the first to examine the gene expression microarray database in PDAC tissues and adjacent non-cancerous tissue samples, considering different ethnic groups. Materials and methods Microarray datasets information PDAC datasets were obtained from the Gene Expression Omnibus, a public functional genomic database containing high-throughput gene expression data, chips, and microarrays. The GEO database was searched using the following criteria: "human-derived pancreatic ductal adenocarcinoma tissues and adjacent non-cancerous tissue samples" (study keyword), "Homo sapiens" (organism), "expression profiling by array" (study type), "tissue" (attribute name), and "sample count" > 50. After a systematic review, five independent PDAC microarray datasets were selected, including GSE62452 11 , GSE28735 12 , GSE15471 13 , GSE62165 14 , GSE102238 15 , with 321 primary tumor samples and 208 adjacent non-cancerous samples. The dataset GSE62452 was based on the GPL6244 platform (HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array) and included 69 tumor and 61 adjacent non-cancerous tissue samples. The dataset GSE28735 was based on the GPL6244 platform (HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array) and had 45 matched tumor and adjacent non-cancerous samples. The GSE15471 dataset was produced using the GPL570 Platform [(HG‐U133_Plus_2) Affymetrix Human Genome U133 Plus 2.0 Array], including 39 matched tumors and adjacent non-cancerous samples. The GSE62165 dataset was based on the GPL13667 Platform [(HG‐U219) Affymetrix Human Genome U219 Array], which contained 118 tumors and 13 adjacent non-cancerous samples. The GSE102238 dataset was based on the GPL19072 Platform [Agilent-052909 CBC_lncRNAmRNA_V3], which included 50 matched tumor and adjacent non-cancerous samples. These five gene expression profiles were respectively from different regions, including North America, Europe, and Asia, thus averting the differences caused by sample heterogeneity of single profiles and revealing universal DEGs that apply to different ethnic groups, as it has been reported that ethnic difference may affect disease-associated gene expression profiles 9 , 10 . The clinical datasets included 321 tumors and 208 adjacent non-cancerous tissues diagnosed as PDAC (Table 1 ). Of note, pancreatic tissue samples in microarray datasets were obtained from the patients who underwent surgical resection for PDAC. Subsequently, tissue samples were stored in liquid nitrogen and/or at − 80 °C until further use. Total RNA was extracted from the snap-frozen tissue samples, and further analysis was carried out. The clinicopathological characteristics of the microarray datasets are briefly shown in Supplementary Table 1 . Table 1 Characteristics of datasets used in meta-analysis of PDAC tissues vs. adjacent non-cancerous tissues. Author, year GEO accession Region Platform Tissue types and sample numbers PDAC Adjacent Count Yang et al. (2016) 11 GSE62452 USA GPL6244 69 61 130 Zhang et al. (2012) 12 GSE28735 USA GPL6244 45 45 90 Badea et al. (2008) 13 GSE15471 Romania GPL570 39 39 78 Janky et al. (2016) 14 GSE62165 Belgium GPL13667 118 13 131 Yang et al. (2020) 8 GSE102238 China GPL19072 50 50 100 PDAC pancreatic ductal adenocarcinoma. Identification of DEGs DEGs between PDAC and adjacent non-cancerous tissue samples were screened by GEO2R ( http://www.ncbi.nlm.nih.gov/geo/geo2r ) 16 , an online tool that can be used to compare two or more datasets in a GEO series to identify DEGs according to the experimental conditions. Adjusted p values (adj. p ) and Benjamini and Hochberg false discovery rates were employed as criteria for statistically significant genes and to limit false positives. The data normalization was applied for the five datasets (Supplementary Fig. 1 ). Probe sets with no corresponding gene symbols were removed, while genes with multiple gene probe sets were averaged. Log2 FC (fold change) ≥ 1.5 or ≥ − 1.5 and adj. p  0.4 were set as significant. Subsequently, the protein–protein interaction (PPI) networks were visualized using Cytoscape 3.8.2 ( https://cytoscape.org/ ), an open-source bioinformatics software platform 23 . A combined score of 0.5 and a tissue-specific (pancreas) filter score of 1 was considered for the construction of the PPI network. Subsequently, the MCODE (Molecular Complex Detection) plugin was used to identify hub genes in the constructed network. The standard for selection was set as follows: MCODE scores ≥ 10, degree cut-off = 2, node score cut-off = 0.2, max depth = 100 and k-score = 2 24 . Oncomine analysis of hub genes in pancreatic cancer An independent database, namely Oncomine ( https://www.oncomine.org/resource/login.html ; last access: 14th February 2021), was used to validate hub gene expression. In the Oncomine database, the gene name "COL12A1", "FN1", "ITGA2", "LAMB3", "LAMC2", "THBS2" or "VCAN" was entered. The differential gene analysis module (cancer vs. normal analysis) was selected to retrieve the results. This analysis presented a series of pancreatic cancer studies and related COL12A1, FN1, ITGA2, LAMB3, LAMC2, THBS2, and VCAN mRNA expression in cancer and normal tissues. The filters were set as follows: (1) Gene: COL12A1 or FN1 or ITGA2 or LAMB3 or LAMC2 or THBS2 or VCAN. (2) Analysis type: cancer vs. normal analysis. (3) Cancer type: pancreatic carcinoma. (4) Sample type: clinical specimen. (5) Data type: mRNA. (6) Threshold settings: p  2; gene rank, top 10%. Finding prognostic genes for PDAC To explore the expression correlation of hub genes in PDAC, the Spearman coefficient correlation was analyzed using the GEPIA2 tool 19 . The interaction efficiency was represented as an R score. An R score of > 0.8 was considered a significant correlation. Next, the expression levels of hub genes and pathological stages in PDAC tissues were assessed using the GEPIA2 platform. The GEPIA2 was also utilized for overall survival and disease-free survival analyses of the hub genes using the TCGA and GTEx databases. The plots were considered significant when showed in both overall and disease-free survival states. Beta-actin was used to normalize the expression of genes, and the median was selected for group cut-off criteria. p  50. After a systematic review, five independent PDAC microarray datasets were selected, including GSE62452 11 , GSE28735 12 , GSE15471 13 , GSE62165 14 , GSE102238 15 , with 321 primary tumor samples and 208 adjacent non-cancerous samples. The dataset GSE62452 was based on the GPL6244 platform (HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array) and included 69 tumor and 61 adjacent non-cancerous tissue samples. The dataset GSE28735 was based on the GPL6244 platform (HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array) and had 45 matched tumor and adjacent non-cancerous samples. The GSE15471 dataset was produced using the GPL570 Platform [(HG‐U133_Plus_2) Affymetrix Human Genome U133 Plus 2.0 Array], including 39 matched tumors and adjacent non-cancerous samples. The GSE62165 dataset was based on the GPL13667 Platform [(HG‐U219) Affymetrix Human Genome U219 Array], which contained 118 tumors and 13 adjacent non-cancerous samples. The GSE102238 dataset was based on the GPL19072 Platform [Agilent-052909 CBC_lncRNAmRNA_V3], which included 50 matched tumor and adjacent non-cancerous samples. These five gene expression profiles were respectively from different regions, including North America, Europe, and Asia, thus averting the differences caused by sample heterogeneity of single profiles and revealing universal DEGs that apply to different ethnic groups, as it has been reported that ethnic difference may affect disease-associated gene expression profiles 9 , 10 . The clinical datasets included 321 tumors and 208 adjacent non-cancerous tissues diagnosed as PDAC (Table 1 ). Of note, pancreatic tissue samples in microarray datasets were obtained from the patients who underwent surgical resection for PDAC. Subsequently, tissue samples were stored in liquid nitrogen and/or at − 80 °C until further use. Total RNA was extracted from the snap-frozen tissue samples, and further analysis was carried out. The clinicopathological characteristics of the microarray datasets are briefly shown in Supplementary Table 1 . Table 1 Characteristics of datasets used in meta-analysis of PDAC tissues vs. adjacent non-cancerous tissues. Author, year GEO accession Region Platform Tissue types and sample numbers PDAC Adjacent Count Yang et al. (2016) 11 GSE62452 USA GPL6244 69 61 130 Zhang et al. (2012) 12 GSE28735 USA GPL6244 45 45 90 Badea et al. (2008) 13 GSE15471 Romania GPL570 39 39 78 Janky et al. (2016) 14 GSE62165 Belgium GPL13667 118 13 131 Yang et al. (2020) 8 GSE102238 China GPL19072 50 50 100 PDAC pancreatic ductal adenocarcinoma. Identification of DEGs DEGs between PDAC and adjacent non-cancerous tissue samples were screened by GEO2R ( http://www.ncbi.nlm.nih.gov/geo/geo2r ) 16 , an online tool that can be used to compare two or more datasets in a GEO series to identify DEGs according to the experimental conditions. Adjusted p values (adj. p ) and Benjamini and Hochberg false discovery rates were employed as criteria for statistically significant genes and to limit false positives. The data normalization was applied for the five datasets (Supplementary Fig. 1 ). Probe sets with no corresponding gene symbols were removed, while genes with multiple gene probe sets were averaged. Log2 FC (fold change) ≥ 1.5 or ≥ − 1.5 and adj. p  0.4 were set as significant. Subsequently, the protein–protein interaction (PPI) networks were visualized using Cytoscape 3.8.2 ( https://cytoscape.org/ ), an open-source bioinformatics software platform 23 . A combined score of 0.5 and a tissue-specific (pancreas) filter score of 1 was considered for the construction of the PPI network. Subsequently, the MCODE (Molecular Complex Detection) plugin was used to identify hub genes in the constructed network. The standard for selection was set as follows: MCODE scores ≥ 10, degree cut-off = 2, node score cut-off = 0.2, max depth = 100 and k-score = 2 24 . Oncomine analysis of hub genes in pancreatic cancer An independent database, namely Oncomine ( https://www.oncomine.org/resource/login.html ; last access: 14th February 2021), was used to validate hub gene expression. In the Oncomine database, the gene name "COL12A1", "FN1", "ITGA2", "LAMB3", "LAMC2", "THBS2" or "VCAN" was entered. The differential gene analysis module (cancer vs. normal analysis) was selected to retrieve the results. This analysis presented a series of pancreatic cancer studies and related COL12A1, FN1, ITGA2, LAMB3, LAMC2, THBS2, and VCAN mRNA expression in cancer and normal tissues. The filters were set as follows: (1) Gene: COL12A1 or FN1 or ITGA2 or LAMB3 or LAMC2 or THBS2 or VCAN. (2) Analysis type: cancer vs. normal analysis. (3) Cancer type: pancreatic carcinoma. (4) Sample type: clinical specimen. (5) Data type: mRNA. (6) Threshold settings: p  2; gene rank, top 10%. Finding prognostic genes for PDAC To explore the expression correlation of hub genes in PDAC, the Spearman coefficient correlation was analyzed using the GEPIA2 tool 19 . The interaction efficiency was represented as an R score. An R score of > 0.8 was considered a significant correlation. Next, the expression levels of hub genes and pathological stages in PDAC tissues were assessed using the GEPIA2 platform. The GEPIA2 was also utilized for overall survival and disease-free survival analyses of the hub genes using the TCGA and GTEx databases. The plots were considered significant when showed in both overall and disease-free survival states. Beta-actin was used to normalize the expression of genes, and the median was selected for group cut-off criteria. p  0.8 was considered statistically significant. The light blue box represents the correlation coefficient based on R scores. Association of hub genes in PDAC pathological stages Further analysis of the TCGA PDAC data in GEPIA2 showed that the hub genes were significantly correlated with the pathological disease stages, underlying their prognostic value for PDAC. COL12A1, FN1, ITGA2, LAMB3, LAMC2, and THBS2 were observed to be significantly associated with PDAC stages (Fig. 7 ), wherein no significant association on PDAC tumor stages and VCAN was observed (data not shown). Figure 7 Pathological stages of hub genes in PDAC tissues. Association of mRNA expression and pathological tumor stages in patients with PDAC. Violin plots were created using the GEPIA2 platform based on the TCGA PDAC dataset. F-value indicates the statistical value of the F test; Pr (> F) indicates p value. A p value of  0.8 was considered statistically significant. The light blue box represents the correlation coefficient based on R scores. Association of hub genes in PDAC pathological stages Further analysis of the TCGA PDAC data in GEPIA2 showed that the hub genes were significantly correlated with the pathological disease stages, underlying their prognostic value for PDAC. COL12A1, FN1, ITGA2, LAMB3, LAMC2, and THBS2 were observed to be significantly associated with PDAC stages (Fig. 7 ), wherein no significant association on PDAC tumor stages and VCAN was observed (data not shown). Figure 7 Pathological stages of hub genes in PDAC tissues. Association of mRNA expression and pathological tumor stages in patients with PDAC. Violin plots were created using the GEPIA2 platform based on the TCGA PDAC dataset. F-value indicates the statistical value of the F test; Pr (> F) indicates p value. A p value of < 0.05 was considered statistically significant. Survival analysis of hub genes in PDAC The Kaplan–Meier survival plots were used to observe the overall survival and disease free-survival status of the hub genes in PDAC. Elevated expression levels of ITGA2, LAMB3, and LAMC2 were found to be inversely correlated with prolonged patient survival (Fig. 8 ), whereas no significant relationship was observed for other genes (data not shown). Figure 8 Kaplan–Meier survival plots of hub genes in PDAC tissues. The Kaplan–Meier plots were generated by using the GEPIA2 platform. The overall survival and disease-free survival plots compared a high-risk group (in red) and a low-risk group (in blue) in PDAC tissues. p < 0.05 were regarded as statistically significant. Validation of expression of hub genes-encoded proteins The expression levels of proteins encoded by the COL12A1, FN1, ITGA2, LAMB3, LAMC2, THBS2, and VCAN were obtained. The protein expression profiles in pancreatic cancer clinical specimens are shown in Fig. 9 . The antibody intensity for FN1, ITGA2, LAMB3, LAMC2, and VCAN was higher in PDAC tissues, while no staining was observed in corresponding normal tissues. COL12A1 had medium staining intensity with low intensity observed in normal pancreatic tissues. THBS2 had medium staining intensity in both pancreatic cancer and normal pancreatic tissues. Further observations revealed that COL12A1 and FN1 were predominantly expressed by stromal cells. THBS2 and VCAN were expressed in both stromal and pancreatic cancer cells, whereas ITGA2, LAMB3, and LAMC2 were solely expressed by pancreatic cancer cells. Figure 9 Immunohistochemical expression of hub genes in human pancreatic cancer specimens. The immunohistochemical data were obtained from the Human Protein Atlas. Staining demonstrated that the protein expression of hub genes was higher in pancreatic cancer tissue than in normal pancreatic tissue samples. The light blue box represents antibodies information. Image courtesy: Human Protein Atlas ( http://www.proteinatlas.org ). Identification of hub genes in previous bioinformatics studies associated with pancreatic cancer The literature review was done to investigate hub genes from previous bioinformatics studies in pancreatic cancer. Nine bioinformatics studies were chosen after a comprehensive analysis based on the criteria which we set. The hub genes, their associated pathways, and potential clinical relevance were explored, which is shown in Table 5 . In brief, collagens (COL1A1, COL1A2, COL3A1, COL3A2, and COL5A2), integrins (ITGA2 and ITGB2), laminins (LAMA3, LAMB3, and LAMC2), and fibronectin were the most common hub genes found in those studies. Further, the cell cycle regulation, tissue remodeling, ribosomal protein, and nuclear pore complex-related genes were found to be altered in those studies. The pathways analysis has shown that ECM–receptor interaction, focal adhesion, pathways in cancer, and altered metabolic pathways have been the most commonly involved with those hub genes. Table 5 Literature review of the existing bioinformatics studies associated with pancreatic cancer. Author, year Hub genes Pathways Observations Yang et al. (2020) 8 AHNAK2, CDH3, IFI27, ITGA2, LAMB3, SFN, SLC6A14, and TMPRSS4 Pancreatic secretion, pathways in cancer, p53 signaling pathway, MAPK signaling pathway, Insulin signaling pathway, and pancreatic cancer AHNAK2, CDH3, IFI27, ITGA2, LAMB3, SLC6A14, and TMPRSS4 may have great diagnostic and prognostic value for pancreatic cancer Jin et al. (2020) 7 TOP2A, CDK1, PRM2, PRC1, NEK2 , ZWINNT1, DTL, MELK, CENPF, CEP55, ANLN, ASPM, and ECT2 ECM–receptor interaction, focal adhesion, pathways in cancer, proteoglycans in cancer, p53 signaling pathway, and PI3-Akt signaling pathway CDK1, TOP2A, and CEP55 may play pleiotropic roles in the progression of pancreatic cancer Li et al. (2019) 26 ALB, COL1A2, EGF, COL3A1, FN1, CEL, ITGA2, COL5A2, MMP1, and CELA3B Protein digestion and absorption, ECM–receptor interaction, pancreatic secretion, and fat digestion and absorption COL1A2, COL3A1, and COL5A2 may promote pancreatic fibrosis and EMT via the ECM–receptor interaction pathway in the early stages of pancreatic cancer Liu et al. (2019) 27 HN1, ITGA2, S100A6, KIF1A, DYM, and BACE1 Ubiquitin-mediated proteolysis and pathways in cancer HN1, ITGA2, and S100A6 may be promising potential targets for diagnosing and treating pancreatic cancer Shen et al. (2018) 31 RPL13, RPL17 , RPL21, RPL22, RPL23, RPL26, RPL31 , RPL35A , RPL36A , RPL37, RPL39 , RPL7 , RPS17, RPS23 , RPS3A,RPS6, RPS7, NUP107, NUP160, and HNRNPU Ribosome pathway and the spliceosome pathway Nup170 , Nup160 , and HNRNPU may be used as possible molecular markers for early diagnosis of pancreatic cancer Pan et al. (2018) 28 CXCL8, ADCY7, ITGAM, ITGB2, ITGB1, IL1A, ICAM1, ITGA2, THBS2, SDC1, COL3A1, COL1A2, COL1A1, MYL9, LAMA3, LAMB3, LAMC2, COL4A1 and FN1 ECM–receptor interaction, focal adhesion, pathways in cancer, and small cell lung cancer LAMA3, LAMB3, LAMC2, COL4A1, and FN1 may involve the malignant progression of pancreatic cancer Tang et al. (2018) 6 DKK1 and HMGA2 Glycine, serine, and threonine metabolism DKK1 and HMGA2 may be important in the progression of pancreatic cancer Li et al. (2018) 29 ALB, EGF, FN1, ITGA2, COL1A2, SPARC, COL3A1, TIMP1, COL5A1, COL11A1, and MMP7 ECM–receptor interaction, cell adhesion, and transforming growth factor-beta receptor signaling pathway ITGA2 and MMP7 may act as potential diagnostic and therapeutic biomarkers for pancreatic cancer Wang et al. (2015) 30 VCAN, SULF1, COL8A1, FAP, COL1A1, THBS2, CTHRC1, COL1A2, COL6A3, FN1, COL10A1, COL3A1, TIMP, AEBP1, and COL5A1 ECM–receptor interaction, focal adhesion, and complement and coagulation cascades The collagen family genes and FN1 may play an essential role in the progression of pancreatic cancer Discussion In the present study, 20 DEGs were identified (19 upregulated and 1 downregulated), which were differentially expressed in PDAC tissue compared to the adjacent non-cancerous pancreatic tissue samples. By using an online tool, the mRNA expression levels of DEGs in PDAC tissue samples were validated. The GO and KEGG pathway analysis revealed that DEGs were primarily enriched with ECM-organization, cell adhesion, ECM–receptor interaction, and focal adhesion, especially for the upregulated genes. The PPI network was constructed, and hub genes were selected. COL12A1, FN1, ITGA2, LAMB3, LAMC2, THBS2, and VCAN were identified as hub genes. To verify the expression level of hub genes, an independent database was then used. This confirmed that, compared to normal pancreatic tissues, identified hub genes were highly expressed in pancreatic cancer samples. The correlation analysis revealed that the hub genes in PDAC tissue samples are significantly interconnected. The interaction of hub genes with pathological stages in patients with PDAC showed that the expression of COL12A1, FN1, ITGA2, LAMB3, LAMC2, and THBS2 is negatively associated with disease progression. The survival plots of Kaplan–Meier showed that ITGA2, LAMB3, and LAMC2 expression are inversely correlated with prolonged patient survival. Using histopathological images from the Human Protein Atlas platform, the protein expression profiles of hub genes were validated. It was found that proteins encoded by hub genes are highly expressed in pancreatic cancer tissue compared to normal pancreatic tissue samples. It was also observed that ITGA2, LAMB3, and LAMC2 were the only proteins expressed in pancreatic cancer cells but not in stromal cells. The cancer cells specific expression of these three proteins might be crucial for PDAC pathogenesis and progression. Together, this data suggested that ITGA2, LAMB3, and LAMC2 individually might have high prognostic and diagnostic values, as well as the potential to be therapeutic targets for PDAC. ITGA2 is a collagen receptor expressed on cell membranes and forms a heterodimer α2β1 with a β subunit, which mediates cell-to-ECM attachment 32 . The increased ITGA2 level was reported in pancreatic cancer and others, including gastric, liver, prostate, and breast cancer 33 . The increased ITGA2 expression promotes pancreatic cancer cell migration, invasion, metastasis, and chemoresistance 34 , 35 . In contrast, inhibition of ITGA2 abrogated these functions 33 . Although the exact mechanism by which ITGA2 is involved in pancreatic carcinogenesis remains unclear, it has been suggested that ITGA2 promotes pancreatic cancer progression through ECM remodeling 36 , 37 . The reconstituted ECM triggers pancreatic cancer progression by directly promoting cellular transformation and enhancing tumorigenic microenvironment formation by affecting stromal-cell behavior 38 . In this process, ITGA2 activates fibroblasts to cancer-associated fibroblasts (CAFs), resulting in extensive desmoplasia with ECM deposition 39 , wherein desmoplasia is a characteristic feature of PDAC and constitutes up to 90% of the tumor volume. Mainly ECM and CAF, immune cells, and vascular components form the desmoplastic microenvironment 40 , 41 . ECM is a three-dimensional structural complex consisting of structural and non-structural proteins 42 , 43 . ECM-proteins can affect PDAC progression and patient survival by promoting cancer cell proliferation and metastatic spread 44 . Even though stromal cells produce over 90% of the ECM mass in PDAC, cancer cells produce elevated ECM-proteins, and cancer cell-derived ECM-proteins play important roles in PDAC carcinogenesis 45 , 46 . A previous report suggested that ECM proteins originating from cancer cells were the most strongly connected to poor patient survival. In contrast, ECM-proteins derived from stromal cells, include both proteins linked to good and poor patient outcomes 47 . Hence, using the Human Protein Atlas database, the protein expression profiles and cellular origins of hub genes encoded proteins in pancreatic cancer tissues were observed. ITGA2 is the transmembrane receptor for collagens and related proteins, as mentioned above 32 , while COL12A1, FN1, LAMB3, LAMC2, THBS2, and VCAN are ECM-related proteins 47 . Our histopathological evidence has shown that COL12A1 and FN1 are expressed from stromal cells, THBS2, and VCAN from stromal and cancer cells, while ITGA2, LAMB3, and LAMC2 are expressed solely from the cancer cells. The Kaplan–Meier survival plots showed that ITGA2, among the ECM-proteins LAMB3 and LAMC2 expression, is inversely correlated with the overall and disease-free survival status in PDAC. Interestingly, a previous report confirmed that LAMB3 and LAMC2 were exclusively derived from pancreatic cancer cells 47 . This study reached a similar conclusion that increased levels of ECM-proteins originated from cancer cells, rather than being solely produced by stromal cells, correlate with poor patient outcomes. However, further studies are needed to clarify this phenomenon. Meanwhile, these results may explain why previous non-selective ECM depletion strategies led to poor patient outcomes and suggest more accurate ECM manipulations as PDAC treatments 48 . Together, the present data and the previous report suggested that cancer-cell-derived ECM-proteins may be potential therapeutic targets 47 . Therefore, sorting out the composition and changes of the ECM during PDAC progression would guide the development and application of more effective PDAC therapies. It is worth noting that DEGs in PDAC have already been demonstrated in several studies 6 – 8 , 26 – 31 . However, the results were not consistent, which could be due to the differences in the selection of datasets and statistical procedures. Then, using effective search engines, we performed a literature review of existing pancreatic cancer bioinformatics studies and explored hub genes. In brief, the hub genes were mainly involved with ECM remodeling and organization. The predominant expression of collagen, integrin, and laminin family genes was observed in those studies, clarifying their role in ECM remodeling. The reconstituted ECM was reported to promote pancreatic fibrosis and epithelial-mesenchymal transition (EMT) in early stages of PDAC pathogenesis 38 . Thus, ECM manipulation is an appealing therapeutic strategy for PDAC patients. While the occurrence of PDAC has been observed to differ between racial/ethnic subpopulations, this disparity may be partially explained by the prevalence of risk factors (smoking and drinking alcohol, obesity, diabetes, and family history) among ethnic groups 49 , 50 . These racial/ethnic variations might result in tumor biology differences in PDAC 50 . Biomarkers that could be useful regardless of racial differences are thus urgently needed. In this study, we selected the datasets from different regions, thus averting the differences caused by the samples heterogeneity and revealing universal DEGs that apply to different ethnic groups. The identified DEGs in this analysis might be applicable irrespective of the ethnicities and may allow the development of more targeted prevention strategies. However, a lack of adequate validation in vitro or in vivo is a limitation of this study. Moreover, due to GEO limitations, the clinicopathological data and demographic variables within this study datasets were not detailed enough. Thus, we failed to consider factors such as the presence of different ethnicities within datasets. Our future research will include experimental verification of this meta-analysis results using different laboratory approaches. In conclusion, the present meta-analysis identified 20 DEGs. The hub genes are COL12A1, FN1, ITGA2, LAMB3, LAMC2, THBS2, and VCAN. The Kaplan–Meier survival plots indicate that ITGA2, LAMB3, and LAMC2 are inversely correlated with prolonged patient survival. Histopathological evidence shows that ITGA2, LAMB3, and LAMC2 are expressed exclusively from pancreatic cancer cells. The specific expression of these three proteins by cancer cells could make them promising potential targets for diagnosing and treating pancreatic cancer. Supplementary Information Supplementary Information. Supplementary Information. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-021-90077-x.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962973/

Immunomodulation of T H 2 biased immunity with mucosal administration of nanoemulsion adjuvant

Highlights • Nanoemulsion vaccine adjuvant induces robust antigen specific T H 1-biased immunity. • Nanoemulsion vaccine adjuvant suppresses established T H 2 immunity. • Efficacy of nanoemulsion vaccine in mice with pre-existing immunity to same antigen. • Nanoemulsion vaccines induce IL-10 and regulatory T cells. 1 Introduction CD4 + effector T cell responses are classified according to their cytokine and transcription factor profiles, with T H 1 and T H 2 cells being the most widely studied types [1] . Differentiation of T H 1 cells is driven by IL-12 secreted by macrophages and IFN-γ from T cells or NK cells, and results in the production of T H 1-type cytokines including IFN-γ, IL-2 and TNF-α. In mice, IFN-γ enhances immunoglobulin class switching to increase production of IgG2a and IgG2b subclasses as well as activation of other T H 1 cell-mediated effector responses [2] . Alternatively, T H 2 responses can be initiated by IL-4-dependent differentiation of T H 2 effector CD4 + cells that produce T H 2-type cytokines, including IL-4, IL-5, IL-9 and IL-13, which can culminate in the increased production of IgG1 subclass and IgE antibodies. The T H 1/T H 2 paradigm is useful for classification of immune responses and becomes better defined as mechanisms of action of CD4 + effector T cells are further elucidated. The type of cell-mediated immunity affects the induction of specific protective immunity to infectious diseases, inflammatory responses, allergy or autoimmunity and even can increase susceptibility to certain infections [3] , [4] . This is of particular importance in vaccine development because adjuvants are able to skew helper T cell profiles, and choosing the appropriate adjuvant may influence efficacy [5] . The most widely used adjuvant alum induces strong T H 2-associated immune responses which are less effective against pathogens for which T H 1 cell-mediated immunity is required for clearance [6] , [7] , [8] . Because of this, a number of vaccines based on new T H 1 polarizing adjuvants including liposomes, CpG-containing oligodinucleotides, monophosphoryl lipid A, and QS-21 are being evaluated both in animal studies and in clinical trials [9] , [10] , [11] , [12] . Many of these adjuvants are under development for production of vaccines that may be used in people who have already been exposed to the same antigen or pathogen, either through prior vaccination or infection. In the case of individuals previously primed to have a T H 2 skewed immune response from an alum-adjuvanted vaccine, it is unclear if boosting with a T H 1-polarizing vaccine adjuvant would redirect the immune response towards a T H 1 response, or if it would simply boost the T H 2 responses for which the immune system had already been primed. Additionally, a considerable interest has been directed towards development of strategies for modulation of existing T H 2 immune responses, especially for the alleviation of T H 2-biased allergic responses [13] , [14] . Adjuvants capable of redirecting established antigen-specific T H 2 responses to induce T H 1 while suppressing T H 2 immunity have the potential for impacting a variety of diseases driven by aberrant T H 2 immune responses. Our group has developed a nanoscale oil-in-water emulsion (nanoemulsion, NE) vaccine adjuvant platform that when delivered intranasally (i.n.) induces robust systemic and mucosal responses without local inflammatory effects [15] , [16] , [17] , [18] , [19] , [20] . In animal studies, i.n. immunizations with NE mixed with a variety of viral and bacteria-derived antigens, including influenza, hepatitis B, respiratory syncytial virus, vaccinia and anthrax, yields high protective antibody titers. In contrast to adjuvants like alum that induce T H 2-biased immune responses [6] , [21] , [22] , nasally administered NE vaccines result in T H 1 and T H 17 polarized immune responses [18] , [23] , [24] . Regardless of the model tested, T H 2 cytokine responses in animals immunized with NE are always low, and no significant production of IgE has been observed. This is true even in BALB/c mice that are inherently biased towards a T H 2-type response [25] . Because this NE adjuvant is a robust T H 1-polarizing adjuvant, we hypothesized that it would be a good candidate for redirecting established T H 2 immune responses to a more balanced T H 1/T H 2/T H 17. In proof-of-concept studies presented here, we have investigated the effect of nasal administration of a NE vaccine in mice previously vaccinated with an alum-adjuvanted vaccine. 2 Material and methods 2.1 Antigen and adjuvants The recombinant hepatitis B surface antigen (HBs) was supplied by Human Biologicals Institute (Indian Immunologics, Ltd, Hyderabad, India). The endotoxin level was determined to be <7.5 EU/20 μg of HBs, which is significantly below the internationally accepted standard of ⩽30 EU/20 μg of protein. Ovalbumin (ova) was purchased from Sigma–Aldrich. Ova peptides, class I-restricted ova 257–264 (SIINFEKL, ova I) and class II-restricted ova 323–339 (ova II) were purchased from Invitrogen. Nanoemulsion adjuvant (NE) was supplied by NanoBio Corporation, Ann Arbor, MI. NE was produced by a high speed emulsification of ultra pure soybean oil with cetyl pyridinium chloride, Tween 80 and ethanol in water, with resultant NE droplets with average 350–400 nm diameter [18] . Aluminum hydroxide (alum) was purchased from Sigma–Aldrich, Inc. All reagents were tested for the presence of endotoxin using RAW-Blue cell-based assay in vitro (InvivoGen, San Diego, CA). 2.2 Mice and immunizations Pathogen-free CD-1 mice (females 6–8 weeks old) were purchased from the Charles River Laboratories. All animal procedures were performed according to the University Committee on the Use and Care of Animals at the University of Michigan. Immunization schedule is shown in Fig. 1 . For all immunizations, mice were anesthetized under isoflurane anesthesia using the IMPAC6 precision vaporizer. Intranasal (i.n.) immunizations were done using a pipette tip by administration of 5 μl/nare of formulation containing 20 μg of antigen mixed with 20% NE. Antigen mixed with PBS alone served as a control. Intramuscular immunizations (i.m.) were performed by injection of 50 μl containing 20 μg of antigen adsorbed on 0.5 mg/ml alum into the epaxial muscle as described previously [18] . Sera were obtained by saphenous vein bleeding, and splenocytes were harvested at the end of the experiment. In IL-10 depletion experiments, mice were injected i.p. with 1 mg anti-IL-10 (purified from rabbit serum [26] ) or control rabbit IgG 12 h before and 2 days after NE immunization. 2.3 Measurement of serum IgG subclasses Serum antibody and IgG subclasses titers were determined by ELISA, with plates coated with 5 μg/ml of HBs as described previously [18] . 2.4 Analysis of cytokine expression Single cell suspensions of freshly isolated mouse splenocytes were cultured at 4 × 10 6 cells/ml with or without antigen (10 μg/ml). After 48 h, supernatants were collected and analyzed for the presence of cytokines using Milliplex Mouse Cytokine/Chemokine Immunoassay Kit (Millipore, Billerica, MA). 2.5 Measurement of the induction of regulatory T cells (Tregs) after NE immunization Mice were immunized i.n. with ova and NE (ova-NE) or non-adjuvated ova (ova-PBS) at weeks 0 and 4. Splenocytes were harvested at 1 and 7 weeks after the first immunization. Red blood cell depleted single cell suspensions were stained by flow cytometry to quantify regulatory T cells. Fc receptors were blocked with purified anti-CD16/32 (clone 93, BioLegend) and surface markers were stained using antibodies against CD3 (145-2C11), CD4 (RM4-5) and CD25 (7D4) (all from eBioscience or BD Biosciences), permeabilized, fixed and labeled for intracellular Foxp3 (FJK-16s). Samples were acquired on an Accuri C6 flow cytometer (BD Biosciences). Data were analyzed using FlowJo (Treestar). 2.6 Statistics Results are presented as the geometric mean ± 95% confidence interval. Statistical comparisons were assessed by the Mann–Whitney test using GraphPad Prism version 6 (GraphPad Software). The p value < 0.05 was considered as significant. In every reported result the data shown are representative of at least 2 experiments. 2.1 Antigen and adjuvants The recombinant hepatitis B surface antigen (HBs) was supplied by Human Biologicals Institute (Indian Immunologics, Ltd, Hyderabad, India). The endotoxin level was determined to be <7.5 EU/20 μg of HBs, which is significantly below the internationally accepted standard of ⩽30 EU/20 μg of protein. Ovalbumin (ova) was purchased from Sigma–Aldrich. Ova peptides, class I-restricted ova 257–264 (SIINFEKL, ova I) and class II-restricted ova 323–339 (ova II) were purchased from Invitrogen. Nanoemulsion adjuvant (NE) was supplied by NanoBio Corporation, Ann Arbor, MI. NE was produced by a high speed emulsification of ultra pure soybean oil with cetyl pyridinium chloride, Tween 80 and ethanol in water, with resultant NE droplets with average 350–400 nm diameter [18] . Aluminum hydroxide (alum) was purchased from Sigma–Aldrich, Inc. All reagents were tested for the presence of endotoxin using RAW-Blue cell-based assay in vitro (InvivoGen, San Diego, CA). 2.2 Mice and immunizations Pathogen-free CD-1 mice (females 6–8 weeks old) were purchased from the Charles River Laboratories. All animal procedures were performed according to the University Committee on the Use and Care of Animals at the University of Michigan. Immunization schedule is shown in Fig. 1 . For all immunizations, mice were anesthetized under isoflurane anesthesia using the IMPAC6 precision vaporizer. Intranasal (i.n.) immunizations were done using a pipette tip by administration of 5 μl/nare of formulation containing 20 μg of antigen mixed with 20% NE. Antigen mixed with PBS alone served as a control. Intramuscular immunizations (i.m.) were performed by injection of 50 μl containing 20 μg of antigen adsorbed on 0.5 mg/ml alum into the epaxial muscle as described previously [18] . Sera were obtained by saphenous vein bleeding, and splenocytes were harvested at the end of the experiment. In IL-10 depletion experiments, mice were injected i.p. with 1 mg anti-IL-10 (purified from rabbit serum [26] ) or control rabbit IgG 12 h before and 2 days after NE immunization. 2.3 Measurement of serum IgG subclasses Serum antibody and IgG subclasses titers were determined by ELISA, with plates coated with 5 μg/ml of HBs as described previously [18] . 2.4 Analysis of cytokine expression Single cell suspensions of freshly isolated mouse splenocytes were cultured at 4 × 10 6 cells/ml with or without antigen (10 μg/ml). After 48 h, supernatants were collected and analyzed for the presence of cytokines using Milliplex Mouse Cytokine/Chemokine Immunoassay Kit (Millipore, Billerica, MA). 2.5 Measurement of the induction of regulatory T cells (Tregs) after NE immunization Mice were immunized i.n. with ova and NE (ova-NE) or non-adjuvated ova (ova-PBS) at weeks 0 and 4. Splenocytes were harvested at 1 and 7 weeks after the first immunization. Red blood cell depleted single cell suspensions were stained by flow cytometry to quantify regulatory T cells. Fc receptors were blocked with purified anti-CD16/32 (clone 93, BioLegend) and surface markers were stained using antibodies against CD3 (145-2C11), CD4 (RM4-5) and CD25 (7D4) (all from eBioscience or BD Biosciences), permeabilized, fixed and labeled for intracellular Foxp3 (FJK-16s). Samples were acquired on an Accuri C6 flow cytometer (BD Biosciences). Data were analyzed using FlowJo (Treestar). 2.6 Statistics Results are presented as the geometric mean ± 95% confidence interval. Statistical comparisons were assessed by the Mann–Whitney test using GraphPad Prism version 6 (GraphPad Software). The p value < 0.05 was considered as significant. In every reported result the data shown are representative of at least 2 experiments. 3 Results 3.1 Mucosal immunization with NE adjuvant modifies T H 2 polarized immune response To elicit the T H 2 response, mice were immunized with two i.m. injections of 20 μg HBs adsorbed on alum [21] . Analysis of serum IgG subclass and cytokine expression confirmed that HBs-alum immunization yielded predominantly IgG1 antibody subclass ( Fig. 2 A) and induction of T H 2-type cytokines IL-4 and IL-5 ( Fig. 2 B). There was no change in antibody titers in mice receiving only the HBs-alum vaccine from weeks 6–12 (data not shown). To investigate whether NE adjuvant can modify this T H 2 bias, the mice were subsequently immunized with a single intranasal administration of HBs-NE at 2 or 6 weeks after the second HBs-alum sensitization ( Fig. 1 ). Serum IgG analysis showed significant increases in IgG2a and IgG2b subclasses following HBs-NE immunization, with antibody titers comparable to the HBs-NE immunization in mice that did not receive the HBs-alum vaccine ( Fig. 2 A). Antigen-specific cytokine expression in splenic lymphocytes after the 6 week NE immunization showed significant induction of T H 1-type IFN-γ and TNF-α and the T H 17 cytokine IL-17( Fig. 2 B) and decreased IL-4 and IL-5 production in mice immunized with HBs-NE six weeks after HBs-alum sensitization. This effect was not significant in mice immunized with HBs-NE at an earlier time point (2 weeks). Nasal immunization with HBs-NE alone was used as a control to assess modulation of established T H 2 immunity with NE adjuvant, and antibody and cytokine patterns were similar after HBs-NE immunization regardless of whether the mice had been previously T H 2 sensitized or not ( Fig. 2 A and B). There was a slight decrease in IFN-γ and IL-17 in mice that received both vaccines compared with mice immunized with NE only, however these differences were not statistically significant ( p = 0.70 and 0.41, respectively). To investigate a potential role of regulatory T cells (Tregs) in the mechanism of NE adjuvant, mice were immunized i.n. with ova-NE or with non-adjuvanted ova in PBS (ova-PBS) as a control. Treg frequency (CD4 + Foxp3 + ) was measured after 6 days both in the nasal draining lymph nodes (cervical lymph nodes, cLN) and in the periphery in splenic lymphocytes. Analysis of CD4 + Foxp3 + T cells showed that mucosal administration of ova-NE induced significantly more Treg expansion in both cLN and spleen compared to ova-PBS and PBS administration ( p < 0.03) ( Fig. 3 A). Interestingly, by 6 weeks after immunization the frequency of Tregs was elevated in both ova-NE and ova-PBS groups ( Fig. 3 B). Consistent with results documented previously, the i.n. immunization with ova-NE induced a potent IgG response, while no significant titers were detected in ova-PBS immunized mice ( Fig. 3 C). Further analysis revealed that production of IL-10, a suppressive cytokine associated with Treg function [27] , [28] , was increased in cells from mice immunized with i.n. ova-NE ( Fig. 4 A). Furthermore, IL-10 production was only significantly induced with stimulation with ova protein or a MHC II-restricted ova peptide, not a MHC I ova peptide, suggesting that the IL-10 is produced by CD4 + T cells. Correlates of Treg frequency vs. IL-10 expression show no IL-10 production in mice immunized with ova-PBS despite the increase in frequency of Treg frequency. In contrast, in mice treated with ova-NE there was a significant increase in IL-10 levels that closely correlated with increased Treg frequency ( Fig. 4 B). In order to determine the effects of IL-10 on NE-mediated suppression of the alum-induced T H 2 immune response, mice were immunized i.m. with HBs-alum and IL-10 was depleted at the time of HBs-NE immunization. There was no balancing of IgG subclasses when IL-10 was depleted during NE immunization ( Fig 5 A), and the subclass pattern was similar to that observed from mice only receiving the HBs-alum vaccine. The suppression of T H 2 cytokines (IL-4, IL-5, IL-13) did not occur upon HBs-NE immunization with simultaneous IL-10 depletion. The induction of the T H 1 cytokine, IFN-γ, was not inhibited by IL-10 depletion. IL-10 depletion did not significantly change the percentage of Tregs induced by NE immunization. 3.1 Mucosal immunization with NE adjuvant modifies T H 2 polarized immune response To elicit the T H 2 response, mice were immunized with two i.m. injections of 20 μg HBs adsorbed on alum [21] . Analysis of serum IgG subclass and cytokine expression confirmed that HBs-alum immunization yielded predominantly IgG1 antibody subclass ( Fig. 2 A) and induction of T H 2-type cytokines IL-4 and IL-5 ( Fig. 2 B). There was no change in antibody titers in mice receiving only the HBs-alum vaccine from weeks 6–12 (data not shown). To investigate whether NE adjuvant can modify this T H 2 bias, the mice were subsequently immunized with a single intranasal administration of HBs-NE at 2 or 6 weeks after the second HBs-alum sensitization ( Fig. 1 ). Serum IgG analysis showed significant increases in IgG2a and IgG2b subclasses following HBs-NE immunization, with antibody titers comparable to the HBs-NE immunization in mice that did not receive the HBs-alum vaccine ( Fig. 2 A). Antigen-specific cytokine expression in splenic lymphocytes after the 6 week NE immunization showed significant induction of T H 1-type IFN-γ and TNF-α and the T H 17 cytokine IL-17( Fig. 2 B) and decreased IL-4 and IL-5 production in mice immunized with HBs-NE six weeks after HBs-alum sensitization. This effect was not significant in mice immunized with HBs-NE at an earlier time point (2 weeks). Nasal immunization with HBs-NE alone was used as a control to assess modulation of established T H 2 immunity with NE adjuvant, and antibody and cytokine patterns were similar after HBs-NE immunization regardless of whether the mice had been previously T H 2 sensitized or not ( Fig. 2 A and B). There was a slight decrease in IFN-γ and IL-17 in mice that received both vaccines compared with mice immunized with NE only, however these differences were not statistically significant ( p = 0.70 and 0.41, respectively). To investigate a potential role of regulatory T cells (Tregs) in the mechanism of NE adjuvant, mice were immunized i.n. with ova-NE or with non-adjuvanted ova in PBS (ova-PBS) as a control. Treg frequency (CD4 + Foxp3 + ) was measured after 6 days both in the nasal draining lymph nodes (cervical lymph nodes, cLN) and in the periphery in splenic lymphocytes. Analysis of CD4 + Foxp3 + T cells showed that mucosal administration of ova-NE induced significantly more Treg expansion in both cLN and spleen compared to ova-PBS and PBS administration ( p < 0.03) ( Fig. 3 A). Interestingly, by 6 weeks after immunization the frequency of Tregs was elevated in both ova-NE and ova-PBS groups ( Fig. 3 B). Consistent with results documented previously, the i.n. immunization with ova-NE induced a potent IgG response, while no significant titers were detected in ova-PBS immunized mice ( Fig. 3 C). Further analysis revealed that production of IL-10, a suppressive cytokine associated with Treg function [27] , [28] , was increased in cells from mice immunized with i.n. ova-NE ( Fig. 4 A). Furthermore, IL-10 production was only significantly induced with stimulation with ova protein or a MHC II-restricted ova peptide, not a MHC I ova peptide, suggesting that the IL-10 is produced by CD4 + T cells. Correlates of Treg frequency vs. IL-10 expression show no IL-10 production in mice immunized with ova-PBS despite the increase in frequency of Treg frequency. In contrast, in mice treated with ova-NE there was a significant increase in IL-10 levels that closely correlated with increased Treg frequency ( Fig. 4 B). In order to determine the effects of IL-10 on NE-mediated suppression of the alum-induced T H 2 immune response, mice were immunized i.m. with HBs-alum and IL-10 was depleted at the time of HBs-NE immunization. There was no balancing of IgG subclasses when IL-10 was depleted during NE immunization ( Fig 5 A), and the subclass pattern was similar to that observed from mice only receiving the HBs-alum vaccine. The suppression of T H 2 cytokines (IL-4, IL-5, IL-13) did not occur upon HBs-NE immunization with simultaneous IL-10 depletion. The induction of the T H 1 cytokine, IFN-γ, was not inhibited by IL-10 depletion. IL-10 depletion did not significantly change the percentage of Tregs induced by NE immunization. 4 Discussion The development of new materials and adjuvants that can modulate the immune system is an emerging field in immunology, with interests in multiple settings, including vaccine development and allergy [13] , [29] , [30] , [31] . In this proof of concept study we present a new adjuvant-based approach to immunomodulation in mice. We have demonstrated that immunization with novel oil-in-water nanoemulsion adjuvant not only produced robust cellular and humoral immunity but also redirected existing T H 2-biased responses towards a more balanced T H 1/T H 2 phenotype in a model of established antigen-specific T H 2 immunity. In contrast to the commonly used aluminum adjuvant(s), NE is not associated with the T H 2 phenotype. Consistent with our previous results [16] , [17] , [18] , immunization with NE adjuvant produced T H 1 biased immunity with IFN-γ and TNF-α production ( Fig. 2 B). The significant increase of IgG2a and IgG2b antibodies and T H 1 type cytokines and simultaneous reduction of IgG1 antibodies and T H 2 cytokines demonstrates that NE adjuvant is capable of shifting an established T H 2 response towards a more balanced cell-mediated immunity both through the induction of T H 1 and suppression of T H 2. In mice, IgG1 is regulated via a T H 2/IL-4 pathway, and in numerous studies IgG1 has been used as a robust indicator for the assessment of a T H 2 response [32] , [33] . Mucosal HBs-NE immunization of HBs-alum sensitized mice diminished IgG1/IgG2a and IgG1/IgG2b ratios from 10.46 and 8.67, to 1.2 and 2.1, respectively, clearly demonstrating modulation of the HBs-specific immune response. Analysis of cytokine expression provided direct assessment of T cell activation. Elevated IFN-γ and diminished IL-4 levels after antigen stimulation of splenic lymphocytes indicated that HBs-NE immunization resulted in T H 2 cell suppression and a shift to T H 1 response ( Figs. 2 B and 3 B). This effect was not detected in splenocytes from mice immunized with NE at an earlier time point (2 weeks), despite increase in IgG2a and IgG2b antibodies in comparison to HBs-alum controls ( Fig. 2 ). This result indicates that while NE has the potential to modify established T H 2 immunity, effective modification of an ongoing immune response may depend on the schedule and number of immunizations. Intranasal immunization with NE adjuvant induces a T H 17 immune response [23] . The antigen-specific IL-17 expression was also detected in splenocytes of mice with T H 1 redirected immune response ( Fig. 2 B). Despite association with various autoimmune disorders, T H 17 also contributes to host defense as a T cell subset involved in protection against extracellular pathogens [34] and has been shown to play a critical role in the efficacy of several vaccines [35] , [36] , [37] , [38] , [39] . Although excessive prolonged IL-17 production may contribute to pathophysiology of respiratory infections or asthma and allergy, the degree of T H 17 induction with NE immunization is much lower than levels typically observed in diseases in which IL-17 contributes to pathology [40] , [41] . The effect of NE-induced IL-17 production on T H 2/T H 1 immunomodulation remains to be investigated; however, T H 17 cell-mediated immunity may suppress IgE responses, as has been recently indicated for T H 17 immunity associated with human autoimmune disease [42] . The exact mechanism of action of NE adjuvant is not yet fully elucidated. NE is formulated using ultrapure and endotoxin-free components and does not contain any commonly recognized TLR agonists or ligands [18] . However, our recent results demonstrate involvement of the TLR pathway in immunogenicity of NE adjuvant both in vivo and in vitro [24] . NE facilitates antigen uptake and trafficking into lymphoid tissue while not causing either nasal irritation or disruption of mucosal epithelial architecture [43] , [44] . NE-mediated enhancement of antigen internalization and processing by the antigen presenting cells could be important for the optimal antigen presentation to T cells and development of T H 1 biased immunity [45] , [46] , [47] . We have shown that intranasal treatment with NE adjuvant does not produce significant amounts of IFN-γ, TNF-α, IL-12, IL-4, IL-5, IL-9, IL-13 or inflammatory cytokines such as IL-1β in the nasal mucosa [43] . Based on the absence of inflammatory mediators, rhinitis or cellular infiltrates at the high 20% concentration, NE appears to be non-inflammatory and is generally biocompatible with mucosal and pulmonary tissue in mice, rats, guinea pigs, dogs and humans (not shown and [18] , [44] , [48] ). While data from mouse models clearly show that alum drives T H 2 immunity, the evidence for T H 2 skewing by alum based vaccines in humans is not entirely clear. A few clinical studies have shown that alum induces a mixed T H 2 and T H 1 response, but the overall effect across various antigens in humans as compared to mice is poorly defined [49] , [50] , [51] , [52] . Additionally, studies assessing immune polarization induced by alum mainly have been performed in adults. Given that neonatal immune systems are inherently biased towards T H 2 [53] , [54] , the immunization of newborns with an alum-based Hepatitis B vaccine raises concerns about the role vaccines might play in the growing issue of allergic disease in young people [55] , [56] . Moving forward, it may be advantageous to consider vaccine adjuvants that induce required protective immunity without activating T H 2 polarized responses. While the ability of NE adjuvant to shift towards T H 1 in humans is not explored in this study, in a Phase I clinical trial a flu vaccine containing this NE adjuvant formulation induced T H 1 antigen-specific IgG, neutralizing antibody, as well as mucosal IgA, demonstrating the immunogenicity of this adjuvant in humans [44] . Here, we demonstrate that NE immunization resulted in the induction of Tregs in both the draining lymph nodes and the periphery. The correlating increase in IL-10, suggests that these Tregs may have suppressive function, and likely play a role in the immune responses induced by NE [57] . Tregs are considered essential for the maintenance of immunological homeostasis and for the control of exacerbated immune responses. Numerous studies have demonstrated a role for Tregs in restraining exacerbated immune responses during natural infection, suggesting that Treg depletion and/or inactivation could improve efficacy of vaccines [58] , [59] . Much less is known regarding the role of Tregs in the induction and maintenance of protective immune response with various adjuvant-based vaccines; however the data presented here suggest that for NE the induction of Tregs does not inhibit overall vaccine efficacy but may be responsible for the suppression of the T H 2 response. It has previously been reported that antigen-specific T H 1 and regulatory T cells can mediate modification of IgG subclass pattern [60] , consistent with the data presented here. Since Tregs induced with antigen alone are often considered as immune suppressors in the process of immune tolerance, our results may suggest a functional difference between the Treg populations in mice immunized with antigen alone compared with antigen and NE. Similarly, these results may suggest a functional difference between Treg populations generated in various modes of i.n. immunization. Further characterization of Treg function and direct functional assessment of their suppressive potential will help to clarify their role in NE induced immune response. IL-10 production is one mechanism by which immune responses can be suppressed. Not only does NE induce IL-10, but depletion of IL-10 during NE immunization alters the ability of NE to suppress T H 2 immunity ( Fig. 5 ). Interestingly, IL-10 depletion did not alter T H 1 induction by NE, so IL-10 does not appear to be involved in the induction of immune responses by NE. IL-10 was depleted during immunization but not at the time of sacrifice when the recall response to antigen was determined, suggesting that IL-10 is critical for priming of cellular immune responses that result in a shift from T H 2 to T H 1 in this model. 5 Conclusions Our initial results suggest the usefulness of NE-based delivery systems in the development of therapeutic vaccines to modify T H 2 immune responses, as well as the ability of NE-based vaccines to retain their immune phenotype even in individuals that received previous vaccinations with the same antigen and other adjuvants. This novel approach to immunomodulation using i.n. delivery of NE adjuvant to produce mucosal immunity and a systemic T H 1-biased immune response could be useful for the development of vaccines to induce antigen-specific T H 1 immune responses even in individuals with pre-existing T H 2 biased immunity. This suggests that NE adjuvant may be especially useful in situations where pathologies are due to aberrant T H 2 immune response, such as allergy. Grant support This project has been funded by the Bill and Melinda Gates Foundation , under award 37868 and the National Institute for Allergy and Infectious Disease , National Institutes of Health under Contract No. HHSN272200900031C.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7126925/

Microwave-assisted tissue processing for same-day EM-diagnosis of potential bioterrorism and clinical samples

The purpose of this study was to explore the turnaround times, section and image quality of a number of more "difficult" specimens destined for rapid diagnostic electron microscopy (EM) after microwave-assisted processing. The results were assessed and compared with those of conventionally processed samples. A variety of infectious agents, some with a potential for bioterrorism, and liver biopsies serving as an example for routine histopathology samples were studied. The samples represented virus-producing cell cultures (such as SARS-coronavirus, West Nile virus , Orthopox virus ), bacteria suspensions (cultures of Escherichia coli and genetically knockout apathogenic Bacillus anthracis ), suspensions of parasites (malaria Plasmodium falciparum , Leishmania major , Microsporidia cuniculi , Caenorhabditis elegans ), and whole Drosophila melanogaster flies infected with microsporidia. Fresh liver samples and infected flies were fixed in Karnovsky-fixative by microwaving (20 min), all other samples were fixed in buffered glutaraldehyde or Karnovsky-fixative overnight or longer. Subsequently, all samples were divided to evaluate alternative processing protocols: one part of the sample was OsO 4 -postfixed, ethanol-dehydrated, Epon-infiltrated (overnight) in an automated tissue processor (LYNX, Leica), and polymerized at 60 °C for 48 h; in parallel the other part was microwave-assisted processed in the bench microwave device (REM, Milestone), including post-osmication and the resin block polymerization. The microwave-assisted processing protocol required at minimum 3 h 20 min: the respective epon resin blocks were uniformly polymerized allowing an easy sectioning of semi- and ultrathin sections. Sections collected on non-coated 200 mesh grids were stable in the electron beam and showed an excellent preservation of the ultrastructure and high contrast, thus allowing an easy, unequivocal and rapid assessment of specimens. Compared with conventional routine methods, microwave technology facilitates a significant reduction in sample processing time from days to hours without any loss in ultrastructural details. Microwave-assisted processing could, therefore, be a substantial benefit for the routine electron microscopic diagnostic workload. Due to its speed and robust performance it could be applied wherever a rapid electron microscopy diagnosis is required, e.g., if bioterrorism or emerging agents are suspected. Combining microwave technology with digital image acquisition, the 1-day diagnosis based on ultrathin section electron microscopy will become possible, with crucial or interesting findings being consulted or shared worldwide with experts using modern telemicroscopy tools via Internet. 1 Introduction Diagnostic electron microscopy (EM) can contribute decisively to the laboratory diagnosis of infectious diseases and will be of particular importance if a rapid diagnosis is required, as in a potential bioterror scenario or if emerging infectious agents are to be characterized ( O'Toole, 1999 , Hazelton and Gelderblom, 2003 , Madeley, 2003 , Miller, 2003 , Ng et al., 2003 , Goldsmith et al., 2004 , Morens et al., 2004 ). Likewise EM should be applied in critical clinical setting of controversial or urgent pathological cases ( Kamondi et al., 2000 , Nordhausen and Barr, 2001 , Turbat-Herrera et al., 2004 ). In many instances, in infectious diseases a rapid and accurate diagnosis is achieved after negative staining of virus or bacterial suspensions ( Madeley and Field, 1988 , Biel and Madeley, 2001 , Gelderblom, 2001 , Gelderblom, 2003 , Gentile and Gelderblom, 2005 ; Robert Koch Institute http://www.rki.de ). However, in other cases, if infected cell cultures or tissues (e.g., biopsies taken to complement material for the negative staining approach) have to be examined, the need for rapid embedding methods enabling a same-day diagnosis from ultrathin sections is evident. In the past a number of simplified tissue embedding procedures using reduced dehydration times and increased polymerization temperatures were proposed ( Bencosme and Tsutsumi, 1970 , Johannessen, 1973 , Doane et al., 1974 , Rowden and Lewis, 1974 , Miller, 1982 , Bozzola and Russell, 1992 ). Rapid diagnostic EM could probably benefit from the microwave employment, a technology that has been successfully applied in organic chemistry ( De la Hoz et al., 2005 ), industrial food processing ( Vaid and Bishop, 1998 ), sterilization of hospital waste ( Celandroni et al., 2004 ), and also in the clinical laboratory. Here a great number of microwave accelerated procedures for light microscopy and EM tissue processing, staining reactions, and immunolabelling was developed ( Mayers, 1970 , Leong et al., 1985 , Login and Dvorak, 1988 , Login and Dvorak, 1994 , Kok and Boon, 1990 , Kok and Boon, 2003 , Shi et al., 1991 , Leong and Sormunen, 1998 , Cavusoglu et al., 2001 , Giberson and Demaree, 2001 , Giberson et al., 2003 , Leria et al., 2004 , Munoz et al., 2004 ), all of them resulting in a significant time reduction. The effect of microwave irradiation on polar substances is mainly well understood. It is attributed to dielectric heating, also called "thermal effect" ( Leonard and Shepardson, 1994 , Giberson and Demaree, 1995 , Kok and Boon, 2003 ) causing a temperature rise in the whole sample ("internal heating"; in contrast to conventional heating which starts at the specimen surface). The existence of an additional "non-thermal" effect of microwave energy ( Marani and Feirabend, 1993 , De la Hoz et al., 2005 ), which may be particularly effective in biological hydrated material in influencing the fixation of cells ( Ruijgrok et al., 1993 , Leria et al., 2004 , Wendt et al., 2004 ), is still unproven. A number of efforts has been undertaken to standardize the microwave-assisted procedures ( Login, 1998 ). Different microwave bench "ovens" designed for the laboratory requirements are now commercially available. Assuming the rise in internal temperature as the primary mechanism responsible for the enhanced diffusion of reagents and protein cross-linking during fixation, microwave device manufacturers continually add features into their equipment to provide the end user with more control over the irradiated reaction process. We report here on the application of a microwave device (REM, Milestone, Sorisole, Italy) recently developed for EM rapid tissue processing equipped with a customized software and a sample basket handling system compatible to our routinely used tissue processor (LYNX, Leica, Bensheim, Germany). Using this system, various specimens representing critical infectious or potential bioterrorism agents and liver biopsies which substitute pathological samples were processed in parallel following both a conventional and a microwave-assisted protocol. Ultrathin sections were evaluated, images were documented digitally and the results of both techniques compared. 2 Materials and methods 2.1 Sample collection and primary fixation The following specimens in the primary fixative were sent by post to the EM laboratory in Regensburg for subsequent comparative processing and evaluation: cell suspensions infected with SARS-coronavirus, West Nile virus , an Orthopox virus isolated from an elephant (fixed in 2.5% phosphate buffered glutaraldehyde (GA), pH 7.2: Robert Koch Institute, Berlin), suspensions of Escherichia coli and Acanthamoeba castellanii (fixed in 4% cacodylate buffered GA, pH 7.3: Medical Service of Federal Armed Forces, Koblenz), suspensions containing Plasmodium falciparum , Leishmania major , Microsporidia cuniculi and the worm Caenorhabditis elegans (fixed in 2% cacodylate buffered GA, pH 7.2: Bernhard-Nocht-Institute for Tropical Medicine, Hamburg). A part of each sample kept at the collaborative site was processed following the respective in-house protocols. Grids with stained ultrathin sections were sent to the EM laboratory in Regensburg for evaluation and documentation. Agar culture (6-day-old) of apathogenic genetically knockout pX-02 Bacillus anthracis samples (courtesy of Dr. H.-J. Linde, Microbiology Department, University Regensburg), Drosophila melanogaster flies infected with microsporidia Tubulinosema ratisbonensis (courtesy of Dr. C. Franzen, Internal Medical Department, University Regensburg), and murine and human surgical liver samples were fixed in modified Karnovsky-fixative (1% paraformaldehdyde/2.5% GA in cacodylate buffer, pH 7.3) overnight as well as 20 min under microwave irradiation (Central EM Laboratory, Regensburg). Subsequently, cells and microorganisms were sedimented by centrifugation (800 × g for 5 min), enclosed in low-melting-point agar at 45 °C and chilled on ice. The agar blocks with the agents as well as the solid tissue samples were dissected with a razor blade into cubes of 1-mm edge length. Each sample was processed according to a conventional and a microwave-assisted protocol. 2.2 Conventional processing One part of each sample was processed in one batch exclusively in the automated tissue processor (LYNX) as a control, involving 1% OsO 4 post-fixation, dehydration in graded ethanols, infiltration (overnight) and embedding in the EMbed-812 epoxy resin (all reagents from Science Services, Munich, Germany). After 48 h polymerization at 60 °C, semithin sections were cut, stained with toluidine blue and basic fuchsin, and after selection of areas of interest the Epon blocks were trimmed for ultrathin sectioning. Ultrathin sections 70 nm in thickness were cut with a diamond knife (45°, Diatome, Biel, Switzerland) on a Reichert Ultracut-S ultramicrotome, mounted on non-coated 200 mesh copper grids, and double stained with aqueous 2% uranyl acetate and lead citrate solutions for 10 min each. 2.3 Microwave processing In parallel, the other part of each sample was processed using the Rapid Electron Microscope microwave device (REM, Fig. 1 ) utilizing the same specimen baskets ( Fig. 2 ) as for the LYNX-processor. This microwave bench unit is claimed by the manufacturer to have an even microwave distribution within the device resonant "cavity" ( Visinoni et al., 1998 ) during the processing. Each vial, containing the samples immersed in the process solutions, is placed in a special designed safety carrier, which locates the vial in a defined position in the microwave "cavity". A non-contact infra-red temperature sensor measures the current solution temperature in the vial, while a magnetic stirrer is ensuring uniform heat distribution in the vial solution. The solution temperature is the critical parameter to monitor the magnetron wattage (maximum 700 W) power output, which is controlled via a feedback loop during the continuous microwave irradiation of the sample. The graphical display of the slope of temperature rise, irradiation temperature stabilization and time for each processing step, could be easily defined on a dedicated touch screen monitor. The whole microwave-assisted processing is controlled by a microprocessor and dedicated software package. This software also allows routine documentation of the entire microwave process for Quality Assurance and Quality Control. Fig. 1 Milestone rapid electron microscopy (REM) histoprocessor. Note the vial with processed samples placed in a predefined position in the microwave "oven". Fig. 2 Sample preparation tools for the REM. The baskets are the same as for the automated LYNX-processor. As outlined in Fig. 3 , also the primary fixation of the "fresh" samples ( Drosophila flies infected with microsporidia and liver tissue samples) with an adjusted temperature of 50 °C, not merely the OsO 4 -post-fixation, ethanol-dehydration, Epon-infiltration and polymerization (BEEM-capsules, under water) was carried out with microwave irradiation of the samples. This completely microwave-assisted sample processing required 4 h 25 min, except handling time, e.g., for solution exchange. In order to shorten the process further, we tested also an abbreviated microwave-assisted protocol with time reduction in all steps with the SARS-coronavirus infected cells, the B. anthracis , and human surgical liver tissue samples; here the microwave processing time was 3 h 20 min ( Table 1 ). Fig. 3 Protocol outline of the microwave-assisted specimen processing. Table 1 Comparison of processing times for conventional and microwave-assisted protocols Processing step Conventional (LYNX) Microwave Microwave shortened Primary fixation ≥2 h 20 min 15 min Buffer washes 1 h 9 min 3 min Osmium post-fixation 2 h 20 min 10 min Water washes 40 min 6 min 3 min Dehydration 1 h 45 min 20 min 10 min Infiltration 19 h 1 h 25 min 59 min Polymerization 48 h 1 h 45 min 1 h 40 min Total ≥74 h 25 min 4 h 25 min 3 h 20 min The polymerized Epon blocks were sectioned and stained in the same manner as described for the conventionally processed samples. 2.4 Evaluation of the sections and images The sections were examined in a LEO 912AB transmission electron microscope (Zeiss, Oberkochen, Germany) operating at 80 kV in zero-loss mode and equipped with a side-entry and a bottom-mounted fiber-optic coupled CCD-camera (Proscan, Lagerlechfeld, Germany) capable to record images with 1024 × 1024 pixels. Efforts were taken to standardize the conditions of image acquisition (constant beam brightness, exposure time and magnification settings) during the EM examination. Imaging and measuring were done with the analySIS software, version 3.2 (Soft Imaging System GmbH, Muenster, Germany), without any digital image post-processing manipulations. Notes of the section quality and beam stability during the examination were made, and the quality of the images obtained by the conventional versus microwave-assisted procedures was evaluated visually on a calibrated CRT monitor and by high-resolution laser prints. 2.1 Sample collection and primary fixation The following specimens in the primary fixative were sent by post to the EM laboratory in Regensburg for subsequent comparative processing and evaluation: cell suspensions infected with SARS-coronavirus, West Nile virus , an Orthopox virus isolated from an elephant (fixed in 2.5% phosphate buffered glutaraldehyde (GA), pH 7.2: Robert Koch Institute, Berlin), suspensions of Escherichia coli and Acanthamoeba castellanii (fixed in 4% cacodylate buffered GA, pH 7.3: Medical Service of Federal Armed Forces, Koblenz), suspensions containing Plasmodium falciparum , Leishmania major , Microsporidia cuniculi and the worm Caenorhabditis elegans (fixed in 2% cacodylate buffered GA, pH 7.2: Bernhard-Nocht-Institute for Tropical Medicine, Hamburg). A part of each sample kept at the collaborative site was processed following the respective in-house protocols. Grids with stained ultrathin sections were sent to the EM laboratory in Regensburg for evaluation and documentation. Agar culture (6-day-old) of apathogenic genetically knockout pX-02 Bacillus anthracis samples (courtesy of Dr. H.-J. Linde, Microbiology Department, University Regensburg), Drosophila melanogaster flies infected with microsporidia Tubulinosema ratisbonensis (courtesy of Dr. C. Franzen, Internal Medical Department, University Regensburg), and murine and human surgical liver samples were fixed in modified Karnovsky-fixative (1% paraformaldehdyde/2.5% GA in cacodylate buffer, pH 7.3) overnight as well as 20 min under microwave irradiation (Central EM Laboratory, Regensburg). Subsequently, cells and microorganisms were sedimented by centrifugation (800 × g for 5 min), enclosed in low-melting-point agar at 45 °C and chilled on ice. The agar blocks with the agents as well as the solid tissue samples were dissected with a razor blade into cubes of 1-mm edge length. Each sample was processed according to a conventional and a microwave-assisted protocol. 2.2 Conventional processing One part of each sample was processed in one batch exclusively in the automated tissue processor (LYNX) as a control, involving 1% OsO 4 post-fixation, dehydration in graded ethanols, infiltration (overnight) and embedding in the EMbed-812 epoxy resin (all reagents from Science Services, Munich, Germany). After 48 h polymerization at 60 °C, semithin sections were cut, stained with toluidine blue and basic fuchsin, and after selection of areas of interest the Epon blocks were trimmed for ultrathin sectioning. Ultrathin sections 70 nm in thickness were cut with a diamond knife (45°, Diatome, Biel, Switzerland) on a Reichert Ultracut-S ultramicrotome, mounted on non-coated 200 mesh copper grids, and double stained with aqueous 2% uranyl acetate and lead citrate solutions for 10 min each. 2.3 Microwave processing In parallel, the other part of each sample was processed using the Rapid Electron Microscope microwave device (REM, Fig. 1 ) utilizing the same specimen baskets ( Fig. 2 ) as for the LYNX-processor. This microwave bench unit is claimed by the manufacturer to have an even microwave distribution within the device resonant "cavity" ( Visinoni et al., 1998 ) during the processing. Each vial, containing the samples immersed in the process solutions, is placed in a special designed safety carrier, which locates the vial in a defined position in the microwave "cavity". A non-contact infra-red temperature sensor measures the current solution temperature in the vial, while a magnetic stirrer is ensuring uniform heat distribution in the vial solution. The solution temperature is the critical parameter to monitor the magnetron wattage (maximum 700 W) power output, which is controlled via a feedback loop during the continuous microwave irradiation of the sample. The graphical display of the slope of temperature rise, irradiation temperature stabilization and time for each processing step, could be easily defined on a dedicated touch screen monitor. The whole microwave-assisted processing is controlled by a microprocessor and dedicated software package. This software also allows routine documentation of the entire microwave process for Quality Assurance and Quality Control. Fig. 1 Milestone rapid electron microscopy (REM) histoprocessor. Note the vial with processed samples placed in a predefined position in the microwave "oven". Fig. 2 Sample preparation tools for the REM. The baskets are the same as for the automated LYNX-processor. As outlined in Fig. 3 , also the primary fixation of the "fresh" samples ( Drosophila flies infected with microsporidia and liver tissue samples) with an adjusted temperature of 50 °C, not merely the OsO 4 -post-fixation, ethanol-dehydration, Epon-infiltration and polymerization (BEEM-capsules, under water) was carried out with microwave irradiation of the samples. This completely microwave-assisted sample processing required 4 h 25 min, except handling time, e.g., for solution exchange. In order to shorten the process further, we tested also an abbreviated microwave-assisted protocol with time reduction in all steps with the SARS-coronavirus infected cells, the B. anthracis , and human surgical liver tissue samples; here the microwave processing time was 3 h 20 min ( Table 1 ). Fig. 3 Protocol outline of the microwave-assisted specimen processing. Table 1 Comparison of processing times for conventional and microwave-assisted protocols Processing step Conventional (LYNX) Microwave Microwave shortened Primary fixation ≥2 h 20 min 15 min Buffer washes 1 h 9 min 3 min Osmium post-fixation 2 h 20 min 10 min Water washes 40 min 6 min 3 min Dehydration 1 h 45 min 20 min 10 min Infiltration 19 h 1 h 25 min 59 min Polymerization 48 h 1 h 45 min 1 h 40 min Total ≥74 h 25 min 4 h 25 min 3 h 20 min The polymerized Epon blocks were sectioned and stained in the same manner as described for the conventionally processed samples. 2.4 Evaluation of the sections and images The sections were examined in a LEO 912AB transmission electron microscope (Zeiss, Oberkochen, Germany) operating at 80 kV in zero-loss mode and equipped with a side-entry and a bottom-mounted fiber-optic coupled CCD-camera (Proscan, Lagerlechfeld, Germany) capable to record images with 1024 × 1024 pixels. Efforts were taken to standardize the conditions of image acquisition (constant beam brightness, exposure time and magnification settings) during the EM examination. Imaging and measuring were done with the analySIS software, version 3.2 (Soft Imaging System GmbH, Muenster, Germany), without any digital image post-processing manipulations. Notes of the section quality and beam stability during the examination were made, and the quality of the images obtained by the conventional versus microwave-assisted procedures was evaluated visually on a calibrated CRT monitor and by high-resolution laser prints. 3 Results A significant processing time reduction was realized between the conventional (3 days) and microwave-assisted protocol (4 h 25 min or 3 h 20 min, Table 1 ). As the solution exchange in the microwave "oven" is not automated, it had to be performed manually as well as the transfer of the samples into the BEEM-capsules for polymerization (including probe identity tags mounting), which required additional time depending on the number of samples processed in parallel (approximately 1 h). The microwave processed resin blocks were polymerized uniformly and revealed good sectioning properties for semi- and ultrathin sections. The sections examined in the EM were stable in the electron beam. The images were crisp displaying a high and uniform contrast. After microwave-assisted processing the fine structure of virus-infected cells and infectious agents was well preserved ( Fig. 4 , Fig. 5 , Fig. 6 , Fig. 7 , Figs. 8 and 9 , Fig. 10 , Fig. 11 ). The SARS-coronaviruses can be clearly recognized in the dilated perinuclear space and RER-cisternae, also the microtubules of the cytoskeleton and mitochondria of the host cells are visible in detail ( Fig. 4 b). In cells infected with the elephant Orthopox virus artificial extraction of the core material was much less common ( Fig. 5 ). This is a phenomenon often experienced in conventional embedding preparations. In the apathogenic B. anthracis and E. coli culture similar good preservation of the vegetative organisms as well as of mature Anthrax spores were achieved in both conventional LYNX and microwave-assisted embedding ( Fig. 6 , Fig. 7 ); each mature Anthrax spore displayed a clearly delineated endo- and exosporium covered with tiny surface microfilaments ( Fig. 6 b and d). Fig. 4 SARS-coronaviruses produced in Vero. 6 cell culture, conventional fixation (2.5% GA): (a), (b) REM-microwave embedding; (c), (d) LYNX-embedding; (e), (f) external embedding (Robert Koch Institute, Berlin). Original magnification: (a), (c), (e) 1250×; (b), (d), (f) 10,000×. Fig. 5 Orthopox virus isolated from an elephant (cell culture), conventional fixation (2.5% GA): (a), (b) REM-microwave embedding; (c), (d) LYNX-embedding; (e), (f) external embedding (Robert Koch Institute, Berlin). Original magnification: (a), (c), (e) 1250×; (b), (d), (f) 20,000×. Fig. 6 Bacillus anthracis , conventional fixation (4% GA). (a), (b) REM-microwave embedding; (c), (d) LYNX-embedding. Original magnification: (a), (b), (c) 8000×; (d) 10,000×. Fig. 7 Escherichia coli , conventional fixation (4% GA), Medical Service of Federal Armed Forces, Koblenz: (a) REM-microwave embedding; (b) LYNX-embedding. Original magnification: 8000×. Figs. 8 and 9 (8) Leishmania major , conventional fixation (2% GA). (a) REM-microwave embedding; (b) LYNX-embedding; (c) external embedding (Bernhard-Nocht-Institute for Tropical Medicine, Hamburg). Original magnification: 3150×. (9) Malaria Plasmodium falciparum , conventional fixation (2% GA): (a) REM-microwave embedding; (b) LYNX-embedding; (c) external embedding (Bernhard-Nocht-Institute for Tropical Medicine, Hamburg). Original magnification: 3150×. Fig. 10 Caenorhabditis elegans , conventional fixation (2% GA); (a) REM-microwave embedding; (b) LYNX-embedding; (c) external embedding (Bernhard-Nocht-Institute for Tropical Medicine, Hamburg). Original magnification (side-entry camera): 10,000×. Fig. 11 Microsporidia Tubulinosema ratisbonensis from infected Drosophila melanogaster flies (modified Karnovsky-fixative): (a) REM-microwave fixation and embedding; (b) conventional fixation, LYNX-embedding. Original magnification: 6300×. In the protozoon L. major , the causative agent for tropical sore, the kinetochore–mitochondrium complex – which is the ultrastructural hallmark of the flagellated promastigote form – was preserved excellently in all procedures used ( Fig. 8 ). The malaria merozoites of Pl. falciparum had well discernible nuclei and vacuoles with typical pigment inclusions ( Fig. 9 ). The cross-sections through the adult worm C. elegans body displayed very well-preserved layers of the cuticula with underlying muscle cells and the internal organs in all preparations ( Fig. 10 ). The very good ultrastructural preservation of the mitochondria was a notable feature of this specimen ( Fig. 10 a). In the infected Drosophila flies, microsporidia were observed in all body compartments enclosed by the chitine external skeleton (lateral incised for better solution infusion). Well-preserved mature spores were abundant in the completely microwave-assisted fixed and embedded samples ( Fig. 11 ) often showing an uniformly delineated space between the endo- and exosporium ( Fig. 11 a). The mouse liver samples, which were microwave-assisted fixed and thereafter alternatively conventional or microwave-assisted processed and embedded, showed excellently preserved ultrastructural criteria of the hepatocytes as well as the glycogen and lipid inclusions ( Fig. 12 ). At higher magnification fine structural details, e.g., the double membrane profiles of the nucleus and mitochondria as well as the cytoplasmic and mitochondrial matrix, did not appear to differ between the two procedures ( Fig. 12 b and d). This high quality ultrastructure preservation was obtained in samples not exceeding 1-mm edge length. In larger samples, the centres of the tissue blocks showed in semithin sections a reduced staining intensity, in ultrathin sections cell shrinkage (dilated intercellular and Disse space), cytoplasmic matrix disruptions and artificial cavity formation as well as loss of mitochondrial fine structure ( Fig. 13 ). Fig. 12 Liver (mouse), REM-microwave fixation (modified Karnovsky-fixative): (a), (b) REM-microwave embedding; (c), (d) LYNX-embedding. Original magnification: (a), (c) 1250×; (b), (d) 5000×. Fig. 13 Liver (mouse), REM-microwave fixation (modified Karnovsky-fixative). A hepatocyte selected from the centre of a large (2 mm × 2 mm × 3 mm) tissue block displaying artificial cavity formation in the perinuclear cytoplasm and loss of mitochondrial fine structure. Original magnification: 5000×. Noteworthy, also with the application of the shortened (3 h 20 min) microwave-assisted protocol convincing results were achieved: a SARS-virus producing cell ( Fig. 14 a), a mature B. anthracis spore ( Fig. 14 b), and the human surgical liver sample ( Fig. 14 c) displayed good quality of the examined ultrastructure. The cytoplasm and organelles of cells and the pathogenic agents, however, clearly differed in densities ( Fig. 6 , Fig. 12 ), as there was more "compactness" and also the membranes appeared coarser, in comparison with the 4.5 h protocol preparation. Fig. 14 Reduced microwave-assisted processing protocol: 3 h 20 min: (a) SARS-coronavirus; (b) Bacillus anthracis spore; (c) liver (human), REM-microwave fixation. Original magnification: (a) 20,000×; (b) 10,000×; (c) 5000×. 4 Discussion Short turnaround times as well as reliable good section and image quality are important criteria when rapid embedding protocols are considered. Comparing the samples processed according to a microwave-assisted versus a conventional protocol, we obtained very satisfying results with the microwave bench "oven". The experienced reduction in sample processing time from days to hours with no compromised ultrastructural preservation of selected "difficult" specimens is in agreement with other workers using this technology. Microwave processing time of 3–4 h ( Nordhausen and Barr, 2001 , Gerrity and Forbes, 2003 , Laboux et al., 2004 ) and short as 2 h ( Giberson et al., 2003 ) were reported from EM laboratories with similar sample spectrum and workload as those of the authors. The microwave-assisted processed samples were comparable or superior in section and image quality to the conventionally processed samples and compliant to published standards ( Morioka et al., 1992 , Miller, 2003 , Peiris et al., 2003 , Buchanan, 2004 , Celandroni et al., 2004 , Franzen et al., 2005 , Larsson, 2005 ). The completely microwave-assisted processed parenchymal tissue of liver, used in our comparative approach as a substitute for other pathological solid tissues, revealed good results as shown in Fig. 12 in samples not exceeding 1-mm edge length. In the centre of the block where larger samples had been taken, there was a loss of ultrastructural preservation, and artefacts were seen such as have been described previously by Wild et al. (1989) . This phenomenon reflects the continuing discussion about the probably "dichotomous" nature of the non-ionizing microwave radiation in respect to the "heat" and the "non-thermal" or direct microwave effect ( Galvez et al., 2004 ). Despite the complex aspects influencing the aldehyde fixation process of tissues ( Fox et al., 1985 , Leong and Sormunen, 1998 , Izumi et al., 2000 , Werner et al., 2000 , Giberson and Elliott, 2001 ), recent observations tend to support the existence of the direct mechanism of microwave energy action ( Kok and Boon, 2003 , Leria et al., 2004 , Wendt et al., 2004 , De la Hoz et al., 2005 , Gaber et al., 2005 ). This needs to be considered in the microwave-assisted tissue processing because also our results suggest, that an "overheating" inside the solid tissue samples can adversely affect tissue ultrastructure. Therefore, further development work should include efforts to reduce the critical "thermal" effect and increase direct "non-thermal" microwave action during tissue fixation ( Galvez et al., 2004 , Leria et al., 2004 ). The REM Milestone microwave device is run with the sample baskets that are used also in the widely applied automated LYNX-processor for routine conventional tissue embedding. We observed that this shared equipment is a great benefit for the EM laboratory, e.g., a sample received late afternoon can be rapidly fixed in the microwave (e.g., 10–20 min in place of at least 2 h at room temperature) and added (without basket change) to other tissues which are to be processed automatically in the LYNX overnight anyway, and if urgent, rapidly polymerized in the microwave in 1 h 45 min the next morning (instead of 48 h). This option generates more flexibility of the laboratory workflow in handling clinical urgent samples and improves the economic employment of the available manpower. Thus, a "same-day" electron microscopy diagnosis for cells and tissues becomes reality. The results obtained with the shortened protocol appear especially promising. As judged from sectioning properties and section quality, the samples were uniformly polymerized, i.e., comparable to the results with the longer microwave protocol. The observed denser appearance of cell cytoplasm could be explained by less extraction of cell material during the shorter course of the complete procedure. Therefore, in view of the acceptable results with the approximately 3.5 h protocol, attempts to cut down further the preparation schedule appear worthwhile. Clearly, urgent samples reaching the diagnostic laboratory at unpredictable daytime need to be examined immediately in potential bioterrorism scenario or in case of an emerging agent disease ( Lane et al., 2001 , Morens et al., 2004 ). The microwave-assisted sample preparation enables EM to evaluate also thin sections for rapid diagnosis. Basing on our experience, the subsequent section ultramicrotomy, staining and examination in the EM can be completed in about 2 h. In consequence, using the potential of accelerated section preparation, EM-diagnosis from biopsy material can be rendered on the same workday. Thus, both negative staining of small particulate suspensions – from the smallest viruses up to bacteria, spores and parasites – as well as tissues and diagnostic cell cultures can be scrutinized by the "open view" of EM ( Hazelton and Gelderblom, 2003 ) as part of the "frontline" diagnostic methods guiding further measures, e.g., containment and therapeutic activities. In combination with digital image acquisition and telemicroscopy networking tools ( Schroeder et al., 2001 ) crucial or interesting findings could be consulted or shared worldwide in very short time.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124246/

Public health and clinical laboratories: Partners in the age of emerging infections

Clinical and public health laboratories have experienced unprecedented challenges in the form of demands to comply with revised regulations and economic pressures to be more efficient while preparing to respond to everything from pandemic influenza to bioterrorism. These forces have been an impetus for laboratorians to communicate, cooperate, and collaborate as never before and to seek the common ground where knowledge and resources can be shared to weather the profound economic and political forces at work today. The appearance of newly emerging and reemergent infections caused by agents of foodborne illness, anthrax, smallpox, plague, influenza, and other diseases has fostered cooperative network enterprises between clinical and public health laboratories, allowing the early detection of outbreaks of common and unusual pathogens and the measurement of the effectiveness of public health measures. Introduction The pace of change in the laboratory community continues to accelerate. Both clinical and public health laboratories have experienced unprecedented challenges. Demands for compliance with revised regulations have arrived at the same time as the need for laboratories to be prepared to respond to everything from pandemic influenza to bioterrorism. These forces have swept together clinical and public health laboratorians, as well as laboratory workers from other arenas. Laboratorians are finding the need to communicate, cooperate, and collaborate as never before and are seeking the common ground in which to share knowledge and resources to weather the profound economic and political forces at work today. In 1997, a Health and Human Services-commissioned report by the Lewin Group ( 1 ) identified the trends that were affecting public health and clinical laboratories. Those forces are still at work. Table 1 is a summary of some of the economic forces, including the rise of managed care organizations and their attendant accumulation of bargaining power with reference laboratories. In the face of ever-rising health care costs, hospitals are enduring unrelenting pressure to reduce laboratory costs, resulting in the formation of consortium laboratories among hospital groups, referrals of specimens to private reference laboratories, closings of smaller hospital laboratories, and reductions in laboratory staff. Table 1 Market economic forces Managed care organizations and a few private laboratory companies dominate market Cost reductions are the driving force Reimbursement rates for laboratory services are falling Consolidation of reference laboratories Consolidation of hospital laboratories Table 2 shows the forces at work for clinical laboratories, which are fighting battles on two fronts to maintain staff levels against further erosion. Management is asking for workforce reductions, while fewer qualified applicants are available for open positions. With the closing of many medical technology schools over the past two decades, graduating medical technologists, clinical laboratory scientists, and technicians number less than half of projected workforce needs. At the same time, the federal Health Insurance Portability and Accountability Act (HIPAA) has placed new demands for secure patient information handling, adding another burden on laboratory staff ( 1 , 2 ). Table 2 What is happening to clinical laboratories? Staff shortages, aging workforce, fewer choosing laboratory for career Downsizing, consolidation, partnerships Laboratory management focused on cost control Reimbursement driving changes in laboratory activity Increasing regulatory demands from HIPAA, CLIA Table 3 lists the forces at work with public health laboratories. While engaged in traditional public health activities, these laboratories are experiencing multiple new pressures, including HIPAA and CLIA regulatory compliance issues, the need to be prepared for analytic response to bioterrorism acts, loss of state funding with a parallel need to bill Medicaid for patient testing and provide testing on a fee-for-service basis. While a significant amount of funding support and technology transfer has accompanied the charge to state and local public health laboratories to develop capability for bioterrorism testing, state funding for traditional public health testing has withered ( 1 ). Table 3 What is happening to public health laboratories? Fee-for-service testing on the rise, state support decreasing Federal support for bioterrorism preparedness Medicaid-reimbursed laboratory testing Emerging infectious diseases Regulatory demands increasing, such as HIPAA, CLIA Select agent certification and security issues In this setting, new, emerging infections continue to arise. Emerging infections have required public health laboratories to be able to test accurately for many new agents, either confirming clinical laboratory findings or testing specimens referred from clinical laboratories. The prospect of a continued emergence of newly recognized pathogens or reemergent agents is waxing strong. In spite of all these issues, or because of them, public health and clinical laboratories are working together to surmount the challenges and provide quality in both patient care and public health laboratory testing. Several new initiatives have been launched to facilitate the collaboration of laboratorians, but while most public health laboratorians know what clinical laboratories do, the reverse is often not true. Table 4 illustrates the parallel, but different, roles each plays. Clinical laboratories are focused on patient care, while public health laboratories are focused on the health of the entire population. Clinical laboratory information is directed to physicians, and from them, ends at patient care. In contrast, public health laboratories gather information for public health experts and epidemiologists, who must determine the cause of illnesses and outbreaks affecting patients in many hospitals, detect new and unusual threats to the health of the community, and respond to these threats and prevent them. These roles are complementary, and as many laboratory professionals are discovering, mutually supportive. Table 4 Comparison of clinical and public health laboratory roles Clinical Public health Health (sick) care Health of public Provide information regarding a PATIENT to PHYSICIAN Provide information regarding POPULATION to public health PROFESSIONALS Diagnostic, therapeutic, and disease management testing Some diagnostic testing, screening, epidemiologic typing, human and environmental testing Outcome: recovery from illness Outcome: health of population; detection and intervention; prevention The 1988 Institute of Medicine report, The Future of Public Health ( 3 ), defined for the first time the functions of public health as (i) assessment, (ii) policy development, and (iii) assurance. These functions could be translated as follows. (i) What illness is afflicting the population and what is the cause? (ii) After knowing what is wrong and what the cause is, what laws or regulations must be enacted, and what testing must be conducted to ensure remediation and control of the problem? (iii) How can we be assured the problem remains under control? The public health laboratory has a role in all three of these elements. The public health laboratory should be able to respond to outbreaks, provide and manage analytic information, monitor the environment, advocate for high-quality testing, and provide reference testing services, training, and leadership in the overall effort to secure health for all ( 4 ). In recent years, emerging infections have arrived unpredictably but frequently. Table 5 shows some challenges that have confronted medical and pub-lic health authorities and their support laboratories during the last 4 years. Recently, Cockerill and Smith ( 5 ) provided their perspective on the impact of emerging infectious diseases on clinical laboratories, promoting the adoption of advanced nucleic acid amplification methods. While this technical advance is certainly desirable, it may not be economically feasible for most clinical laboratories. The authors point out the recently developed capability of public health laboratories for testing for SARS, coronavirus, and variola as examples of agents that can be identified through cooperation between clinical and public health laboratories. Table 5 Recent emerging infections Disease, year Anthrax, 2001 Multistate foodborne illness outbreaks, ongoing West Nile virus, 1999 to present Smallpox, 2003 Monkeypox, 2003 SARS, 2003 and 2004 Avian influenza, 2004-2005 The need for cooperation and collaboration among laboratories has been long recognized by public health experts. In 1994, a CDC strategy document ( 6 ) outlined the need for the formation of networks to gather information to detect, report, and analyze information regarding infection. This document became the foundation for actions that led to the formation of networks that are cementing bonds between clinical and public health laboratories. PulseNet Clinical laboratories have traditionally referred isolates to public health laboratories for confirmation and serotyping, but beginning in 1998, these isolates were subjected to a new powerful method to type the strains. Public health laboratories began to use pulsed-field gel electrophoresis (PFGE) to type isolates of Escherichia coli O157:H7 and other foodborne-illness pathogens, subdividing these isolates into closely related groups or clones, and allowing epidemiologists to efficiently investigate cases of infections and determine the cause. Often, clusters of cases are seen immediately by public health laboratory scientists when a group of isolates with a unique pattern are observed. Epidemiologists can investigate smaller numbers of cases and spend less time collecting the information that points to a particular food product. This has occurred many times since the initiation of the PulseNet system. Outbreaks caused by Salmonella spp., Shigella spp., E. coli O157, and Camplylobacter and Listeria spp. are detected, and the numbers of cases are limited when the incriminated food product is determined and recalled. Clinical laboratories are being encouraged to refer such pathogens quickly, rather than letting a group of isolates accumulate for convenient transport to a state laboratory, where outbreaks of foodborne illness can be identified rapidly, resulting in limitations on the extent of outbreaks ( 7 ). PFGE patterns uploaded by public health laboratories to the CDC PulseNet database are examined by epidemiologists looking for the appearance of clusters that cross state borders. Numerous multi-state and national foodborne-illness outbreaks have been uncovered that could not have been detected by other means. PulseNet works because clinical laboratories are effective in culturing agents of foodborne illness and referring these isolates quickly to public health laboratories that perform PFGE typing, conduct a statewide analysis, and then upload this information to the CDC database for nationwide analysis. This partnership has raised the level of detection of foodborne illness to unprecedented levels, virtually ensuring that no multistate outbreak of foodborne illness of any significant magnitude will go undetected. When three state laboratories determine a strain to be the same by PFGE typing, the recovery of a single isolate by each of three clinical microbiologists in three different states might be enough to detect a nationwide food problem. Emerging infections program The recovery and referral of isolates have done more than detect outbreaks of foodborne illnesses. By extremely accurate measurements of the number of infections in 10 states, the CDC's Emerging Infections Program (EIP), a program for surveillance of invasive bacterial infections, has shown that vaccines, such as the pneumococcal vaccine, are highly effective and virtually eliminate illness in vaccinated groups ( 8 ). FoodNet An EIP program called FoodNet is an alliance of 10 state public health programs and clinical laboratories, which, year-to-year, has determined the overall rates of sporadic cases (not outbreaks) caused by a number of foodborne pathogens and the factors associated with infection. The isolation of agents such as Salmonella, Shigella , and Listeria spp. and E. coli O157, which are recovered by clinical laboratories and referred to state public health laboratories, is doubly important because it provides critical information in revealing outbreaks and allows measurement of the overall disease burden of sporadic cases. Sporadic cases are those in which an outbreak has not occurred but some part of the food system has failed. Over the past 8 years, extremely accurate data have shown that the numbers of cases of illness due to some foodborne pathogens have declined ( 9 , 10 ). These data provide hard evidence that food safety measures are actually protecting people from foodborne illness. The new networks Initiatives in several states have spawned a new model for progressive collaboration between clinical and public health laboratories. With support from the CDC, states, and other agencies, cooperative state-based networks, such as the Colorado Laboratory Forum, the Minnesota Laboratory System, the Nebraska Laboratory System, the Washington State Clinical Laboratory Initiative, the Michigan Laboratory System, and groups in 10 other states, have been set up. Table 6 shows a broad array of activities in which these new networks are engaged, including improving communication, providing education and training, enhancing the quality of laboratory data, responding to emergencies, and sharing information. As an example, the Colorado Laboratory Forum consists of laboratorians from clinical, public health, veterinary, agricultural, forensic, environmental, federal, military, food, and research agencies, with the following goals: to facilitate communications, improve analytic capabilities of all member laboratories, and facilitate education and training. The goal is to solidify these state groups into a National Laboratory System ( 11 ). Table 6 New network activities Communications Education and training Quality assessment Emergency response Advisory group Information sharing Response to bioterrorism Clinical laboratorians can capture the first evidence of biologic-agent use by culturing the agent and conducting presumptive identification tests. During the anthrax spore attacks of 2001, it was a clinical microbiologist who, shortly after taking training provided by public health laboratories, recognized and referred the isolate from the first case of anthrax ( 12 ). Public health groups have provided training for clinical laboratorians to ensure that agents of anthrax, plague, and tularemia, rarely seen by any laboratory worker in the United States, are recognized and referred as soon as possible. The partnership is evident. Clinical microbiologists are at the front lines, handling, culturing, and testing primary specimens and providing the information needed to make the primary diagnosis. Public health microbiologists are in the best position to train clinical microbiologists about bioterrorism agents and then to rapidly confirm the identity of referred isolates. Laboratory response network Trained and equipped, public health laboratories are using leading-edge methods developed at the CDC, such as real-time PCR, luminex-type assays, and time-resolved fluorescence antigen immunoassays ( 13 ). These new methods have elevated identification accuracy to new heights while reducing the time needed for confirmation to a few hours rather than the many days conventional techniques typically require. Clinical laboratory microbiologists, by serving in the role of sentinels, have responded to the call, rapidly referring suspicious isolates to their partner public health laboratories, which can then either confirm the identity of or rule out a possible agent of bioterrorism. Conclusion Clinical and public health laboratories each have an indispensable function, and neither can be successful without the other. Many public health laboratory scientists have had to adopt a new leadership role for the state or city to which they are responsible, providing training; establishing safe and secure laboratory facilities for possessing, using, and maintaining select agents; channeling funding to clinical laboratories for bioterrorism preparedness and communications; and maintaining links between laboratories. The end result of these collaborative efforts is difficult to ascertain, but collectively, factors such as the reduction in the size and frequency of outbreaks and sporadic cases of infection, proof that food safety practices are effective, and validation of the efficacy of new vaccines can result in a healthier population, the goal of both medical care and public health. A healthier population means a reduction in the overall cost of health care related to infections. The alliance of clinical and public health laboratories promises to be a key component in slowing the rise of health insurance costs and managed care expenses.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4481173/

Neutrophils negatively regulate induction of mucosal IgA responses after sublingual immunization

Induction of mucosal IgA capable of providing a first line of defense against bacterial and viral pathogens remains a major goal of needle-free vaccines given via mucosal routes. Innate immune cells are known to play a central role in induction of IgA responses by mucosal vaccines, but the relative contribution of myeloid cell subsets to these responses has not firmly been established. Using an in vivo model of sublingual vaccination with Bacillus anthracis edema toxin (EdTx) as adjuvant, we examined the role of myeloid cell subsets for mucosal secretory IgA responses. Sublingual immunization of wild-type mice resulted in a transient increase of neutrophils in sublingual tissues and cervical lymph nodes. These mice later developed Ag-specific serum IgG responses, but not serum or mucosal IgA. Interestingly, EdTx failed to increase neutrophils in sublingual tissues of IKKβ ΔMye mice, and these mice developed IgA responses. Partial depletion of neutrophils before immunization of wild-type mice allowed the development of both mucosal and serum IgA responses. Finally, co-culture of B cells with neutrophils from either wild-type or IKKβ ΔMye mice suppressed production of IgA, but not IgM or IgG. These results identify a new role for neutrophils as negative regulators of IgA responses. INTRODUCTION Mucosal surfaces are constantly exposed to microorganisms and represent the main portal of entry of pathogens and toxins. Mucosal IgA or secretory IgA (SIgA) neutralizes pathogenic microorganisms and toxins, interferes with bacterial or viral colonization of the epithelium, and participates in homeostasis of mucosal tissues 1 . Ideally, vaccines capable of promoting both IgG in the bloodstream and SIgA in mucosal tissues would provide two layers of defense for optimal protection against infectious agents. Injected vaccines containing alum, the most widely used adjuvant, induce serum IgG responses, but unlike experimental mucosal adjuvants, fails to promote SIgA responses 2 , 3 . Cholera toxin (CT) and the related heat labile toxin I of E. coli (LT) are the most studied experimental adjuvants for induction of SIgA 4 , however, their inherent toxicity precludes their use in oral or nasal vaccines. Cytokines play a crucial role in shaping the profile of T helper cytokine responses as well as the Ig isotype and subclass responses. Previous studies have shown that the mucosal adjuvant CT induces pro-inflammatory cytokine ( i.e. , IL-6 or IL-1β) secretion by antigen presenting cells ( i.e., macrophages and dendritic cells) 5 , 6 . Cholera toxin also induces TGF-β and IL-10, two anti-inflammatory cytokines that play a central role in the induction of SIgA 6 – 8 . Studies with live bacterial and viral vectors as well as immunization studies with Th1-inducing cytokines ( i.e., IL-12 and IL-18) have now established that SIgA can also be induced in the context of Th1-biased responses 4 . More recently, the ability of CT as adjuvant to promote SIgA responses was impaired in mice lacking IL-17A, suggesting a role for IL-17A or related signaling in SIgA responses 6 . In this regard, differentiation of Th17 cells requires IL-1β, IL-6 and TGF-β 6 , 9 , which are cytokines that support IgA responses. Unlike Th1 and Th2 cytokines, which activate JAK–STAT signaling pathways, signaling through IL-17R activates Act1 for subsequent activation of the classical NF-κB signaling pathway 10 . Furthermore, IL-17A directly triggers Ig class switching to IgG2a and IgG3, but not to IgG1 11 . To our knowledge, it is still unclear whether production of IgA is directly regulated by IL-17A/IL-17RA signaling in B cells. The nuclear factor κB (NF-κB) pathway plays an important role in inflammatory responses and a number of stimuli can lead to NF-κB translocation to the nucleus 12 . Previous studies have shown that the NF-κB pathway can mediate both pro- and anti-inflammatory effects 13 , 14 depending on the immune cells in which the IKKβ-NF-κB signaling occurs 15 and stimuli to which they are exposed. A recent study showed a link between activation of the non-canonical NF-kB pathway in B cells and their ability to undergo immunoglobulin class switch for production of IgA 16 . However, it remains unclear if IKKβ-dependent signaling in myeloid cells (IKKβ ΔMye ) regulates IgA responses to mucosal vaccination. Sublingual tissues have been used as a delivery site for bacterial and viral vaccines 17 , 18 , and cervical lymph nodes (CLNs) were identified as the primary site of antigen presentation after sublingual immunization 19 . However, how innate immune cells in sublingual tissues and/or CLNs regulate antibody production remains unknown. Edema toxin (EdTx) is one of the exotoxins produced by the Gram-positive, spore-forming rod Bacillus anthracis 20 . EdTx is composed of two subunits: a binding subunit and an enzymatic subunit. The binding subunit, or protective Ag (PA), allows the binding of these toxins to the anthrax toxin receptors that are expressed by most cells. The enzymatic subunit, or edema factor (EF), is a calmodulin- and calcium-dependent adenylate cyclase that catalyzes the conversion of ATP to cAMP 20 , 21 . We previously showed that EdTx is a mucosal adjuvant that promotes mucosal and systemic immunity to intranasally co-administered vaccine antigens 22 , 23 . These studies addressed the contribution of monocytes/macrophages to mucosal SIgA responses to sublingual immunization. Using Bacillus anthracis edema toxin (EdTx) as a model of vaccine adjuvant to target anthrax toxin receptors, we show a previously unknown role of neutrophils as negative regulators of IgA responses. Thus, recruitment of neutrophils into sublingual tissues shortly after sublingual immunization impaired the development of IgA responses. The negative role of neutrophils in IgA responses was confirmed in vivo by depletion of neutrophils before immunization with EdTx and in vitro , by co-culture of B cells with neutrophils. RESULTS Toxin adjuvants differentially recruit myeloid-lineage cells into sublingual tissues Both CT 24 and EdTx 22 , 23 are mucosal adjuvants that promote mucosal SIgA responses via the nasal route. CT can also promote IgA responses when used as adjuvant for vaccines given by the epicutaneous route or topically on the sublingual mucosa 19 , 25 . In contrast, EdTx was not effective at inducing IgA when used as an epicutaneous (Duverger et al, unpublished observation) or sublingual adjuvant. To elucidate the mechanism underlying the inability of EdTx to induce IgA by sublingual route, we first analyzed innate cell subsets present in sublingual tissues after sublingual application of EdTx. The number of CD11b + myeloid cells increased in the sublingual tissues of mice 3 hours after application of EdTx, but not CT ( Figure 1A ). Flow cytometric 26 and morphologic analysis of the myeloid cell subsets ( Figures S1, 1B, 1C, and 1D ) in sublingual tissues 3 hours after application of EdTx showed high a frequency (32%) of CD11b + F4/80 − Gr-1 high cells (neutrophils), and a lower frequency (12%) of CD11b + F4/80 + Gr-1 − cells (non-inflammatory monocytes). The frequencies of CD11b + F4/80 + Gr-1 low cells (macrophages or DCs) and CD11b + F4/80 + Gr-1 high (inflammatory monocytes) were not affected by EdTx. In contrast with EdTx, CT increased the frequency (34% vs. 26%) of macrophages/DC ( Figure 1C and 1D ). Edema toxin does not recruit neutrophils into sublingual tissues of mice lacking IKKβ in myeloid cells The adjuvant activities of CT and EdTx involve pro-inflammatory responses and acquisition of antigen-presenting cell functions by myeloid cells 22 , 23 , 27 , 28 . The transcription factor NF-κB is a master regulator of cytokine responses and migration of innate cells 29 . We previously showed that activation of NF-κB in mouse epithelial cells lacking IKKβ, and with impaired ability for nuclear translocation of phospoNF-κB p65, resulted in increased pSTAT3 responses in gut tissues 30 . Thus, we examined how EdTx affects the expression of STAT3 in sublingual tissues of control and IKKβ ΔMye mice, which lack IKKβ in myeloid cells. As depicted in Figures 2A and S2 , pSTAT3 levels were low in tissues of mice that received saline and increased after application of EdTx. In contrast, pNF-κB levels were higher in tissues of PBS-than in EdTx-treated mice. Although the transcription factors appeared to be regulated in the opposite direction after EdTx-treatment, the difference in their levels of expression failed to reach statistical significance during the time frame analyzed ( Figures 2A and S2 ). We also analyzed myeloid cells in sublingual tissues at 3 and 6 hours after application of EdTx ( Figures 2B, 2C and S3 ). Unlike control C57BL/6 mice, the IKKβ ΔMye mice did not exhibit an important increase in the frequency of CD11b + cells in the sublingual tissues 3 hours after application of EdTx ( Figure 2B ). Control C57BL/6 and IKKβ ΔMye mice exhibited a similar proportion of myeloid cell subsets before application of EdTx, except for non-inflammatory monocytes, which were higher in IKKβ ΔMye than in control C57BL/6 mice ( Figure 2C and Figure S3 ). Three hours after application of EdTx, sublingual tissues of IKKβ ΔMye mice showed significantly lower frequencies of neutrophils when compared to C57BL/6 ( Figure 2C and Figure S3 ). Edema toxin was reported to differentially affect the recruitment and cytokine secretion by some immune cells 31 . Therefore, we also examined the expression of CCL2 and CXCL2, two chemokines known to recruit inflammatory monocytes and neutrophils, respectively. Sublingual tissue cells of control C57BL/6 and IKKβ ΔMye mice had similar basal levels of CCL2 and CXCL2 mRNA, and exhibited similar kinetics and magnitude of responses after exposure to EdTx ( Figure S4 ). We also examined the expression of CCR2 and CXCR2, and CCL2 and CXCL2 receptors in myeloid cell subsets found in sublingual tissues 3 hours after application of EdTx in vivo ( Figure 3 ). Since leukotriene B4 could mediate chemotaxis of macrophages and granulocytes 32 , 33 , the expression of the leukotriene B4 receptor (LTB4R2) was also investigated. Neutrophils collected in sublingual tissues of C57BL/6 and IKKβ ΔMye mice exhibited similar profiles of receptor expression ( Figures 3B, 3C ). On the other hand, macrophages/DCs and non-inflammatory monocytes collected in sublingual tissues of IKKβ ΔMye mice exhibited higher frequencies of CCR2 + , CXCR2 + and LTB4R2 + cells. Alone, these results cannot explain the higher number of neutrophils in the sublingual tissues of C57BL/6. The pie diagram ( Figure 3C ), which summarizes the relative contribution of each receptor in myeloid cell subsets shows a broader profile of receptor expression in macrophages/DCs and non-inflammatory monocytes of IKKβ ΔMye mice. Thus, these cells may have a competitive advantage for responding to chemo-attractant signals via ligand binding to these receptors. IKKβ deficiency in myeloid cells enhances the adjuvant activity of EdTx for sublingual immunization and promotes Ag-specific SIgA responses We next asked whether the broader expression of chemokine and leukotriene B4 receptors by macrophage/DC and non-inflammatory monocytes in sublingual tissues of IKKβ ΔMye mice after application of EdTx could affect the profile of immune responses induced by this adjuvant. For this purpose, mice were immunized via the sublingual route with recombinant Yersinia pestis F1-V antigen and Bacillus anthracis EdTx as adjuvant. Sublingual co-application of EdTx enhanced antigen-specific serum IgG responses (IgG and IgG1) and no difference was seen between control C57BL/6 and IKKβ ΔMye mice ( Figure 4A ). The same levels of IgE responses were seen in control C57BL/6 and IKKβ ΔMye mice, suggesting that Th2- dependent Abs were not affected in IKKβ ΔMye mice. On the other hand, IKKβ ΔMye mice exhibited enhanced IgG2c responses ( Figure 4A ). Interestingly, unlike control C57BL/6 mice, IKKβ ΔMye mice developed antigen-specific serum IgA responses, ( Figure 4A ). The increase in serum IgA responses in IKKβ ΔMye mice was associated with antigen-specific SIgA in the saliva, vaginal washes, and fecal extracts ( Figure 4B ). We also asked whether the specificity and function of Ab induced by EdTx as sublingual adjuvant were affected by the absence of IKKβ-dependent signaling in myeloid cells. After sublingual immunization with F1-V alone, control C57BL/6 and IKKβ ΔMye mice developed IgG Abs, which were directed against the same peptide ( i.e. , P 1 – 17 or P1) of the F1-capsular antigen ( Figure 4C and Table 1 ). EdTx as adjuvant promoted Abs that reacted to two additional epitope peptides in control C57BL/6 and IKKβ ΔMye mice. However, only one of the additional peptides (P19) was shared by Abs from control C57BL/6 and IKKβ ΔMye mice ( Figure 4C and Table 1 ). We also wondered whether the enhanced IgA responses seen in IKKβ ΔMye mice were restricted to FI-V as antigen and EdTx as adjuvant. Nasal immunization with EdTx is known to promote immunity against the EdTx binding subunit PA 22 , 23 . Sublingual immunization with EdTx also enhanced PA-specific serum IgG Ab titers in IKKβ ΔMye mice ( Figure S5A ) and this was consistent with the enhanced levels of PA-specific neutralizing Abs ( Figure S5B ). In addition, we found that serum and mucosal IgA responses induced by cholera toxin as a sublingual adjuvant were enhanced in IKKβ ΔMye mice ( Figure S6 ). Finally, we analyzed antigen- (i.e., F1-V)-specific T helper cytokine responses supported by EdTx as an adjuvant for sublingual vaccination. In wild-type C57BL/6 mice, the sublingual adjuvant EdTx enhanced the frequency of antigen-specific IFN-γ producing T helper (Th) cells in the spleen ( Figure 5 ). On the other hand, the IKKβ ΔMye mice exhibited a broader profile of Th cell-responses with a significant increase of antigen-specific IFN-γ + (Th1), IL-4 + (Th2), and IL- 17A + producing Th cells ( Figure 5 ). The frequency of neutrophils inversely correlates with production of IgA in cervical lymph nodes Cervical lymph nodes (CLNs) are considered inductive sites for adaptive immune responses after sublingual 19 and nasal 34 immunization. We have shown that 6 hours after application of EdTx, the frequency of CD11b + cells returned toward basal levels in sublingual tissues ( Figure 2B ). We hypothesized that cells had migrated to CLN and analyzed myeloid cell subsets in these lymphoid tissues. The frequency of neutrophils was significantly reduced in CLNs of IKKβ ΔMye compared to control C57BL/6 mice, while the other myeloid cell subsets remained unchanged ( Figure 6A ). CLN cells from EdTx-treated mice were then cultured in the presence of LPS. Three days later, we found a significantly higher number of IgA-secreting cells in IKKβ ΔMye than in control C57BL/6 mice ( Figure 6B ). Of interest, the number of IgA-secreting cells in the CLNs of both control C57BL/6 and IKKβ ΔMye mice were inversely correlated (r= −0.8) with the numbers of neutrophils in these tissues ( Figure 6C ). Reduction of neutrophils augmented the adjuvant effect of EdTx on Ag-specific IgA responses In order to further establish that an inverse correlation exists between the frequency of neutrophils in sublingual tissues and CLNs, and antigen-specific IgA responses, wild-type C57BL/6 were injected (i.p) with a neutrophil Ly6G-specific 1A8 monoclonal Ab 2 days before sublingual immunization with F1-V and EdTx as adjuvant. This treatment reduced the frequency of neutrophils in CLNs of wild-type C57BL/6 mice ( Figure S7A ) and C57BL/6 mice pre-treated with 1A8 (1A8+C57BL/6) contained virtually no neutrophils in sublingual tissues after application of EdTx ( Figure S7B ). These 1A8+C57BL/6 mice also gradually developed F1-V-specific serum IgA titers over the time points tested and reached higher serum IgA titers than non-treated C57BL/6 or IKKβ ΔMye mice at Day 28 ( Figure 6D ). Interestingly, depletion of neutrophils also enhanced mucosal IgA Ab-responses; 1A8+C57BL/6 mice produced high levels of F1-V-specific fecal IgA Abs, which were comparable to those measured in IKKβ ΔMye mice ( Figure 6D ). Neutrophils suppress production of IgA by B cells Our results clearly show that the ability to generate IgA responses is enhanced in the absence of IKKβ in myeloid cells or when the number of neutrophils is reduced. In addition, the ability of EdTx to induce systemic and mucosal IgA responses in IKKβ Δ Mye mice is associated with increased Th17 responses and production of IL-17A ( Figure 5 ). Thus, we next examined how alteration of canonical NF-κB mediated-signaling via IKKβ-deletion in myeloid cells (IKKβ Δ Mye ) could support Ig class switch and antibody production by B cells. For this purpose, CD11b-depleted spleen cells from C57BL/6 mice were co-cultured with 20% autologous CD11b + cells (C57BL/6 CD11b + ) or CD11b + cells from IKKβ Δ Mye mice (IKKβ Δ Mye CD11b + ) with or without EdTx in the presence of LPS 35 . After 5 days of culture, cells were segregated into IL-17RA low and IL-17RA high cells ( Figure S8A ). Co-culture with IKKβ Δ Mye CD11b + cells significantly increased the frequency of B220 + IL-17RA high B cells ( Figure S8B ). As shown in Figure 7A , in these cultures contained low frequencies of surface IgA cells among IL-17RA low B cells regardless of the presence of IKKβ Δ Mye CD11b + cells. Interestingly, high frequencies of IL- 17RA high B cells expressed surface IgA and co-culture with IKKβ Δ Mye CD11b + cells further increased these frequencies. To further elucidate signals that supported IgA responses, we analyzed mRNA levels of the B cell activators APRIL (a proliferation-inducing ligand) and BAFF (B cell activation factor of the TNF family) and activation-induced deaminase (AID). Addition of EdTx to cultures of spleen cells enhanced mRNA levels of APRIL, BAFF and AID, and the presence of IKKβ ΔMye CD11b + further enhanced BAFF- and AID-specific mRNA expression ( Figure S8C ). The presence of IKKβ ΔMye CD11b + did not affect EdTx-induced IL-1β and IL-6 mRNA levels, but increased EdTx-induced TNF-α, IL-23, IL-10, and Caspase-1 mRNA levels ( Figure S9 ). Taken together, these results show that myeloid cells lacking IKKβ provide a microenvironment favorable for Ig class switch and B cell production of IgA. To gain insight into the mechanism of how neutrophils affect IgA responses, B cells from C57BL/6 mice were co-cultured with or without neutrophils from C57BL/6 or IKKβ ΔMye mice for 5 days. The addition of neutrophils from either C57BL/6 or IKKβ ΔMye mice to cultures of B cells did not affect the secretion of IgM or IgG Abs into culture supernatants ( Figure 7B ). Interestingly, co-culture with neutrophils significantly reduced the amounts of IgA Abs secreted by B cells and this inhibitory effect was independent of the presence of functional IKKβ in neutrophils ( Figure 7B ). Finally, mRNA analysis of B cells co-cultured with neutrophils showed that neutrophils reduced the level of IgA heavy chain transcripts in B cells ( Figure 7C ). Toxin adjuvants differentially recruit myeloid-lineage cells into sublingual tissues Both CT 24 and EdTx 22 , 23 are mucosal adjuvants that promote mucosal SIgA responses via the nasal route. CT can also promote IgA responses when used as adjuvant for vaccines given by the epicutaneous route or topically on the sublingual mucosa 19 , 25 . In contrast, EdTx was not effective at inducing IgA when used as an epicutaneous (Duverger et al, unpublished observation) or sublingual adjuvant. To elucidate the mechanism underlying the inability of EdTx to induce IgA by sublingual route, we first analyzed innate cell subsets present in sublingual tissues after sublingual application of EdTx. The number of CD11b + myeloid cells increased in the sublingual tissues of mice 3 hours after application of EdTx, but not CT ( Figure 1A ). Flow cytometric 26 and morphologic analysis of the myeloid cell subsets ( Figures S1, 1B, 1C, and 1D ) in sublingual tissues 3 hours after application of EdTx showed high a frequency (32%) of CD11b + F4/80 − Gr-1 high cells (neutrophils), and a lower frequency (12%) of CD11b + F4/80 + Gr-1 − cells (non-inflammatory monocytes). The frequencies of CD11b + F4/80 + Gr-1 low cells (macrophages or DCs) and CD11b + F4/80 + Gr-1 high (inflammatory monocytes) were not affected by EdTx. In contrast with EdTx, CT increased the frequency (34% vs. 26%) of macrophages/DC ( Figure 1C and 1D ). Edema toxin does not recruit neutrophils into sublingual tissues of mice lacking IKKβ in myeloid cells The adjuvant activities of CT and EdTx involve pro-inflammatory responses and acquisition of antigen-presenting cell functions by myeloid cells 22 , 23 , 27 , 28 . The transcription factor NF-κB is a master regulator of cytokine responses and migration of innate cells 29 . We previously showed that activation of NF-κB in mouse epithelial cells lacking IKKβ, and with impaired ability for nuclear translocation of phospoNF-κB p65, resulted in increased pSTAT3 responses in gut tissues 30 . Thus, we examined how EdTx affects the expression of STAT3 in sublingual tissues of control and IKKβ ΔMye mice, which lack IKKβ in myeloid cells. As depicted in Figures 2A and S2 , pSTAT3 levels were low in tissues of mice that received saline and increased after application of EdTx. In contrast, pNF-κB levels were higher in tissues of PBS-than in EdTx-treated mice. Although the transcription factors appeared to be regulated in the opposite direction after EdTx-treatment, the difference in their levels of expression failed to reach statistical significance during the time frame analyzed ( Figures 2A and S2 ). We also analyzed myeloid cells in sublingual tissues at 3 and 6 hours after application of EdTx ( Figures 2B, 2C and S3 ). Unlike control C57BL/6 mice, the IKKβ ΔMye mice did not exhibit an important increase in the frequency of CD11b + cells in the sublingual tissues 3 hours after application of EdTx ( Figure 2B ). Control C57BL/6 and IKKβ ΔMye mice exhibited a similar proportion of myeloid cell subsets before application of EdTx, except for non-inflammatory monocytes, which were higher in IKKβ ΔMye than in control C57BL/6 mice ( Figure 2C and Figure S3 ). Three hours after application of EdTx, sublingual tissues of IKKβ ΔMye mice showed significantly lower frequencies of neutrophils when compared to C57BL/6 ( Figure 2C and Figure S3 ). Edema toxin was reported to differentially affect the recruitment and cytokine secretion by some immune cells 31 . Therefore, we also examined the expression of CCL2 and CXCL2, two chemokines known to recruit inflammatory monocytes and neutrophils, respectively. Sublingual tissue cells of control C57BL/6 and IKKβ ΔMye mice had similar basal levels of CCL2 and CXCL2 mRNA, and exhibited similar kinetics and magnitude of responses after exposure to EdTx ( Figure S4 ). We also examined the expression of CCR2 and CXCR2, and CCL2 and CXCL2 receptors in myeloid cell subsets found in sublingual tissues 3 hours after application of EdTx in vivo ( Figure 3 ). Since leukotriene B4 could mediate chemotaxis of macrophages and granulocytes 32 , 33 , the expression of the leukotriene B4 receptor (LTB4R2) was also investigated. Neutrophils collected in sublingual tissues of C57BL/6 and IKKβ ΔMye mice exhibited similar profiles of receptor expression ( Figures 3B, 3C ). On the other hand, macrophages/DCs and non-inflammatory monocytes collected in sublingual tissues of IKKβ ΔMye mice exhibited higher frequencies of CCR2 + , CXCR2 + and LTB4R2 + cells. Alone, these results cannot explain the higher number of neutrophils in the sublingual tissues of C57BL/6. The pie diagram ( Figure 3C ), which summarizes the relative contribution of each receptor in myeloid cell subsets shows a broader profile of receptor expression in macrophages/DCs and non-inflammatory monocytes of IKKβ ΔMye mice. Thus, these cells may have a competitive advantage for responding to chemo-attractant signals via ligand binding to these receptors. IKKβ deficiency in myeloid cells enhances the adjuvant activity of EdTx for sublingual immunization and promotes Ag-specific SIgA responses We next asked whether the broader expression of chemokine and leukotriene B4 receptors by macrophage/DC and non-inflammatory monocytes in sublingual tissues of IKKβ ΔMye mice after application of EdTx could affect the profile of immune responses induced by this adjuvant. For this purpose, mice were immunized via the sublingual route with recombinant Yersinia pestis F1-V antigen and Bacillus anthracis EdTx as adjuvant. Sublingual co-application of EdTx enhanced antigen-specific serum IgG responses (IgG and IgG1) and no difference was seen between control C57BL/6 and IKKβ ΔMye mice ( Figure 4A ). The same levels of IgE responses were seen in control C57BL/6 and IKKβ ΔMye mice, suggesting that Th2- dependent Abs were not affected in IKKβ ΔMye mice. On the other hand, IKKβ ΔMye mice exhibited enhanced IgG2c responses ( Figure 4A ). Interestingly, unlike control C57BL/6 mice, IKKβ ΔMye mice developed antigen-specific serum IgA responses, ( Figure 4A ). The increase in serum IgA responses in IKKβ ΔMye mice was associated with antigen-specific SIgA in the saliva, vaginal washes, and fecal extracts ( Figure 4B ). We also asked whether the specificity and function of Ab induced by EdTx as sublingual adjuvant were affected by the absence of IKKβ-dependent signaling in myeloid cells. After sublingual immunization with F1-V alone, control C57BL/6 and IKKβ ΔMye mice developed IgG Abs, which were directed against the same peptide ( i.e. , P 1 – 17 or P1) of the F1-capsular antigen ( Figure 4C and Table 1 ). EdTx as adjuvant promoted Abs that reacted to two additional epitope peptides in control C57BL/6 and IKKβ ΔMye mice. However, only one of the additional peptides (P19) was shared by Abs from control C57BL/6 and IKKβ ΔMye mice ( Figure 4C and Table 1 ). We also wondered whether the enhanced IgA responses seen in IKKβ ΔMye mice were restricted to FI-V as antigen and EdTx as adjuvant. Nasal immunization with EdTx is known to promote immunity against the EdTx binding subunit PA 22 , 23 . Sublingual immunization with EdTx also enhanced PA-specific serum IgG Ab titers in IKKβ ΔMye mice ( Figure S5A ) and this was consistent with the enhanced levels of PA-specific neutralizing Abs ( Figure S5B ). In addition, we found that serum and mucosal IgA responses induced by cholera toxin as a sublingual adjuvant were enhanced in IKKβ ΔMye mice ( Figure S6 ). Finally, we analyzed antigen- (i.e., F1-V)-specific T helper cytokine responses supported by EdTx as an adjuvant for sublingual vaccination. In wild-type C57BL/6 mice, the sublingual adjuvant EdTx enhanced the frequency of antigen-specific IFN-γ producing T helper (Th) cells in the spleen ( Figure 5 ). On the other hand, the IKKβ ΔMye mice exhibited a broader profile of Th cell-responses with a significant increase of antigen-specific IFN-γ + (Th1), IL-4 + (Th2), and IL- 17A + producing Th cells ( Figure 5 ). The frequency of neutrophils inversely correlates with production of IgA in cervical lymph nodes Cervical lymph nodes (CLNs) are considered inductive sites for adaptive immune responses after sublingual 19 and nasal 34 immunization. We have shown that 6 hours after application of EdTx, the frequency of CD11b + cells returned toward basal levels in sublingual tissues ( Figure 2B ). We hypothesized that cells had migrated to CLN and analyzed myeloid cell subsets in these lymphoid tissues. The frequency of neutrophils was significantly reduced in CLNs of IKKβ ΔMye compared to control C57BL/6 mice, while the other myeloid cell subsets remained unchanged ( Figure 6A ). CLN cells from EdTx-treated mice were then cultured in the presence of LPS. Three days later, we found a significantly higher number of IgA-secreting cells in IKKβ ΔMye than in control C57BL/6 mice ( Figure 6B ). Of interest, the number of IgA-secreting cells in the CLNs of both control C57BL/6 and IKKβ ΔMye mice were inversely correlated (r= −0.8) with the numbers of neutrophils in these tissues ( Figure 6C ). Reduction of neutrophils augmented the adjuvant effect of EdTx on Ag-specific IgA responses In order to further establish that an inverse correlation exists between the frequency of neutrophils in sublingual tissues and CLNs, and antigen-specific IgA responses, wild-type C57BL/6 were injected (i.p) with a neutrophil Ly6G-specific 1A8 monoclonal Ab 2 days before sublingual immunization with F1-V and EdTx as adjuvant. This treatment reduced the frequency of neutrophils in CLNs of wild-type C57BL/6 mice ( Figure S7A ) and C57BL/6 mice pre-treated with 1A8 (1A8+C57BL/6) contained virtually no neutrophils in sublingual tissues after application of EdTx ( Figure S7B ). These 1A8+C57BL/6 mice also gradually developed F1-V-specific serum IgA titers over the time points tested and reached higher serum IgA titers than non-treated C57BL/6 or IKKβ ΔMye mice at Day 28 ( Figure 6D ). Interestingly, depletion of neutrophils also enhanced mucosal IgA Ab-responses; 1A8+C57BL/6 mice produced high levels of F1-V-specific fecal IgA Abs, which were comparable to those measured in IKKβ ΔMye mice ( Figure 6D ). Neutrophils suppress production of IgA by B cells Our results clearly show that the ability to generate IgA responses is enhanced in the absence of IKKβ in myeloid cells or when the number of neutrophils is reduced. In addition, the ability of EdTx to induce systemic and mucosal IgA responses in IKKβ Δ Mye mice is associated with increased Th17 responses and production of IL-17A ( Figure 5 ). Thus, we next examined how alteration of canonical NF-κB mediated-signaling via IKKβ-deletion in myeloid cells (IKKβ Δ Mye ) could support Ig class switch and antibody production by B cells. For this purpose, CD11b-depleted spleen cells from C57BL/6 mice were co-cultured with 20% autologous CD11b + cells (C57BL/6 CD11b + ) or CD11b + cells from IKKβ Δ Mye mice (IKKβ Δ Mye CD11b + ) with or without EdTx in the presence of LPS 35 . After 5 days of culture, cells were segregated into IL-17RA low and IL-17RA high cells ( Figure S8A ). Co-culture with IKKβ Δ Mye CD11b + cells significantly increased the frequency of B220 + IL-17RA high B cells ( Figure S8B ). As shown in Figure 7A , in these cultures contained low frequencies of surface IgA cells among IL-17RA low B cells regardless of the presence of IKKβ Δ Mye CD11b + cells. Interestingly, high frequencies of IL- 17RA high B cells expressed surface IgA and co-culture with IKKβ Δ Mye CD11b + cells further increased these frequencies. To further elucidate signals that supported IgA responses, we analyzed mRNA levels of the B cell activators APRIL (a proliferation-inducing ligand) and BAFF (B cell activation factor of the TNF family) and activation-induced deaminase (AID). Addition of EdTx to cultures of spleen cells enhanced mRNA levels of APRIL, BAFF and AID, and the presence of IKKβ ΔMye CD11b + further enhanced BAFF- and AID-specific mRNA expression ( Figure S8C ). The presence of IKKβ ΔMye CD11b + did not affect EdTx-induced IL-1β and IL-6 mRNA levels, but increased EdTx-induced TNF-α, IL-23, IL-10, and Caspase-1 mRNA levels ( Figure S9 ). Taken together, these results show that myeloid cells lacking IKKβ provide a microenvironment favorable for Ig class switch and B cell production of IgA. To gain insight into the mechanism of how neutrophils affect IgA responses, B cells from C57BL/6 mice were co-cultured with or without neutrophils from C57BL/6 or IKKβ ΔMye mice for 5 days. The addition of neutrophils from either C57BL/6 or IKKβ ΔMye mice to cultures of B cells did not affect the secretion of IgM or IgG Abs into culture supernatants ( Figure 7B ). Interestingly, co-culture with neutrophils significantly reduced the amounts of IgA Abs secreted by B cells and this inhibitory effect was independent of the presence of functional IKKβ in neutrophils ( Figure 7B ). Finally, mRNA analysis of B cells co-cultured with neutrophils showed that neutrophils reduced the level of IgA heavy chain transcripts in B cells ( Figure 7C ). DISCUSSION Recent studies have identified sublingual immunization as a potentially safer alternative to nasal immunization. However, inductive sites for generating immune responses to sublingual immunization, the identity and function of the cells involved, and the signaling pathways for induction of SIgA via this mucosal route are poorly understood. Here we show that the ability of a sublingual vaccine to mount an SIgA response inversely correlates with the presence of neutrophils in sublingual tissue and CLNs. We also show that depletion of Gr1 + cells improves the development of IgA responses after sublingual immunization and that neutrophils impair the transcription of IgA heavy chain by B cells. This work also shows that myeloid cells lacking IKKβ-dependent NF-κB signaling provide an environment that supports the production of IgA by B cells. Alum is the most widely used adjuvant for injected vaccines. However, attempts to include alum in mucosal vaccines aimed at prompting SIgA responses have been unsuccessful because this adjuvant fails to effectively induce IgA 2 . Studies that addressed mechanisms underlying the adjuvant activity of alum have shown that alum acts via Gr1 + splenic myeloid cells expressing IL-4 to stimulate early B cell priming 36 . Other studies have shown that the NALP3 inflammasome was a crucial element in the adjuvant activity of alum by promoting the maturation of inflammatory cytokines 37 ; and furthermore, alum recruits inflammatory monocytes 38 . In other studies, intranasal co-administration of human neutrophil proteins enhanced antigen-specific serum IgG responses, but failed to promote SIgA responses 39 . These reports are consistent with our finding that less recruitment of neutrophils into sublingual tissues and CLNs of IKKβ ΔMye mice is a reliable indication of the ability of EdTx as adjuvant to promote SIgA responses. Because IgG production is not impaired by the recruitment of neutrophils, it is unlikely that neutrophils limit SIgA responses by limiting antigen access to antigen presenting cells or interactions between the latter and T cells as was previously suggested 40 . Induction of SIgA is well-known to require priming of effector cells in unique inductive sites 4 . Thus, our finding that the lower proportion of Gr-1 + inflammatory monocytes and/or higher proportion of Gr-1 − non-inflammatory monocytes in the sublingual tissue correlates with induction of broad Ab responses consisting of both serum IgG and SIgA responses is in agreement with the recent report that neutrophils also control the spread of T cell responses to distant lymph nodes 41 . The Gr-1 − monocytes, also described as tissue resident myeloid cells, have been classified as alternatively activated macrophages (M2 macrophages) capable of producing IL-10 and TGF-β 26 , 42 . Interestingly, these two cytokines are central for Ig class switch in B cells and for production of IgA. Experiments using IKKβ ΔMye mice provided new insights into signaling in the induction of SIgA responses. Previous studies have shown that the NF-κB pathway can mediate both pro-and anti-inflammatory effects 13 , 14 . Our data suggest that activation of IKKβ-NF-κB signaling in myeloid cells may in fact reduce their capacity to help B cells undergo Ig class switch for production of IgA. This finding is interesting in light of the recent report that the kinase TBK1 in B cells limits IgA class switch by negative regulation of the non-canonical NF-κB pathway 16 . Thus, stimulation of non-canonical NF-κB signaling either directly in B cells or in other antigen presenting cells could represent a major pathway for induction of IgA Abs. In this regard, we have recently shown that IKKβ deficiency in intestinal epithelial cells increases IgA responses induced by cholera toxin used as an oral adjuvant 43 . The notion that IKKβ can reduce or suppress the functions of macrophages or DCs is consistent with previous studies by others suggesting that IKKβ may suppress activation of M1 macrophages during infections through inhibition of STAT-1 15 . In those studies, deletion of IKKβ in macrophages increased STAT-1 activation and promoted a shift toward the M1 phenotype, characterized by increased production of pro-inflammatory and inflammatory cytokines, i.e., IL-1β, TNF-α, IL-12 and IFN-γ and iNOS in response to intraperitoneal injection of Group B streptococcus or E. coli LPS 13 , 15 . While our studies showed enhanced antigen-specific Th1 cytokine responses in IKKβ ΔMye mice after sublingual immunization, the most striking observation was the enhanced IL-17 response. The IKKβ ΔMye mice were useful tools that helped us identify the repressive effect of neutrophils on IgA responses. Analysis of chemokine receptors on myeloid cell subsets in sublingual tissues revealed a broader expression of CCR2, CxCR2 and LTB4R2 on macrophages/DC and non-inflammatory monocytes from IKKβ ΔMye mice. One can speculate that this pattern of receptor expression could improve cellular responses to corresponding ligands and facilitate migration to inductive sites and support IgA responses. Previous studies have shown that injection of alum recruits neutrophils and induces the formation of nodules consistent with those of extracellular DNA traps 44 . A recent report showed that formation of neutrophil extracellular traps (NETs) requires phosphorylation of p65 NFκB 45 . However, NETs are primarily known to be involved in the killing of pathogens 46 . Furthermore, our results showing that neutrophils from both wild-type and IKKβ ΔMye mice suppress transcription of IgA heavy chain suggest the involvement of other mechanisms, which will be addressed in future studies. Nasal immunization with the cAMP-inducing adjuvant CT 6 or E.coli heat labile toxin I 47 promotes Th17 responses. Here we show that EdTx as a sublingual adjuvant promotes antigen-specific Th17 responses in CLNs and spleen, and is associated with in vitro induction of IL-1β and IL-6. The hallmark cytokine produced by Th17 cells is IL-17A 48 . Unlike most T helper cell-derived cytokines, IL-17 does not activate JAK-STAT 10 , but engages Act1 leading to activation of IKKβ and downstream NF-κB, C/EBP, and AP-1, which in turn lead to expression of pro-inflammatory cytokines 49 , 50 . Recently, it has been suggested that Th17 cells stimulate B cell proliferation and Ig class switch for enhanced Ab production 11 . We have shown that CD11b + cells from IKKβ ΔMye mice increase specific B cell populations, i.e., IL-17RA high B cells, and that the IL-17RA high B cells express higher levels of surface IgA. IL-17A was reported to act as helper for the development of germinal centers 51 . Our results suggest that IKKβ ΔMye cells stimulate B cells to be more responsive to IL-17A. This pathway could be one of the mechanisms that rescues the mucosal adjuvant EdTx and induction of SIgA Abs. The limited understanding of molecular and cellular mechanisms that regulate IgA responses has hampered the development of safe mucosal vaccines capable to promote mucosal IgA production. Using an experimental vaccine adjuvant that does not normally induce SIgA after sublingual immunization, we showed that IKKβ is one of the key regulatory pathways for induction of SIgA responses by sublingual vaccines. We also showed that neutrophils negatively regulate IgA production by B cells, an effect that can be countered by Gr1 − myeloid cells lacking a functional IKKβ. Our results provide new insights for the development of sublingual vaccines that can promote both IgA at mucosal surfaces and IgG in the blood stream for optimal protection against infectious agents. MATERIALS AND METHODS Mice Control C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) or NCI-Frederick (Frederick, MD) and acclimated to our facility for at least two weeks before being used. IKKβ ΔMye mice were kindly provided by Dr. Karin (University of California at San Diego) and were generated by crossing LysMCre mice expressing Cre down-stream of the lysozyme promoter in myeloid lineage cells with IKKβ f/f mice harboring a lox P-flanked IKKβ gene 13 , 14 Mice were bred in our facility, maintained in a pathogen-free environment and were used at 8–12 weeks of age. All experiments were performed in co-housed mice in accordance with both NIH and Institutional Animal Care and Use Committee guidelines. Immunization and sample collection The F1-V antigen and Bacillus anthracis protective antigen (PA) and edema factor (EF) were obtained from BEI Resources (Manassas, VA). Mice were immunized three times, i.e., days 0, 7, and 14 by sublingual application of 30 μl of PBS containing 50 μg of F1-V antigen alone, or 50 μg of F1-V antigen plus 15 μg EdTx, i.e., 15 μg PA and 15 μg EF. Blood and external secretions (fecal extracts, vaginal washes, and saliva) were collected as previously described 24 . In selected experiments, mice were injected i.p. with 0.5 mg of the neutrophil Ly6G-specific 1A8 monoclonal Ab (BioXCell) 2 days before the sublingual immunization. Neutrophils were isolated from bone marrow and blood using a 62% Percoll gradient, followed by MACS-sorting with the aid of CD11b microbeads. Other materials and methods Other methods were previously reported and are summarized in the supplementary materials . These methods include ELISA 22 , 23 , ELISPOT 24 , macrophage toxicity assay 22 , 23 , i n vitro cultures and flow cytometry analysis, and quantitative real-time RT-PCR 22 , 30 . Mice Control C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) or NCI-Frederick (Frederick, MD) and acclimated to our facility for at least two weeks before being used. IKKβ ΔMye mice were kindly provided by Dr. Karin (University of California at San Diego) and were generated by crossing LysMCre mice expressing Cre down-stream of the lysozyme promoter in myeloid lineage cells with IKKβ f/f mice harboring a lox P-flanked IKKβ gene 13 , 14 Mice were bred in our facility, maintained in a pathogen-free environment and were used at 8–12 weeks of age. All experiments were performed in co-housed mice in accordance with both NIH and Institutional Animal Care and Use Committee guidelines. Immunization and sample collection The F1-V antigen and Bacillus anthracis protective antigen (PA) and edema factor (EF) were obtained from BEI Resources (Manassas, VA). Mice were immunized three times, i.e., days 0, 7, and 14 by sublingual application of 30 μl of PBS containing 50 μg of F1-V antigen alone, or 50 μg of F1-V antigen plus 15 μg EdTx, i.e., 15 μg PA and 15 μg EF. Blood and external secretions (fecal extracts, vaginal washes, and saliva) were collected as previously described 24 . In selected experiments, mice were injected i.p. with 0.5 mg of the neutrophil Ly6G-specific 1A8 monoclonal Ab (BioXCell) 2 days before the sublingual immunization. Neutrophils were isolated from bone marrow and blood using a 62% Percoll gradient, followed by MACS-sorting with the aid of CD11b microbeads. Other materials and methods Other methods were previously reported and are summarized in the supplementary materials . These methods include ELISA 22 , 23 , ELISPOT 24 , macrophage toxicity assay 22 , 23 , i n vitro cultures and flow cytometry analysis, and quantitative real-time RT-PCR 22 , 30 . Supplementary Material

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150219/

Biology and Diseases of Ruminants: Sheep, Goats, and Cattle

I. INTRODUCTION Since the first edition of this book, the use of ruminants as research subjects has changed dramatically. Formerly, large animals were primarily used for agricultural research or as models of human diseases. Over the past decade, ruminants have continued in their traditional agricultural research role but are now extensively used for studies in molecular biology, genetic engineering, and biotechnology for basic science, agricultural, and clinical applications. Concern and interest for the welfare for these species and improved understanding of their biology and behavior have continued during this time, and these are reflected in some respects in the changing husbandry and management. This chapter addresses the basic biology, husbandry, and more common and important diseases of three ruminant species—sheep, goats, and cattle—commonly used in the laboratory. One chapter is simply not adequate, however, to address the many details and complexities of these species' biology, management, and diseases. References noted in the text offer more information to the interested reader. A. Taxonomy Sheep, goats, and cattle are ungulates, "hooved" animals that are members of the order Artiodactyla (even-toed ungulates, or animals with cloven hooves), suborder Ruminantia (ruminants, or cud-chewing animals) and family Bovidae. Members of the Bovidae group of mammals are distinguished by characteristics such as an even number of toes, a compartmentalized forestomach, and horns. These animals are obligate herbivores and, as adults, derive all their glucose from gluconeogenesis. The subfamily Caprinae includes sheep and goats. The genus and subgenus Ovis includes domestic sheep as well as wild Asian and European sheep species. Domestic sheep are Ovis aries. The subgenus Pachyceros includes the wild North American species as well as snow sheep ( O. nivicola ) of northeastern Siberia. Capra hircus is the domestic goat that originated from western Asian goats. Capra pyrenaica (Spanish goat), C. ibex (goats of the Red Sea and Caucasus area), and C. falconiere (wild goat of Afghanistan and Pakistan) are other members of the genus. The subfamily Bovinae and genus Bos include all domestic and wild cattle. The subgenus taurus contains all of today's domestic cattle. Common genus and species terminology for modern-day cattle includes Bos taurus and B. indicus. Bos taurus (domestic cattle), originally from the European continent, have no hump over the withers. Bos indicus, also known as Zebu cattle, have a hump over the withers and drooping ears. These cattle include breeds found in the tropics and are extremely heat tolerant, and some breeds are known for parasite resistance. Bos taurus and B. indicus have been crossed, and new breeds have been developed during this century ( Briggs and Briggs, 1980 ; Walker et al., 1983 ). There are several hundred breeds of sheep worldwide that are distinguished as "meat," "wool" or "hair," or "dual-purpose." Some wool or hair breeds have varying coat colors. Some breeds are raised for milk (cheese) production. Common breeds of European origin that are raised for meat in the United States include the larger breeds such as Dorset, Columbia, Suffolk, and Hampshire. Slightly smaller breeds include Southdown and Border Cheviot. Wool breeds include Merino, Rambouillet, Lincoln, and Romney; wool breeds are subclassified according to the properties of the wool. The Barbados is known as a "hair" breed. Newer breeds that have been developed in the United States include Polypay and Targhee ( Briggs and Briggs, 1980 ). Goat breeds are numerous and are usually classified according to use as dairy, meat, fiber, or skin-type breeds. The major dairy breeds are the Alpine, Nubian, Toggenburg, La Mancha, Saanen, and Oberhaslie; all have origins on the European continent. The Nubian breed was developed from crossbreeding British stock with Egyptian and Indian goats. This breed is relatively heat tolerant and produces milk with the highest butter-fat (about 4–5%). Fiber breeds include the Angora and the Cashmere. The Angora, the source of mohair, originated in Turkey. The Cashmere breed is found primarily in mountainous areas of Central Asia. The La Mancha, a newer breed of dairy goat first registered in the United States in 1958, has rudimentary ears that are a genetically dominant distinguishing characteristic of the breed. The meat breeds include the Boer, Sapel, Ma Tou, Kambling, and Pygmy. The Pygmy goat is small and is sometimes used for both meat and milk. The Mubend of Uganda and the Red Sokoto of West Africa produce quality skins for fine leather ( Smith and Sherman, 1994 ). Most breeds of cattle are classified as "dairy" or "beef"; a few breeds are considered "dual-purpose." Common dairy breeds in the United States include Holstein-Friesian, Brown Swiss, Jersey, Ayrshire, Guernsey, and Milking Shorthorn. Holsteins have the largest body size, whereas Jerseys have the smallest. Of breeds in temperate regions, Jerseys have been considered to be the most heat tolerant, but Holsteins have been found to adapt to warmer climates. There are many beef breeds. The more common in the United States include Angus (also called Aberdeen-Angus), Hereford (both polled and horned), and Simmental ( Briggs and Briggs, 1980 ; Schmidt et al., 1988 ). Breeds indigenous to other continents, such as the Cape Buffalo, have been found to have unique innate immune characteristics that protect them from endemic trypanosomiasis ( Muranjan et al., 1997 ). More detailed information regarding these and other ruminant breeds is available in Briggs and Briggs (1980) . "Rare" or "minor" breeds of sheep, goats, and cattle are studied for their genetic and production characteristics. Discussions of these and efforts at conservation are described in detail elsewhere ( National Research Council, 1993 ). Several terms are unique to ruminants. In relation to sheep, a ewe is the female, and a ram is the adult intact male. A lamb is the young animal, and ram lamb and ewe lamb are commonly used terms. A wether is a castrated male. The birthing process is referred to as lambing. With respect to goats, a doe or nanny is the female. A buck or billy is the adult intact male. A kid or goatling is a young goat. A young male may be referred to as a buckling, and a young female may be referred to as a doeling. A castrated male in this species is also called a wether. The birthing process is called kidding. With respect to cattle, an adult female is a cow, and an adult male is a bull. A calf is a young animal. A heifer is a female who has not had her first calf. A steer is a castrated male. Calving refers to the act of giving birth. B. Comments about and Examples of Use in Research Ruminants have been used as research models since the inception of the land grant college system, first in production agriculture and now also in basic and applied studies for the anatomic and physiologic sciences and in biomedical research for a variety of purposes. Healthy, normal young ruminants serve as models of cardiac transplantation and as preclinical models for evaluation of cardiac assist or prosthetic devices, such as vascular stents and cardiac valves ( Salerno et al., 1998 ). For many years, ruminants have been useful research subjects for reproductive research, such as research on embryo transfer, artificial insemination, and control of the reproductive cycle ( Wall et al., 1997 ). Several important milestones in gene transfer, cloning, nuclear transfer, and genetic engineering techniques have been developed or demonstrated using these species ( Ebert et al., 1994 ; Schnieke, 1997 ; Cibelli et al., 1998a , b ) (see Fig. 1 ). One of many proposed uses of genetically engineered ruminants is the production of proteins that will be secreted in the milk and later isolated ( Ebert et al., 1994 ; Memon and Ebert, 1992 ). Healthy sheep and goats are also often used for antibody production ( Hanly et al., 1995 ). Genome mapping developed rapidly during the 1990s; extensive information is available and is increasing for sheep and cattle ( Broad et al., 1998 ; Womack, 1998 ). Fig. 1 The production of cloned cattle reflects the changing use of ruminants in research. Sheep are often selected for studying areas such as ruminant physiology and nutrition. These animals provide obvious benefits over the use of cattle in research from the standpoint of size, ease of handling, cost of maintenance, and docile behavior. Sheep are also widely used models for basic and applied fetal and reproductive research ( Buttar, 1997 ; Rees et al., 1998 ; Ross and Nijland, 1998 ). The species is used for investigating circadian rhythms related to day length ( Lehman et al., 1997 ), and the interaction between olfactory cues and behavior ( Kendrick et al., 1997 ). The number and diversity of natural- and induced-disease research models in sheep are great and increasing. Natural models include congenital hyperbilirubinemia/hepatic organic anion excretory defect (Dubin-Johnson syndrome) in the Corriedale breed, congenital hyperbilirubinemia/hepatic organic anion uptake defect (Gilbert syndrome) in the Southdown breed, glucose-6-phosphate dehydrogenase deficiency in the Dorset breed, GM 1 gangliosidosis in the Suffolk breed, and pulmonary adenomatosis (jaagsiekte) in many breeds (Hegreberg, 1981a). Induced models include arteriosclerosis, hemorrhagic shock, copper poisoning (Wilson's disease), and metabolic toxocosis (Hegreberg, 1981b). Goats are used in a wide variety of agricultural and biomedical disciplines such as immunology, mastitis, nutrition, and parasitology research. Vascular researchers select the goat because of the large, readily accessible jugular veins. Goats with inherited caprine myotonia congenita ("fainting goats") have been used as a model for human myotonia congenita (Thomsen's disease) ( Kuhn, 1993 ). A line of inbred Nubians serves as models for the genetic disease β-mannosidosis and prenatal therapeutic cell transplantation strategies ( Lovell et al., 1997 ). (These disorders are discussed in more detail in Section III,B,1.) Goats are used as a model for osteoporosis research ( Welch et al., 1996 ). Cattle are often used as a source of ruminal fluid for research, teaching, or treatment of other cattle, by placing a permanent fistula in the left abdominal wall to allow sampling of ruminal fluid ( Dougherty, 1981 ). Cattle also serve as models of many infectious diseases, including zoonoses, and several inherited metabolic diseases. This species is useful for the basic and comparative research on the pathogenesis and immunology of inherited and infectious diseases. Bovine trichomoniasis, caused by Tritrichomonas (Trichomonas) fetus, has been identified as a useful model for the human infection by Trichomonas vaginalis ( Corbeil, 1995 ). Inherited cardiomyopathies have been found in the Holstein-Friesian, Simmental-Red Holstein, Black Spotted Friesian, and Polled Hereford with woolly coat ( Weil et al., 1997 ). Lipofuscinosis has been identified in Ayrshires and Friesians, and glycogenesis in Shorthorns and Brahmans. Metabolic diseases such as hereditary orotic aciduria and hereditary zinc deficiency have been characterized in Holstein-Friesian or Friesian cattle. Holstein cattle also serve as a model for leukocyte adhesion deficiency syndrome ( AFIP, 1995 ). C. Availability and Sources Common breeds of normal, healthy ruminants are usually readily available, although seasonality may play a role, as noted below. Agricultural sources and reputable farms may be located through land-grant universities or agricultural schools, cooperative extension and 4-H networks, regional ruminant breeders' associations, and farm bureaus. Commercial sources of purpose-bred animals are found in technical publications and annual listings of research animal vendors. Breeds carrying genetic traits of interest, either as animal models or as valuable production characteristics, may be located through literature or Internet searches, animal science societies, breed or livestock conservation associations, and information resources such as the Armed Forces Institute of Pathology. Organizations such as the Institute for Laboratory Animal Research (ILAR), National Center for Research Resources (NCRR), or the Animal Welfare Information Center (AWIC) may also serve as information sources about the animals needed. Purpose-bred research sheep and goats are available from commercial vendors and are usually maintained in registered facilities under federal standards that are also acceptable to research animal accrediting agencies. These commercial animals are frequently described as specific pathogen-free (SPF) and housed as biosecure or closed flocks. Animal health programs are in place, and health reports or other quality assurance reports are usually available on request. Agricultural sources of either small ruminant may be acceptable, but specific research needs may not have been addressed or may not be understood. Lambs, kids, and milking goats may be difficult to locate in fall and winter months because most breeds of sheep and goats are seasonal breeders. Management practices exist, however, to extend the breeding and milking seasons. Most cattle used as animal models in research in the United States are from one of the dairy breeds, usually Holstein, because this breed is now the most common. Purpose-bred, specific pathogen-free research cattle are not typically available. Because of selection and the management of dairy production units, calves and young stock are available year-round. Availability of young beef cattle is more seasonal, according to production cycles typically followed by that industry. Auction barns or sales are not appropriate sources for research ruminants. Many of these animals are culls and will be poor-quality research subjects. They may be in poor body condition and stressed, may be sources of disease, and may contaminate other healthy animals, as well as the research facility. Selection of the suppliers should be made only after research needs have been carefully considered. Consistently working with and buying directly from as few sources as possible are best. Certain types of research (i.e., agricultural nutrition studies) may better be served by selecting animals from local agricultural suppliers rather than commercial vendors located in a different geographical area. The selection of sources for research ruminants includes scrutiny of flock or herd record keeping; health monitoring, vaccination, and preventive medicine programs (including hoof care); production standards and management practices consistent with the industry; management of the breeding flock or herd; sanitation and waste handling programs; vermin and insect control measures (especially for flies and other flying insects); rearing programs for and condition of young stock; the location, health, and condition of the other animals on the premises; intensity of housing; and animal housing facilities. Preliminary and periodic visits to the source farms should be conducted. It is important to establish a good relationship with the local attending large-animal veterinarians, who will be valuable resources for current approved therapies and practices. They may need to be oriented on the specific requirements of animal research. Creative ways can be used to initiate and foster a good working relationship between the agricultural supplier and the research facility. Supplying the vaccines or de-wormers required for flock health programs, providing services such as quarterly serological testing or fecal examinations for the herd or flock, and paying a premium (rather than market price) for animals that meet the quality criteria established for the research animals are often helpful. A set of testing standards can be developed based on one high-quality supplier, and then flocks or herds can be "qualified" based on those standards. Qualifying entails evaluations utilizing the facility and management aspects mentioned above and testing either a percentage of the herd or flock or the entire herd or flock for a number of infectious agents. The testing regimen itself should be carefully developed and evaluated. Once qualified, each source farm should be reevaluated periodically to maintain its status. Slaughter checks may be appropriate; otherwise necropsy of sentinel animals may be required. Selected animals undergoing screening tests should be quarantined from the rest of flock or herd while awaiting test results. Vaccination and deworming regimens can be instituted during these quarantine periods. A second quarantine should occur when animals arrive at the research facility. The animal screening process also depends on the origin of the animal (state, country) and the scientific program. Federal and state regulations must be followed. Socialization of the animals at the source facility should also be considered in terms of ease of handling and safety for personnel in the confinement of the research lab, barn, or farm. For example, frequently handled calves will be easier to manage, and adult dairy goats that have been acclimated to human contact are preferable. Several texts provide information on industry standards for flock and herd management and preventive medicine strategies that can provide helpful orientation to those unfamiliar with these aspects. These references also provide information regarding vaccination products licensed for use in ruminants and typical herd and flock vaccination parasite control schedules ( "Current Veterinary Therapy," 1986 , 1993 , 1999 ; "Council report," 1994 ; "Large Animal Internal Medicine," 1996 ; Smith and Sherman, 1994 ) When designing a vaccination program during qualification of a source or at the research facility, it is important to evaluate the local disease incidence and the potential for exposure. Vaccination programs should be conducted with an awareness of duration of passive immunity and stresses in ruminants' lives (e.g., weaning, grouping, management changes, and shipping) that may impair immunity or increase susceptibility to infectious diseases. It is also prudent to evaluate the cost-effectiveness of vaccination; labor and vaccine expenses may be much higher than the potential animal morbidity or mortality for diseases in a particular locality. Not all of the vaccines mentioned subsequently will be necessary in all herds or flocks. Vaccination needs for research animals will also depend on the local disease history, intent of the research, the age of the animals needed for research, and the length of time the animals will be housed. Typical health screening programs for sheep include Q fever ( Coxiella burnetii); contagious ecthyma; caseous lymphadenitis ( Corynebacteriumpseudotuberculosis); Johne's disease ( Mycobacterium paratuberculosis); ovine progressive pneumonia; internal parasitism such as nasal bots, lungworms, and intestinal worms; and external parasitism such as sheep keds. Each supplier should be queried about vaccination programs for blue-tongue, Brucella ovis, Campylobacter spp., Chlamydia (enzootic abortion of ewes), clostridial diseases, pneumonia complex (parainfluenza 3, Pasteurella haemolytica, and P. multocida), ovine ecthyma, rabies, Dichelobacter (Bacteroides) nodosus, Arcanobacterium pseudotuberculosis, Bacillus anthracis, and Fusobacterium necrophorum. Because of the limited number of biologics approved for small ruminants, products licensed for cattle have been used with success in sheep, and some licensed for sheep are used in goats ( "Council report," JAVMA, 1994 ). In some cases, approved feed additives, such as coccidiostats, are fed to sheep. The basic screening profile for goats should include Q fever ( Coxiella burnettii), caprine arthritis encephalitis (CAE), brucellosis, tuberculosis, and Johne's disease ( Mycobacterium paratuberculosis). Goats may also be tested for caseous lymphadenitis, contagious ecthyma, or Mycoplasma as needed. Herd vaccination programs may include immunizations against tetanus and other clostridial diseases, Chlamydia, Campylobacter, contagious ecthyma, caseous lymphadenitis, Corynebacterium pseudotuberculosis, and Escherichia coli. Cattle herds should be screened for Johne's disease, brucellosis, tuberculosis, respiratory diseases, internal and external parasitism, and foot conditions such as hairy heel warts and foot rot. Determination of the status of the herd with respect to bovine leukemia virus (BLV) may be worthwhile. Herd programs may include essential or highly recommended vaccines against bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), bovine respiratory syncytial virus (BRSV), parainfluenza 3 (PI-3), Leptospira pomona, Tritrichomonas fetus, rotavirus, coronavirus, Campylobacter (Vibrio), Pasteurella haemolytica and P. multocida, and Brucella abortus. Other vaccination programs, dependent on herd status, endemic diseases, or geographic location, may include immunizations against the Clostridial diseases, Moraxella bovis (pinkeye), Fusobacterium necrophorum (foot rot), Staphylococcus aureus (mastitis), Haemophilus somnus, rabies, tetanus, Bacillus anthracis, enterotoxigenic E. coli, Anaplasma, and other Leptospira species. Some products considered to have limited efficacy include vaccines against Salmonella dublin and S. typhimurium. Some autogenous vaccines may be more effective than the commercially available products—for example, the bovine papillomavirus (warts) vaccines. Rearing programs for dairy calves differ from those for the smaller ruminants, including the withdrawal of calves from their dams immediately or by 24 hours after birth. In the cattle industry, antibiotics, ionophores (antibiotics that control selected populations of ruminai organisms), coccidiostats, probiotics, and other approved additives may be part of the milk replacers, grain and concentrate formulations, and/or creep feeding regimens. Use varies by the segment of the industry, and regulations vary by country. Subcutaneous hormonal implants (such as estradiol benzoate and progesterone combined, zeranol, or 17β-estradiol) are administered, especially to beef calves destined for market rather than breeding, to promote growth. Transportation of the animals from the source to the research facility must be carefully planned, and all applicable livestock travel regulations followed. It is best to have the animals transported in vehicles regularly utilized by the source farm. If commercial haulers are used, then disinfecting trucks, trailers, and associated equipment, such as ramps and chutes, beforehand is particularly important. The loading, footing, and distribution of the animals in the trailers and trucks, as well as environmental conditions during shipping, are important to consider to minimize stress and injury to the animals. Sufficient time for acclimation to the facility, pens, handlers, feed, and water must be allowed once at the destination ("Livestock Handling and Transport," 1998). D. Laboratory Management and Husbandry Recent publications address many general considerations as well as specifics about the facilities, husbandry, space requirements, and standard practices for research and production ruminants. Institutions, private entities, researchers, and facility staff must also be aware of the recent adoption by the U.S. Department of Agriculture (USDA) of specific guidelines for regulation of farm animals, such as ruminants, that are used in biomedical and other nonagricultural research. The USDA Animal Care Policy 29 notes that the "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching" and the "Guide for the Care and Use of Laboratory Animals" provide additional information to supplement the existing Animal Welfare Act regulations ( CFR, 1985 ; FASS, 1999 ; Hays et al., 1998 ; NRC, 1996a; USDA, 2000 ). In all cases, stress should be considered and minimized in the husbandry and handling of ruminants. Animals need to be provided adequate time to adapt to new surroundings. Stress decreases feed intake, and the resulting energy, vitamin, and mineral deficiencies will affect the growth and development in younger animals. Reproductive soundness and rumen function are affected by transport and similar stresses. Standard practices such as weaning, castration, dehorning, vaccinations, deworming and treatments for external parasites, shipping and the associated feed and water deprivation, introduction to a new housing environment and new personnel, and intercurrent disease are all stressors ( Houpt, 1998 ). Animals should be acclimated to the use of halters and leads, temporary restraint devices, and other handling equipment associated with the research program. Personnel in the research facility who are unfamiliar with ruminants should be trained in appropriate handling techniques. Appreciation for ruminant behaviors has grown in recent years, and refined ruminant handling techniques have been published ( Houpt, 1998 ; Grandin, 1998 ). When ruminants are confinement-housed, care should be taken to provide adequate but draft-free ventilation. Ammonia buildup and other waste gases may induce respiratory problems. In cold weather, if the ceiling, walls, or water pipes condense water, then the ventilation should be increased even at the expense of lower temperatures. Even adult goats and younger cattle are quite comfortable in cold, even subfreezing temperatures, if provided with adequate amounts of dry dust-free bedding and draft protection. Sheep, because of their wool, are remarkably tolerant to both hot and cold extremes. Newborn lambs and recently shorn adults are susceptible to hypothermia, hyperthermia, and sunburn. Therefore, in outside housing areas, sheep should be provided with shelters to minimize exposure to sun and inclement weather. Animals housed under intensive confinement should be kept clean, and excreta should be removed from the pens or enclosures daily. Feed and water equipment should be maintained in sound, clean condition and should be constructed to prevent fecal contamination. Waterers should not create a muddy environment in paddocks or pens. There should be sufficient continuous-access waterers placed around the area to prevent competition or fighting. Feeders should be constructed to conform to species size and feeding characteristics and to prevent entrapment of head and limbs. Pens, other enclosures, passageways, chutes, and floors must be very sturdy to withstand such factors as the frequent cleaning; the strength, weight, and curiosity of all ages of animals; and the investigative and climbing behaviors of goats. Chain-link fences are dangerous because goats (as well as some breeds and ages of sheep) are curious and tend to stand on their hind legs against fencing or walls. Fore-limbs may be caught easily in the mesh. Floors in any areas where animals will be housed, led, or herded must ensure secure footing at all times to prevent slipping injuries. All ruminants are social and herding animals. Therefore, they should be housed in groups or at least within eyesight and hearing of other animals. Singly housed animals should have regular human contact. Environmental enrichment should be governed by the experimental protocol or standard operating procedures, and durable play objects should be supplied to those animals that are housed in confinement. Calves, in particular, that must be singly housed or that have been recently weaned, need play objects ( Morrow-Tesch, 1997 ). Because sheep and goats are sensitive to changes in light cycle (especially reproductive parameters), photoperiod must be taken into account. Normally, sheep and goats should be maintained on a cycle comparable to natural conditions. Light intensity should be maintained at about 220 lux ( ILAR, 1996 ; FASS, 1999 ). Light cycles can be manipulated for experimental reasons. A. Taxonomy Sheep, goats, and cattle are ungulates, "hooved" animals that are members of the order Artiodactyla (even-toed ungulates, or animals with cloven hooves), suborder Ruminantia (ruminants, or cud-chewing animals) and family Bovidae. Members of the Bovidae group of mammals are distinguished by characteristics such as an even number of toes, a compartmentalized forestomach, and horns. These animals are obligate herbivores and, as adults, derive all their glucose from gluconeogenesis. The subfamily Caprinae includes sheep and goats. The genus and subgenus Ovis includes domestic sheep as well as wild Asian and European sheep species. Domestic sheep are Ovis aries. The subgenus Pachyceros includes the wild North American species as well as snow sheep ( O. nivicola ) of northeastern Siberia. Capra hircus is the domestic goat that originated from western Asian goats. Capra pyrenaica (Spanish goat), C. ibex (goats of the Red Sea and Caucasus area), and C. falconiere (wild goat of Afghanistan and Pakistan) are other members of the genus. The subfamily Bovinae and genus Bos include all domestic and wild cattle. The subgenus taurus contains all of today's domestic cattle. Common genus and species terminology for modern-day cattle includes Bos taurus and B. indicus. Bos taurus (domestic cattle), originally from the European continent, have no hump over the withers. Bos indicus, also known as Zebu cattle, have a hump over the withers and drooping ears. These cattle include breeds found in the tropics and are extremely heat tolerant, and some breeds are known for parasite resistance. Bos taurus and B. indicus have been crossed, and new breeds have been developed during this century ( Briggs and Briggs, 1980 ; Walker et al., 1983 ). There are several hundred breeds of sheep worldwide that are distinguished as "meat," "wool" or "hair," or "dual-purpose." Some wool or hair breeds have varying coat colors. Some breeds are raised for milk (cheese) production. Common breeds of European origin that are raised for meat in the United States include the larger breeds such as Dorset, Columbia, Suffolk, and Hampshire. Slightly smaller breeds include Southdown and Border Cheviot. Wool breeds include Merino, Rambouillet, Lincoln, and Romney; wool breeds are subclassified according to the properties of the wool. The Barbados is known as a "hair" breed. Newer breeds that have been developed in the United States include Polypay and Targhee ( Briggs and Briggs, 1980 ). Goat breeds are numerous and are usually classified according to use as dairy, meat, fiber, or skin-type breeds. The major dairy breeds are the Alpine, Nubian, Toggenburg, La Mancha, Saanen, and Oberhaslie; all have origins on the European continent. The Nubian breed was developed from crossbreeding British stock with Egyptian and Indian goats. This breed is relatively heat tolerant and produces milk with the highest butter-fat (about 4–5%). Fiber breeds include the Angora and the Cashmere. The Angora, the source of mohair, originated in Turkey. The Cashmere breed is found primarily in mountainous areas of Central Asia. The La Mancha, a newer breed of dairy goat first registered in the United States in 1958, has rudimentary ears that are a genetically dominant distinguishing characteristic of the breed. The meat breeds include the Boer, Sapel, Ma Tou, Kambling, and Pygmy. The Pygmy goat is small and is sometimes used for both meat and milk. The Mubend of Uganda and the Red Sokoto of West Africa produce quality skins for fine leather ( Smith and Sherman, 1994 ). Most breeds of cattle are classified as "dairy" or "beef"; a few breeds are considered "dual-purpose." Common dairy breeds in the United States include Holstein-Friesian, Brown Swiss, Jersey, Ayrshire, Guernsey, and Milking Shorthorn. Holsteins have the largest body size, whereas Jerseys have the smallest. Of breeds in temperate regions, Jerseys have been considered to be the most heat tolerant, but Holsteins have been found to adapt to warmer climates. There are many beef breeds. The more common in the United States include Angus (also called Aberdeen-Angus), Hereford (both polled and horned), and Simmental ( Briggs and Briggs, 1980 ; Schmidt et al., 1988 ). Breeds indigenous to other continents, such as the Cape Buffalo, have been found to have unique innate immune characteristics that protect them from endemic trypanosomiasis ( Muranjan et al., 1997 ). More detailed information regarding these and other ruminant breeds is available in Briggs and Briggs (1980) . "Rare" or "minor" breeds of sheep, goats, and cattle are studied for their genetic and production characteristics. Discussions of these and efforts at conservation are described in detail elsewhere ( National Research Council, 1993 ). Several terms are unique to ruminants. In relation to sheep, a ewe is the female, and a ram is the adult intact male. A lamb is the young animal, and ram lamb and ewe lamb are commonly used terms. A wether is a castrated male. The birthing process is referred to as lambing. With respect to goats, a doe or nanny is the female. A buck or billy is the adult intact male. A kid or goatling is a young goat. A young male may be referred to as a buckling, and a young female may be referred to as a doeling. A castrated male in this species is also called a wether. The birthing process is called kidding. With respect to cattle, an adult female is a cow, and an adult male is a bull. A calf is a young animal. A heifer is a female who has not had her first calf. A steer is a castrated male. Calving refers to the act of giving birth. B. Comments about and Examples of Use in Research Ruminants have been used as research models since the inception of the land grant college system, first in production agriculture and now also in basic and applied studies for the anatomic and physiologic sciences and in biomedical research for a variety of purposes. Healthy, normal young ruminants serve as models of cardiac transplantation and as preclinical models for evaluation of cardiac assist or prosthetic devices, such as vascular stents and cardiac valves ( Salerno et al., 1998 ). For many years, ruminants have been useful research subjects for reproductive research, such as research on embryo transfer, artificial insemination, and control of the reproductive cycle ( Wall et al., 1997 ). Several important milestones in gene transfer, cloning, nuclear transfer, and genetic engineering techniques have been developed or demonstrated using these species ( Ebert et al., 1994 ; Schnieke, 1997 ; Cibelli et al., 1998a , b ) (see Fig. 1 ). One of many proposed uses of genetically engineered ruminants is the production of proteins that will be secreted in the milk and later isolated ( Ebert et al., 1994 ; Memon and Ebert, 1992 ). Healthy sheep and goats are also often used for antibody production ( Hanly et al., 1995 ). Genome mapping developed rapidly during the 1990s; extensive information is available and is increasing for sheep and cattle ( Broad et al., 1998 ; Womack, 1998 ). Fig. 1 The production of cloned cattle reflects the changing use of ruminants in research. Sheep are often selected for studying areas such as ruminant physiology and nutrition. These animals provide obvious benefits over the use of cattle in research from the standpoint of size, ease of handling, cost of maintenance, and docile behavior. Sheep are also widely used models for basic and applied fetal and reproductive research ( Buttar, 1997 ; Rees et al., 1998 ; Ross and Nijland, 1998 ). The species is used for investigating circadian rhythms related to day length ( Lehman et al., 1997 ), and the interaction between olfactory cues and behavior ( Kendrick et al., 1997 ). The number and diversity of natural- and induced-disease research models in sheep are great and increasing. Natural models include congenital hyperbilirubinemia/hepatic organic anion excretory defect (Dubin-Johnson syndrome) in the Corriedale breed, congenital hyperbilirubinemia/hepatic organic anion uptake defect (Gilbert syndrome) in the Southdown breed, glucose-6-phosphate dehydrogenase deficiency in the Dorset breed, GM 1 gangliosidosis in the Suffolk breed, and pulmonary adenomatosis (jaagsiekte) in many breeds (Hegreberg, 1981a). Induced models include arteriosclerosis, hemorrhagic shock, copper poisoning (Wilson's disease), and metabolic toxocosis (Hegreberg, 1981b). Goats are used in a wide variety of agricultural and biomedical disciplines such as immunology, mastitis, nutrition, and parasitology research. Vascular researchers select the goat because of the large, readily accessible jugular veins. Goats with inherited caprine myotonia congenita ("fainting goats") have been used as a model for human myotonia congenita (Thomsen's disease) ( Kuhn, 1993 ). A line of inbred Nubians serves as models for the genetic disease β-mannosidosis and prenatal therapeutic cell transplantation strategies ( Lovell et al., 1997 ). (These disorders are discussed in more detail in Section III,B,1.) Goats are used as a model for osteoporosis research ( Welch et al., 1996 ). Cattle are often used as a source of ruminal fluid for research, teaching, or treatment of other cattle, by placing a permanent fistula in the left abdominal wall to allow sampling of ruminal fluid ( Dougherty, 1981 ). Cattle also serve as models of many infectious diseases, including zoonoses, and several inherited metabolic diseases. This species is useful for the basic and comparative research on the pathogenesis and immunology of inherited and infectious diseases. Bovine trichomoniasis, caused by Tritrichomonas (Trichomonas) fetus, has been identified as a useful model for the human infection by Trichomonas vaginalis ( Corbeil, 1995 ). Inherited cardiomyopathies have been found in the Holstein-Friesian, Simmental-Red Holstein, Black Spotted Friesian, and Polled Hereford with woolly coat ( Weil et al., 1997 ). Lipofuscinosis has been identified in Ayrshires and Friesians, and glycogenesis in Shorthorns and Brahmans. Metabolic diseases such as hereditary orotic aciduria and hereditary zinc deficiency have been characterized in Holstein-Friesian or Friesian cattle. Holstein cattle also serve as a model for leukocyte adhesion deficiency syndrome ( AFIP, 1995 ). C. Availability and Sources Common breeds of normal, healthy ruminants are usually readily available, although seasonality may play a role, as noted below. Agricultural sources and reputable farms may be located through land-grant universities or agricultural schools, cooperative extension and 4-H networks, regional ruminant breeders' associations, and farm bureaus. Commercial sources of purpose-bred animals are found in technical publications and annual listings of research animal vendors. Breeds carrying genetic traits of interest, either as animal models or as valuable production characteristics, may be located through literature or Internet searches, animal science societies, breed or livestock conservation associations, and information resources such as the Armed Forces Institute of Pathology. Organizations such as the Institute for Laboratory Animal Research (ILAR), National Center for Research Resources (NCRR), or the Animal Welfare Information Center (AWIC) may also serve as information sources about the animals needed. Purpose-bred research sheep and goats are available from commercial vendors and are usually maintained in registered facilities under federal standards that are also acceptable to research animal accrediting agencies. These commercial animals are frequently described as specific pathogen-free (SPF) and housed as biosecure or closed flocks. Animal health programs are in place, and health reports or other quality assurance reports are usually available on request. Agricultural sources of either small ruminant may be acceptable, but specific research needs may not have been addressed or may not be understood. Lambs, kids, and milking goats may be difficult to locate in fall and winter months because most breeds of sheep and goats are seasonal breeders. Management practices exist, however, to extend the breeding and milking seasons. Most cattle used as animal models in research in the United States are from one of the dairy breeds, usually Holstein, because this breed is now the most common. Purpose-bred, specific pathogen-free research cattle are not typically available. Because of selection and the management of dairy production units, calves and young stock are available year-round. Availability of young beef cattle is more seasonal, according to production cycles typically followed by that industry. Auction barns or sales are not appropriate sources for research ruminants. Many of these animals are culls and will be poor-quality research subjects. They may be in poor body condition and stressed, may be sources of disease, and may contaminate other healthy animals, as well as the research facility. Selection of the suppliers should be made only after research needs have been carefully considered. Consistently working with and buying directly from as few sources as possible are best. Certain types of research (i.e., agricultural nutrition studies) may better be served by selecting animals from local agricultural suppliers rather than commercial vendors located in a different geographical area. The selection of sources for research ruminants includes scrutiny of flock or herd record keeping; health monitoring, vaccination, and preventive medicine programs (including hoof care); production standards and management practices consistent with the industry; management of the breeding flock or herd; sanitation and waste handling programs; vermin and insect control measures (especially for flies and other flying insects); rearing programs for and condition of young stock; the location, health, and condition of the other animals on the premises; intensity of housing; and animal housing facilities. Preliminary and periodic visits to the source farms should be conducted. It is important to establish a good relationship with the local attending large-animal veterinarians, who will be valuable resources for current approved therapies and practices. They may need to be oriented on the specific requirements of animal research. Creative ways can be used to initiate and foster a good working relationship between the agricultural supplier and the research facility. Supplying the vaccines or de-wormers required for flock health programs, providing services such as quarterly serological testing or fecal examinations for the herd or flock, and paying a premium (rather than market price) for animals that meet the quality criteria established for the research animals are often helpful. A set of testing standards can be developed based on one high-quality supplier, and then flocks or herds can be "qualified" based on those standards. Qualifying entails evaluations utilizing the facility and management aspects mentioned above and testing either a percentage of the herd or flock or the entire herd or flock for a number of infectious agents. The testing regimen itself should be carefully developed and evaluated. Once qualified, each source farm should be reevaluated periodically to maintain its status. Slaughter checks may be appropriate; otherwise necropsy of sentinel animals may be required. Selected animals undergoing screening tests should be quarantined from the rest of flock or herd while awaiting test results. Vaccination and deworming regimens can be instituted during these quarantine periods. A second quarantine should occur when animals arrive at the research facility. The animal screening process also depends on the origin of the animal (state, country) and the scientific program. Federal and state regulations must be followed. Socialization of the animals at the source facility should also be considered in terms of ease of handling and safety for personnel in the confinement of the research lab, barn, or farm. For example, frequently handled calves will be easier to manage, and adult dairy goats that have been acclimated to human contact are preferable. Several texts provide information on industry standards for flock and herd management and preventive medicine strategies that can provide helpful orientation to those unfamiliar with these aspects. These references also provide information regarding vaccination products licensed for use in ruminants and typical herd and flock vaccination parasite control schedules ( "Current Veterinary Therapy," 1986 , 1993 , 1999 ; "Council report," 1994 ; "Large Animal Internal Medicine," 1996 ; Smith and Sherman, 1994 ) When designing a vaccination program during qualification of a source or at the research facility, it is important to evaluate the local disease incidence and the potential for exposure. Vaccination programs should be conducted with an awareness of duration of passive immunity and stresses in ruminants' lives (e.g., weaning, grouping, management changes, and shipping) that may impair immunity or increase susceptibility to infectious diseases. It is also prudent to evaluate the cost-effectiveness of vaccination; labor and vaccine expenses may be much higher than the potential animal morbidity or mortality for diseases in a particular locality. Not all of the vaccines mentioned subsequently will be necessary in all herds or flocks. Vaccination needs for research animals will also depend on the local disease history, intent of the research, the age of the animals needed for research, and the length of time the animals will be housed. Typical health screening programs for sheep include Q fever ( Coxiella burnetii); contagious ecthyma; caseous lymphadenitis ( Corynebacteriumpseudotuberculosis); Johne's disease ( Mycobacterium paratuberculosis); ovine progressive pneumonia; internal parasitism such as nasal bots, lungworms, and intestinal worms; and external parasitism such as sheep keds. Each supplier should be queried about vaccination programs for blue-tongue, Brucella ovis, Campylobacter spp., Chlamydia (enzootic abortion of ewes), clostridial diseases, pneumonia complex (parainfluenza 3, Pasteurella haemolytica, and P. multocida), ovine ecthyma, rabies, Dichelobacter (Bacteroides) nodosus, Arcanobacterium pseudotuberculosis, Bacillus anthracis, and Fusobacterium necrophorum. Because of the limited number of biologics approved for small ruminants, products licensed for cattle have been used with success in sheep, and some licensed for sheep are used in goats ( "Council report," JAVMA, 1994 ). In some cases, approved feed additives, such as coccidiostats, are fed to sheep. The basic screening profile for goats should include Q fever ( Coxiella burnettii), caprine arthritis encephalitis (CAE), brucellosis, tuberculosis, and Johne's disease ( Mycobacterium paratuberculosis). Goats may also be tested for caseous lymphadenitis, contagious ecthyma, or Mycoplasma as needed. Herd vaccination programs may include immunizations against tetanus and other clostridial diseases, Chlamydia, Campylobacter, contagious ecthyma, caseous lymphadenitis, Corynebacterium pseudotuberculosis, and Escherichia coli. Cattle herds should be screened for Johne's disease, brucellosis, tuberculosis, respiratory diseases, internal and external parasitism, and foot conditions such as hairy heel warts and foot rot. Determination of the status of the herd with respect to bovine leukemia virus (BLV) may be worthwhile. Herd programs may include essential or highly recommended vaccines against bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), bovine respiratory syncytial virus (BRSV), parainfluenza 3 (PI-3), Leptospira pomona, Tritrichomonas fetus, rotavirus, coronavirus, Campylobacter (Vibrio), Pasteurella haemolytica and P. multocida, and Brucella abortus. Other vaccination programs, dependent on herd status, endemic diseases, or geographic location, may include immunizations against the Clostridial diseases, Moraxella bovis (pinkeye), Fusobacterium necrophorum (foot rot), Staphylococcus aureus (mastitis), Haemophilus somnus, rabies, tetanus, Bacillus anthracis, enterotoxigenic E. coli, Anaplasma, and other Leptospira species. Some products considered to have limited efficacy include vaccines against Salmonella dublin and S. typhimurium. Some autogenous vaccines may be more effective than the commercially available products—for example, the bovine papillomavirus (warts) vaccines. Rearing programs for dairy calves differ from those for the smaller ruminants, including the withdrawal of calves from their dams immediately or by 24 hours after birth. In the cattle industry, antibiotics, ionophores (antibiotics that control selected populations of ruminai organisms), coccidiostats, probiotics, and other approved additives may be part of the milk replacers, grain and concentrate formulations, and/or creep feeding regimens. Use varies by the segment of the industry, and regulations vary by country. Subcutaneous hormonal implants (such as estradiol benzoate and progesterone combined, zeranol, or 17β-estradiol) are administered, especially to beef calves destined for market rather than breeding, to promote growth. Transportation of the animals from the source to the research facility must be carefully planned, and all applicable livestock travel regulations followed. It is best to have the animals transported in vehicles regularly utilized by the source farm. If commercial haulers are used, then disinfecting trucks, trailers, and associated equipment, such as ramps and chutes, beforehand is particularly important. The loading, footing, and distribution of the animals in the trailers and trucks, as well as environmental conditions during shipping, are important to consider to minimize stress and injury to the animals. Sufficient time for acclimation to the facility, pens, handlers, feed, and water must be allowed once at the destination ("Livestock Handling and Transport," 1998). D. Laboratory Management and Husbandry Recent publications address many general considerations as well as specifics about the facilities, husbandry, space requirements, and standard practices for research and production ruminants. Institutions, private entities, researchers, and facility staff must also be aware of the recent adoption by the U.S. Department of Agriculture (USDA) of specific guidelines for regulation of farm animals, such as ruminants, that are used in biomedical and other nonagricultural research. The USDA Animal Care Policy 29 notes that the "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching" and the "Guide for the Care and Use of Laboratory Animals" provide additional information to supplement the existing Animal Welfare Act regulations ( CFR, 1985 ; FASS, 1999 ; Hays et al., 1998 ; NRC, 1996a; USDA, 2000 ). In all cases, stress should be considered and minimized in the husbandry and handling of ruminants. Animals need to be provided adequate time to adapt to new surroundings. Stress decreases feed intake, and the resulting energy, vitamin, and mineral deficiencies will affect the growth and development in younger animals. Reproductive soundness and rumen function are affected by transport and similar stresses. Standard practices such as weaning, castration, dehorning, vaccinations, deworming and treatments for external parasites, shipping and the associated feed and water deprivation, introduction to a new housing environment and new personnel, and intercurrent disease are all stressors ( Houpt, 1998 ). Animals should be acclimated to the use of halters and leads, temporary restraint devices, and other handling equipment associated with the research program. Personnel in the research facility who are unfamiliar with ruminants should be trained in appropriate handling techniques. Appreciation for ruminant behaviors has grown in recent years, and refined ruminant handling techniques have been published ( Houpt, 1998 ; Grandin, 1998 ). When ruminants are confinement-housed, care should be taken to provide adequate but draft-free ventilation. Ammonia buildup and other waste gases may induce respiratory problems. In cold weather, if the ceiling, walls, or water pipes condense water, then the ventilation should be increased even at the expense of lower temperatures. Even adult goats and younger cattle are quite comfortable in cold, even subfreezing temperatures, if provided with adequate amounts of dry dust-free bedding and draft protection. Sheep, because of their wool, are remarkably tolerant to both hot and cold extremes. Newborn lambs and recently shorn adults are susceptible to hypothermia, hyperthermia, and sunburn. Therefore, in outside housing areas, sheep should be provided with shelters to minimize exposure to sun and inclement weather. Animals housed under intensive confinement should be kept clean, and excreta should be removed from the pens or enclosures daily. Feed and water equipment should be maintained in sound, clean condition and should be constructed to prevent fecal contamination. Waterers should not create a muddy environment in paddocks or pens. There should be sufficient continuous-access waterers placed around the area to prevent competition or fighting. Feeders should be constructed to conform to species size and feeding characteristics and to prevent entrapment of head and limbs. Pens, other enclosures, passageways, chutes, and floors must be very sturdy to withstand such factors as the frequent cleaning; the strength, weight, and curiosity of all ages of animals; and the investigative and climbing behaviors of goats. Chain-link fences are dangerous because goats (as well as some breeds and ages of sheep) are curious and tend to stand on their hind legs against fencing or walls. Fore-limbs may be caught easily in the mesh. Floors in any areas where animals will be housed, led, or herded must ensure secure footing at all times to prevent slipping injuries. All ruminants are social and herding animals. Therefore, they should be housed in groups or at least within eyesight and hearing of other animals. Singly housed animals should have regular human contact. Environmental enrichment should be governed by the experimental protocol or standard operating procedures, and durable play objects should be supplied to those animals that are housed in confinement. Calves, in particular, that must be singly housed or that have been recently weaned, need play objects ( Morrow-Tesch, 1997 ). Because sheep and goats are sensitive to changes in light cycle (especially reproductive parameters), photoperiod must be taken into account. Normally, sheep and goats should be maintained on a cycle comparable to natural conditions. Light intensity should be maintained at about 220 lux ( ILAR, 1996 ; FASS, 1999 ). Light cycles can be manipulated for experimental reasons. II. BIOLOGY A. Unique Physiological Characteristics and Attributes, with Emphasis on Comparative Physiology The development of the digestive system and the unique function of the rumen are among the most notable comparative anatomic and physiologic characteristics of ruminants. There is a three-compartment forestomach (rumen, reticulum, and omasum) and a true stomach (abomasum). The mature rumen functions as an anaerobic fermentation chamber in which the enzymes, such as cellulase, of the resident bacteria allow the animals to prosper as herbivores. Digestion is also aided by other microorganisms, such as protozoa (10 5 –10 6 /ml) and bacteria (10 9 –10 10 /ml), that contribute to rumen fermentation. The result is the production of volatile fatty acids (acetic, propionic, and butyric). Unlike in the monogastrics, fermentative digestion and volatile fatty acid absorption also occur in the large intestines. The main sources of energy for ruminants are volatile fatty acids (VFAs) rather than glucose. Glucose is formed from propionic acid (or from amino acids) for metabolism in the central nervous system (CNS), uterus, and mammary glands. Plasma glucose in ruminants is much lower than and is regulated differently from that in nonruminants. The rumen microorganisms also synthesize vitamins, such as B and K, and provide protein that is used by the animals' systems. Large amounts of fermentation gases such as CO 2 and methane, and small amounts of nitrogen, are naturally eructed ( Hecker, 1983 ; Schimdt et al., 1988). Intestinal immunoglobulin absorption by pinocytosis in the neonates is crucial to the success of passive transfer. This transfer mechanism is functional for approximately the first 36 hr after birth. Neonatal ruminants are immunocompetent, however, and this condition is used to advantage for vaccinations against some common diseases of the neonatal and later juvenile periods, such as infectious bovine rhinotracheitis (IBR) vaccine (using modified live virus vaccines) to calves when their dams' colostrum is lacking antibody against this virus. Unlike hepatic lipogenesis in humans, lipogenesis in sheep primarily occurs in adipose tissue and the mammary gland ( Hecker, 1983 ). In addition to normal lymph node chains, and as in other ruminants, sheep have small red "nodes" associated with blood vessels. Inadvertently named hemal "lymph nodes," they contain numerous red blood cells. Sheep have a relatively large pituitary gland, and accessory adrenal medullary tissue may be interspersed throughout the abdominal cavity. Three major ovine histocompatability classes have been identified and designated as OVAR ( Ovis aries ) classes I, II, and III ( Franz-Werner et al., 1996 ). Bovines are recognized as having several unique aspects involving their immune systems. The bovine lymphocyte antigen (BoLA) system ranks after the human (HLA) and murine (H-2) systems in terms of depth of knowledge ( Lewin, 1996 ). Cattle are considered free of autoimmune diseases ( Schook and Lamont, 1996 ). The complexity of the immunobiology of the bovine mammary gland is being studied extensively because mastitis is the most prevalent disease in the dairy industry. Several innate immune mechanisms and cellular defenses, and their variation throughout lactation, have been described ( Sordillo et al., 1997 ). B. Normal Values: Growth, Longevity, Hematology, Clinical Chemistry Hematology and clinical reference texts are available for the ruminant species and include overviews of normal values for age, sex, and breed-specific ranges, as well as discussions regarding the influences on the hemogram of many management, nutritional, geographic, metabolic, physiologic (including lactation), medication, and iatrogenic variables ( Duncan and Prasse, 1986 ; Jain, 1986 ; Kanekoe/a/., 1997). These references should be consulted when preparing to include blood collection data in research protocols and when reviewing hematologic findings. In addition, most veterinary diagnostic laboratories have also developed databases for normal ranges for hematologic and clinical chemistry values based on subjects from their service areas, and these may be useful as local and breed references. Appropriate control groups must be incorporated into each research plan, however, to establish the normal values (see Table I ) for the particular locale, diagnostic facilities, breed, age, sex, and research circumstances. Normal hematologic and clinical biochemistry data are presented in Tables II and III . Table I Normal Values for Sheep, Goats, and Cattle: Vital Signs, Life Spans, and Weights a Parameter and age Sheep Goats Cattle Chromosome number 54 60 60 Body temperature (° C) Young 39.5–40.5 39–40.5 39–40.5 Adult 39–40 38.5–39.5 38–39 Heart rate (beats/min) Young 140(120–160) 140 (120–160) 120(100–140) Adult 75 (60–120) 85 (70–110) 60 (40–80) Respiration rate (breaths/min) Young 50 (30–70) 50(40–65) 48 (30–60) Adult 36 (12–72) 28 (15–40) 24(12–36) Life span (years) 10–15 8–12 20–25 Body weights (lb) Birth 3–25 1 month 25 3 months 55 400 6 months 110 85 9 months 110 12 months 130 720 18 months 155 24 months 300 (ram), 200 (ewe) 170 1100 36 months 205 Deciduous dental formula 2(Di 0/3, Dc 0/1, Dp 3/3) = 20 2(Di 0/3, Dc 0/1, Dp 3/3) = 20 2(Di 0/3, Dc 0/1, Dp 3/3) = 20 Permanent dental formula 2(10/3, C 0/1, M 3/3) = 32 2(10/3, C 0/1, M 3/3) = 32 2(10/3, C0/1, M 3/3) = 32 a Vital sign data for goats are from "Large Animal Internal Medicine" (1996). Sheep weight data represent weights of feeder lamb and adult dry ewe (Federation of Animal Science Societies [FASS], 1998). Goat weight data are for a large-breed male goat. Cattle weight data represent weights of female Holstein or Guernsey dairy cattle (FASS, 1998). Life span data for sheep and cattle are from Brooks et al. (1984). Table II Normal Values for Sheep, Goats, and Cattle: Hematology Parameter (units) Sheep Goats Cattle Packed cell volume (%) 27–45 22–38 24–46 Hemoglobin (g/dl) 9–15 8–12 8–15 Red blood cells (RBC) (×10 6 /μ1) 9–15 8–18 5–10 White blood cells (WBC) (× 10 3 ) 4–12 4–13 4–12 Total protein (g/dl) 6.0–7.5 6–7.5 7–8.5 Mean corpuscular volume (fL) 28–40 16–25 40–60 Mean corpuscular hemoglobin (pg) 8–12 5.2–8 11–17 Mean corpuscular hemoglobin concentration (g/dl) 31–34 30–36 30–36 Reticulocytes (%) 0 0 0 RBC diameter (μm) 3.2–6 2.5–3.9 4.8 RBC life (days) 140–150 125 160 Myeloid: Erythroid ratio 0.77–1.68:10 0.69:10 0.31–1.85:10 Platelets (×10 3 /μ1) 250–750 300–600 100–800 Fibrinogen (mg/dl) 100–500 100–400 300–700 WBC differential: absolute count/μ1 (% of total) Stabs, bands Rare Rare 0–250(0–2) Segmented neutrophils 400–6000(10–50) 1200–6250 (30–48) 600–5400(15–45) Lymphocytes 1600–9000(40–75) 2000–9100(50–70) 1800–9000(45–75) Monocytes 0–750 (0–6) 0–550 (0–4) 80–850 (2–7) Eosinophils 0–1200(0–10) 50–1050(1–8) 80–2400 (2–20) Basophils 0–350 (0–3) 0–150 (0–1) 0–250 (0–2) Coagulation tests (sec) Prothrombin time 13.5–15.9 9.0–14.0 6.8–8.4 Partial thromboplastin time 27.9–40.7 11.0–17.4 Thrombin time 4.8–8.0 20.9–33.4 4.3–7.1 Table III Normal Values for Sheep, Goats, and Cattle: Clinical Biochemistry a Parameter (units) Source Sheep Goat Cattle Alanine aminotransferase (ALT, SGPT; U/liter) s, hp 30 ± 4 6–19 11–40(27 ± 14) Albumin (g/liter) 24–3.0(27 ± 1.9) 27.0–39.0 (33.0 ± 3.3) 30.3–35.5 (32.9 ± 1.3) Alkaline phosphatase (AP; U/liter) 68–387(178 ± 102) 93–387 (219 ± 76) 0–488 (194 ± 126) Aspartate aminotransferase (AST, SGOT; U/liter) s, hp 60–280 (307 ± 43) 167–513 78–132(105 ± 27) Bicarbonate (HCO 3 ; mmol/liter) 20–25 17–29 Bilirubin Conjugated (mg/dl) s, p, hp 0–0.27 (0.12) 0.04–0.44 (0.18) Unconjugated (mg/dl) 0–0.12 0.03 Total (mg/dl) 0.1–0.5 (0.23 ± 0.01) 0.01 0.01–0.5 (0.2) Blood urea nitrogen (BUN; mg/dl) s, p, hp 8–20 10–20(15 ± 2.0) 20–30 Calcium, total (mg/dl) s, hp 11.5–12.8 8.9–11.7 9.7–12.4 Carbon dioxide, total (mmol/L) s, hp 21–28 (26.2) 25.6–29.6 (27.4 ± 1.4) 21.2–32.2(26.5) Chloride (CI; mmol/liter) s, hp 95–103 99–110.3 (105.1 ± 2.9) 97–111 (104) Creatine kinase (CK) U/liter) s, hp 8.1–12.9(10.3 ± 1.6) 0.8–8.9 (4.5 ± 2.8) 4.8–12.1 (7.4 ± 2.4) Creatinine (mg/dl) s, p, hp 1.2–1.9 1.0–1.8 1.0–2.0 γ-Glutamyltransferase (GGT; U/liter) s,p 20–52 (33.5 ± 4.3) 20–56(38 ± 13) 6.1–17.4(15.7 ± 4.0) Globulin (g/liter) s 35.0–57.0 (44.0 ± 5.3) 27.0–41.0 (36.0 ± 5.0) 30.0–34.8 (32.4 ± 2.4) Glucose (mg/dl) s, ρ, hp 50–80 (68.4 ± 6.0) 50–75 (62.8 ± 7.1) 45–75 (57.4 ± 6.8) Lactate dehydrogenase (U/liter) s, hp 238–440(352 ± 59) 692–1445 (1061 ± 222) Magnesium (mg/dl) s 2.2–2.8 2.8–3.6 1.8–2.3 Phosphorus (P; mg/dl) hp 5.0–7.3 (6.4 ± 0.2) 4.2–9.1 (6.5) 5.6–6.5 Potassium (K; mmol/L) hp 3.9–5.4 (4.8) 3.5–6.7 (4.3 ± 0.5) 3.9–5.8 (4.8) Sorbitol dehydrogenase (SDH; U/liter) hp 5.8–27.9(15.7 ± 7.5) 14.0–23.6(19.4 ± 3.6) 4.3–15.3 (9.2 + 3.1) Sodium (Na; mmol/liter) hp 139–152 142–155 (150 ± 3.1) 132–152(142) Total protein (TP, g/liter) s 60.0–79.0 (72.0 ± 5.2) 64.0–70.0 (69.0 ± 4.8) 67.4–74.6(71.0 ± 1.8) a Data presented as ranges with mean and standard deviation in parentheses, s, Serum; p, plasma; hp, heparinized plasma. Clinical biochemistry data from Kaneko et al. (1997) . Some general statements apply to most ruminants. Most ruminants have fewer neutrophils than lymphocytes. The blood urea nitrogen (BUN) values cannot be used as an indicator of renal function because of the metabolism of urea nitrogen by rumen microflora. Because of the large volume of rumen water, ruminants can generally go several days without drinking before significant dehydration occurs. Erythrocytes may become more fragile during rehydration, resulting in some degree of hemolysis and hemoglobinuria. Severe dehydration can occur quickly, however, in animals that are ill. Urine pH is generally alkaline in adult ruminants. Ruminant erythrocytes are smaller than those in other mammals, and hematocrits tend to be overestimated unless blood samples are centrifuged for longer amounts of time for packing of the cell pellet. Increased red-cell fragility is also associated with the smaller erythrocyte. Rouleau formation does not occur in cattle but does to a limited extent in sheep and goats. In addition to fetal hemoglobin, sheep are reported to have at least six different hemoglobins ( Hecker, 1983 ). Blood coagulation in sheep is similar to that in humans. Erythrocytes in Pygmy and Toggenburg goats tend to be more fragile than erythrocytes from other goat breeds. Normal caprine erythrocytes lack central pallor because they are flat and lack biconcavity. Normal caprine erythrocytes may exhibit poikilocytosis. At least five blood groups have been reported in goats: B, C, M, R-O, and X. Because transfusion reaction rates may be as high as 2–3%, cross-matching is advisable although not always practical ( Smith and Sherman, 1994 ). Blood loss of up to 25% of the red cell mass at a single time point can be tolerated by healthy goats. Blood may safely be obtained in volumes of 10 ml/kg body weight and given in volumes of 10–20 ml/kg. In general, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) are not liver-specific in goats, and alanine aminotransferase (ALT; formally serum glutamic-pyruvic transaminase, or SGPT) cannot be used to evaluate hepatic disease in goats. γ-Glutamyltransferase (GGT) and alkaline phosphatase (AP) are associated with biliary stasis, and elevations in GGT are generally associated with hepatic damage. C. Nutrition The nutritional needs of ruminants vary considerably according to the species, breed type, different phases of development, the use of the animals, location, and different stresses in their lives. For example, mineral requirements and other nutritional requirements vary even among breeds of cattle. Several references are available that describe the varying requirements and are useful for determining the requirements of ruminants consistent with the parameters noted above and the type of feeds available ( Jurgens, 1988 ; "Large Animal Clinical Nutrition," 1991 ; NRC, 1981, 1989, 1993, 1996b; "Large Animal Internal Medicine," 1996 ). Preformulated commercial feeds, concentrates, and supplements are available specifically for the different species of ruminants. Some of these provide complete energy and protein requirements or may be used as supplements for what cannot be provided entirely by pasture, forage, hay, or silage. Concentrate mixtures contain salt, minerals, and other elements. Concentrates should contain a protein source such as soybean meal, cottonseed meal, or linseed meal. Computer programs are also readily available for those who may need to formulate and balance rations. The palatability of feeds should be taken into account. Mineral deficiencies and supplementation have been shown to influence several physiologic parameters such as immune function. Introduction of young stock should include continuation of the feeding program of the source or gradual transition to appropriate feed for the animals available in the region of the research facility ( NRC, 1996 ). Good-quality pasture can support ruminants under certain circumstances. Lush spring pastures, especially pastures containing alfalfa, can induce bloat, diarrhea, grass tetany, or nitrate poisoning. Ruminants not acclimated to lush pasture should be fed good-quality hay and slowly introduced to pasture environments. When ruminants have access to pasture, it is important to be aware of different eating habits. Sheep and cattle are grazers. Goats are browsers and will readily eat grasses, as well as seeds, nuts, fruit, and woody-stemmed plants. Goats, however, can also be selective eaters and will only eat the leafy, more nutritious parts of the plant. Therefore, goats have a tendency to "waste" hay. Other eating habits should also be considered. Finely ground concentrates are not tolerated well by goats; pelleted concentrates are preferred because the goat will pick out large particles in mixes. Generally, goats do not prefer "sweet" feeds that contain molasses and do not need supplemental concentrates if a good-quality pasture or hay is fed. When given access to a salt block, goats generally are self-regulating. Grass-fed goats and lactating goats may need supplementation with calcium and phosphorus, whereas alfalfa-fed goats do not ( Bretzlaff et al., 1991 ). Horse and sheep feeds may be fed to goats provided that the feed does not contain much molasses ( Bretzlaff et al., 1991 ). The copper content of horse feed is not excessive for goats, as it is for sheep. Pelleted horse feeds with 25–28% fiber and 12–14% protein are good goat rations. Goats will consume 5–8% of body weight in dry-matter intake (whereas cattle will usually consume only 4% of body weight). Goats enjoy human contact, and small alfalfa cubes make tasty treats for the goat. Rations that have excessive calcium-phosphorus ratios or elevated magnesium levels may induce urinary calculi in male ruminants. These may also occur when forage grasses are high in silicates and oxalates. To increase ovulation rate in does, some producers "flush" females by feeding 0.5–1 lb concentrate per head per day for several weeks before and after the initiation of the breeding season. Thin pregnant dairy goats should be fed 1 lb concentrate per day, with the amount increasing to 1.5 lb per head per day during the last 6 weeks of gestation. Forage should be fed ad libitum during this time. All newborn ruminants must receive passive immunity from colostrum, the first postpartum milk of a dam that contains concentrated protective maternal antibodies (most as IgG 1 ), functional leukocytes, cytokines, vitamins, minerals, and protein. Colostrum also has laxative properties. Trypsin inhibitors in the colostrum allow the passage of intact antibody molecules, by pinocytosis, through the neonate's gut wall and into the bloodstream during the first few days after birth. The quality of the colostrum is directly related to herd or flock management, vaccination programs, and the dam's overall condition and nutrition throughout gestation and at the time of parturition. Ensuring effective colostrum transfer is also dependent on the timing and amount taken by the neonate. Most neonatal ruminants can suckle well within 3 hr of birth. Those that do so have been shown to have significantly less diarrhea ( Naylor, 1996 ). Neonates weakened by dystocia or hypothermia, for example, should be hand-fed or tube-fed colostrum. If necessary, the dam should be hand-milked and the newborn fed colostrum (for example, 20–40 ml for kids) every 2–4 hr for the first 1–2 days. In typical management situations, dairy calves either are separated from their dams immediately after birth and bottle-fed colostrum, or they remain with their dams for only about 24 hr and suckle fresh colostrum during this time. Dairy producers then refrigerate and/or freeze the colostrum that cannot be consumed by the calf during that time and then feed this diluted 50:50 with warm water 3 times a day to the calves during the next 2–3 days. Extra frozen colostrum for emergencies may be obtained from dairy farmers; it is advantageous to obtain colostrum from well-managed herds and from the multiparous cows in the herd (not heifers) in the same geographic locale. Holstein calves, for example, should receive a minimum of 3–5 liters within 12 hr of birth and then be fed about 10–15% of body weight in colostrum by 24 hr of age. After 3 days, calves are then placed on milk replacers. Although young ruminants generally do well receiving their dams' milk, commercially available milk replacers are available and should generally be prepared and fed according to the manufacturer's recommendations. Containers used to prepare and feed these replacers should be sanitized daily. The fat content of both calf and lamb milk replacers is excessive; however, calf milk replacers can be used for kids if care is taken not to overfeed. Young ruminants can be offered good-quality hay (such as second cutting) to nibble on by 1 week of age. Calves may be provided with calf starter, a commercially available concentrate with appropriate levels of energy and protein, fed according to the manufacturer's recommendations at 2–3 weeks of age. They can be weaned off milk replacer by 4–7 weeks of age. Young ruminants (4–12 months of age) need good-quality forage as well as grain and concentrate supplementation to promote development of the rumen. In farm management situations, forage can be silage, pasture, and hay. In a confinement situation like a research unit, good-quality hay, such as second cutting, is desirable. Animals should not be overfed and should be offered a mineral mix free-choice. In contrast to dairy calves, beef calves remain with their mother cows until weaning at 7 months of age. Calves tend to suckle many times per day. As they mature, calves are creep-fed, with the energy and protein content of the ration determined by the milk production of the dams and by the available forage, such as pasture. D. Reproduction Several useful references addressing ruminant reproduction in detail are available ( "Current Veterinary Therapy: Food Animal Practice," 1986 , 1993 , 1999 ; "Large Animal Internal Medicine," 1996 ; "Current Therapy in Large Animal Theriogenology," 1997 ; Hafez, 1987 ). 1. Reproductive Physiology Sheep are seasonally polyestrous; most breeds will express estrus in the fall (Northern Hemisphere) and subsequently lamb in the spring. Some breeds of sheep may cycle in both the fall and the spring. Between seasonal periods of receptivity, the females undergo a long period of sexual quiescence called anestrus. In a research environment, ewes can be artificially stimulated to progress from anestrous to estrous cyclicity by maintaining the females in 8 hr of light and 16 hr of dark for 8–10 weeks. Puberty is reached at about 7–8 months (or earlier) in both rams and ewes; rams will typically reach puberty before their female counterparts. Ewes will display signs of estrus for about 24–30 hr and will ovulate spontaneously at the end of estrus. The estrous cycle length is 14–19 days, with an average of about 17 days. Following breeding, the average length of gestation is 147–150 days. Slightly longer gestations are observed in animals carrying single lambs (singlets), in animals carrying rams, and in certain breeds such as those derived from Merinos. Prolificacy, or the number of lambs produced per gestation, tends to be dependent on the maturity of the dam (older dams tend to have multiple lambs) and on breed characteristics (some fine-wool breeds have fewer multiple births). The Finn and Dorset breeds are especially prolific. Lambs vary in size at birth from about 3–4 lb up to 25 lb. Factors that affect birthweight include parental size, number of lambs in the litter (fewer lambs or singlets tend to be larger), age of the ewe (younger ewes have smaller lambs), lamb gender (males tend to be heavier), nutrition, and season or temperature (spring lambs tend to be larger than fall lambs). Goats are seasonally polyestrous in temperate regions, so that young are born in favorable times of the year. They are short-day breeders, in that estrus (heat) is brought about by the decreasing light of shorter days. In temperate climates of the Northern Hemisphere, goats are normally anestrous during the summer and begin cycling in the fall. The actual length of the sexual cycle depends on day length, breed, and nutrition. Most dairy goats cycle between August and February or March. Nubians often have extended breeding cycles, and the sexual season of some breeds, including the Alpine, can be extended by artificial means. The caprine gestation length averages 150 days with a variation of 145–155 days. Does bear singletons, twins, and triplets, with slightly shorter gestation when the doe is carrying triplets. Cows are polyestrous. Domestication of cattle has included selection against seasonality of the breeding season, particularly in dairy breeds but to some extent also in the beef breeds. In spite of this, cattle have been found to be still sensitive, in varying manifestations, to photoperiodicity. Reproductive physiology in cattle is influenced by many factors. The reproductive programs in source herds and at well-managed facilities will be production-related. Extensive coverage of both physiologic basics and specific industry-related criteria—for retention of a cow as a breeder, for example—are addressed in detail in texts and references oriented toward herd and production management ( "Current Veterinary Therapy," 1986 ). Gestation in cattle is approximately 280 days, with a range of 270–292 days. The length of gestation in cattle is influenced by fetal sex; fetal numbers; age and parity of the cow; breed; genotype of cow, bull, or fetus; nutrition; and local environmental factors. As noted, these factors are also important in sheep and goats. Cows usually bear single calves, although twin births do occur. When twins are combinations of male and female calves, the female should be evaluated for freemartinism. 2. Detection of Estrus and Pregnancy Ovine estrus detection is usually accomplished by the ram. Nonetheless, because artificial insemination is achievable in ewes, clinical signs of estrus are important. Typically, ewes in heat will show a mild enlargement of the vulva, with slight increases of mucus secretion. Ewes may isolate from the flock and appear anxious. It is often better and clearly more reliable to employ the help of a sterile ram to mark females when they are in standing heat. Two mating systems commonly employed include hand mating and group mating. With hand mating, ewes are placed either singly or in small groups with the ram of choice. Ewes are removed as serviced. Group mating involves placement of a mature ram with approximately 50–60 ewes for the entire 6-week breeding season. In either mating system, it is best to attach a marking harness to the male so that individual ewes can be identified as serviced. This is important so that parturition dates can be calculated. An easy, natural way to estimate pregnancy is by placing sterile teaser rams with the ewes at the end of the breeding season. Any animal marked by the ram probably has not conceived. Ultrasound scanners are also used for pregnancy detection. The ultrasound transducer is placed against the right abdomen; presence of a fetus is indicated on the machine. Claims of 98% accuracy at 6 weeks postbreeding have been made, although accuracy is generally best beyond 60 days of gestation. Interrectal Doppler ultrasound probes detect fetal pulses. Fetal heart rate is in the range of 130–160 beats per minute, whereas maternal heart rates tend to be 90–110 beats per minute. Accuracy is best beyond 60 days of pregnancy. Rectal-abdominal palpation is an inexpensive alternative. A plastic probe is introduced intrarectally into the ewe, which is restrained on her back in a cradle. The plastic probe is then manipulated toward the abdomen while palpating for the fetus with the opposite hand. The age of the doe when she first expresses heat varies with breed. Some does will express signs of heat between 3 and 4 months old. However, does should be 7–10 months old or at least 80–90 lb in weight before being bred. The caprine estrous cycle lasts 18–24 days. The duration of estrus is 24–96 hr but averages about 40 hr. The estrous cycle can be more erratic in the beginning than in the end of the breeding season ( Smith, 1997 ). "Standing heat" is usually 12–24 hr but can be as short as a few hours. Signs of estrus in goats include uneasiness, tail switching or "flagging," redness and swelling of the vulva, clear vaginal discharge that becomes white by the end of estrus, vocalization such as continuous bleating, and occasionally riding and standing with other does. A doe that is not in heat will not stand to back pressure or for attempts to hold her tail. Does can be induced to show signs of heat by buck exposure and will ovulate within 7–10 days after introduction of the buck. Goats ovulate during the later part of the estrous cycle, most between 24–36 hr after the onset of estrus. Nevertheless, goats should be mated once signs of estrus are recognized and every 12 hr until the end of estrus. Most goats kid only once a year, although some goats near the equator may kid twice. Once bred successfully, a goat will only rarely show signs of heat again. In fact, the first sign of pregnancy is usually a failure to return to heat, so animals should be carefully watched. Pregnancy can be affirmed by a variety of means. Goats will generally decrease milk production with pregnancy and should have at least a 6- to 8-week dry period for the udder to fully involute and prepare for the next milking period. In cattle, age of first estrus is dependent on the breed, the season (with winter delaying), and the level of nutrition (with higher levels hastening puberty). In some cases, the presence of mature cycling cows influences heifer puberty. With adequate nutrition, dairy breeds will reach puberty at 10–12 months and beef breeds at 11–15 months, and estrous cycles will occur regularly after the pubertal (first) estrus. Maturing heifers will often have one or more ovulations before showing overt signs of estrus. Only one follicle usually ovulates per estrous cycle ( Hafez, 1987 ) Estrus, or standing heat, in cattle averages 12–16 hr in length, with a range of 6–24 hr ( "Large Animal Internal Medicine," 1996 ). Detection of standing heat is important because it is closely related to the time of ovulation. Ovulation occurs approximately 25–32 hr after estrus. Detection of estrus is usually accomplished by visual observation of vaginal mucous discharge, mounting behavior by other females (i.e., the cow standing to be mounted is the individual in estrus), and receptivity to a bull (willingness to stand). Successful visual detection of standing heat is dependent on observation skills of handlers, knowledge of the herd, stresses (e.g., detection decreased in Bos taurus during heat stress), barn and yard surfaces (estrus detected better on dirt than on concrete), and maintaining a consistent observation schedule. Teaser animals outfitted with marking devices are also used. Other methods of detecting estrus include monitoring progesterone levels; glass slide and other evaluations of cervical mucus; change in vaginal pH; and body temperature changes ( Hafez, 1987 ). Estrous cycles are usually 21 days in length, with a range of 17–25 days. It is recommended that a heifer deliver her first calf by 2 years of age. After successful conception, progesterone levels in the cow remain elevated for most of the pregnancy, as the result of the corpus luteum of pregnancy, and they decline only during the final month. Conceptus implantation occurs beginning at about day 17. If the pregnancy fails before this time, the cow will begin to cycle again between days 18–24, but if the pregnancy ends after day 17, there may be a delayed return to estrus. Realtime ultrasonography can be used to determine pregnancy as early as 9 days after insemination, with embyros seen by days 26–29. Fetal gender can also be determined by experienced personnel by this method by about day 55. Detection of pregnancy can be successful by 25–40 days after conception by observation of failure to return to estrus or by palpation per rectum (detecting fetal membrane slip by days 30–35 and/or amniotic vesicle by days 28–35). Palpation of the fetus is possible by day 65 and placentomes by approximately days 100–110. Palpation later in presumed pregnancy will provide information based on differences in size of the two uterine horns, changes in the uterine wall, and fremitus in the miduterine artery. Pregnancy can also be determined with reasonable success rates by determining if progesterone levels are elevated at days 20–24 after insemination. Levels of bovine pregnancy-specific protein B may also be measured; this is produced by trophoblastic cells and is detectable by days 15–24 and elevated throughout pregnancy. Placentation in sheep, goats, and cattle is epitheliochorial and cotyledonary, in contrast to the diffuse or microcotyledonary placentas of horses and pigs. The placentomes, the infolded functional units of the placenta, are formed as the result of fusion of the villi of the fetal cotyledons projecting into the crypts of the maternal caruncles (specialized projections of uterine mucosa). Caruncles of sheep and goats are concave in shape, whereas those of cows are convex. The placentomes are distributed between the pregnant and nonpregant horns of the uterus in sheep, and there are 90–100. In cattle, although the placentomes initially develop around the fetus, they will eventually be distributed to the limit of the chorioallantoic membrane even in the nongravid horn. The placentomes in the nongravid horn will be smaller than in the gravid horn. The total number will be 70–120. 3. Husbandry Needs The best birthing preparation for all dams is to ensure a proper plane of nutrition (not overnutrition) and adequate exercise. If possible, the dam should be confined to a birthing pasture or sanitized maternity pen a few days prior to parturition. The birthing environment will be very important in the overall health of the dam and offspring; stress minimization and a clean environment will benefit the immune health of both in the short and long term. Outdoor parturition in a small birthing pasture has advantages. There is less stress and less intensity of pathogens. Indoor maternity pens should be clean, dry, warm, well bedded, well ventilated but draft-free, and well lighted. Adequate space per pen minimizes losses of neonates from being stepped and sat on by the dam. Management of these pens, especially if concentrated in an area, is important to minimize pathogens to which dam and young are exposed. Water troughs or buckets should be elevated or placed outside the pen, because lambs and kids have a tendency to fall or be pushed into them. Soiled bedding should be removed from the birthing pen between dams, the area sanitized and allowed to dry, and fresh bedding installed for the next occupant. Moving the female immediately before or during parturition may delay the birthing process. In goats, furthermore, in utero death may occur if parturition is unduly delayed. Dams should be monitored closely during parturition for dystocias; these may result in loss of young or in young severely weakened from the prolonged birthing process. Prior to parturition, ewes should be sheared or crutched. Crutching refers to removing wool around the perineal and mammary areas; this minimizes fetal contamination during the birth process. Foot trimming can be done at this time as well. The tail and perineal area of the doe should be clipped and cleaned to improve postbirth sanitation. In general, the pregnant doe needs a 14ft 2 (1.2 m × 1.2 m) area for the birthing process, and area needs to be increased after birthing to allow spacing for kids. Each cow should have a minimum pen area of 10 ft × 10 ft. Evaluation of a cow's udder prior to breeding and especially as parturition approaches is important in order to assure adequate nutrition and success of passive transfer by the neonate. If the udder is edematous or if mastitis is present, for example, an alternate source of colostrum (such as frozen reserves) must made be available. Poor udder conformation may also be problematic; contingency plans should be made to ensure adequate support for the young if they cannot suckle from those udders. Inexperienced heifers may react indifferently or aggressively to their offspring and should be monitored more closely than older, multiparous cows with uneventful calving histories. 4. Parturition Ewes approaching parturition generally isolate themselves from the flock, become restless, stamp their feet, blat, and periodically turn and look at their abdomen. The pelvic region will appear relaxed, and milk will be present in the udder. Once hard labor contractions begin, lambs will usually be born quickly. Animals that do not appear to be progressing correctly should be examined for dystocia. Most cases of fetal malpresentation or malpositioning can be corrected via vagino-uterine manipulation. Occasionally cesarean sections will be necessary. Sanitation, cleanliness, and adequate lubrication are of utmost importance when performing obstetrical procedures. For about a week before parturition, rectal temperature of the doe will be above normal, or about 103° F depending on environmental temperatures. Approximately 24 hr prior to birth, rectal temperature will fall to slightly below normal. Many large dairy-goat facilities attempt to control the onset of parturition in order to assist birthing. The drug of choice to induce parturition in the goat is prostaglandin F 2α (PGF 2α ) ( Ott, 1982 ). On day 144 of gestation, goats given PGF 2α (2.5–5 mg) will deliver kids within 28–57 hr. Most goats prefer to kid alone and do so unaided. Human interaction can actually interfere with normal birthing, especially in young or nervous does. Some does may reject kids if extensive human interference occurs. Does nearing parturition have an obviously swollen udder and a red, swollen vulva. Pelvic ligaments at the base of tail relax. The doe may circle to make a bed, get up and down, look at her tail or sides, push other goats away, and bleat softly. Signs of impending parturition include restlessness; vocalization (bleating softly); uneasiness, including getting up and down, pawing, and bedding; and a mucous discharge, leading to a moist tail. Eight to 12 hr prior to parturition, the cervix will dilate and the cervical mucous plug will be evident as a tan, smeared substance on the tail and perineum of the dam. Kids should present within 1–6 hr in either anterior or posterior position. A posterior presentation can be recognized by the presence of upward-pointing feet. Most does will rest between fetuses and are best left alone. However, if labor is prolonged more than 1 hr, a vaginal exam is indicated. If the pregnant goat is housed with other goats, then herdmates will express great interest in the dam. Unless moved prior to parturition, it is best to leave the dam with the group until after parturition, because removal may delay parturition. Goats are not prone to retained placenta. Normal kids will be quite active and will quickly attempt to stand and nurse. Weak kids should be towel-dried, warmed (via heat lamp, heat pad, or warm water bottle), and assisted to nurse or fed colostrum. The goat is one of the few ungulate species that will exhibit "false pregnancy," or pseudopregnancy. This is a fairly common condition. Does may have characteristically distended abdomens and may develop hydrometra and "deliver" large volumes of cloudy fluid at expected due dates. Subsequent pregnancies can be normal. Goats should be tested for pregnancy by 40 days of age. Veterinary use of prostaglandins has been successful in treating this condition. As in other species, parturition in cattle results from a combination of hormonal changes associated with the maturity of the fetus, notably ACTH (adrenocorticotropic hormone) and subsequent increases in fetal corticosteriods within 2 days of birth. Administration of ACTH to a fetus, or administration to the dam, results in premature birth. Pregnancy is extended if fetal pituitary or adrenal glands are removed surgically. The fetal cortisol probably affects placental steroid production, accounting for sharp increases in the estrogens and estrogen precursors. Coincident with this, maternal progesterone levels fall. The rising levels of estrogen cause release of maternal PGF 2α and induction of oxytocin receptors. Most cows will separate themselves from the rest of the herd. A cow will lift her tail and arch her back when she is within a few hours of delivering the calf, and most cows are recumbent when delivering the calf. Typically, the whole birthing process takes about 100 min. The length of labor of cows carrying larger calves also will be longer. Nervous heifers will take longer to deliver, and if they are disturbed, their labor may cease. All postparturient animals should be monitored for successful passage of these fetal membranes within 12 hr of birth. Veterinary intervention is required if not. Cows occasionally eat placentas, which may subsequently obstruct rumen outflow and require surgical correction. For cattle, it is now recommended practice to remove membranes that have passed, in order to prevent ingestion. 5. Early Development of the Newborn Following lambing, it is critical that the newborns be "processed" so that they will have greatest survival chances. In a well-managed flock, many lambs and ewes will not need much assistance. When assistance is given, the newborn lamb's nose and mouth should be wiped free of secretions; gently swinging the lambs, head down, aids in removal of these fluids. The lamb should be dried off and stimulated through rubbing to aid its breathing. The lamb's navel should be dipped in an iodine solution to prevent subsequent navel infections. And the lamb should be identified by the application of an ear tag or ear notch. It is extremely important that the lamb be supplied with high-quality colostrum within the first 12 hr of birth. Lambs that are not nursing on their own should be tube-fed with colostrum that has been collected and saved previously (i.e., frozen in ice cube trays) or collected from the mother after parturition. Passive transfer can be assessed by measuring serum γ-glutamyltransferase (GGT) levels ( Tessman et al., 1997 ). After the first few days, colostrum changes over to milk. Nursing lambs will ingest increasing amounts of milk as they grow. If the ewe cannot produce sufficient milk, the lamb should be "grafted" onto another ewe or fed artificially with a baby bottle. Powdered milk replacers are commercially available; the content of ewe milk is much different from that of cow's milk; thus lamb milk replacer should specifically be used. One report notes that 50–70% of lamb deaths occur during the first week of life and up to 90% occur within the first month. Good management of ewes during gestation, care of the lamb at parturition, application of an appropriate vaccination program, and observation and intervention within the first several weeks of a lamb's life will minimize losses ( Ross, 1989 ). Immediately after birth, the placenta and any birthing materials should be removed from the doe's pen. Kids do not usually need assistance. If kids are to be raised by the dam, they can be left alone; otherwise, kids should be towel-dried and removed from the dam. Kids are cold-sensitive and may require a heat lamp or other source of added warmth in cold weather. Navel cords should be dipped in tincture of iodine, and kids should be dehorned and castrated within the first several days of life. To control caprine arthritis encephalitis (CAE), kids should be immediately removed from the dam and hand-fed heat-treated colostrum. Colostrum should be heat-treated for 1 hr at 131°F. The first feeding can be up to 125 ml of colostrum. Kids should receive a total of 250 ml colostrum within the first 36–48 hr of birth. After day 3, kids can be placed on milk replacer. Milk replacers should contain 16–24% fat and 20–28% milk-based protein. By 14 days of age, kids should be consuming approximately 1.1–1.4 liters of milk per day. Kids should be introduced to forages as soon as possible and may be weaned by 6–10 weeks or 18–25 lb body weight. Milk that is fed can be reduced by 4 weeks of age by decreasing either the volume fed or the number of feedings. As with other dams, a cow is usually very attentive to her newborn calf, cleaning and softly vocalizing to the neonate. Calves typically are standing by 1 hr after birth and are suckling within 3 hr. As noted previously, dairy calves may be removed from the cow even before suckling, and the colostrum milked from the dam and given to the calf. Assistance may be required for nervous heifers, after dystocias and in extreme circumstances such as severe cold. Cleaning the newborn's nose and mouth, rubbing down the neonate, assuring that the calf does not get chilled, and assuring that it receives adequate colostrum are all important under any of these circumstances. A stressed calf's umbilical may be treated with an iodine or chlorhexidine solution, although some authors note no benefit of navel treatment, specifying that successful transfer of passive immunity and sound sanitary management of birthing area are the most crucial factors in preventing omphalitis (navel ill) ( House, 1996 ; Kersting, 1997 ; Kasari and Roussel, 1999 ). Because newborn calves can be deficient in vitamin A and iron, these may be injected to improve disease resistance ( Wikse and Baker, 1996 ). In cases in which the dams' colostrum is known to be deficient in antibodies against common diseases, vaccinations may be administered at 1 day old and followed with boosters at regular intervals. Dehorning is performed when horn buds appear. Castration is performed between 2 and 9 weeks of age or later. 6. Sexing Sexing the young in any of the ruminant species is straightforward. The vulva of the female young is located just ventral to the anus. The genitalia of the male include a penis, located along the ventral midline, and a scrotum, located in the inguinal region. The phenomenon of the freemartin, a genetic female born as a twin to a male, is the result of anastomoses between placental circulations of the twin fetuses; the mixing of blood-forming cells and germ cells results in the XX/XY chimeras. This occurs in 85–90% of phenotypic bovine females born as co-twins with males. The female will often have abnormal vulva and clitoris, and the vagina will be a blind end because of the lack of a cervix. Sometimes singleton freemartins are born if the male fetus is lost after 30 days' gestation. Multiple births are selected for and are common in sheep; the freemartin phenomenon is regarded as rare. Twinning is common in goats, and freemartinism occurs in about 6% of male-female pairs of twins. Intersexes are seen in some goat breeds and when polled goats are mated. Proof is usually based on evidence of abnormal genital development and reports of abnormal sexual behavior. 7. Weaning Prior to weaning, it must be established that lambs can nutritionally survive without mother's milk. Thus, grain, and later roughage, should be offered to lambs well in advance of the day of weaning so that they can adjust to the feedstuff. To prevent the ewes from ingesting the lamb ration, a "creep" should be set up by building an area adjacent to the ewe-lamb pen and devising a slatted entry for the lambs to enter but not the ewes. Therefore, the lambs will be accustomed to the new ration through this creep-feeding process. If lambs and ewes will be pastured later in the spring, it is still beneficial to creep-feed lambs until pasture growth is adequate enough to fulfill the requirements of the growing lambs. Lambs that are consuming 1.5–2 lb of creep feed per day may be weaned. Depending on the individual program, lambs may be weaned as early as 4 weeks of age, although 6–8 weeks of age is more common. If ewes are of a breed that will cycle twice a year, and if it is expected that they will be rebred, then the lambs must be weaned as early as possible so that lactational anestrus will resolve and ewes will recycle. Another factor is the cost of lactation rations for the ewes; if lamb grain is more economical than ewe grain, then lambs should be weaned. About 4–5 days prior to weaning, feeding of the lactation ration to the ewes should be discontinued, and only roughage fed. At weaning, the lambs should be removed in the creep, and the ewes removed to an area that is not within sight (and preferably sound) of the lambs. The ewes should be monitored for postweaning mastitis and treated as necessary. Ewes that have physical or disease problems or that have not been productive at lambing or feeding their lambs should be culled. The lambs should be monitored to assure that they continue to gain weight and are eating the new ration. Kids should be introduced to forages within the first week of life because the natural curiosity of these animals will cause them to investigate sources of feed. Kids can be weaned by 6–10 weeks or 18–25 lb. Hand-fed milk should be reduced by 4 weeks of age by reducing the volume fed or by decreasing the number of feedings. Dairy calves are now usually removed from their dams immediately after birth. It is less common now to allow the calves to remain with their dams for about 24 hr and suckle fresh colostrum during this time, because their intake will be inadequate. Dairy producers refrigerate and/or freeze the colostrum produced during the first 24 hr and feed this, diluted 50:50 with warm water, twice a day to the calves during the next 2–3 days. Holstein calves, for example, should receive a minimum of 3–5 liters within 12 hr of birth and then be fed about 10–15% of body weight in colostrum by 24 hr of age. After 3 days, calves are then placed on milk replacers, preformulated powders reconstituted with water that provide complete nutrition. Milk replacers are commercially available and should be fed according to manufacturer's recommendations Vaccination programs for calves vary with the preventive medicine program for the overall herd. Passive immunity provided by colostrum from cows on sound management programs will last until a calf is about 6–7 months old; normally vaccinations are not necessary and are contraindicated during those first 6 months. The duration of passive immunity varies considerably among calves, however; some producers choose to begin vaccinating calves at 1–2 months of age and continue with monthly booster immunizations until the animals are 7 months old, when passive immunity is no longer a possibility. 8. Artificial Insemination Artificial insemination (AI) in sheep is more difficult than in cattle because sheep are smaller and cannot be reproductively manipulated via the rectum and because the cervix of sheep is more difficult to traverse with the insemination pipette. Breeding animals artificially with fresh semen produces pregnancy rates averaging 50% (not unlike that of cattle); artificial insemination with frozen semen is less successful. Several artificial insemination techniques have been used. Laparoscopic AI involves the surgical instillation of semen into the uterus through a small abdominal opening. The procedure is successful but is technically involved and costly. Cervical AI involves the transvaginal introduction of semen into the cervix. A modification of this technique (transcervical AI) allows for penetration through the cervix into the uterus. This method (called the Guelph system for transcervical AI) leads to successful penetration into the uterus in up to 75% of ewes when performed by an experienced inseminator. Artificial insemination is now an integral part of dairy herding; natural insemination as a management practice is relatively rare. Technicians performing the AI technique are available through commercial enterprises. Dairy production employees are also trained. Information regarding the management of the donors and recipients, the storage and handling of the semen, and the skills and record keeping required is covered extensively elsewhere ( Nebel, 1997 ). 9. Synchronization Because sheep are hormonally similar to other ruminants, estrous synchronization techniques are comparable. Progesterone suppresses follicle-stimulating hormone (FSH) secretion, preventing animals from developing follicles and exhibiting estrus. Artificial or natural progesterone can be administered in the feed, through parenteral injection, subcuticular implants, and vaginal pessaries. The progesterone is withdrawn in about 12–14 days, after which the FSH secretion will initiate the process of follicle development ( Trower, 1993 ). Estrus usually will occur in 36–60 hr (average is 48 hr). A natural method of synchronization, often applied to promote flock breeding within a short period of time (and thus parturition will be within a narrow window as well), is the introduction of sterile rams with the ewes before the beginning of the normal fall mating period. Pheromones released from males naturally stimulate the females to cycle and to synchronize their heats. It should be noted that introduction of a male during late anestrus will often stimulate ovulation in about 6 days; however, this cycle will generally be without clinical signs of estrus (silent heat). Vasectomy of rams is one method of producing sterile "teaser rams." Introduction of the buck to a group of does will induce ovulation and may even synchronize does. Does that are kept separate from the buck will show signs of estrus, will ovulate within 6–10 days, and will have normal pregnancies when introduced to a buck. Bucks with horns and intact scent glands are better able to induce ovulation than dehorned bucks, whose scent glands often been removed. Control of breeding in the goat has been studied mostly in dairy breeds in order to produce milk throughout the year and to reduce kidding labor. Goats in the luteal phase of the estrous cycle, days 4–16, are sensitive to PGF 2α (2.5–5 mg IM) and will show estrus in 36–60 hr postinjection ( Bretzlaff, 1997 ). Dosing cycling animals twice 11 days apart will synchronize goats, and artificial insemination using this method has resulted in 40–60% conception rates ( Bretzlaff, 1997 ; Greyling and Van Niekerk, 1986 ). Programs for timed breeding have been described and involve administering progestogens ( Bretzlaff, 1997 ). Vaginal pessaries of fluorogestone acetate left in place for 21 days in the doe followed by an injection of pregnant mare serum gonadotropin (PMSG) at the time of pessary removal have proven successful. Also, when primed by PGF 2α an 11-day regimen of fluorogestone acetate with PMSG given on day 9 has been successful. Synchronization of cattle estrous cycles and superovulation are used as management techniques in certain commercial cattle and dairy production settings where estrus synchronization or embryo transfer is advantageous to production and management. The methodology is also used in the research setting for coordinating donors and recipients of embryos or other genetically manipulated tissues for implantation. The options and dosing regimens are described in detail in veterinary clinical texts ( Wenzel, 1997 ; Vanderboom et al., 1997 ). In synchronization, the principle is lysis of the existing corpus luteum. The more common practices involve the use of products approved for use in cattle such as PGF 2α, one of its analogs, or products containing estradiol valerate. Progestogens are also used in conjunction with estradiol valerate. Other approaches, involving management techniques combined with pharmacologic interventions, are considered less successful. Superovulation regimens involve injections of FSH either alone or with PGF 2α at timed internals. Estrus is expected 48 hr after the final injection, and two inseminations are performed at 12 hr intervals after estrus detection. Preparation of recipients involves injection of PGF 2α or progestogens with gonadotropins such as PMSG. For greatest success as management tools, these must be combined with a consistent program that provides appropriate nutrition for all cattle involved. Synchronization of animals is also influenced by several other factors, however, such as time in the cycle when hormones are administered, response by each individual animal, whether the cow is a dairy or beef animal, parity and maturity of the cows, success of heat detection after the luteolysis, and accurate record keeping. 10. Embryo Transfer Embryo transfer involves the removal of multiple embryos from a superovulated embryo donor and transferring them to synchronized recipients. This method maximizes the genetic potential of the donor animal. The donor animal is hormonally superovulated and inseminated. In sheep, about 1 week after breeding, the embryos are surgically removed from the donor's uterus. In cattle, the procedure is nonsurgical. About 75% of expected embryos (determined by counting corpora lutea) can be recovered; successful recovery is affected by factors such as age of the donor, reproductive health, and experience of the surgeon or technician. Furthermore, not all collected embryos are of transferable quality. Recipients are hormonally synchronized with the donor animals. On the day of embryo collection, transferable embryos are implanted into the uterus of the recipient; laparoscopy has been used in the past and is now being replaced by nonsurgical methods. Pregnancy rates average about 70%. If recipients are not available, embryos, like sperm, can be frozen and kept for later transfer. Embryo transfer is commonly practiced in cattle as a herd improvement technique and as a research technique for engineered embyros. Disease screening programs for all animals involved are important because several pathogens can be transmitted directly or indirectly, such as bovine viral diarrhea virus, bluetongue virus, infectious bovine rhinotracheitis virus, and mycoplasmal species. 11. Miscellaneous Management Considerations a. Management of Male Animals In sheep flocks and goat herds, as noted, male young are usually castrated by 1 month of age. The elastrator method is the more popular for animals less than 1 week of age. Other methods include the emasculatome (crushing) and surgical removal ("knife method"). The distress associated with castration and tail docking in lambs is the subject of debate and has been researched recently ( Kent et al., 1995 ). As noted, male calves are usually castrated as early as possible and no later than 3 month of age. In some production situations, however, where maximum hormone responsive muscle development and grouping animals together for procedures dictate scheduling, the procedure may be performed on older males. Open and closed techniques are used, depending on the age of animals and on veterinary or farm practice. Breeding and vasectomized rams and bucks are usually maintained by medium to large production farms. Smaller farms often borrow breeding males. Breeding males are typically selected by production record, pedigree, and/or breed. Vasectomized males are often retired breeders and should be tattooed or identified clearly to avoid any wasted breeding time. The vasectomy technique for both species is comparable ( Smith and Sherman, 1994 ). Rams may be housed together for most of the year, whereas bucks are penned separately. Because ewes will exhibit only a limited number of estrous cycles before becoming reproductively quiescent, it is critical that the male be capable of successfully breeding the female in an expeditious manner. Any defects in the external genitalia, reproductive diseases, or musculoskeletal abnormalities may prevent successful copulatory behaviors. Furthermore, it is important to know the semen quality of the ram as one indicator of fertility. Semen can be collected via electroejaculation or by use of a teaser mount. Once semen is collected, it should be handled carefully and kept warm to prevent sperm death, leading to improper conclusions about the male. Typically, the characteristics usually evaluated as a determinate of sperm quality are volume (normal between 0.7 and 2.0 ml); motility (% of sperm moving in a forward wave; high quality is associated with motility of approximately 90%); concentration (sperm count per unit of volume as measured by a hemocytometer; high-quality semen should contain 1.8 × 10 9 sperm per ml); morphology (live versus dead cells, as determined by special stains and the percentage of abnormal-appearing sperm; neither the abnormalities nor the dead sperm should exceed 10% in high-quality semen). The extensive use of artificial insemination in the dairy cattle industry has minimized the use of bulls on many farms, although a farm may maintain a few bulls for heat detection and for "cleanup" breeding. Breeding bulls are maintained in beef production establishments. Breeding bulls must be part of the herd vaccination program, with special attention to appropriate timing of immunizations for the commonly transmitted venereal diseases campylobacteriosis and trichomoniasis. b. Cattle Tail Docking Tail docking is a relatively recent development in dairy herd management and is practiced in the belief that it will minimize bacterial contamination of the udder and therefore the milk. Tails are typically docked to about 10 inches in length. The practice is more popular in certain regions in the United States. To date, there is no published study indicating that this technique provides any distinctive advantage over keeping the tail switch hair clipped short. E. Behavior Healthy ruminants have good appetites, chew cud, are alert and curious, have healthy intact coats, move without hindrance, and have clear, bright, clean eyes and cool dry noses. Even adult animals, when provided sufficient space, will play. Sheep and goats have tidy "pelleted" dark green feces. Cattle have pasty, moist, dark green-brown feces. Ruminants normally vocalize, and handlers will learn to recognize normal communication among the group or directed at caregivers in contrast to that when animals are stressed. Excessive, strained vocalizations are often a sign of stress in cattle. "Bruxism," or grinding of the teeth by a ruminant, is usually associated with discomfort or pain. Other signs of discomfort, stress, or illness include decreased time spent eating and cud chewing, restlessness, prolonged recumbency with outstretched neck and head, and hunched back when standing. Unhealthy ruminants may be thin, may arch their backs or favor a limb, or may have external lumps or swollen joints, an unusual abdominal profile, or rough or dull coats. All ruminants are herd animals to some extent and social individuals; therefore, every effort should be made to allow contact among animals, in terms either of direct contact or of sound, smell, or sight. Human contact and handling should be initiated promptly and maintained regularly and consistently throughout the animal's stay in the research facilities. Animals should be provided sufficient time to acclimate to handlers and research staff. Cattle and sheep can hear at higher frequencies than humans can and may react to sounds not perceived by handlers. Knowledge of the peculiarities of sheep behavior will increase the ease of handling and decrease stress-related effects in research. Generally, fine-wooled breeds, such as Rambouillet, are the most gregarious and are best handled in groups. The meat, or "downs," breeds tend to be less gregarious, and the long-wooled breeds tend to be solitary ( Ross, 1989 ; ASIA, 1996 ). Nonetheless, movement of animals is simplified by proper facility design. Sheep have a wide-angle visual field and are easily scared by activities that are taking place behind them. Sheep should be moved slowly and gently. To capture individuals within a flock, it is best to confine the flock to a smaller space and use a shepherd's crook or to gently catch the animal in front of the neck/thorax. Grabbing the wool can injure the animals, as well as damage the wool and the underlying tissues. Sheep move best in chutes that have solid walls, and individual animals will generally follow a lead animal. Any escape route will be challenged and, if successfully breached, will disrupt the entire flock movement. Sheep movement is also disrupted by contrasts such as light and shadows that impinge on a chute or corral. Finally, like most animals, sheep have a flight zone (minimum zone of comfort), the penetration of which will result in sheep scattering. This minimal flight distance can be modified by increasing handling of the animals and working at the edge of the zone, but it should always be considered when working with animals in chutes, pens, or other confined areas. Goats exhibit behavioral characteristics that make them quite distinct from other ruminants. Their browsing activity makes them quite orally investigative. Goats will readily nibble or chew just about anything they come in contact with, so researchers should keep all paperwork and equipment out of reach. A herd of goats will readily chew through wood gates and fencing, especially when confined in areas without alternatives for chewing behavior. Goats are also inquisitive, restless, agile jumpers and climbers, and quite mischievous. If maintained in paddocks, strong high fences are essential, as are adequate spaces for exercise or boulders or rock piles for hoof maintenance and recreational climbing. Goats are more tolerant of isolation and are more easily acclimated to human contact than sheep are, but goats will confront unfamiliar intruders and make sneezing noises. Goats with horns will use them to advantage, and horns may also become entangled in fencing. Although less strongly affected by flock behavior, goats are social animals. Most goats raised in close human contact are personable and cooperative and can easily be taught to stand for various procedures, including blood collection. An understanding of breed behaviors, sources of stress in cattle, play behaviors, calf behaviors, and dominance determinants will contribute to prevention of injuries to handlers and better health and welfare of the animals. Ruminants of all ages, especially cattle of all ages, should be handled with an appreciation of the serious injury to human handlers that may result ( Houpt, 1998 ). Cattle have a wide visual field, as sheep do, and a flight zone that varies in size, according to previous handling experiences (gentle handling and animal tameness make the flight zone smaller) and the circumstances of the moment (Grandin, 1993). Groups of cattle are moved effectively around a facility by utilizing chute systems, with sequences of gates, that minimize chances of animals turning around. Dairy cattle have been bred and selected over centuries for their docile, tractable characters and production characteristics. In contrast, beef breeds have not been selected for docility and are generally more difficult to handle and restrain. Beef breeds, such as Angus, are known for their independent natures and protective maternal instincts. All cattle respond well to feed as a reward for desired behavior. Healthy cattle typically are very curious and watchful and are alert to sounds and smells. When not grazing or eating, they hold their heads up. When sleeping, the head and neck may be tucked back. Because of ruminant digestive and metabolic needs, much of the day is spent eating or cud chewing. Occasionally, adult cows sit upright like dogs. Cattle maintained inside tend to be more docile. In addition to forced isolation from other cattle, sources of stress include rough attitudes of handlers and unfamiliar visual patterns, routines, or environments. These stressors may exacerbate signs of systemic illnesses. Calves are known for non-nutritive suckling, bar licking, and tongue rolling. Non-nutritive suckling behavior is greater in hungry calves and also right after a milk meal. It is best to provide nipples and other clean noninjurious materials for the animals to suck. Non-nutritive suckling can be detrimental in group-housed calves because it can result in disease transmission and hair ball formation. Environmental enrichment devices have been developed to cope with this behavior. The behavior diminishes as the animals are weaned onto solid food ( Morrow-Tesch, 1997 ). Play activity and vocalizations of calves mimic adult dominance behaviors. Play activity by young adult cattle is more common in males, can be quite rough, and is often triggered by a change in the environment. Dominance behaviors are dependent on direct physical contact among the cattle, and dominance hierarchies are established within a herd. Horns, age, and weight have been reported to be the most important determinants. Aggressive behaviors in cattle may be triggered by newly introduced animals or unfamiliar visual patterns and by feeding when animals are very hungry. Aggression is more common among intact adult males. A. Unique Physiological Characteristics and Attributes, with Emphasis on Comparative Physiology The development of the digestive system and the unique function of the rumen are among the most notable comparative anatomic and physiologic characteristics of ruminants. There is a three-compartment forestomach (rumen, reticulum, and omasum) and a true stomach (abomasum). The mature rumen functions as an anaerobic fermentation chamber in which the enzymes, such as cellulase, of the resident bacteria allow the animals to prosper as herbivores. Digestion is also aided by other microorganisms, such as protozoa (10 5 –10 6 /ml) and bacteria (10 9 –10 10 /ml), that contribute to rumen fermentation. The result is the production of volatile fatty acids (acetic, propionic, and butyric). Unlike in the monogastrics, fermentative digestion and volatile fatty acid absorption also occur in the large intestines. The main sources of energy for ruminants are volatile fatty acids (VFAs) rather than glucose. Glucose is formed from propionic acid (or from amino acids) for metabolism in the central nervous system (CNS), uterus, and mammary glands. Plasma glucose in ruminants is much lower than and is regulated differently from that in nonruminants. The rumen microorganisms also synthesize vitamins, such as B and K, and provide protein that is used by the animals' systems. Large amounts of fermentation gases such as CO 2 and methane, and small amounts of nitrogen, are naturally eructed ( Hecker, 1983 ; Schimdt et al., 1988). Intestinal immunoglobulin absorption by pinocytosis in the neonates is crucial to the success of passive transfer. This transfer mechanism is functional for approximately the first 36 hr after birth. Neonatal ruminants are immunocompetent, however, and this condition is used to advantage for vaccinations against some common diseases of the neonatal and later juvenile periods, such as infectious bovine rhinotracheitis (IBR) vaccine (using modified live virus vaccines) to calves when their dams' colostrum is lacking antibody against this virus. Unlike hepatic lipogenesis in humans, lipogenesis in sheep primarily occurs in adipose tissue and the mammary gland ( Hecker, 1983 ). In addition to normal lymph node chains, and as in other ruminants, sheep have small red "nodes" associated with blood vessels. Inadvertently named hemal "lymph nodes," they contain numerous red blood cells. Sheep have a relatively large pituitary gland, and accessory adrenal medullary tissue may be interspersed throughout the abdominal cavity. Three major ovine histocompatability classes have been identified and designated as OVAR ( Ovis aries ) classes I, II, and III ( Franz-Werner et al., 1996 ). Bovines are recognized as having several unique aspects involving their immune systems. The bovine lymphocyte antigen (BoLA) system ranks after the human (HLA) and murine (H-2) systems in terms of depth of knowledge ( Lewin, 1996 ). Cattle are considered free of autoimmune diseases ( Schook and Lamont, 1996 ). The complexity of the immunobiology of the bovine mammary gland is being studied extensively because mastitis is the most prevalent disease in the dairy industry. Several innate immune mechanisms and cellular defenses, and their variation throughout lactation, have been described ( Sordillo et al., 1997 ). B. Normal Values: Growth, Longevity, Hematology, Clinical Chemistry Hematology and clinical reference texts are available for the ruminant species and include overviews of normal values for age, sex, and breed-specific ranges, as well as discussions regarding the influences on the hemogram of many management, nutritional, geographic, metabolic, physiologic (including lactation), medication, and iatrogenic variables ( Duncan and Prasse, 1986 ; Jain, 1986 ; Kanekoe/a/., 1997). These references should be consulted when preparing to include blood collection data in research protocols and when reviewing hematologic findings. In addition, most veterinary diagnostic laboratories have also developed databases for normal ranges for hematologic and clinical chemistry values based on subjects from their service areas, and these may be useful as local and breed references. Appropriate control groups must be incorporated into each research plan, however, to establish the normal values (see Table I ) for the particular locale, diagnostic facilities, breed, age, sex, and research circumstances. Normal hematologic and clinical biochemistry data are presented in Tables II and III . Table I Normal Values for Sheep, Goats, and Cattle: Vital Signs, Life Spans, and Weights a Parameter and age Sheep Goats Cattle Chromosome number 54 60 60 Body temperature (° C) Young 39.5–40.5 39–40.5 39–40.5 Adult 39–40 38.5–39.5 38–39 Heart rate (beats/min) Young 140(120–160) 140 (120–160) 120(100–140) Adult 75 (60–120) 85 (70–110) 60 (40–80) Respiration rate (breaths/min) Young 50 (30–70) 50(40–65) 48 (30–60) Adult 36 (12–72) 28 (15–40) 24(12–36) Life span (years) 10–15 8–12 20–25 Body weights (lb) Birth 3–25 1 month 25 3 months 55 400 6 months 110 85 9 months 110 12 months 130 720 18 months 155 24 months 300 (ram), 200 (ewe) 170 1100 36 months 205 Deciduous dental formula 2(Di 0/3, Dc 0/1, Dp 3/3) = 20 2(Di 0/3, Dc 0/1, Dp 3/3) = 20 2(Di 0/3, Dc 0/1, Dp 3/3) = 20 Permanent dental formula 2(10/3, C 0/1, M 3/3) = 32 2(10/3, C 0/1, M 3/3) = 32 2(10/3, C0/1, M 3/3) = 32 a Vital sign data for goats are from "Large Animal Internal Medicine" (1996). Sheep weight data represent weights of feeder lamb and adult dry ewe (Federation of Animal Science Societies [FASS], 1998). Goat weight data are for a large-breed male goat. Cattle weight data represent weights of female Holstein or Guernsey dairy cattle (FASS, 1998). Life span data for sheep and cattle are from Brooks et al. (1984). Table II Normal Values for Sheep, Goats, and Cattle: Hematology Parameter (units) Sheep Goats Cattle Packed cell volume (%) 27–45 22–38 24–46 Hemoglobin (g/dl) 9–15 8–12 8–15 Red blood cells (RBC) (×10 6 /μ1) 9–15 8–18 5–10 White blood cells (WBC) (× 10 3 ) 4–12 4–13 4–12 Total protein (g/dl) 6.0–7.5 6–7.5 7–8.5 Mean corpuscular volume (fL) 28–40 16–25 40–60 Mean corpuscular hemoglobin (pg) 8–12 5.2–8 11–17 Mean corpuscular hemoglobin concentration (g/dl) 31–34 30–36 30–36 Reticulocytes (%) 0 0 0 RBC diameter (μm) 3.2–6 2.5–3.9 4.8 RBC life (days) 140–150 125 160 Myeloid: Erythroid ratio 0.77–1.68:10 0.69:10 0.31–1.85:10 Platelets (×10 3 /μ1) 250–750 300–600 100–800 Fibrinogen (mg/dl) 100–500 100–400 300–700 WBC differential: absolute count/μ1 (% of total) Stabs, bands Rare Rare 0–250(0–2) Segmented neutrophils 400–6000(10–50) 1200–6250 (30–48) 600–5400(15–45) Lymphocytes 1600–9000(40–75) 2000–9100(50–70) 1800–9000(45–75) Monocytes 0–750 (0–6) 0–550 (0–4) 80–850 (2–7) Eosinophils 0–1200(0–10) 50–1050(1–8) 80–2400 (2–20) Basophils 0–350 (0–3) 0–150 (0–1) 0–250 (0–2) Coagulation tests (sec) Prothrombin time 13.5–15.9 9.0–14.0 6.8–8.4 Partial thromboplastin time 27.9–40.7 11.0–17.4 Thrombin time 4.8–8.0 20.9–33.4 4.3–7.1 Table III Normal Values for Sheep, Goats, and Cattle: Clinical Biochemistry a Parameter (units) Source Sheep Goat Cattle Alanine aminotransferase (ALT, SGPT; U/liter) s, hp 30 ± 4 6–19 11–40(27 ± 14) Albumin (g/liter) 24–3.0(27 ± 1.9) 27.0–39.0 (33.0 ± 3.3) 30.3–35.5 (32.9 ± 1.3) Alkaline phosphatase (AP; U/liter) 68–387(178 ± 102) 93–387 (219 ± 76) 0–488 (194 ± 126) Aspartate aminotransferase (AST, SGOT; U/liter) s, hp 60–280 (307 ± 43) 167–513 78–132(105 ± 27) Bicarbonate (HCO 3 ; mmol/liter) 20–25 17–29 Bilirubin Conjugated (mg/dl) s, p, hp 0–0.27 (0.12) 0.04–0.44 (0.18) Unconjugated (mg/dl) 0–0.12 0.03 Total (mg/dl) 0.1–0.5 (0.23 ± 0.01) 0.01 0.01–0.5 (0.2) Blood urea nitrogen (BUN; mg/dl) s, p, hp 8–20 10–20(15 ± 2.0) 20–30 Calcium, total (mg/dl) s, hp 11.5–12.8 8.9–11.7 9.7–12.4 Carbon dioxide, total (mmol/L) s, hp 21–28 (26.2) 25.6–29.6 (27.4 ± 1.4) 21.2–32.2(26.5) Chloride (CI; mmol/liter) s, hp 95–103 99–110.3 (105.1 ± 2.9) 97–111 (104) Creatine kinase (CK) U/liter) s, hp 8.1–12.9(10.3 ± 1.6) 0.8–8.9 (4.5 ± 2.8) 4.8–12.1 (7.4 ± 2.4) Creatinine (mg/dl) s, p, hp 1.2–1.9 1.0–1.8 1.0–2.0 γ-Glutamyltransferase (GGT; U/liter) s,p 20–52 (33.5 ± 4.3) 20–56(38 ± 13) 6.1–17.4(15.7 ± 4.0) Globulin (g/liter) s 35.0–57.0 (44.0 ± 5.3) 27.0–41.0 (36.0 ± 5.0) 30.0–34.8 (32.4 ± 2.4) Glucose (mg/dl) s, ρ, hp 50–80 (68.4 ± 6.0) 50–75 (62.8 ± 7.1) 45–75 (57.4 ± 6.8) Lactate dehydrogenase (U/liter) s, hp 238–440(352 ± 59) 692–1445 (1061 ± 222) Magnesium (mg/dl) s 2.2–2.8 2.8–3.6 1.8–2.3 Phosphorus (P; mg/dl) hp 5.0–7.3 (6.4 ± 0.2) 4.2–9.1 (6.5) 5.6–6.5 Potassium (K; mmol/L) hp 3.9–5.4 (4.8) 3.5–6.7 (4.3 ± 0.5) 3.9–5.8 (4.8) Sorbitol dehydrogenase (SDH; U/liter) hp 5.8–27.9(15.7 ± 7.5) 14.0–23.6(19.4 ± 3.6) 4.3–15.3 (9.2 + 3.1) Sodium (Na; mmol/liter) hp 139–152 142–155 (150 ± 3.1) 132–152(142) Total protein (TP, g/liter) s 60.0–79.0 (72.0 ± 5.2) 64.0–70.0 (69.0 ± 4.8) 67.4–74.6(71.0 ± 1.8) a Data presented as ranges with mean and standard deviation in parentheses, s, Serum; p, plasma; hp, heparinized plasma. Clinical biochemistry data from Kaneko et al. (1997) . Some general statements apply to most ruminants. Most ruminants have fewer neutrophils than lymphocytes. The blood urea nitrogen (BUN) values cannot be used as an indicator of renal function because of the metabolism of urea nitrogen by rumen microflora. Because of the large volume of rumen water, ruminants can generally go several days without drinking before significant dehydration occurs. Erythrocytes may become more fragile during rehydration, resulting in some degree of hemolysis and hemoglobinuria. Severe dehydration can occur quickly, however, in animals that are ill. Urine pH is generally alkaline in adult ruminants. Ruminant erythrocytes are smaller than those in other mammals, and hematocrits tend to be overestimated unless blood samples are centrifuged for longer amounts of time for packing of the cell pellet. Increased red-cell fragility is also associated with the smaller erythrocyte. Rouleau formation does not occur in cattle but does to a limited extent in sheep and goats. In addition to fetal hemoglobin, sheep are reported to have at least six different hemoglobins ( Hecker, 1983 ). Blood coagulation in sheep is similar to that in humans. Erythrocytes in Pygmy and Toggenburg goats tend to be more fragile than erythrocytes from other goat breeds. Normal caprine erythrocytes lack central pallor because they are flat and lack biconcavity. Normal caprine erythrocytes may exhibit poikilocytosis. At least five blood groups have been reported in goats: B, C, M, R-O, and X. Because transfusion reaction rates may be as high as 2–3%, cross-matching is advisable although not always practical ( Smith and Sherman, 1994 ). Blood loss of up to 25% of the red cell mass at a single time point can be tolerated by healthy goats. Blood may safely be obtained in volumes of 10 ml/kg body weight and given in volumes of 10–20 ml/kg. In general, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) are not liver-specific in goats, and alanine aminotransferase (ALT; formally serum glutamic-pyruvic transaminase, or SGPT) cannot be used to evaluate hepatic disease in goats. γ-Glutamyltransferase (GGT) and alkaline phosphatase (AP) are associated with biliary stasis, and elevations in GGT are generally associated with hepatic damage. C. Nutrition The nutritional needs of ruminants vary considerably according to the species, breed type, different phases of development, the use of the animals, location, and different stresses in their lives. For example, mineral requirements and other nutritional requirements vary even among breeds of cattle. Several references are available that describe the varying requirements and are useful for determining the requirements of ruminants consistent with the parameters noted above and the type of feeds available ( Jurgens, 1988 ; "Large Animal Clinical Nutrition," 1991 ; NRC, 1981, 1989, 1993, 1996b; "Large Animal Internal Medicine," 1996 ). Preformulated commercial feeds, concentrates, and supplements are available specifically for the different species of ruminants. Some of these provide complete energy and protein requirements or may be used as supplements for what cannot be provided entirely by pasture, forage, hay, or silage. Concentrate mixtures contain salt, minerals, and other elements. Concentrates should contain a protein source such as soybean meal, cottonseed meal, or linseed meal. Computer programs are also readily available for those who may need to formulate and balance rations. The palatability of feeds should be taken into account. Mineral deficiencies and supplementation have been shown to influence several physiologic parameters such as immune function. Introduction of young stock should include continuation of the feeding program of the source or gradual transition to appropriate feed for the animals available in the region of the research facility ( NRC, 1996 ). Good-quality pasture can support ruminants under certain circumstances. Lush spring pastures, especially pastures containing alfalfa, can induce bloat, diarrhea, grass tetany, or nitrate poisoning. Ruminants not acclimated to lush pasture should be fed good-quality hay and slowly introduced to pasture environments. When ruminants have access to pasture, it is important to be aware of different eating habits. Sheep and cattle are grazers. Goats are browsers and will readily eat grasses, as well as seeds, nuts, fruit, and woody-stemmed plants. Goats, however, can also be selective eaters and will only eat the leafy, more nutritious parts of the plant. Therefore, goats have a tendency to "waste" hay. Other eating habits should also be considered. Finely ground concentrates are not tolerated well by goats; pelleted concentrates are preferred because the goat will pick out large particles in mixes. Generally, goats do not prefer "sweet" feeds that contain molasses and do not need supplemental concentrates if a good-quality pasture or hay is fed. When given access to a salt block, goats generally are self-regulating. Grass-fed goats and lactating goats may need supplementation with calcium and phosphorus, whereas alfalfa-fed goats do not ( Bretzlaff et al., 1991 ). Horse and sheep feeds may be fed to goats provided that the feed does not contain much molasses ( Bretzlaff et al., 1991 ). The copper content of horse feed is not excessive for goats, as it is for sheep. Pelleted horse feeds with 25–28% fiber and 12–14% protein are good goat rations. Goats will consume 5–8% of body weight in dry-matter intake (whereas cattle will usually consume only 4% of body weight). Goats enjoy human contact, and small alfalfa cubes make tasty treats for the goat. Rations that have excessive calcium-phosphorus ratios or elevated magnesium levels may induce urinary calculi in male ruminants. These may also occur when forage grasses are high in silicates and oxalates. To increase ovulation rate in does, some producers "flush" females by feeding 0.5–1 lb concentrate per head per day for several weeks before and after the initiation of the breeding season. Thin pregnant dairy goats should be fed 1 lb concentrate per day, with the amount increasing to 1.5 lb per head per day during the last 6 weeks of gestation. Forage should be fed ad libitum during this time. All newborn ruminants must receive passive immunity from colostrum, the first postpartum milk of a dam that contains concentrated protective maternal antibodies (most as IgG 1 ), functional leukocytes, cytokines, vitamins, minerals, and protein. Colostrum also has laxative properties. Trypsin inhibitors in the colostrum allow the passage of intact antibody molecules, by pinocytosis, through the neonate's gut wall and into the bloodstream during the first few days after birth. The quality of the colostrum is directly related to herd or flock management, vaccination programs, and the dam's overall condition and nutrition throughout gestation and at the time of parturition. Ensuring effective colostrum transfer is also dependent on the timing and amount taken by the neonate. Most neonatal ruminants can suckle well within 3 hr of birth. Those that do so have been shown to have significantly less diarrhea ( Naylor, 1996 ). Neonates weakened by dystocia or hypothermia, for example, should be hand-fed or tube-fed colostrum. If necessary, the dam should be hand-milked and the newborn fed colostrum (for example, 20–40 ml for kids) every 2–4 hr for the first 1–2 days. In typical management situations, dairy calves either are separated from their dams immediately after birth and bottle-fed colostrum, or they remain with their dams for only about 24 hr and suckle fresh colostrum during this time. Dairy producers then refrigerate and/or freeze the colostrum that cannot be consumed by the calf during that time and then feed this diluted 50:50 with warm water 3 times a day to the calves during the next 2–3 days. Extra frozen colostrum for emergencies may be obtained from dairy farmers; it is advantageous to obtain colostrum from well-managed herds and from the multiparous cows in the herd (not heifers) in the same geographic locale. Holstein calves, for example, should receive a minimum of 3–5 liters within 12 hr of birth and then be fed about 10–15% of body weight in colostrum by 24 hr of age. After 3 days, calves are then placed on milk replacers. Although young ruminants generally do well receiving their dams' milk, commercially available milk replacers are available and should generally be prepared and fed according to the manufacturer's recommendations. Containers used to prepare and feed these replacers should be sanitized daily. The fat content of both calf and lamb milk replacers is excessive; however, calf milk replacers can be used for kids if care is taken not to overfeed. Young ruminants can be offered good-quality hay (such as second cutting) to nibble on by 1 week of age. Calves may be provided with calf starter, a commercially available concentrate with appropriate levels of energy and protein, fed according to the manufacturer's recommendations at 2–3 weeks of age. They can be weaned off milk replacer by 4–7 weeks of age. Young ruminants (4–12 months of age) need good-quality forage as well as grain and concentrate supplementation to promote development of the rumen. In farm management situations, forage can be silage, pasture, and hay. In a confinement situation like a research unit, good-quality hay, such as second cutting, is desirable. Animals should not be overfed and should be offered a mineral mix free-choice. In contrast to dairy calves, beef calves remain with their mother cows until weaning at 7 months of age. Calves tend to suckle many times per day. As they mature, calves are creep-fed, with the energy and protein content of the ration determined by the milk production of the dams and by the available forage, such as pasture. D. Reproduction Several useful references addressing ruminant reproduction in detail are available ( "Current Veterinary Therapy: Food Animal Practice," 1986 , 1993 , 1999 ; "Large Animal Internal Medicine," 1996 ; "Current Therapy in Large Animal Theriogenology," 1997 ; Hafez, 1987 ). 1. Reproductive Physiology Sheep are seasonally polyestrous; most breeds will express estrus in the fall (Northern Hemisphere) and subsequently lamb in the spring. Some breeds of sheep may cycle in both the fall and the spring. Between seasonal periods of receptivity, the females undergo a long period of sexual quiescence called anestrus. In a research environment, ewes can be artificially stimulated to progress from anestrous to estrous cyclicity by maintaining the females in 8 hr of light and 16 hr of dark for 8–10 weeks. Puberty is reached at about 7–8 months (or earlier) in both rams and ewes; rams will typically reach puberty before their female counterparts. Ewes will display signs of estrus for about 24–30 hr and will ovulate spontaneously at the end of estrus. The estrous cycle length is 14–19 days, with an average of about 17 days. Following breeding, the average length of gestation is 147–150 days. Slightly longer gestations are observed in animals carrying single lambs (singlets), in animals carrying rams, and in certain breeds such as those derived from Merinos. Prolificacy, or the number of lambs produced per gestation, tends to be dependent on the maturity of the dam (older dams tend to have multiple lambs) and on breed characteristics (some fine-wool breeds have fewer multiple births). The Finn and Dorset breeds are especially prolific. Lambs vary in size at birth from about 3–4 lb up to 25 lb. Factors that affect birthweight include parental size, number of lambs in the litter (fewer lambs or singlets tend to be larger), age of the ewe (younger ewes have smaller lambs), lamb gender (males tend to be heavier), nutrition, and season or temperature (spring lambs tend to be larger than fall lambs). Goats are seasonally polyestrous in temperate regions, so that young are born in favorable times of the year. They are short-day breeders, in that estrus (heat) is brought about by the decreasing light of shorter days. In temperate climates of the Northern Hemisphere, goats are normally anestrous during the summer and begin cycling in the fall. The actual length of the sexual cycle depends on day length, breed, and nutrition. Most dairy goats cycle between August and February or March. Nubians often have extended breeding cycles, and the sexual season of some breeds, including the Alpine, can be extended by artificial means. The caprine gestation length averages 150 days with a variation of 145–155 days. Does bear singletons, twins, and triplets, with slightly shorter gestation when the doe is carrying triplets. Cows are polyestrous. Domestication of cattle has included selection against seasonality of the breeding season, particularly in dairy breeds but to some extent also in the beef breeds. In spite of this, cattle have been found to be still sensitive, in varying manifestations, to photoperiodicity. Reproductive physiology in cattle is influenced by many factors. The reproductive programs in source herds and at well-managed facilities will be production-related. Extensive coverage of both physiologic basics and specific industry-related criteria—for retention of a cow as a breeder, for example—are addressed in detail in texts and references oriented toward herd and production management ( "Current Veterinary Therapy," 1986 ). Gestation in cattle is approximately 280 days, with a range of 270–292 days. The length of gestation in cattle is influenced by fetal sex; fetal numbers; age and parity of the cow; breed; genotype of cow, bull, or fetus; nutrition; and local environmental factors. As noted, these factors are also important in sheep and goats. Cows usually bear single calves, although twin births do occur. When twins are combinations of male and female calves, the female should be evaluated for freemartinism. 2. Detection of Estrus and Pregnancy Ovine estrus detection is usually accomplished by the ram. Nonetheless, because artificial insemination is achievable in ewes, clinical signs of estrus are important. Typically, ewes in heat will show a mild enlargement of the vulva, with slight increases of mucus secretion. Ewes may isolate from the flock and appear anxious. It is often better and clearly more reliable to employ the help of a sterile ram to mark females when they are in standing heat. Two mating systems commonly employed include hand mating and group mating. With hand mating, ewes are placed either singly or in small groups with the ram of choice. Ewes are removed as serviced. Group mating involves placement of a mature ram with approximately 50–60 ewes for the entire 6-week breeding season. In either mating system, it is best to attach a marking harness to the male so that individual ewes can be identified as serviced. This is important so that parturition dates can be calculated. An easy, natural way to estimate pregnancy is by placing sterile teaser rams with the ewes at the end of the breeding season. Any animal marked by the ram probably has not conceived. Ultrasound scanners are also used for pregnancy detection. The ultrasound transducer is placed against the right abdomen; presence of a fetus is indicated on the machine. Claims of 98% accuracy at 6 weeks postbreeding have been made, although accuracy is generally best beyond 60 days of gestation. Interrectal Doppler ultrasound probes detect fetal pulses. Fetal heart rate is in the range of 130–160 beats per minute, whereas maternal heart rates tend to be 90–110 beats per minute. Accuracy is best beyond 60 days of pregnancy. Rectal-abdominal palpation is an inexpensive alternative. A plastic probe is introduced intrarectally into the ewe, which is restrained on her back in a cradle. The plastic probe is then manipulated toward the abdomen while palpating for the fetus with the opposite hand. The age of the doe when she first expresses heat varies with breed. Some does will express signs of heat between 3 and 4 months old. However, does should be 7–10 months old or at least 80–90 lb in weight before being bred. The caprine estrous cycle lasts 18–24 days. The duration of estrus is 24–96 hr but averages about 40 hr. The estrous cycle can be more erratic in the beginning than in the end of the breeding season ( Smith, 1997 ). "Standing heat" is usually 12–24 hr but can be as short as a few hours. Signs of estrus in goats include uneasiness, tail switching or "flagging," redness and swelling of the vulva, clear vaginal discharge that becomes white by the end of estrus, vocalization such as continuous bleating, and occasionally riding and standing with other does. A doe that is not in heat will not stand to back pressure or for attempts to hold her tail. Does can be induced to show signs of heat by buck exposure and will ovulate within 7–10 days after introduction of the buck. Goats ovulate during the later part of the estrous cycle, most between 24–36 hr after the onset of estrus. Nevertheless, goats should be mated once signs of estrus are recognized and every 12 hr until the end of estrus. Most goats kid only once a year, although some goats near the equator may kid twice. Once bred successfully, a goat will only rarely show signs of heat again. In fact, the first sign of pregnancy is usually a failure to return to heat, so animals should be carefully watched. Pregnancy can be affirmed by a variety of means. Goats will generally decrease milk production with pregnancy and should have at least a 6- to 8-week dry period for the udder to fully involute and prepare for the next milking period. In cattle, age of first estrus is dependent on the breed, the season (with winter delaying), and the level of nutrition (with higher levels hastening puberty). In some cases, the presence of mature cycling cows influences heifer puberty. With adequate nutrition, dairy breeds will reach puberty at 10–12 months and beef breeds at 11–15 months, and estrous cycles will occur regularly after the pubertal (first) estrus. Maturing heifers will often have one or more ovulations before showing overt signs of estrus. Only one follicle usually ovulates per estrous cycle ( Hafez, 1987 ) Estrus, or standing heat, in cattle averages 12–16 hr in length, with a range of 6–24 hr ( "Large Animal Internal Medicine," 1996 ). Detection of standing heat is important because it is closely related to the time of ovulation. Ovulation occurs approximately 25–32 hr after estrus. Detection of estrus is usually accomplished by visual observation of vaginal mucous discharge, mounting behavior by other females (i.e., the cow standing to be mounted is the individual in estrus), and receptivity to a bull (willingness to stand). Successful visual detection of standing heat is dependent on observation skills of handlers, knowledge of the herd, stresses (e.g., detection decreased in Bos taurus during heat stress), barn and yard surfaces (estrus detected better on dirt than on concrete), and maintaining a consistent observation schedule. Teaser animals outfitted with marking devices are also used. Other methods of detecting estrus include monitoring progesterone levels; glass slide and other evaluations of cervical mucus; change in vaginal pH; and body temperature changes ( Hafez, 1987 ). Estrous cycles are usually 21 days in length, with a range of 17–25 days. It is recommended that a heifer deliver her first calf by 2 years of age. After successful conception, progesterone levels in the cow remain elevated for most of the pregnancy, as the result of the corpus luteum of pregnancy, and they decline only during the final month. Conceptus implantation occurs beginning at about day 17. If the pregnancy fails before this time, the cow will begin to cycle again between days 18–24, but if the pregnancy ends after day 17, there may be a delayed return to estrus. Realtime ultrasonography can be used to determine pregnancy as early as 9 days after insemination, with embyros seen by days 26–29. Fetal gender can also be determined by experienced personnel by this method by about day 55. Detection of pregnancy can be successful by 25–40 days after conception by observation of failure to return to estrus or by palpation per rectum (detecting fetal membrane slip by days 30–35 and/or amniotic vesicle by days 28–35). Palpation of the fetus is possible by day 65 and placentomes by approximately days 100–110. Palpation later in presumed pregnancy will provide information based on differences in size of the two uterine horns, changes in the uterine wall, and fremitus in the miduterine artery. Pregnancy can also be determined with reasonable success rates by determining if progesterone levels are elevated at days 20–24 after insemination. Levels of bovine pregnancy-specific protein B may also be measured; this is produced by trophoblastic cells and is detectable by days 15–24 and elevated throughout pregnancy. Placentation in sheep, goats, and cattle is epitheliochorial and cotyledonary, in contrast to the diffuse or microcotyledonary placentas of horses and pigs. The placentomes, the infolded functional units of the placenta, are formed as the result of fusion of the villi of the fetal cotyledons projecting into the crypts of the maternal caruncles (specialized projections of uterine mucosa). Caruncles of sheep and goats are concave in shape, whereas those of cows are convex. The placentomes are distributed between the pregnant and nonpregant horns of the uterus in sheep, and there are 90–100. In cattle, although the placentomes initially develop around the fetus, they will eventually be distributed to the limit of the chorioallantoic membrane even in the nongravid horn. The placentomes in the nongravid horn will be smaller than in the gravid horn. The total number will be 70–120. 3. Husbandry Needs The best birthing preparation for all dams is to ensure a proper plane of nutrition (not overnutrition) and adequate exercise. If possible, the dam should be confined to a birthing pasture or sanitized maternity pen a few days prior to parturition. The birthing environment will be very important in the overall health of the dam and offspring; stress minimization and a clean environment will benefit the immune health of both in the short and long term. Outdoor parturition in a small birthing pasture has advantages. There is less stress and less intensity of pathogens. Indoor maternity pens should be clean, dry, warm, well bedded, well ventilated but draft-free, and well lighted. Adequate space per pen minimizes losses of neonates from being stepped and sat on by the dam. Management of these pens, especially if concentrated in an area, is important to minimize pathogens to which dam and young are exposed. Water troughs or buckets should be elevated or placed outside the pen, because lambs and kids have a tendency to fall or be pushed into them. Soiled bedding should be removed from the birthing pen between dams, the area sanitized and allowed to dry, and fresh bedding installed for the next occupant. Moving the female immediately before or during parturition may delay the birthing process. In goats, furthermore, in utero death may occur if parturition is unduly delayed. Dams should be monitored closely during parturition for dystocias; these may result in loss of young or in young severely weakened from the prolonged birthing process. Prior to parturition, ewes should be sheared or crutched. Crutching refers to removing wool around the perineal and mammary areas; this minimizes fetal contamination during the birth process. Foot trimming can be done at this time as well. The tail and perineal area of the doe should be clipped and cleaned to improve postbirth sanitation. In general, the pregnant doe needs a 14ft 2 (1.2 m × 1.2 m) area for the birthing process, and area needs to be increased after birthing to allow spacing for kids. Each cow should have a minimum pen area of 10 ft × 10 ft. Evaluation of a cow's udder prior to breeding and especially as parturition approaches is important in order to assure adequate nutrition and success of passive transfer by the neonate. If the udder is edematous or if mastitis is present, for example, an alternate source of colostrum (such as frozen reserves) must made be available. Poor udder conformation may also be problematic; contingency plans should be made to ensure adequate support for the young if they cannot suckle from those udders. Inexperienced heifers may react indifferently or aggressively to their offspring and should be monitored more closely than older, multiparous cows with uneventful calving histories. 4. Parturition Ewes approaching parturition generally isolate themselves from the flock, become restless, stamp their feet, blat, and periodically turn and look at their abdomen. The pelvic region will appear relaxed, and milk will be present in the udder. Once hard labor contractions begin, lambs will usually be born quickly. Animals that do not appear to be progressing correctly should be examined for dystocia. Most cases of fetal malpresentation or malpositioning can be corrected via vagino-uterine manipulation. Occasionally cesarean sections will be necessary. Sanitation, cleanliness, and adequate lubrication are of utmost importance when performing obstetrical procedures. For about a week before parturition, rectal temperature of the doe will be above normal, or about 103° F depending on environmental temperatures. Approximately 24 hr prior to birth, rectal temperature will fall to slightly below normal. Many large dairy-goat facilities attempt to control the onset of parturition in order to assist birthing. The drug of choice to induce parturition in the goat is prostaglandin F 2α (PGF 2α ) ( Ott, 1982 ). On day 144 of gestation, goats given PGF 2α (2.5–5 mg) will deliver kids within 28–57 hr. Most goats prefer to kid alone and do so unaided. Human interaction can actually interfere with normal birthing, especially in young or nervous does. Some does may reject kids if extensive human interference occurs. Does nearing parturition have an obviously swollen udder and a red, swollen vulva. Pelvic ligaments at the base of tail relax. The doe may circle to make a bed, get up and down, look at her tail or sides, push other goats away, and bleat softly. Signs of impending parturition include restlessness; vocalization (bleating softly); uneasiness, including getting up and down, pawing, and bedding; and a mucous discharge, leading to a moist tail. Eight to 12 hr prior to parturition, the cervix will dilate and the cervical mucous plug will be evident as a tan, smeared substance on the tail and perineum of the dam. Kids should present within 1–6 hr in either anterior or posterior position. A posterior presentation can be recognized by the presence of upward-pointing feet. Most does will rest between fetuses and are best left alone. However, if labor is prolonged more than 1 hr, a vaginal exam is indicated. If the pregnant goat is housed with other goats, then herdmates will express great interest in the dam. Unless moved prior to parturition, it is best to leave the dam with the group until after parturition, because removal may delay parturition. Goats are not prone to retained placenta. Normal kids will be quite active and will quickly attempt to stand and nurse. Weak kids should be towel-dried, warmed (via heat lamp, heat pad, or warm water bottle), and assisted to nurse or fed colostrum. The goat is one of the few ungulate species that will exhibit "false pregnancy," or pseudopregnancy. This is a fairly common condition. Does may have characteristically distended abdomens and may develop hydrometra and "deliver" large volumes of cloudy fluid at expected due dates. Subsequent pregnancies can be normal. Goats should be tested for pregnancy by 40 days of age. Veterinary use of prostaglandins has been successful in treating this condition. As in other species, parturition in cattle results from a combination of hormonal changes associated with the maturity of the fetus, notably ACTH (adrenocorticotropic hormone) and subsequent increases in fetal corticosteriods within 2 days of birth. Administration of ACTH to a fetus, or administration to the dam, results in premature birth. Pregnancy is extended if fetal pituitary or adrenal glands are removed surgically. The fetal cortisol probably affects placental steroid production, accounting for sharp increases in the estrogens and estrogen precursors. Coincident with this, maternal progesterone levels fall. The rising levels of estrogen cause release of maternal PGF 2α and induction of oxytocin receptors. Most cows will separate themselves from the rest of the herd. A cow will lift her tail and arch her back when she is within a few hours of delivering the calf, and most cows are recumbent when delivering the calf. Typically, the whole birthing process takes about 100 min. The length of labor of cows carrying larger calves also will be longer. Nervous heifers will take longer to deliver, and if they are disturbed, their labor may cease. All postparturient animals should be monitored for successful passage of these fetal membranes within 12 hr of birth. Veterinary intervention is required if not. Cows occasionally eat placentas, which may subsequently obstruct rumen outflow and require surgical correction. For cattle, it is now recommended practice to remove membranes that have passed, in order to prevent ingestion. 5. Early Development of the Newborn Following lambing, it is critical that the newborns be "processed" so that they will have greatest survival chances. In a well-managed flock, many lambs and ewes will not need much assistance. When assistance is given, the newborn lamb's nose and mouth should be wiped free of secretions; gently swinging the lambs, head down, aids in removal of these fluids. The lamb should be dried off and stimulated through rubbing to aid its breathing. The lamb's navel should be dipped in an iodine solution to prevent subsequent navel infections. And the lamb should be identified by the application of an ear tag or ear notch. It is extremely important that the lamb be supplied with high-quality colostrum within the first 12 hr of birth. Lambs that are not nursing on their own should be tube-fed with colostrum that has been collected and saved previously (i.e., frozen in ice cube trays) or collected from the mother after parturition. Passive transfer can be assessed by measuring serum γ-glutamyltransferase (GGT) levels ( Tessman et al., 1997 ). After the first few days, colostrum changes over to milk. Nursing lambs will ingest increasing amounts of milk as they grow. If the ewe cannot produce sufficient milk, the lamb should be "grafted" onto another ewe or fed artificially with a baby bottle. Powdered milk replacers are commercially available; the content of ewe milk is much different from that of cow's milk; thus lamb milk replacer should specifically be used. One report notes that 50–70% of lamb deaths occur during the first week of life and up to 90% occur within the first month. Good management of ewes during gestation, care of the lamb at parturition, application of an appropriate vaccination program, and observation and intervention within the first several weeks of a lamb's life will minimize losses ( Ross, 1989 ). Immediately after birth, the placenta and any birthing materials should be removed from the doe's pen. Kids do not usually need assistance. If kids are to be raised by the dam, they can be left alone; otherwise, kids should be towel-dried and removed from the dam. Kids are cold-sensitive and may require a heat lamp or other source of added warmth in cold weather. Navel cords should be dipped in tincture of iodine, and kids should be dehorned and castrated within the first several days of life. To control caprine arthritis encephalitis (CAE), kids should be immediately removed from the dam and hand-fed heat-treated colostrum. Colostrum should be heat-treated for 1 hr at 131°F. The first feeding can be up to 125 ml of colostrum. Kids should receive a total of 250 ml colostrum within the first 36–48 hr of birth. After day 3, kids can be placed on milk replacer. Milk replacers should contain 16–24% fat and 20–28% milk-based protein. By 14 days of age, kids should be consuming approximately 1.1–1.4 liters of milk per day. Kids should be introduced to forages as soon as possible and may be weaned by 6–10 weeks or 18–25 lb body weight. Milk that is fed can be reduced by 4 weeks of age by decreasing either the volume fed or the number of feedings. As with other dams, a cow is usually very attentive to her newborn calf, cleaning and softly vocalizing to the neonate. Calves typically are standing by 1 hr after birth and are suckling within 3 hr. As noted previously, dairy calves may be removed from the cow even before suckling, and the colostrum milked from the dam and given to the calf. Assistance may be required for nervous heifers, after dystocias and in extreme circumstances such as severe cold. Cleaning the newborn's nose and mouth, rubbing down the neonate, assuring that the calf does not get chilled, and assuring that it receives adequate colostrum are all important under any of these circumstances. A stressed calf's umbilical may be treated with an iodine or chlorhexidine solution, although some authors note no benefit of navel treatment, specifying that successful transfer of passive immunity and sound sanitary management of birthing area are the most crucial factors in preventing omphalitis (navel ill) ( House, 1996 ; Kersting, 1997 ; Kasari and Roussel, 1999 ). Because newborn calves can be deficient in vitamin A and iron, these may be injected to improve disease resistance ( Wikse and Baker, 1996 ). In cases in which the dams' colostrum is known to be deficient in antibodies against common diseases, vaccinations may be administered at 1 day old and followed with boosters at regular intervals. Dehorning is performed when horn buds appear. Castration is performed between 2 and 9 weeks of age or later. 6. Sexing Sexing the young in any of the ruminant species is straightforward. The vulva of the female young is located just ventral to the anus. The genitalia of the male include a penis, located along the ventral midline, and a scrotum, located in the inguinal region. The phenomenon of the freemartin, a genetic female born as a twin to a male, is the result of anastomoses between placental circulations of the twin fetuses; the mixing of blood-forming cells and germ cells results in the XX/XY chimeras. This occurs in 85–90% of phenotypic bovine females born as co-twins with males. The female will often have abnormal vulva and clitoris, and the vagina will be a blind end because of the lack of a cervix. Sometimes singleton freemartins are born if the male fetus is lost after 30 days' gestation. Multiple births are selected for and are common in sheep; the freemartin phenomenon is regarded as rare. Twinning is common in goats, and freemartinism occurs in about 6% of male-female pairs of twins. Intersexes are seen in some goat breeds and when polled goats are mated. Proof is usually based on evidence of abnormal genital development and reports of abnormal sexual behavior. 7. Weaning Prior to weaning, it must be established that lambs can nutritionally survive without mother's milk. Thus, grain, and later roughage, should be offered to lambs well in advance of the day of weaning so that they can adjust to the feedstuff. To prevent the ewes from ingesting the lamb ration, a "creep" should be set up by building an area adjacent to the ewe-lamb pen and devising a slatted entry for the lambs to enter but not the ewes. Therefore, the lambs will be accustomed to the new ration through this creep-feeding process. If lambs and ewes will be pastured later in the spring, it is still beneficial to creep-feed lambs until pasture growth is adequate enough to fulfill the requirements of the growing lambs. Lambs that are consuming 1.5–2 lb of creep feed per day may be weaned. Depending on the individual program, lambs may be weaned as early as 4 weeks of age, although 6–8 weeks of age is more common. If ewes are of a breed that will cycle twice a year, and if it is expected that they will be rebred, then the lambs must be weaned as early as possible so that lactational anestrus will resolve and ewes will recycle. Another factor is the cost of lactation rations for the ewes; if lamb grain is more economical than ewe grain, then lambs should be weaned. About 4–5 days prior to weaning, feeding of the lactation ration to the ewes should be discontinued, and only roughage fed. At weaning, the lambs should be removed in the creep, and the ewes removed to an area that is not within sight (and preferably sound) of the lambs. The ewes should be monitored for postweaning mastitis and treated as necessary. Ewes that have physical or disease problems or that have not been productive at lambing or feeding their lambs should be culled. The lambs should be monitored to assure that they continue to gain weight and are eating the new ration. Kids should be introduced to forages within the first week of life because the natural curiosity of these animals will cause them to investigate sources of feed. Kids can be weaned by 6–10 weeks or 18–25 lb. Hand-fed milk should be reduced by 4 weeks of age by reducing the volume fed or by decreasing the number of feedings. Dairy calves are now usually removed from their dams immediately after birth. It is less common now to allow the calves to remain with their dams for about 24 hr and suckle fresh colostrum during this time, because their intake will be inadequate. Dairy producers refrigerate and/or freeze the colostrum produced during the first 24 hr and feed this, diluted 50:50 with warm water, twice a day to the calves during the next 2–3 days. Holstein calves, for example, should receive a minimum of 3–5 liters within 12 hr of birth and then be fed about 10–15% of body weight in colostrum by 24 hr of age. After 3 days, calves are then placed on milk replacers, preformulated powders reconstituted with water that provide complete nutrition. Milk replacers are commercially available and should be fed according to manufacturer's recommendations Vaccination programs for calves vary with the preventive medicine program for the overall herd. Passive immunity provided by colostrum from cows on sound management programs will last until a calf is about 6–7 months old; normally vaccinations are not necessary and are contraindicated during those first 6 months. The duration of passive immunity varies considerably among calves, however; some producers choose to begin vaccinating calves at 1–2 months of age and continue with monthly booster immunizations until the animals are 7 months old, when passive immunity is no longer a possibility. 8. Artificial Insemination Artificial insemination (AI) in sheep is more difficult than in cattle because sheep are smaller and cannot be reproductively manipulated via the rectum and because the cervix of sheep is more difficult to traverse with the insemination pipette. Breeding animals artificially with fresh semen produces pregnancy rates averaging 50% (not unlike that of cattle); artificial insemination with frozen semen is less successful. Several artificial insemination techniques have been used. Laparoscopic AI involves the surgical instillation of semen into the uterus through a small abdominal opening. The procedure is successful but is technically involved and costly. Cervical AI involves the transvaginal introduction of semen into the cervix. A modification of this technique (transcervical AI) allows for penetration through the cervix into the uterus. This method (called the Guelph system for transcervical AI) leads to successful penetration into the uterus in up to 75% of ewes when performed by an experienced inseminator. Artificial insemination is now an integral part of dairy herding; natural insemination as a management practice is relatively rare. Technicians performing the AI technique are available through commercial enterprises. Dairy production employees are also trained. Information regarding the management of the donors and recipients, the storage and handling of the semen, and the skills and record keeping required is covered extensively elsewhere ( Nebel, 1997 ). 9. Synchronization Because sheep are hormonally similar to other ruminants, estrous synchronization techniques are comparable. Progesterone suppresses follicle-stimulating hormone (FSH) secretion, preventing animals from developing follicles and exhibiting estrus. Artificial or natural progesterone can be administered in the feed, through parenteral injection, subcuticular implants, and vaginal pessaries. The progesterone is withdrawn in about 12–14 days, after which the FSH secretion will initiate the process of follicle development ( Trower, 1993 ). Estrus usually will occur in 36–60 hr (average is 48 hr). A natural method of synchronization, often applied to promote flock breeding within a short period of time (and thus parturition will be within a narrow window as well), is the introduction of sterile rams with the ewes before the beginning of the normal fall mating period. Pheromones released from males naturally stimulate the females to cycle and to synchronize their heats. It should be noted that introduction of a male during late anestrus will often stimulate ovulation in about 6 days; however, this cycle will generally be without clinical signs of estrus (silent heat). Vasectomy of rams is one method of producing sterile "teaser rams." Introduction of the buck to a group of does will induce ovulation and may even synchronize does. Does that are kept separate from the buck will show signs of estrus, will ovulate within 6–10 days, and will have normal pregnancies when introduced to a buck. Bucks with horns and intact scent glands are better able to induce ovulation than dehorned bucks, whose scent glands often been removed. Control of breeding in the goat has been studied mostly in dairy breeds in order to produce milk throughout the year and to reduce kidding labor. Goats in the luteal phase of the estrous cycle, days 4–16, are sensitive to PGF 2α (2.5–5 mg IM) and will show estrus in 36–60 hr postinjection ( Bretzlaff, 1997 ). Dosing cycling animals twice 11 days apart will synchronize goats, and artificial insemination using this method has resulted in 40–60% conception rates ( Bretzlaff, 1997 ; Greyling and Van Niekerk, 1986 ). Programs for timed breeding have been described and involve administering progestogens ( Bretzlaff, 1997 ). Vaginal pessaries of fluorogestone acetate left in place for 21 days in the doe followed by an injection of pregnant mare serum gonadotropin (PMSG) at the time of pessary removal have proven successful. Also, when primed by PGF 2α an 11-day regimen of fluorogestone acetate with PMSG given on day 9 has been successful. Synchronization of cattle estrous cycles and superovulation are used as management techniques in certain commercial cattle and dairy production settings where estrus synchronization or embryo transfer is advantageous to production and management. The methodology is also used in the research setting for coordinating donors and recipients of embryos or other genetically manipulated tissues for implantation. The options and dosing regimens are described in detail in veterinary clinical texts ( Wenzel, 1997 ; Vanderboom et al., 1997 ). In synchronization, the principle is lysis of the existing corpus luteum. The more common practices involve the use of products approved for use in cattle such as PGF 2α, one of its analogs, or products containing estradiol valerate. Progestogens are also used in conjunction with estradiol valerate. Other approaches, involving management techniques combined with pharmacologic interventions, are considered less successful. Superovulation regimens involve injections of FSH either alone or with PGF 2α at timed internals. Estrus is expected 48 hr after the final injection, and two inseminations are performed at 12 hr intervals after estrus detection. Preparation of recipients involves injection of PGF 2α or progestogens with gonadotropins such as PMSG. For greatest success as management tools, these must be combined with a consistent program that provides appropriate nutrition for all cattle involved. Synchronization of animals is also influenced by several other factors, however, such as time in the cycle when hormones are administered, response by each individual animal, whether the cow is a dairy or beef animal, parity and maturity of the cows, success of heat detection after the luteolysis, and accurate record keeping. 10. Embryo Transfer Embryo transfer involves the removal of multiple embryos from a superovulated embryo donor and transferring them to synchronized recipients. This method maximizes the genetic potential of the donor animal. The donor animal is hormonally superovulated and inseminated. In sheep, about 1 week after breeding, the embryos are surgically removed from the donor's uterus. In cattle, the procedure is nonsurgical. About 75% of expected embryos (determined by counting corpora lutea) can be recovered; successful recovery is affected by factors such as age of the donor, reproductive health, and experience of the surgeon or technician. Furthermore, not all collected embryos are of transferable quality. Recipients are hormonally synchronized with the donor animals. On the day of embryo collection, transferable embryos are implanted into the uterus of the recipient; laparoscopy has been used in the past and is now being replaced by nonsurgical methods. Pregnancy rates average about 70%. If recipients are not available, embryos, like sperm, can be frozen and kept for later transfer. Embryo transfer is commonly practiced in cattle as a herd improvement technique and as a research technique for engineered embyros. Disease screening programs for all animals involved are important because several pathogens can be transmitted directly or indirectly, such as bovine viral diarrhea virus, bluetongue virus, infectious bovine rhinotracheitis virus, and mycoplasmal species. 11. Miscellaneous Management Considerations a. Management of Male Animals In sheep flocks and goat herds, as noted, male young are usually castrated by 1 month of age. The elastrator method is the more popular for animals less than 1 week of age. Other methods include the emasculatome (crushing) and surgical removal ("knife method"). The distress associated with castration and tail docking in lambs is the subject of debate and has been researched recently ( Kent et al., 1995 ). As noted, male calves are usually castrated as early as possible and no later than 3 month of age. In some production situations, however, where maximum hormone responsive muscle development and grouping animals together for procedures dictate scheduling, the procedure may be performed on older males. Open and closed techniques are used, depending on the age of animals and on veterinary or farm practice. Breeding and vasectomized rams and bucks are usually maintained by medium to large production farms. Smaller farms often borrow breeding males. Breeding males are typically selected by production record, pedigree, and/or breed. Vasectomized males are often retired breeders and should be tattooed or identified clearly to avoid any wasted breeding time. The vasectomy technique for both species is comparable ( Smith and Sherman, 1994 ). Rams may be housed together for most of the year, whereas bucks are penned separately. Because ewes will exhibit only a limited number of estrous cycles before becoming reproductively quiescent, it is critical that the male be capable of successfully breeding the female in an expeditious manner. Any defects in the external genitalia, reproductive diseases, or musculoskeletal abnormalities may prevent successful copulatory behaviors. Furthermore, it is important to know the semen quality of the ram as one indicator of fertility. Semen can be collected via electroejaculation or by use of a teaser mount. Once semen is collected, it should be handled carefully and kept warm to prevent sperm death, leading to improper conclusions about the male. Typically, the characteristics usually evaluated as a determinate of sperm quality are volume (normal between 0.7 and 2.0 ml); motility (% of sperm moving in a forward wave; high quality is associated with motility of approximately 90%); concentration (sperm count per unit of volume as measured by a hemocytometer; high-quality semen should contain 1.8 × 10 9 sperm per ml); morphology (live versus dead cells, as determined by special stains and the percentage of abnormal-appearing sperm; neither the abnormalities nor the dead sperm should exceed 10% in high-quality semen). The extensive use of artificial insemination in the dairy cattle industry has minimized the use of bulls on many farms, although a farm may maintain a few bulls for heat detection and for "cleanup" breeding. Breeding bulls are maintained in beef production establishments. Breeding bulls must be part of the herd vaccination program, with special attention to appropriate timing of immunizations for the commonly transmitted venereal diseases campylobacteriosis and trichomoniasis. b. Cattle Tail Docking Tail docking is a relatively recent development in dairy herd management and is practiced in the belief that it will minimize bacterial contamination of the udder and therefore the milk. Tails are typically docked to about 10 inches in length. The practice is more popular in certain regions in the United States. To date, there is no published study indicating that this technique provides any distinctive advantage over keeping the tail switch hair clipped short. 1. Reproductive Physiology Sheep are seasonally polyestrous; most breeds will express estrus in the fall (Northern Hemisphere) and subsequently lamb in the spring. Some breeds of sheep may cycle in both the fall and the spring. Between seasonal periods of receptivity, the females undergo a long period of sexual quiescence called anestrus. In a research environment, ewes can be artificially stimulated to progress from anestrous to estrous cyclicity by maintaining the females in 8 hr of light and 16 hr of dark for 8–10 weeks. Puberty is reached at about 7–8 months (or earlier) in both rams and ewes; rams will typically reach puberty before their female counterparts. Ewes will display signs of estrus for about 24–30 hr and will ovulate spontaneously at the end of estrus. The estrous cycle length is 14–19 days, with an average of about 17 days. Following breeding, the average length of gestation is 147–150 days. Slightly longer gestations are observed in animals carrying single lambs (singlets), in animals carrying rams, and in certain breeds such as those derived from Merinos. Prolificacy, or the number of lambs produced per gestation, tends to be dependent on the maturity of the dam (older dams tend to have multiple lambs) and on breed characteristics (some fine-wool breeds have fewer multiple births). The Finn and Dorset breeds are especially prolific. Lambs vary in size at birth from about 3–4 lb up to 25 lb. Factors that affect birthweight include parental size, number of lambs in the litter (fewer lambs or singlets tend to be larger), age of the ewe (younger ewes have smaller lambs), lamb gender (males tend to be heavier), nutrition, and season or temperature (spring lambs tend to be larger than fall lambs). Goats are seasonally polyestrous in temperate regions, so that young are born in favorable times of the year. They are short-day breeders, in that estrus (heat) is brought about by the decreasing light of shorter days. In temperate climates of the Northern Hemisphere, goats are normally anestrous during the summer and begin cycling in the fall. The actual length of the sexual cycle depends on day length, breed, and nutrition. Most dairy goats cycle between August and February or March. Nubians often have extended breeding cycles, and the sexual season of some breeds, including the Alpine, can be extended by artificial means. The caprine gestation length averages 150 days with a variation of 145–155 days. Does bear singletons, twins, and triplets, with slightly shorter gestation when the doe is carrying triplets. Cows are polyestrous. Domestication of cattle has included selection against seasonality of the breeding season, particularly in dairy breeds but to some extent also in the beef breeds. In spite of this, cattle have been found to be still sensitive, in varying manifestations, to photoperiodicity. Reproductive physiology in cattle is influenced by many factors. The reproductive programs in source herds and at well-managed facilities will be production-related. Extensive coverage of both physiologic basics and specific industry-related criteria—for retention of a cow as a breeder, for example—are addressed in detail in texts and references oriented toward herd and production management ( "Current Veterinary Therapy," 1986 ). Gestation in cattle is approximately 280 days, with a range of 270–292 days. The length of gestation in cattle is influenced by fetal sex; fetal numbers; age and parity of the cow; breed; genotype of cow, bull, or fetus; nutrition; and local environmental factors. As noted, these factors are also important in sheep and goats. Cows usually bear single calves, although twin births do occur. When twins are combinations of male and female calves, the female should be evaluated for freemartinism. 2. Detection of Estrus and Pregnancy Ovine estrus detection is usually accomplished by the ram. Nonetheless, because artificial insemination is achievable in ewes, clinical signs of estrus are important. Typically, ewes in heat will show a mild enlargement of the vulva, with slight increases of mucus secretion. Ewes may isolate from the flock and appear anxious. It is often better and clearly more reliable to employ the help of a sterile ram to mark females when they are in standing heat. Two mating systems commonly employed include hand mating and group mating. With hand mating, ewes are placed either singly or in small groups with the ram of choice. Ewes are removed as serviced. Group mating involves placement of a mature ram with approximately 50–60 ewes for the entire 6-week breeding season. In either mating system, it is best to attach a marking harness to the male so that individual ewes can be identified as serviced. This is important so that parturition dates can be calculated. An easy, natural way to estimate pregnancy is by placing sterile teaser rams with the ewes at the end of the breeding season. Any animal marked by the ram probably has not conceived. Ultrasound scanners are also used for pregnancy detection. The ultrasound transducer is placed against the right abdomen; presence of a fetus is indicated on the machine. Claims of 98% accuracy at 6 weeks postbreeding have been made, although accuracy is generally best beyond 60 days of gestation. Interrectal Doppler ultrasound probes detect fetal pulses. Fetal heart rate is in the range of 130–160 beats per minute, whereas maternal heart rates tend to be 90–110 beats per minute. Accuracy is best beyond 60 days of pregnancy. Rectal-abdominal palpation is an inexpensive alternative. A plastic probe is introduced intrarectally into the ewe, which is restrained on her back in a cradle. The plastic probe is then manipulated toward the abdomen while palpating for the fetus with the opposite hand. The age of the doe when she first expresses heat varies with breed. Some does will express signs of heat between 3 and 4 months old. However, does should be 7–10 months old or at least 80–90 lb in weight before being bred. The caprine estrous cycle lasts 18–24 days. The duration of estrus is 24–96 hr but averages about 40 hr. The estrous cycle can be more erratic in the beginning than in the end of the breeding season ( Smith, 1997 ). "Standing heat" is usually 12–24 hr but can be as short as a few hours. Signs of estrus in goats include uneasiness, tail switching or "flagging," redness and swelling of the vulva, clear vaginal discharge that becomes white by the end of estrus, vocalization such as continuous bleating, and occasionally riding and standing with other does. A doe that is not in heat will not stand to back pressure or for attempts to hold her tail. Does can be induced to show signs of heat by buck exposure and will ovulate within 7–10 days after introduction of the buck. Goats ovulate during the later part of the estrous cycle, most between 24–36 hr after the onset of estrus. Nevertheless, goats should be mated once signs of estrus are recognized and every 12 hr until the end of estrus. Most goats kid only once a year, although some goats near the equator may kid twice. Once bred successfully, a goat will only rarely show signs of heat again. In fact, the first sign of pregnancy is usually a failure to return to heat, so animals should be carefully watched. Pregnancy can be affirmed by a variety of means. Goats will generally decrease milk production with pregnancy and should have at least a 6- to 8-week dry period for the udder to fully involute and prepare for the next milking period. In cattle, age of first estrus is dependent on the breed, the season (with winter delaying), and the level of nutrition (with higher levels hastening puberty). In some cases, the presence of mature cycling cows influences heifer puberty. With adequate nutrition, dairy breeds will reach puberty at 10–12 months and beef breeds at 11–15 months, and estrous cycles will occur regularly after the pubertal (first) estrus. Maturing heifers will often have one or more ovulations before showing overt signs of estrus. Only one follicle usually ovulates per estrous cycle ( Hafez, 1987 ) Estrus, or standing heat, in cattle averages 12–16 hr in length, with a range of 6–24 hr ( "Large Animal Internal Medicine," 1996 ). Detection of standing heat is important because it is closely related to the time of ovulation. Ovulation occurs approximately 25–32 hr after estrus. Detection of estrus is usually accomplished by visual observation of vaginal mucous discharge, mounting behavior by other females (i.e., the cow standing to be mounted is the individual in estrus), and receptivity to a bull (willingness to stand). Successful visual detection of standing heat is dependent on observation skills of handlers, knowledge of the herd, stresses (e.g., detection decreased in Bos taurus during heat stress), barn and yard surfaces (estrus detected better on dirt than on concrete), and maintaining a consistent observation schedule. Teaser animals outfitted with marking devices are also used. Other methods of detecting estrus include monitoring progesterone levels; glass slide and other evaluations of cervical mucus; change in vaginal pH; and body temperature changes ( Hafez, 1987 ). Estrous cycles are usually 21 days in length, with a range of 17–25 days. It is recommended that a heifer deliver her first calf by 2 years of age. After successful conception, progesterone levels in the cow remain elevated for most of the pregnancy, as the result of the corpus luteum of pregnancy, and they decline only during the final month. Conceptus implantation occurs beginning at about day 17. If the pregnancy fails before this time, the cow will begin to cycle again between days 18–24, but if the pregnancy ends after day 17, there may be a delayed return to estrus. Realtime ultrasonography can be used to determine pregnancy as early as 9 days after insemination, with embyros seen by days 26–29. Fetal gender can also be determined by experienced personnel by this method by about day 55. Detection of pregnancy can be successful by 25–40 days after conception by observation of failure to return to estrus or by palpation per rectum (detecting fetal membrane slip by days 30–35 and/or amniotic vesicle by days 28–35). Palpation of the fetus is possible by day 65 and placentomes by approximately days 100–110. Palpation later in presumed pregnancy will provide information based on differences in size of the two uterine horns, changes in the uterine wall, and fremitus in the miduterine artery. Pregnancy can also be determined with reasonable success rates by determining if progesterone levels are elevated at days 20–24 after insemination. Levels of bovine pregnancy-specific protein B may also be measured; this is produced by trophoblastic cells and is detectable by days 15–24 and elevated throughout pregnancy. Placentation in sheep, goats, and cattle is epitheliochorial and cotyledonary, in contrast to the diffuse or microcotyledonary placentas of horses and pigs. The placentomes, the infolded functional units of the placenta, are formed as the result of fusion of the villi of the fetal cotyledons projecting into the crypts of the maternal caruncles (specialized projections of uterine mucosa). Caruncles of sheep and goats are concave in shape, whereas those of cows are convex. The placentomes are distributed between the pregnant and nonpregant horns of the uterus in sheep, and there are 90–100. In cattle, although the placentomes initially develop around the fetus, they will eventually be distributed to the limit of the chorioallantoic membrane even in the nongravid horn. The placentomes in the nongravid horn will be smaller than in the gravid horn. The total number will be 70–120. 3. Husbandry Needs The best birthing preparation for all dams is to ensure a proper plane of nutrition (not overnutrition) and adequate exercise. If possible, the dam should be confined to a birthing pasture or sanitized maternity pen a few days prior to parturition. The birthing environment will be very important in the overall health of the dam and offspring; stress minimization and a clean environment will benefit the immune health of both in the short and long term. Outdoor parturition in a small birthing pasture has advantages. There is less stress and less intensity of pathogens. Indoor maternity pens should be clean, dry, warm, well bedded, well ventilated but draft-free, and well lighted. Adequate space per pen minimizes losses of neonates from being stepped and sat on by the dam. Management of these pens, especially if concentrated in an area, is important to minimize pathogens to which dam and young are exposed. Water troughs or buckets should be elevated or placed outside the pen, because lambs and kids have a tendency to fall or be pushed into them. Soiled bedding should be removed from the birthing pen between dams, the area sanitized and allowed to dry, and fresh bedding installed for the next occupant. Moving the female immediately before or during parturition may delay the birthing process. In goats, furthermore, in utero death may occur if parturition is unduly delayed. Dams should be monitored closely during parturition for dystocias; these may result in loss of young or in young severely weakened from the prolonged birthing process. Prior to parturition, ewes should be sheared or crutched. Crutching refers to removing wool around the perineal and mammary areas; this minimizes fetal contamination during the birth process. Foot trimming can be done at this time as well. The tail and perineal area of the doe should be clipped and cleaned to improve postbirth sanitation. In general, the pregnant doe needs a 14ft 2 (1.2 m × 1.2 m) area for the birthing process, and area needs to be increased after birthing to allow spacing for kids. Each cow should have a minimum pen area of 10 ft × 10 ft. Evaluation of a cow's udder prior to breeding and especially as parturition approaches is important in order to assure adequate nutrition and success of passive transfer by the neonate. If the udder is edematous or if mastitis is present, for example, an alternate source of colostrum (such as frozen reserves) must made be available. Poor udder conformation may also be problematic; contingency plans should be made to ensure adequate support for the young if they cannot suckle from those udders. Inexperienced heifers may react indifferently or aggressively to their offspring and should be monitored more closely than older, multiparous cows with uneventful calving histories. 4. Parturition Ewes approaching parturition generally isolate themselves from the flock, become restless, stamp their feet, blat, and periodically turn and look at their abdomen. The pelvic region will appear relaxed, and milk will be present in the udder. Once hard labor contractions begin, lambs will usually be born quickly. Animals that do not appear to be progressing correctly should be examined for dystocia. Most cases of fetal malpresentation or malpositioning can be corrected via vagino-uterine manipulation. Occasionally cesarean sections will be necessary. Sanitation, cleanliness, and adequate lubrication are of utmost importance when performing obstetrical procedures. For about a week before parturition, rectal temperature of the doe will be above normal, or about 103° F depending on environmental temperatures. Approximately 24 hr prior to birth, rectal temperature will fall to slightly below normal. Many large dairy-goat facilities attempt to control the onset of parturition in order to assist birthing. The drug of choice to induce parturition in the goat is prostaglandin F 2α (PGF 2α ) ( Ott, 1982 ). On day 144 of gestation, goats given PGF 2α (2.5–5 mg) will deliver kids within 28–57 hr. Most goats prefer to kid alone and do so unaided. Human interaction can actually interfere with normal birthing, especially in young or nervous does. Some does may reject kids if extensive human interference occurs. Does nearing parturition have an obviously swollen udder and a red, swollen vulva. Pelvic ligaments at the base of tail relax. The doe may circle to make a bed, get up and down, look at her tail or sides, push other goats away, and bleat softly. Signs of impending parturition include restlessness; vocalization (bleating softly); uneasiness, including getting up and down, pawing, and bedding; and a mucous discharge, leading to a moist tail. Eight to 12 hr prior to parturition, the cervix will dilate and the cervical mucous plug will be evident as a tan, smeared substance on the tail and perineum of the dam. Kids should present within 1–6 hr in either anterior or posterior position. A posterior presentation can be recognized by the presence of upward-pointing feet. Most does will rest between fetuses and are best left alone. However, if labor is prolonged more than 1 hr, a vaginal exam is indicated. If the pregnant goat is housed with other goats, then herdmates will express great interest in the dam. Unless moved prior to parturition, it is best to leave the dam with the group until after parturition, because removal may delay parturition. Goats are not prone to retained placenta. Normal kids will be quite active and will quickly attempt to stand and nurse. Weak kids should be towel-dried, warmed (via heat lamp, heat pad, or warm water bottle), and assisted to nurse or fed colostrum. The goat is one of the few ungulate species that will exhibit "false pregnancy," or pseudopregnancy. This is a fairly common condition. Does may have characteristically distended abdomens and may develop hydrometra and "deliver" large volumes of cloudy fluid at expected due dates. Subsequent pregnancies can be normal. Goats should be tested for pregnancy by 40 days of age. Veterinary use of prostaglandins has been successful in treating this condition. As in other species, parturition in cattle results from a combination of hormonal changes associated with the maturity of the fetus, notably ACTH (adrenocorticotropic hormone) and subsequent increases in fetal corticosteriods within 2 days of birth. Administration of ACTH to a fetus, or administration to the dam, results in premature birth. Pregnancy is extended if fetal pituitary or adrenal glands are removed surgically. The fetal cortisol probably affects placental steroid production, accounting for sharp increases in the estrogens and estrogen precursors. Coincident with this, maternal progesterone levels fall. The rising levels of estrogen cause release of maternal PGF 2α and induction of oxytocin receptors. Most cows will separate themselves from the rest of the herd. A cow will lift her tail and arch her back when she is within a few hours of delivering the calf, and most cows are recumbent when delivering the calf. Typically, the whole birthing process takes about 100 min. The length of labor of cows carrying larger calves also will be longer. Nervous heifers will take longer to deliver, and if they are disturbed, their labor may cease. All postparturient animals should be monitored for successful passage of these fetal membranes within 12 hr of birth. Veterinary intervention is required if not. Cows occasionally eat placentas, which may subsequently obstruct rumen outflow and require surgical correction. For cattle, it is now recommended practice to remove membranes that have passed, in order to prevent ingestion. 5. Early Development of the Newborn Following lambing, it is critical that the newborns be "processed" so that they will have greatest survival chances. In a well-managed flock, many lambs and ewes will not need much assistance. When assistance is given, the newborn lamb's nose and mouth should be wiped free of secretions; gently swinging the lambs, head down, aids in removal of these fluids. The lamb should be dried off and stimulated through rubbing to aid its breathing. The lamb's navel should be dipped in an iodine solution to prevent subsequent navel infections. And the lamb should be identified by the application of an ear tag or ear notch. It is extremely important that the lamb be supplied with high-quality colostrum within the first 12 hr of birth. Lambs that are not nursing on their own should be tube-fed with colostrum that has been collected and saved previously (i.e., frozen in ice cube trays) or collected from the mother after parturition. Passive transfer can be assessed by measuring serum γ-glutamyltransferase (GGT) levels ( Tessman et al., 1997 ). After the first few days, colostrum changes over to milk. Nursing lambs will ingest increasing amounts of milk as they grow. If the ewe cannot produce sufficient milk, the lamb should be "grafted" onto another ewe or fed artificially with a baby bottle. Powdered milk replacers are commercially available; the content of ewe milk is much different from that of cow's milk; thus lamb milk replacer should specifically be used. One report notes that 50–70% of lamb deaths occur during the first week of life and up to 90% occur within the first month. Good management of ewes during gestation, care of the lamb at parturition, application of an appropriate vaccination program, and observation and intervention within the first several weeks of a lamb's life will minimize losses ( Ross, 1989 ). Immediately after birth, the placenta and any birthing materials should be removed from the doe's pen. Kids do not usually need assistance. If kids are to be raised by the dam, they can be left alone; otherwise, kids should be towel-dried and removed from the dam. Kids are cold-sensitive and may require a heat lamp or other source of added warmth in cold weather. Navel cords should be dipped in tincture of iodine, and kids should be dehorned and castrated within the first several days of life. To control caprine arthritis encephalitis (CAE), kids should be immediately removed from the dam and hand-fed heat-treated colostrum. Colostrum should be heat-treated for 1 hr at 131°F. The first feeding can be up to 125 ml of colostrum. Kids should receive a total of 250 ml colostrum within the first 36–48 hr of birth. After day 3, kids can be placed on milk replacer. Milk replacers should contain 16–24% fat and 20–28% milk-based protein. By 14 days of age, kids should be consuming approximately 1.1–1.4 liters of milk per day. Kids should be introduced to forages as soon as possible and may be weaned by 6–10 weeks or 18–25 lb body weight. Milk that is fed can be reduced by 4 weeks of age by decreasing either the volume fed or the number of feedings. As with other dams, a cow is usually very attentive to her newborn calf, cleaning and softly vocalizing to the neonate. Calves typically are standing by 1 hr after birth and are suckling within 3 hr. As noted previously, dairy calves may be removed from the cow even before suckling, and the colostrum milked from the dam and given to the calf. Assistance may be required for nervous heifers, after dystocias and in extreme circumstances such as severe cold. Cleaning the newborn's nose and mouth, rubbing down the neonate, assuring that the calf does not get chilled, and assuring that it receives adequate colostrum are all important under any of these circumstances. A stressed calf's umbilical may be treated with an iodine or chlorhexidine solution, although some authors note no benefit of navel treatment, specifying that successful transfer of passive immunity and sound sanitary management of birthing area are the most crucial factors in preventing omphalitis (navel ill) ( House, 1996 ; Kersting, 1997 ; Kasari and Roussel, 1999 ). Because newborn calves can be deficient in vitamin A and iron, these may be injected to improve disease resistance ( Wikse and Baker, 1996 ). In cases in which the dams' colostrum is known to be deficient in antibodies against common diseases, vaccinations may be administered at 1 day old and followed with boosters at regular intervals. Dehorning is performed when horn buds appear. Castration is performed between 2 and 9 weeks of age or later. 6. Sexing Sexing the young in any of the ruminant species is straightforward. The vulva of the female young is located just ventral to the anus. The genitalia of the male include a penis, located along the ventral midline, and a scrotum, located in the inguinal region. The phenomenon of the freemartin, a genetic female born as a twin to a male, is the result of anastomoses between placental circulations of the twin fetuses; the mixing of blood-forming cells and germ cells results in the XX/XY chimeras. This occurs in 85–90% of phenotypic bovine females born as co-twins with males. The female will often have abnormal vulva and clitoris, and the vagina will be a blind end because of the lack of a cervix. Sometimes singleton freemartins are born if the male fetus is lost after 30 days' gestation. Multiple births are selected for and are common in sheep; the freemartin phenomenon is regarded as rare. Twinning is common in goats, and freemartinism occurs in about 6% of male-female pairs of twins. Intersexes are seen in some goat breeds and when polled goats are mated. Proof is usually based on evidence of abnormal genital development and reports of abnormal sexual behavior. 7. Weaning Prior to weaning, it must be established that lambs can nutritionally survive without mother's milk. Thus, grain, and later roughage, should be offered to lambs well in advance of the day of weaning so that they can adjust to the feedstuff. To prevent the ewes from ingesting the lamb ration, a "creep" should be set up by building an area adjacent to the ewe-lamb pen and devising a slatted entry for the lambs to enter but not the ewes. Therefore, the lambs will be accustomed to the new ration through this creep-feeding process. If lambs and ewes will be pastured later in the spring, it is still beneficial to creep-feed lambs until pasture growth is adequate enough to fulfill the requirements of the growing lambs. Lambs that are consuming 1.5–2 lb of creep feed per day may be weaned. Depending on the individual program, lambs may be weaned as early as 4 weeks of age, although 6–8 weeks of age is more common. If ewes are of a breed that will cycle twice a year, and if it is expected that they will be rebred, then the lambs must be weaned as early as possible so that lactational anestrus will resolve and ewes will recycle. Another factor is the cost of lactation rations for the ewes; if lamb grain is more economical than ewe grain, then lambs should be weaned. About 4–5 days prior to weaning, feeding of the lactation ration to the ewes should be discontinued, and only roughage fed. At weaning, the lambs should be removed in the creep, and the ewes removed to an area that is not within sight (and preferably sound) of the lambs. The ewes should be monitored for postweaning mastitis and treated as necessary. Ewes that have physical or disease problems or that have not been productive at lambing or feeding their lambs should be culled. The lambs should be monitored to assure that they continue to gain weight and are eating the new ration. Kids should be introduced to forages within the first week of life because the natural curiosity of these animals will cause them to investigate sources of feed. Kids can be weaned by 6–10 weeks or 18–25 lb. Hand-fed milk should be reduced by 4 weeks of age by reducing the volume fed or by decreasing the number of feedings. Dairy calves are now usually removed from their dams immediately after birth. It is less common now to allow the calves to remain with their dams for about 24 hr and suckle fresh colostrum during this time, because their intake will be inadequate. Dairy producers refrigerate and/or freeze the colostrum produced during the first 24 hr and feed this, diluted 50:50 with warm water, twice a day to the calves during the next 2–3 days. Holstein calves, for example, should receive a minimum of 3–5 liters within 12 hr of birth and then be fed about 10–15% of body weight in colostrum by 24 hr of age. After 3 days, calves are then placed on milk replacers, preformulated powders reconstituted with water that provide complete nutrition. Milk replacers are commercially available and should be fed according to manufacturer's recommendations Vaccination programs for calves vary with the preventive medicine program for the overall herd. Passive immunity provided by colostrum from cows on sound management programs will last until a calf is about 6–7 months old; normally vaccinations are not necessary and are contraindicated during those first 6 months. The duration of passive immunity varies considerably among calves, however; some producers choose to begin vaccinating calves at 1–2 months of age and continue with monthly booster immunizations until the animals are 7 months old, when passive immunity is no longer a possibility. 8. Artificial Insemination Artificial insemination (AI) in sheep is more difficult than in cattle because sheep are smaller and cannot be reproductively manipulated via the rectum and because the cervix of sheep is more difficult to traverse with the insemination pipette. Breeding animals artificially with fresh semen produces pregnancy rates averaging 50% (not unlike that of cattle); artificial insemination with frozen semen is less successful. Several artificial insemination techniques have been used. Laparoscopic AI involves the surgical instillation of semen into the uterus through a small abdominal opening. The procedure is successful but is technically involved and costly. Cervical AI involves the transvaginal introduction of semen into the cervix. A modification of this technique (transcervical AI) allows for penetration through the cervix into the uterus. This method (called the Guelph system for transcervical AI) leads to successful penetration into the uterus in up to 75% of ewes when performed by an experienced inseminator. Artificial insemination is now an integral part of dairy herding; natural insemination as a management practice is relatively rare. Technicians performing the AI technique are available through commercial enterprises. Dairy production employees are also trained. Information regarding the management of the donors and recipients, the storage and handling of the semen, and the skills and record keeping required is covered extensively elsewhere ( Nebel, 1997 ). 9. Synchronization Because sheep are hormonally similar to other ruminants, estrous synchronization techniques are comparable. Progesterone suppresses follicle-stimulating hormone (FSH) secretion, preventing animals from developing follicles and exhibiting estrus. Artificial or natural progesterone can be administered in the feed, through parenteral injection, subcuticular implants, and vaginal pessaries. The progesterone is withdrawn in about 12–14 days, after which the FSH secretion will initiate the process of follicle development ( Trower, 1993 ). Estrus usually will occur in 36–60 hr (average is 48 hr). A natural method of synchronization, often applied to promote flock breeding within a short period of time (and thus parturition will be within a narrow window as well), is the introduction of sterile rams with the ewes before the beginning of the normal fall mating period. Pheromones released from males naturally stimulate the females to cycle and to synchronize their heats. It should be noted that introduction of a male during late anestrus will often stimulate ovulation in about 6 days; however, this cycle will generally be without clinical signs of estrus (silent heat). Vasectomy of rams is one method of producing sterile "teaser rams." Introduction of the buck to a group of does will induce ovulation and may even synchronize does. Does that are kept separate from the buck will show signs of estrus, will ovulate within 6–10 days, and will have normal pregnancies when introduced to a buck. Bucks with horns and intact scent glands are better able to induce ovulation than dehorned bucks, whose scent glands often been removed. Control of breeding in the goat has been studied mostly in dairy breeds in order to produce milk throughout the year and to reduce kidding labor. Goats in the luteal phase of the estrous cycle, days 4–16, are sensitive to PGF 2α (2.5–5 mg IM) and will show estrus in 36–60 hr postinjection ( Bretzlaff, 1997 ). Dosing cycling animals twice 11 days apart will synchronize goats, and artificial insemination using this method has resulted in 40–60% conception rates ( Bretzlaff, 1997 ; Greyling and Van Niekerk, 1986 ). Programs for timed breeding have been described and involve administering progestogens ( Bretzlaff, 1997 ). Vaginal pessaries of fluorogestone acetate left in place for 21 days in the doe followed by an injection of pregnant mare serum gonadotropin (PMSG) at the time of pessary removal have proven successful. Also, when primed by PGF 2α an 11-day regimen of fluorogestone acetate with PMSG given on day 9 has been successful. Synchronization of cattle estrous cycles and superovulation are used as management techniques in certain commercial cattle and dairy production settings where estrus synchronization or embryo transfer is advantageous to production and management. The methodology is also used in the research setting for coordinating donors and recipients of embryos or other genetically manipulated tissues for implantation. The options and dosing regimens are described in detail in veterinary clinical texts ( Wenzel, 1997 ; Vanderboom et al., 1997 ). In synchronization, the principle is lysis of the existing corpus luteum. The more common practices involve the use of products approved for use in cattle such as PGF 2α, one of its analogs, or products containing estradiol valerate. Progestogens are also used in conjunction with estradiol valerate. Other approaches, involving management techniques combined with pharmacologic interventions, are considered less successful. Superovulation regimens involve injections of FSH either alone or with PGF 2α at timed internals. Estrus is expected 48 hr after the final injection, and two inseminations are performed at 12 hr intervals after estrus detection. Preparation of recipients involves injection of PGF 2α or progestogens with gonadotropins such as PMSG. For greatest success as management tools, these must be combined with a consistent program that provides appropriate nutrition for all cattle involved. Synchronization of animals is also influenced by several other factors, however, such as time in the cycle when hormones are administered, response by each individual animal, whether the cow is a dairy or beef animal, parity and maturity of the cows, success of heat detection after the luteolysis, and accurate record keeping. 10. Embryo Transfer Embryo transfer involves the removal of multiple embryos from a superovulated embryo donor and transferring them to synchronized recipients. This method maximizes the genetic potential of the donor animal. The donor animal is hormonally superovulated and inseminated. In sheep, about 1 week after breeding, the embryos are surgically removed from the donor's uterus. In cattle, the procedure is nonsurgical. About 75% of expected embryos (determined by counting corpora lutea) can be recovered; successful recovery is affected by factors such as age of the donor, reproductive health, and experience of the surgeon or technician. Furthermore, not all collected embryos are of transferable quality. Recipients are hormonally synchronized with the donor animals. On the day of embryo collection, transferable embryos are implanted into the uterus of the recipient; laparoscopy has been used in the past and is now being replaced by nonsurgical methods. Pregnancy rates average about 70%. If recipients are not available, embryos, like sperm, can be frozen and kept for later transfer. Embryo transfer is commonly practiced in cattle as a herd improvement technique and as a research technique for engineered embyros. Disease screening programs for all animals involved are important because several pathogens can be transmitted directly or indirectly, such as bovine viral diarrhea virus, bluetongue virus, infectious bovine rhinotracheitis virus, and mycoplasmal species. 11. Miscellaneous Management Considerations a. Management of Male Animals In sheep flocks and goat herds, as noted, male young are usually castrated by 1 month of age. The elastrator method is the more popular for animals less than 1 week of age. Other methods include the emasculatome (crushing) and surgical removal ("knife method"). The distress associated with castration and tail docking in lambs is the subject of debate and has been researched recently ( Kent et al., 1995 ). As noted, male calves are usually castrated as early as possible and no later than 3 month of age. In some production situations, however, where maximum hormone responsive muscle development and grouping animals together for procedures dictate scheduling, the procedure may be performed on older males. Open and closed techniques are used, depending on the age of animals and on veterinary or farm practice. Breeding and vasectomized rams and bucks are usually maintained by medium to large production farms. Smaller farms often borrow breeding males. Breeding males are typically selected by production record, pedigree, and/or breed. Vasectomized males are often retired breeders and should be tattooed or identified clearly to avoid any wasted breeding time. The vasectomy technique for both species is comparable ( Smith and Sherman, 1994 ). Rams may be housed together for most of the year, whereas bucks are penned separately. Because ewes will exhibit only a limited number of estrous cycles before becoming reproductively quiescent, it is critical that the male be capable of successfully breeding the female in an expeditious manner. Any defects in the external genitalia, reproductive diseases, or musculoskeletal abnormalities may prevent successful copulatory behaviors. Furthermore, it is important to know the semen quality of the ram as one indicator of fertility. Semen can be collected via electroejaculation or by use of a teaser mount. Once semen is collected, it should be handled carefully and kept warm to prevent sperm death, leading to improper conclusions about the male. Typically, the characteristics usually evaluated as a determinate of sperm quality are volume (normal between 0.7 and 2.0 ml); motility (% of sperm moving in a forward wave; high quality is associated with motility of approximately 90%); concentration (sperm count per unit of volume as measured by a hemocytometer; high-quality semen should contain 1.8 × 10 9 sperm per ml); morphology (live versus dead cells, as determined by special stains and the percentage of abnormal-appearing sperm; neither the abnormalities nor the dead sperm should exceed 10% in high-quality semen). The extensive use of artificial insemination in the dairy cattle industry has minimized the use of bulls on many farms, although a farm may maintain a few bulls for heat detection and for "cleanup" breeding. Breeding bulls are maintained in beef production establishments. Breeding bulls must be part of the herd vaccination program, with special attention to appropriate timing of immunizations for the commonly transmitted venereal diseases campylobacteriosis and trichomoniasis. b. Cattle Tail Docking Tail docking is a relatively recent development in dairy herd management and is practiced in the belief that it will minimize bacterial contamination of the udder and therefore the milk. Tails are typically docked to about 10 inches in length. The practice is more popular in certain regions in the United States. To date, there is no published study indicating that this technique provides any distinctive advantage over keeping the tail switch hair clipped short. a. Management of Male Animals In sheep flocks and goat herds, as noted, male young are usually castrated by 1 month of age. The elastrator method is the more popular for animals less than 1 week of age. Other methods include the emasculatome (crushing) and surgical removal ("knife method"). The distress associated with castration and tail docking in lambs is the subject of debate and has been researched recently ( Kent et al., 1995 ). As noted, male calves are usually castrated as early as possible and no later than 3 month of age. In some production situations, however, where maximum hormone responsive muscle development and grouping animals together for procedures dictate scheduling, the procedure may be performed on older males. Open and closed techniques are used, depending on the age of animals and on veterinary or farm practice. Breeding and vasectomized rams and bucks are usually maintained by medium to large production farms. Smaller farms often borrow breeding males. Breeding males are typically selected by production record, pedigree, and/or breed. Vasectomized males are often retired breeders and should be tattooed or identified clearly to avoid any wasted breeding time. The vasectomy technique for both species is comparable ( Smith and Sherman, 1994 ). Rams may be housed together for most of the year, whereas bucks are penned separately. Because ewes will exhibit only a limited number of estrous cycles before becoming reproductively quiescent, it is critical that the male be capable of successfully breeding the female in an expeditious manner. Any defects in the external genitalia, reproductive diseases, or musculoskeletal abnormalities may prevent successful copulatory behaviors. Furthermore, it is important to know the semen quality of the ram as one indicator of fertility. Semen can be collected via electroejaculation or by use of a teaser mount. Once semen is collected, it should be handled carefully and kept warm to prevent sperm death, leading to improper conclusions about the male. Typically, the characteristics usually evaluated as a determinate of sperm quality are volume (normal between 0.7 and 2.0 ml); motility (% of sperm moving in a forward wave; high quality is associated with motility of approximately 90%); concentration (sperm count per unit of volume as measured by a hemocytometer; high-quality semen should contain 1.8 × 10 9 sperm per ml); morphology (live versus dead cells, as determined by special stains and the percentage of abnormal-appearing sperm; neither the abnormalities nor the dead sperm should exceed 10% in high-quality semen). The extensive use of artificial insemination in the dairy cattle industry has minimized the use of bulls on many farms, although a farm may maintain a few bulls for heat detection and for "cleanup" breeding. Breeding bulls are maintained in beef production establishments. Breeding bulls must be part of the herd vaccination program, with special attention to appropriate timing of immunizations for the commonly transmitted venereal diseases campylobacteriosis and trichomoniasis. b. Cattle Tail Docking Tail docking is a relatively recent development in dairy herd management and is practiced in the belief that it will minimize bacterial contamination of the udder and therefore the milk. Tails are typically docked to about 10 inches in length. The practice is more popular in certain regions in the United States. To date, there is no published study indicating that this technique provides any distinctive advantage over keeping the tail switch hair clipped short. E. Behavior Healthy ruminants have good appetites, chew cud, are alert and curious, have healthy intact coats, move without hindrance, and have clear, bright, clean eyes and cool dry noses. Even adult animals, when provided sufficient space, will play. Sheep and goats have tidy "pelleted" dark green feces. Cattle have pasty, moist, dark green-brown feces. Ruminants normally vocalize, and handlers will learn to recognize normal communication among the group or directed at caregivers in contrast to that when animals are stressed. Excessive, strained vocalizations are often a sign of stress in cattle. "Bruxism," or grinding of the teeth by a ruminant, is usually associated with discomfort or pain. Other signs of discomfort, stress, or illness include decreased time spent eating and cud chewing, restlessness, prolonged recumbency with outstretched neck and head, and hunched back when standing. Unhealthy ruminants may be thin, may arch their backs or favor a limb, or may have external lumps or swollen joints, an unusual abdominal profile, or rough or dull coats. All ruminants are herd animals to some extent and social individuals; therefore, every effort should be made to allow contact among animals, in terms either of direct contact or of sound, smell, or sight. Human contact and handling should be initiated promptly and maintained regularly and consistently throughout the animal's stay in the research facilities. Animals should be provided sufficient time to acclimate to handlers and research staff. Cattle and sheep can hear at higher frequencies than humans can and may react to sounds not perceived by handlers. Knowledge of the peculiarities of sheep behavior will increase the ease of handling and decrease stress-related effects in research. Generally, fine-wooled breeds, such as Rambouillet, are the most gregarious and are best handled in groups. The meat, or "downs," breeds tend to be less gregarious, and the long-wooled breeds tend to be solitary ( Ross, 1989 ; ASIA, 1996 ). Nonetheless, movement of animals is simplified by proper facility design. Sheep have a wide-angle visual field and are easily scared by activities that are taking place behind them. Sheep should be moved slowly and gently. To capture individuals within a flock, it is best to confine the flock to a smaller space and use a shepherd's crook or to gently catch the animal in front of the neck/thorax. Grabbing the wool can injure the animals, as well as damage the wool and the underlying tissues. Sheep move best in chutes that have solid walls, and individual animals will generally follow a lead animal. Any escape route will be challenged and, if successfully breached, will disrupt the entire flock movement. Sheep movement is also disrupted by contrasts such as light and shadows that impinge on a chute or corral. Finally, like most animals, sheep have a flight zone (minimum zone of comfort), the penetration of which will result in sheep scattering. This minimal flight distance can be modified by increasing handling of the animals and working at the edge of the zone, but it should always be considered when working with animals in chutes, pens, or other confined areas. Goats exhibit behavioral characteristics that make them quite distinct from other ruminants. Their browsing activity makes them quite orally investigative. Goats will readily nibble or chew just about anything they come in contact with, so researchers should keep all paperwork and equipment out of reach. A herd of goats will readily chew through wood gates and fencing, especially when confined in areas without alternatives for chewing behavior. Goats are also inquisitive, restless, agile jumpers and climbers, and quite mischievous. If maintained in paddocks, strong high fences are essential, as are adequate spaces for exercise or boulders or rock piles for hoof maintenance and recreational climbing. Goats are more tolerant of isolation and are more easily acclimated to human contact than sheep are, but goats will confront unfamiliar intruders and make sneezing noises. Goats with horns will use them to advantage, and horns may also become entangled in fencing. Although less strongly affected by flock behavior, goats are social animals. Most goats raised in close human contact are personable and cooperative and can easily be taught to stand for various procedures, including blood collection. An understanding of breed behaviors, sources of stress in cattle, play behaviors, calf behaviors, and dominance determinants will contribute to prevention of injuries to handlers and better health and welfare of the animals. Ruminants of all ages, especially cattle of all ages, should be handled with an appreciation of the serious injury to human handlers that may result ( Houpt, 1998 ). Cattle have a wide visual field, as sheep do, and a flight zone that varies in size, according to previous handling experiences (gentle handling and animal tameness make the flight zone smaller) and the circumstances of the moment (Grandin, 1993). Groups of cattle are moved effectively around a facility by utilizing chute systems, with sequences of gates, that minimize chances of animals turning around. Dairy cattle have been bred and selected over centuries for their docile, tractable characters and production characteristics. In contrast, beef breeds have not been selected for docility and are generally more difficult to handle and restrain. Beef breeds, such as Angus, are known for their independent natures and protective maternal instincts. All cattle respond well to feed as a reward for desired behavior. Healthy cattle typically are very curious and watchful and are alert to sounds and smells. When not grazing or eating, they hold their heads up. When sleeping, the head and neck may be tucked back. Because of ruminant digestive and metabolic needs, much of the day is spent eating or cud chewing. Occasionally, adult cows sit upright like dogs. Cattle maintained inside tend to be more docile. In addition to forced isolation from other cattle, sources of stress include rough attitudes of handlers and unfamiliar visual patterns, routines, or environments. These stressors may exacerbate signs of systemic illnesses. Calves are known for non-nutritive suckling, bar licking, and tongue rolling. Non-nutritive suckling behavior is greater in hungry calves and also right after a milk meal. It is best to provide nipples and other clean noninjurious materials for the animals to suck. Non-nutritive suckling can be detrimental in group-housed calves because it can result in disease transmission and hair ball formation. Environmental enrichment devices have been developed to cope with this behavior. The behavior diminishes as the animals are weaned onto solid food ( Morrow-Tesch, 1997 ). Play activity and vocalizations of calves mimic adult dominance behaviors. Play activity by young adult cattle is more common in males, can be quite rough, and is often triggered by a change in the environment. Dominance behaviors are dependent on direct physical contact among the cattle, and dominance hierarchies are established within a herd. Horns, age, and weight have been reported to be the most important determinants. Aggressive behaviors in cattle may be triggered by newly introduced animals or unfamiliar visual patterns and by feeding when animals are very hungry. Aggression is more common among intact adult males. III. DISEASES This section focuses primarily on the more common diseases affecting sheep, goats, and cattle in the United States and elsewhere in North America and those that are reportable. For detailed information not included in this limited overview and for diseases of importance internationally, the authors recommend several excellent comprehensive and focused veterinary clinical texts and periodicals that address ruminant diseases, preventive medicine, and individual and flock or herd management. These are listed under "Major References" in the reference list at the end of this chapter. Recommendations for current drug therapies, both approved and off-label use in ruminants, including withholding prior to slaughter, formularies, and related information can be found in the references noted above and in formularies ( Hawk and Leary, 1995 ; Plumb, 1999 ). In addition, the Food Animal Residue Avoidance Databank (FARAD), accessible on the Internet , should be used as a resource. FARAD is a food safety project of the U.S. Department of Agriculture and is an information resource to prevent drug and pesticide residues in food animals and animal products. A. Infectious Diseases 1. Bacterial, Mycoplasmal, and Rickettsial Diseases a. Actinobacillosis ("Wooden Tongue") Etiology. Actinobacillus lignieresii is an aerobic, nonmotile, non-spore-forming, gram-negative rod that is widespread in soil and manure and is found as normal flora of the respiratory, gastrointestinal, and reproductive tracts of ruminants. In sheep and cattle, A. lignieresii causes sporadic, noncontagious, and potentially chronic disease characterized by diffuse abscess and granuloma formation in tissues of the head and occasionally other body organs. This disease, called wooden tongue, has not been documented in goats. Clinical signs. Skin lesions are common. Tongue lesions are more common in cattle than in sheep. Lip lesions are more common in sheep. Soft-tissue or lymph node swelling accompanied by draining tracts is observed in the head and neck regions, as well as other areas. Animals may have difficulty prehending food; may be anorexic, weak, unthrifty and depressed; and may salivate excessively. Diagnosis is made based on clinical signs and is confirmed by culture. Epizootiology and transmission. The organism penetrates wounds of the skin, mouth, nose, gastrointestinal tract, testicles, and mammary gland. Rough feed material and foreign bodies may play a role in causing abrasions. Actino bacillus lignieresii then enters into deeper tissues, where it causes chronic inflammation and abscess formation. Lymphatic spread may occur, leading to abscessation of lymph nodes or infection of other organs. Necropsy findings. Purulent discharges of white-green exudate drain from the tracts that often extend from the area of colonization to the skin surface. Exudates will also contain characteristic small white-gray (sulfurlike) granules. The pus is usually nonodorous. Differential diagnosis. Contagious ecthyma and caseous lymphadenitis are the primary differentials. Diseases or injuries causing oral pain and discomfort, such as dental infections, foreign bodies, and trauma, should be considered. Treatment. Animals should be fed softer feeds. Antibiotics such as sulfonamides, tetracyclines, and ampicillin are effective, although high doses and long durations of therapy are required. Penicillin is not effective. Weekly systemic administration of sodium iodide for several weeks is not as effective as antibiotic therapy. Surgical excision and drainage are not recommended. Prevention and control. Because the organism enters through tissue wounds, especially those associated with oral trauma, feedstuffs should be closely monitored for coarse material and foreign bodies. b. Arcanobacterium Infection (Formerly actinomycosis, or "Lumpy Jaw ") Etiology. Arcanobacterium (formerly known as Actinomyces or Corynebacterium) pyogenes and A. bovis are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Arcanobacterium bovis is a normal part of the ruminant oral microflora and is the organism associated with "lumpy jaw" in cattle; this syndrome is rarely seen in sheep and goats. This organism has also been associated with pharyngitis and mastitis in cattle. Clinical signs and diagnosis. Arcanobacterium bovis causes mandibular lesions primarily. The mass will be firm, non-painful, and immovable. Draining tracts may develop over time. If teeth roots become involved, painful eating and weight loss are evident. Radiographic studies are helpful for determining fistulas. Diagnosis is based on clinical signs, and culture is required to confirm Arcanobacterium. The prognosis is poor for lumpy jaw. Epizootiology and transmission. These organisms are normal flora of the gastrointestinal tracts of ruminants and gain entrance into the tissues through abrasions and penetrating wounds. Necropsy. Draining lesions with sulfurlike granules (as with actinobacillosis) are frequently observed. Pathogenesis. Arcanobacterium pyogenes is known to produce an exotoxin, which may be involved in the pathogenesis. Differential diagnosis. Actinobacillus lignieresii and caseous lymphadenitis are important differentials for draining tracts. A major differential for omphalophlebitis is an umbilical hernia, which will typically not be painful or infected. There are many differentials for septic joints and polyarthritis: Chlamydia spp., Mycoplasma spp., streptococci, coliforms, Erysipelothrix rhusiopathiae, Fusobacterium necrophorum, and Salmonella spp. Tumors, trauma to the affected area, such as the mandible, and dental disease or oral foreign body should also be considered. Prevention and control. Arcanobacterium bovis lesions can be prevented or minimized by feeds without coarse or sharp materials. Treatment. Penicillin or derivatives such as ampicillin or amoxicillin are treatments of choice. Sodium iodides (intravenous) and potassium iodides (orally) have been utilized also. Extended antibiotic therapy may be necessary. Surgical excision is an option. In addition to medications noted above, isoniazid is somewhat effective for A. bovis infections in nonpregnant cattle. Research complications. The possibility of long-term infection and long therapy are factors that will diminish the value of affected research animals. c. Actinomycosis Omphalophlebitis, omphaloarteritis, omphalitis, and navel ill are terms referring to infection of the umbilicus in young animals. Arcanobacterium pyogenes is the most common organism causing omphalophlebitis, an acute localized inflammation and infection of the external umbilicus. Most cases occur within the first 3 months of age, and animals are presented with a painful enlargement of the umbilicus. Animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, hematuria, and so on. Severe sequelae may include septicemia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, and endocarditis. Research complications. Young stock affected by omphalophlebitis may be inappropriate subjects because of growth setbacks and physiologic stresses from the infection. Affected adult animals will not thrive and, even with therapy, may not be appropriate research subjects. d. Anthrax Etiology. Bacillus anthracis is a nonmotile, capsulated, spore-forming, aerobic, gram-positive bacillus that is found in alkaline soil, contaminated feeds (such as bonemeal), and water. Common names for the disease anthrax include woolsorters' disease, splenic fever, charbon, and milzbrand. Clinical signs and diagnosis. Anthrax is a sporadic but very serious infectious disease of cattle, sheep, and goats characterized by septicemia, hyperthermia, anorexia, depression, listlessness, depression, and tremors. Subacute and chronic cases may occur also and are characterized by swelling around the shoulders, ventral neck, and thorax. The incubation period is 1 day to 2 weeks. Bloody secretions such as hematuria and bloody diarrhea often occur. Abortion and blood-tinged milk may also be noted. The disease is usually fatal, especially in sheep and goats, after 1–3 days. Death is the result of shock, renal failure, and anoxia. Diagnosis is based on the clinical signs of peracute deaths and hemorrhage. Stained blood smears may show short, single to chained bacilli. Blood may be collected from a superficial vein and submitted for culture. Epizootiology and transmission. Cattle and sheep tend to be affected more commonly than goats, because of grazing habits. Older animals are more vulnerable than younger, and bulls are more vulnerable than cows. Although the disease occurs worldwide, and even in cold climates, most cases in the United States occur in the central and western states, and outbreaks usually occur as the result of spore release after abrupt climatic changes such as heavy rainfall after droughts or during warmer, dryer months. Spores survive very well in the environment. The anthrax organisms (primarily spores) are generally ingested, sporulate, and replicate in the local tissues. Abrasive forages may play a role in infection. Transmission via insect bites or through skin abrasions rarely occurs. Necropsy. Necropsies should not be done around animal pens or pastures, and definitive diagnoses may be made without opening the animals. Incomplete rigor mortis, rapid putrefaction, and dark, uncoagulated blood exuding from all body orifices are common findings. Blood collected carefully and promptly from peripheral veins of freshly dead animals can be used diagnostically. Splenomegaly, cyanosis, epicardial and subcutaneous hemorrhages, and lymphadenopathy are characterisitic of the disease. Pathogenesis. The rapidly multiplying organisms enter the lymphatics and bloodstream and result in a severe septicemia and neurotoxicosis. Encapsulation protects the organisms from phagocytosis. Liberated toxins cause local edema. Differential diagnosis. Although anthrax should always be considered when an animal healthy the previous day dies acutely, other causes of acute death in ruminants should be considered, e.g., bloat, poisoning, enterotoxemia, malignant edema, blackleg, and black disease. Prevention and control. Outbreaks must be reported to state officials. Anthrax is of particular concern as a bioterrorism agent. Any vaccination programs should also be reviewed with regulatory personnel. Herds in endemic areas and along waterways are usually vaccinated routinely with the Sterne-strain spore vaccine (virulent, nonencapsulated, live). Careful hygiene and quarantine practices are crucial during outbreaks. Dead animals and contaminated materials should be incinerated or buried deeply. Biting insects should be controlled. The disease is zoonotic and a serious public health risk. Treatment. Treatment of animals in early stages with penicillin and anthrax antitoxin (hyperimmune serum, if available) may be helpful. Amoxicillin, erythromycin, oxytetracycline, gentamicin, and fluoroquinolones are also good therapeutic agents. During epidemics, animals should be vaccinated with the Sterne vaccine. Research complications. Natural and experimental anthrax infections are a risk to research personnel; the pathogen may be present in many body fluids and can penetrate intact skin. The organism sporulates when exposed to air, and spores may be inhaled during postmortem examinations. e. Brucellosis Etiology. Brucella is a nonmotile, non-spore-forming, nonencapsulated, gram-negative coccobacillus. Brucella abortus is one of several Brucella species that infects domestic animals but cross-species infections occur rarely. Brucella abortus or B. melitensis may cause brucellosis in sheep, cattle, and goats. Brucella melitensis (biovar 1, 2, or 3) is the primary cause of sheep disease ( Garin-Bastuji et al., 1998 ). Brucella ovis is more commonly associated with ovine epididymitis or orchitis than abortion. In the United States, clusters of brucellosis are still found in western areas contiguous to Yellowstone National Park. Bang's disease is the common name given to the disease in ruminants. Clinical signs and diagnosis. Brucella melitensis in the adult ewe is generally asymptomatic and self-limiting within about 3 months. However, because the organism may enter and cause necrosis of the chorionic villi and fetal organs, abortion or stillbirths may occur. Abortion usually occurs in the third trimester, after which the ewe will appear to recover. It has been reported that up to 20% of infected ewes may abort more than once. Rams will also be infected and may develop orchitis or pneumonia. The disease caused by B. ovis is manifested by clinical or subclinical infection of the epididymis, leading to epididymal enlargement and testicular atrophy. Brucella ovis causes decreased fertility. Brucella melitensis is the more common cause of brucellosis in goats. Brucella abortus has been shown to infect goats in natural and experimental infections, and B. ovis has also been shown to infect goats experimentally. Does infected with B. melitensis will also abort during the third trimester. Infections with B. abortus in cattle produce few clinical signs. There may be a brief septicemia during which organisms are phagocytosed by neutrophils and fixed macrophages in lymph nodes. In cows, the organism localizes in supramammary lymph nodes and udders and in the endometrium and placenta of pregnant cows. Infection may cause abortions after the fifth month, with resulting retained placentas. Permanent infection of the udder is common and results in shedding of organisms in milk. In bulls, the organism may cause unilateral orchitis and epidydimitis and involvement of the secondary sex organs. Organisms may be in the semen. In infected herds, lameness may also be a clinical sign. Diagnosis of brucellosis can be made by bacterial isolation of the Brucella organism from necropsy samples (especially the fetal stomach contents), as well as by supportive serological evidence. Many serological tests are available, such as the tube and plate agglutination tests, the card or rose bengal test, the rivanol precipitation test, complement fixation, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and others. Test selection is often dependent on state requirements in the United States. Epizootiology and transmission. The primary route of transmission of B . abortus is ingestion of the organism from infected tissues and fluids (milk, vaginal and uterine discharges) during and for a few weeks after abortion or parturition; contaminated semen is considered to be a minor source of infection. Exposure to the organism may occur via the gastrointestinal tract (contaminated feed or water), the respiratory tract (droplet infection), or the reproductive tract (contaminated semen) and through other mucous membranes such as the conjunctiva. Brucella ovis is transmitted in the semen, as well as orally or nasally through contaminated feed and bedding. Necropsy findings. A sheep fetus aborted due to Brucella will exhibit generalized edema. The liver and spleen will be swollen, and serosal surfaces will be covered with petecchial hemorrhages. Peritoneal and pleural cavities often contain serofibrinous exudates. The placenta will be leathery. Pathogenesis. Ruminants are considered especially susceptible to Brucella infection, because of higher levels of erythritol (a sugar alcohol), which is a growth stimulant for the organism. Brucella utilizes erythritol preferentially over glucose as an energy source. Placentas and male genitalia also contain high levels of erythritol. Brucella organisms also evade lysis when phagocytosed by macrophages and neutrophils and survive intracellularly in phagosomes. Abortion is the result of placentitis, typically during the third trimester of gestation. Brucella ovis enters the host through the mucous membranes, then passes into the lymphatics, causes hyperplasia of reticuloendothelial cells, and is spread to various organs via the blood. The organism localizes in the epididymides, the seminal vesicles, the bulbourethral glands, and the ampullae. Orchitis may be a sequelae of the disease. Epididymitis can be diagnosed by identifying gross lesions by palpation of the epididymides, by serological evidence of antibodies to B. ovis, and by semen cultures. Differential diagnosis. Differential diagnoses include all other abortion-causing diseases. Many other agents, such as Actinobacillus spp., Arcanobacterium (Actinomyces) pyogenes, Eschericia coli, Pseudomonas spp., Proteus mirabilis, Chlamydia, Mycoplasma, and others may be associated with ovine epididymitis and orchitis. A clinically and pathologically similar agent, Actinobacillus seminis, has been isolated from virgin rams. This organism has morphological and staining characteristics similar to those of B. ovis and complicates the diagnosis ( Genetzky, 1995 ). Prevention and control. The Rev 1 vaccine has been recommended for vaccination of ewe lambs in endemic areas, but this vaccine is not used in the United States. Separating young rams from potentially infected older males, sanitizing facilities, and vaccinating them with B. ovis bacterin can prevent the disease. Over the past 20 years, aggressive federal and state regulatory and cattle herd health programs in the United States have provided control and prevention mechanisms for this pathogen through a combination of serological monitoring of herds, slaughter of diseased animals, herd management, vaccination programs, and monitoring of transported animals. Most states are considered brucellosis-free in the cattle populations; thus, procurement of ruminants that have been exposed to this infectious agent will be unlikely. Cattle vaccination programs can be very successful when conducted on a herd basis to reduce likelihood of exposure. Strain 19 and the recently validated attentuated strain RB51 are live vaccines and can be used in healthy heifer calves 4–12 months old. Vaccination for older animals may be done under certain circumstances. Vaccination of bull calves is not recommended, because of low likelihood of spread through semen and possibility of vaccination-induced orchitis. The strain 19 vaccine induces long-term cell-mediated immunity, protects a herd from abortions, and protects the majority of a herd from reactors during a screening and culling program. The vaccine will not, however, protect the animals from becoming infected with B. abortus. Strain 19 vaccine induces an antibody response in cattle. The RB51 vaccine does not result in antibody titers and therefore is advantageous because infection with Brucella can be determined serologically. The RB51 vaccine has been designated as the official calfhood bovine brucellosis vaccine in the United States by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS) ( Stevens et al., 1997 ). Brucella vaccine should be administered to unstressed, healthy cattle, with attention to particular side effects of the vaccination material and to prevention of compounding stresses associated with weaning, regrouping, other management changes, and shipping. The RB51 is regarded as less pathogenic and abortigenic in cattle. Treatment. Definite confirmation of Brucella infection is important from the standpoint of public and herd health. Culling is considered the treatment of choice in cattle herds. Rams infected with B. ovis should be isolated and treated with tetracyclines. Research complications. Brucellosis represents a research complication as a cause of abortions and of infections in male ruminants. Impairment of the infected host's immune system, especially alteration of phagocytic cells where the bacteria stay in membrane-bound vesicles, should be considered. The potential complications of needle sticks by large-animal veterinarians with the strain 19 vaccine and the public health risks (undulant fever) are well known. Less is known presently regarding the RB51 vaccine effects in humans. Epidemiologic and diagnostic methodologies are being developed to track and monitor these cases. There is also a risk of human infection from handling infected materials during a dystocia or postmortem. Worldwide, B. melitensis is the leading cause of human brucellosis. f. Campylobacteriosis (Vibriosis) i. Campylobacter fetus subsp. intestinalis; C. jejuni infection (ovine vibriosis) Etiology. Campylobacter (Vibrio) fetus subsp. intestinalis, a pleomorphic curved to coccoid, motile, non-spore-forming, gram-negative bacterium, causes campylobacteriosis, the most important cause of ovine abortion in the United States. There are few reports of campylobacteriosis in goats in the United States. Vibriosis is derived from the name formerly given to the genus; the term is still frequently used. Clinical signs and diagnosis. Ovine vibriosis is a contagious disease that causes abortion, stillbirths, and weak lambs. The organism inhabits the intestines and gallbladder in subclinical carriers. Abortion generally occurs in the last trimester, and abortion storms may occur as more susceptible animals, such as maiden ewes, become exposed to the infectious tissues. It is reported that 20–25% of the flock may become infected and up to 5% of the ewes will die ( Jensen and Swift, 1982 ). Some lambs may be born alive but will be weak, and dams will not be able to produce milk. Diagnosis is achieved by microscopic identification or isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharges. Tentative identification of the organism can be made by observing curved ("gull-wing") rods in Giemsa-stained or Ziehl–Neelsen–stained smears from fetal stomach contents, placentomes, or maternal uterine fluids. Epizootiology and transmission. Campylobacteriosis occurs worldwide. Campylobacter spp., such as C. jejuni, normally inhabit ovine gastrointestinal tracts. Transmission of the disease occurs through the gastrointestinal tract, followed by shedding, especially associated with aborted tissues and fluids. In abortion storms, considerable contamination of the environment will occur due to placenta, fetuses, and uterine fluids. Ewes may have active Campylobacter organisms in uterine discharges for several months after abortion. The bacteria will also be shed in feces, and feed and water contamination serve as another source. There is no venereal transmission in the ovine. Necropsy. Aborted fetuses will be edematous, with accumulation of serosanguinous fluids within the subcutis and muscle tissue fascia. The liver may contain 2–3 cm pale foci. Placental tissues will be thickened and edematous and will contain serous fluids similar to those of the fetus. The placental cotyledons may appear gray. Pathogenesis. The organism enters the bloodstream and causes a short-term bacteremia (1–2 weeks) prior to the localizing of the bacteria in the chorionic epithelial cells and finally passing into the fetus. Differential diagnosis. Toxoplasma, Chlamydia, and Listeria should be considered in late gestation ovine abortions. Prevention and control. A bacterin is available to prevent the disease. Carrier states have been cleared by treating with a combination of antibiotics, including penicillin and oral Chlortetracycline. Aborting ewes should be isolated immediately from the rest of the flock. After an outbreak, ewes will develop immunity lasting 2–3 years. Treatment. Infected animals should be isolated and provided with supportive therapy. Prompt decontamination of the area and disposal of the aborted tissues and discharges are important. Research complications. Losses from abortion may be considerable. Campylobacter ssp. are zoonotic agents, and C. fetus subsp. intestinalis may be the cause of "shepherd's scours." ii. Campylobacter fetus subsp. venerealis infection (bovine vibriosis) Etiology. Campylobacter fetus subsp. venerealis is the main cause of bovine campylobacteriosis abortions. It does not cause disease in other ruminant species. Clinical signs and diagnosis. Preliminary signs of a problem in the herd will be a high percentage of cows returning to estrus after breeding and temporary infertility. This will be particularly apparent in virgin heifers that may return to estrus by 40 days after breeding. Long interestrous intervals also serve an indication of a problem. Spontaneous abortions will occur in some cases, typically during the fourth to eighth months of gestation. Severe endometritis may lead to salpingitis and permanent infertility. Demonstration or isolation of the organism, a curved rod with corkscrew motility, is the basis for diagnosis. The vaginal mucous agglutination test is used to survey herds for campylobacteriosis. Serology will not be worthwhile, because the infection does not trigger a sufficient antibody response. Culture from breeding animals may be difficult because Campylobacter will be overgrown by faster-growing species also present in the specimens. Epizootiology and transmission. The bacteria is an obligate, ubiquitous organism of the genital tract. Transmission is from infected bulls to heifers. Older cows develop effective immunity. Necropsy findings. Necrotizing placentitis, dehydration, and fibrinous serositis will be found grossly. In addition, bronchopneumonia and hepatitis will be seen histologically. Pathogenesis. Campylobacter organisms grow readily in the genital tract, and infection is established within days of exposure. The resulting endometritis prevents conception or causes embyronic death. Differential diagnosis. The primary differential diagnosis for campylobacteriosis is trichomoniasis. Other venereal diseases should be considered when infertility problems are noted in a herd. These include brucellosis, mycoplasmosis, ureaplasmosis, infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV), and bovine virus diarrhea (BVD). Leptospirosis should also be considered. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. Killed bacterin vaccines are available, either as oil adjuvant or as aluminum hydroxide adsorbed. The former is preferred because of duration of immunity but causes granulomas. That vaccine also has specific recommendations regarding administration several months before the breeding season. The latter product is administered closer to the breeding season, and the duration of immunity is not as prolonged. In both cases, boosters should be given after the initial immunization and as part of the regular prebreeding regimen. Only one bacterin product is approved for use in bulls. Many combination vaccine products contain only the aluminum hydroxide adsorbed product. Artificial insemination (AI) is particularly useful at controlling the disease, but bulls used for AI must be part of a screening program for this and other venereal diseases such as trichomoniasis. Treatment. Cows will usually recover from the infection, and treatment with antibiotics such as penicillin, administered as an intrauterine infusion, improve the chances of returning to breeding condition. g. Caprine Staphylococcal Dermatitis Etiology. The most common caprine bacterial skin infection is caused by Staphylococcus intermedius or S. aureus and is known as staphylococcal dermatitis ( Smith and Sherman, 1994 ). The Staphylococcus organisms are cocci and are categorized as primary pathogens or ubiquitous skin commensals of humans and animals. Staphylococcus aureus and S. intermedius are classified as primary pathogens and produce coagulase, a virulence factor. Clinical signs and diagnosis. Small pustular lesions, caused by bacterial infection and inflammation of the hair follicle, occur around the teats and perineum. Occasionally, the infection may involve the flanks, underbelly, axilla, inner thigh, and neck. Staphylococcal dermatitis may occur secondary to other skin lesions. Diagnosis is based on lesions. Culture will distinguish S. aureus. Pathogenesis. Simple boredom may cause rubbing, followed by staphylococcal infection of damaged epidermis. Differential diagnosis. The presence of scabs makes contagious ecthyma a differential diagnosis, along with fungal skin infections and nutritional causes of skin disease. Treatment. Severe infections should be treated with antibiotics based on culture and sensitivity. Severe lesions and lesions localized to the underbelly, thighs, and udder benefit by periodic cleaning with an iodophor shampoo and spraying with an antibiotic and an astringent ( Smith and Sherman, 1994 ). h. Clostridial Diseases i. Clostridium perfringens type C infection (enterotoxemia and struck) Etiology. Clostridium perfringens is an anaerobic, gram-positive, nonmotile, spore-forming bacterium that lives in the soil, in contaminated feed, and in gastrointestinal tracts of ruminants. The bacteria is categorized by toxin production. Toxins include alpha (hemolytic), beta (necrotizing), delta (cytotoxic and hemoltyic), epsilon, and iota. Types of C. perfingens are A, B, C, D, and E. This is a common and economically significant disease of sheep, goats, and cattle. Clinical signs and diagnosis. The beta toxin associated with overgrowth of this bacterium results in a fatal hemorrhagic enterocolitis within the first 72 hr of a young ruminant's life. Many animals may be found dead, with no clinical presentation. Affected animals are acutely anemic, dehydrated, anorexic, restless, and depressed and may display tremors or convulsions as well as abdominal pain. Feces may range from loose gray-brown to dark red and malodorous. Morbidity and mortality may be nearly 100%. A similar noncontagious but acutely fatal form of enterotoxemia in adult sheep, called struck, occurs in yearlings and adults. Struck is rare in the United States. The disease is also caused by the beta toxin of C. perfringens type C and is often associated with rapid dietary changes or shearing stresses in sheep. Although affected animals are usually found dead, clinical signs include uneasiness, depression, and convulsions. Mortality is usually less than 15%. Diagnosis is usually based on necropsy findings, although confirmation can be made by culture of the organism. Identification of the beta toxin in intestinal contents may be difficult because of instability of the toxin. Epizootiology and transmission. Clostridial organisms are ubiquitous in the environment as well as in the gastrointestinal tract and contaminated feeds. Confinement and poor sanitation predisposes to infection with C. perfringens. Transmission is by ingestion of contaminated material. Necropsy findings. Necropsy findings include a milk-filled abomasum, and hemorrhage in the distal small intestine and throughout the large intestine. Petechial hemorrhages of the serosal surfaces of many organs, especially the thymus, heart, and gastrointestinal tract, will be visible. Hydropericardium, hydroperitoneum, and hemorrhagic mesenteric lymph nodes will also be present. Pulmonary and brain edema may also be seen. Histologically, the gram-positive C. perfringens organisms may be visible in excess numbers along the mucosal surface of the swollen, congested, necrotic intestines. In cases of struck, necropsy findings include congestion and erosions of the mucosa of the gastrointestinal tract, serosal hemorrhages, and serous peritoneal and pericardial fluids. In late stages of the disease and especially if prompt necropsy is not performed, the organism will infiltrate the muscle fascial layers and produce serohemorrhagic and gaseous infiltration of perimysial and epimysial spaces. Pathogenesis. Hemorrhagic enterotoxemia is an acute, sporadic disease caused by the beta toxin of Clostridium perfringens type C. Neonates ingest the organism, which then proliferates and attaches to the gastrointestinal microvilli and elaborates primarily the beta toxins. The trypsin inhibitors present in colostrum prevent inactivation of the beta toxin. The toxins injure intestinal epithelial cells and then enter the blood, leading to acute toxemia. The intestinal injury may result in diarrhea, with small amounts of hemorrhage. Associated electrolyte and water loss result in dehydration, acidosis, and shock. Differential diagnosis. Differential diagnoses include other clostridial diseases such as blackleg and black disease, as well as coccidiosis, salmonellosis, anthrax, and acute poisoning. Prevention and control. A commercial toxoid is available and should be administered to the pregnant animals prior to parturition. An alternative includes administration of an antitoxin to the newborn lambs. The disease may become endemic once it is on the premises. Treatment. Treatment is difficult and usually unsuccessful. Antitoxin may be useful in milder cases, and the antitoxin and toxoid can also be administered during an outbreak. Research complications. This disease can be costly in losses of neonates and younger animals. ii. Clostridium perfringens type D infection (pulpy kidney disease) Etiology. Clostridium perfringens type D releases epsilon toxin that is proteolytically activated by trypsin. This disease caused by C. perfringens tends to be associated with sheep and is of less importance in goats and cattle. Clinical signs. The peracute condition in younger animals is characterized by sudden deaths, which are occasionally preceded by neurological signs such as incoordination, opisthotonus, and convulsions. Because the disease progresses so rapidly to death (within 1–2 hr), clinical signs are rarely observed. Hypersalivation, rapid respirations, hyperthermia, convulsions, and opisthotonus have been noted. In acute cases, hyperglycemia and glucosuria are considered almost pathognomonic. Clinical signs in chronic cases in older animals, such as adult goats, include soft stools, weight loss, anorexia, depression, and severe diarrhea, sometimes with mucus and blood. Mature affected sheep may be blind and anorectic and may head-press. Necropsy findings. Necropsy findings are similar to those seen with C. perfringens type C. Additionally, extremely necrotic, soft kidneys ("pulpy kidneys") are usually observed immediately following death. (This phenomenon is in contrast to what is normally associated with later stages of postmortem autolysis.) Focal encephalomalacia, and petechial hemorrhages on serosal surfaces of the brain, diaphragm, gastrointestinal tract, and heart are common findings. Diagnosis can be made from the typical clinical signs and necropsy findings as well as the observation of glucose in the urine at necropsy. Pathogenesis. The epsilon toxin causes neuronal death and shock, probably through vascular damage. The noncontagious, peracute form of enterotoxemia occurs in suckling, fast-growing animals, either nursing from their dams or on high-protein, high-energy concentrates. The largest, fastest-growing animals generally are predisposed to this condition; for example, lambs, fat ewe lambs, and usually singleton lambs tend to be most susceptible. The hyperglycemia and glucosuria seen in acute cases are due to epsilon toxin effects on liver glycogen metabolism. Differential diagnosis. Tetanus, enterotoxigenic E. coli, botulism, polioencephalomalacia, grain overload, and listeriosis are differentials. Prevention and control. Vaccination prevents the disease. Maternal antibodies last approximately 5 weeks postpartum; thus young animals should be vaccinated at about this time. Feeding regimens to young, fast-growing animals and feeding of concentrates to adults should be evaluated carefully. Treatment. Treatment consists of support (fluids, warmth), antitoxin administration, oral antibiotics, and diet adjustment. iii. Clostridium tetani infection (tetanus, lockjaw) Etiology. Clostridium tetani is a strictly anaerobic, motile, spore-forming, gram-positive rod that persists in soils and manure and within the gastrointestinal tract. At least 10 serotypes of C. tetani exist. Clinical signs. Infection by C. tetani is characterized by a sporadic, acute, and fatal neuropathy. After an incubation period of 4 days to 3 weeks, the animal exhibits bloat; muscular spasticity; prolapse of the third eyelid; rigidity and extension of the limbs, leading to a stiff gate; an inability to chew; and hyperthermia. Erect or drooped ears, retracted lips, drooling, hypersensitivity to external stimuli, and a "sawhorse" stance are frequent signs. The animal may convulse. Death occurs within 3–10 days, and mortality is nearly 100%, primarily from respiratory failure. Diagnosis is based on clinical signs. Muscle-related serum enzymes such as aspartate aminotransferase (AST), creatinine kinase (CK), and lactate dehydrogenase (LDH) might be elevated. ( Jensen and Swift, 1982 ). Serum cortisol may also be elevated, and stress hyperglycemia may be evident. Permanent lameness may result in survivors. Epizootiology and transmission. Clostridium tetani is a soil contaminant and is often found as part of the gut microflora of herbivores. The organisms sporulate and persist in the environment. All species of livestock are susceptible, but sheep and goats are more susceptible than cattle. Individual cases may occur, or herd outbreaks may follow castration, tail docking, ear tagging, or dehorning. Mouth wounds may also be sites of entry. Pathogenesis. Tetanus, or lockjaw, is caused by the toxins of C. tetani. All serovars produce the same exotoxin, which is a multiunit protein composed of tetanospasmin, which is neurotoxic, and tetanolysin, which is hemolytic. A nonspasmogenic toxin is also produced. Contamination of wounds results in anaerobic proliferation of the bacterium and liberation of the tetanospasmin, which diffuses through motor neurons in a retrograde direction to the spinal cord. The toxin inhibits the release of glycine and γ-aminobutyric acid from Renshaw cells; this results in hypertonia and muscular spasms. Proliferation of C. tetani in the gut of affected animals may also serve as a source and may produce clinical signs. The uterus is the most common site of infection in postparturient dairy cattle with retained placentas. Differential diagnoses. Early in the course of the infection, differential diagnoses include bloat, rabies, hypomagnesemic tetany, polioencephalomalacia, white muscle disease, enterotoxemia in lambs, and lead poisoning. Polyarthritis of cattle is a differential for the gait changes in that species. Necropsy findings. Findings are nonspecific except for the inflammatory reaction associated with the wound. Because of the low number of organisms necessary to cause neurotoxicosis, isolation of C. tetani from the wound may be difficult. Treatment. Treatment consists of cleaning the infected wound; administering tetanus antitoxin (e.g., at least 500 IU in an adult sheep or goat); vaccinating with tetanus toxoid; administering of antibiotics (penicillin, both parenterally [potassium penicillin intravenously and procaine penicillin intramuscularly] and flushed into the cleaned wound), a sedative or tranquilizer (e.g., acepromazine or chlorpromazine) and a muscle relaxant; and keeping the animal in a dark, quiet environment. Supportive fluids and glucose must be administered until the animal is capable of feeding. If the animal survives, revaccination should be done 14 days after the previous dose. Prevention and control. Like other ubiquitous clostridial diseases, tetanus is impossible to eradicate. The disease can be controlled and prevented by following good sanitation measures, aseptic surgical procedures, and vaccination programs. Tetanus toxoid vaccine is available and very effective for stimulating long-term immunity. Tetanus antitoxin can be administered (200 IU in lambs) as a preventive or in the face of disease as an adjunct to therapy. Both the toxoid and the antitoxin can be administered to an animal at the same time, but they should not be mixed in the syringe, and each should be administered at different sites, with a second toxoid dose administered 4 weeks later. Animals should be vaccinated 2 or 3 times during the first year of life. Does and ewes should receive booster vaccinations within 2 months of parturition to ensure colostral antibodies. Research complications. Unprotected, younger ruminants may be affected following routine flock or herd management procedures. Contaminated or inadequately managed open wounds or lesions in older animals may provide anaerobic incubation sites. iv. Clostridium novyi infection (bighead; black disease; bacillary hemoglobinuria, or red water) and C. chauvoei infection (blackleg) Etiology. Clostridium novyi, an anaerobic, motile, spore-forming, gram-positive bacteria, is the agent of bighead and black disease. Clostridium novyi type D (C. hemolyticum) is the cause of bacillary hemoglobinuria, or "red water." Clostridium chauvoei is the causative agent of blackleg. Clinical signs. Bighead is a disease of rams characterized by edema of the head and neck. The edema may migrate to ventral regions such as the throat. Additional clinical signs include swelling of the eyelids and nostrils. Most animals will die within 48–72 hours. Black disease, or infectious necrotic hepatitis, is a peracute, fatal disease associated with C. novyi. It is more common in cattle and sheep but may be seen in goats. The clinical course is 1–2 days in cattle and slightly shorter in sheep. Otherwise healthy-appearing adult animals are often affected. Clinical signs are rarely seen, because of the peracute nature of the disease. Occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency are observed prior to death. Bacillary hemoglobinuria is an acute disease seen primarily in cattle and characterized by fever and anorexia, in addition to the hemoglobinemia and hemoglobinuria indicated by the name. Animals that survive a few days will develop icterus. Mortality may be high. Blackleg, a disease similar to bighead, causes necrosis and emphysema of muscle masses, serohemorrhagic fluid accumulation around the infected area, and edema ( Jackson et al., 1995 ). Blackleg is more common in cattle than in sheep. The incubation period is 2–5 days and is followed by hyperthermia, muscular stiffness and pain, anorexia, and gangrenous myositis. The clinical course is short, 24–48 hr, and untreated animals invariably die. Blackleg in cattle can be associated with subcutaneous edema or crepitation; these do not usually occur in sheep. Most lesions are associated with muscles of the face, neck, perineum, thigh, and back. Epizootiology and transmission. Bighead is caused by the toxins of C. novyi, which enters through wounds often associated with horn injuries during fighting. The C. novyi type B organisms produce alpha and beta toxins, and the alpha toxins are mostly responsible for toxemia, tissue necrosis, and subsequent death. Clostridium novyi type D is endemic in the western United States. It is hypothesized that the C. chauvoei organisms enter through the gastrointestinal tract. Black disease and bacillary hemoglobinuria are associated with concurrent liver disease, often associated with Fasciola infections (liver flukes); it is sometimes seen as a sequela to liver biopsies. The diseases are more common in summer months, and fecal contamination of pastures, flooding, and infected carcasses are sources of the organism. Birds and wild animals may be vectors of the pathogen. Ingested spores are believed to develop in hepatic tissue damaged and anoxic from the fluke migrations. Necropsy. Diagnosis of black disease is usually based on postmortem lesions. Subcutaneous vessels will be engorged with blood, resulting in dried skin with a dark appearance. Carcasses putrefy quickly. In addition, hepatomegaly and endocardial hemorrhages are common, and hepatic damage from flukes may be so severe that diagnosis is difficult. Blood coagulates slowly in affected animals. Pathogenesis. The propagation of the clostridial organisms is self-promoted by the damage caused by the toxins and the increased local anaerobic environment created. Clostridium novyi proliferates in the soft tissues of the head and neck, and the resultant clostridial toxin causes increased capillary permeability and the liberation of serous fluids into the tissues. Mixed infections with related clostridial organisms may lead to increasing hemorrhage and necrosis in the affected tissues. Diagnosis is based on clinical signs. In black disease and bacillary hemoglobinuria disease, the ingested clostridial spores are absorbed, enter the liver, and cause hepatic necrosis. Associated toxemia causes subcutaneous vascular dilatation; increased pericardial, pleural, and peritoneal fluid; and endocardial hemorrhages. The toxins produced by C. novyi, identified as beta, eta, and theta, and each having enzymatic or lytic properties or both, also contribute to the hemolytic disease. Clostridium chauvoei spores proliferate in traumatized muscle areas damaged by transportation, rough handling, or injury. Differential diagnosis. Differential diagnoses include other clostridial diseases as well as photosensitization. Hemolytic diseases such as babesiosis, leptospirosis, and hemobartonellosis should be included as differentials. Treatment. For C. chauvoei infection (blackleg), early treatment with penicillin or tetracycline may be helpful. Treatment for black disease is not rewarding even if the animal is found before death. Carcasses from bacillary hemoglobinuria losses should be burned, buried deeply, or removed from the premises. Prevention and control. Vaccinating animals with multivalent clostridial vaccines can prevent these diseases. Subcutaneous administration of vaccine material is recommended over intramuscular. Vaccinations may be useful in an outbreak. Careful handling of ruminants during shipping and transfers will contribute to fewer muscular injuries. For bighead, mature rams penned together should be monitored for lesions, especially during breeding season. Control of fascioliasis is very important in prevention and control of black disease and in the optimal timing of vaccinations. v. Clostridium septicum infection (malignant edema) Etiology. Clostridium septicum is the species usually associated with malignant edema, but mixed infections involving other clostridial species such as C. chauvoei, C. novyi, C. sordellii, and C. perfringens may occur. Clostridium spp. are motile (C. chauvoei, C. septicum) or nonmotile, anaerobic, spore-forming, gram-positive rods. Clinicial signs. Malignant edema, or gas gangrene, is an acute and often fatal bacterial disease caused by Clostridium spp. The incubation period is approximately 2–4 days. The affected area will be warm and will contain gaseous accumulations that can be palpated as crepitation of the subcutaneous tissue around the infected area. Regional lymphadenopathy and fever may occur. The animal becomes anorexic, severely depressed, and possibly hyperthermic. Edema and crepitation may be noted around the wound; death occurs within 12 hr to 2 days. Epizootiology and transmission. The organisms are ubiquitous in the environment and may survive in the soil for years. The disease is especially prevalent in animals that have had recent wounds such as those that have undergone castration, docking, ear notching, shearing, or dystocia. Necropsy findings. The tissue necrosis and hemorrhagic serous fluid accumulations resemble those of other clostridial diseases. Pathogenesis. In most cases, the clostridial organisms cause a spreading infection through the fascial planes around the area of the injury; vegetative organisms then produce potent exotoxins, which result in necrosis (alpha toxin) and/or hemolysis (beta toxin). Furthermore, the toxins enter the bloodstream and central nervous system, resulting in systemic collapse and high mortality. Necropsy. Spreading, crepitant lesions around wounds are suggestive of malignant edema. Affected tissues are inflamed and necrotic. Gas and serosanguineous fluids with foul odors infiltrate the tissue planes. Large rod-shaped bacteria may be observed on histopathology; confirmation is made through culture and identification. Intramuscular inoculation of guinea pigs causes a necrotizing myositis and death. Organisms can be cultured from guinea pig tissues. Treatment. Infected animals can be treated with large doses of penicillin and fenestration of the wound is recommended. Prevention and control. Proper preparation of surgical sites, correct sanitation of instruments and the housing environment, and attention to postoperative wounds will help prevent this disease. Multivalent clostridial vaccines are available. Research complications. Morbidity or loss of animals from lack of or unsuccessful vaccination and from contaminated surgical sites or wounds may be consequences of this disease. i. Colibacillosis Etiology. Escherichia coli is a motile, aerobic, gram-negative, non-spore-forming coccobacillus commonly found in the environment and gastrointestinal tracts of ruminants. Escherichia coli organisms have three areas of surface antigenic complexes (O, somatic; K, envelope or pili; and H, flagellar), which are used to "group" or classify the serotypes. Colibacillosis is the common term for infections in younger animals caused by this bacteria. Clinical signs. Presentation of E. coli infections vary with the animal's age and the type of E. coli involved. Enterotoxigenic E. coli infection causes gastroenteritis and/or septicemia in lambs and calves. Colibacillosis generally develops within the first 72 hr of life when newborn animals are exposed to the organism. The enteric infection causes a semifluid, yellow to gray diarrhea. Occasionally blood streaking of the feces may be observed. The animal may demonstrate abdominal pain, evidenced by arching of the back and extension of the tail, classically described as "tucked up." Hyperthermia is rare. Severe acidosis, depression, and recumbancy ensue, and mortality may be as high as 75%. The septicemic form generally occurs between 2 and 6 weeks of age. Animals display an elevated body temperature and show signs suggestive of nervous system involvement such as incoordination, head pressing, circling, and the appearance of blindness. Opisthotonos, depression, and death follow. Occasionally, swollen, painful joints may be observed with septicemic colibacillosis. Blood cultures may be helpful in identifying the septicemic form. In ruminants, E. coli is is a less common cause of cystitis and pyelonephritis. The cystitis is characterized by dysuria and pollakiuria; gross hematuria and pyuria may be present. The infection may or may not be restricted to the bladder; in the later presentation, and in cases of pyelonephritis, a cow will be acutely depressed, have a fever and ruminal stasis, and be anorexic. In chronic cases, animals will be polyuric and undergo weight loss. Escherichia coli may also cause in utero disease in cattle, resulting in abortion or weakened offspring. Epizootiology and transmission. Escherichia coli is one of the most common gram-negative pathogens isolated from ruminant neonates. Zeman et al. (1989 ) classify E. coli infections into four groups: enterotoxigenic, enterohemorrhagic, enteropathogenic, and enteroinvasive. Enterotoxigenic E. coli (ETEC) attach to the enterocytes via pili, produce enterotoxins, and are the primary cause of colibacillosis in animals and humans. Fimbrial (pili) antigens associated with ovine disease include K99 and F41. Enterohemorrhagic E. coli (EHEC) attach and efface the microvillus, produce verotoxins, and occasionally cause disease in humans and animals. Enteropathogenic E. coli (EPEC) colonize and efface the microvillus but do not produce verotoxins. EPEC are associated with disease in humans and rabbits and cause a secretory diarrhea. Enteroinvasive E. coli (EIEC) invade the enterocytes of humans and cause a shigella-like disease. Overcrowding and poor sanitation contribute significantly to the development of this disease in young animals. The organism will be endemic in a contaminated environment and present on dams' udders. The bacteria rapidly proliferate in the neonates' small intestines. The bacteria and associated toxins cause a secretory diarrhea, resulting in the loss of water and electrolytes. If the bacteria infiltrate the intestinal barrier and enter the blood, septicemia results. Diagnosis of the enteric form can be made by observation of clinical signs, including diarrhea and staining of the tail and wool. Necropsy findings. Swollen, yellow to gray, fluid-filled small and large intestines, swollen and hemorrhagic mesenteric lymph nodes, and generalized tissue dehydration are common. Septicemic lambs may have serofibrinous fluid in the peritoneal, thoracic, and pericardial cavities; enlarged joints containing fibrinopurulent exudates; and congested and inflamed meninges. Isolation and serotyping of E. coli confirm the diagnosis. ELISA and latex agglutination tests are available diagnostic tools. Differential diagnosis. Differential diagnoses include the enterotoxemias caused by C. perfringens type A, B, or C; Campylobacter jejuni; Coccidia, rotavirus, coronavirus, Salmonella, and Cryptosporidia. Other contributing causes of abomasal tympany in young ruminants, such as dietary changes, copper deficiency, excessive intervals between feedings of milk replacer, or feeding large volumes should be considered. Prevention and control. The best preventive measures are maintenance of proper housing conditions, limiting overcrowding, and frequently sanitizing lambing areas. Attention to colostrum feeding techniques and colostral quality are important means of preventing disease. Treatment must include intravenous fluid hydration and reestablishment of acid-base and electrolyte abnormalities. Treatment. Antibiotics such as trimethoprim-sulfadiazine, enrofloxacin, cephalothin, amikacin, and apramycin may be helpful; oral antibiotics are not recommended. Vaccines are available for prevention of colibacillosis in cattle. j. Corynebacterium pseudotuberculosis Infection (Caseous Lymphadenitis) Etiology. Corynebacterium pseudotuberculosis (previously C. ovis) are nonmotile, non-spore-forming, aerobic, short and curved, gram-positive coccobacilli. Caseous lymphadenitis (CLA) is such a common, chronic contagious disease of sheep and goats that any presentation of abscessing and draining lymph nodes should be presumed to be this disease until proven otherwise. The disease has been reported occasionally in cattle. Clinical signs and diagnosis. Abscessation of superficial lymph nodes, such as the superficial cervical, retropharyngeal, subiliacs (prefemoral), mammary, superficial inguinals, and popliteal nodes, and of deep nodes, such as mediastinal and mesenteric lymph nodes, is typical. Radiographs may be helpful in identifying affected central nodes. Peripheral lymph nodes may erode and drain caseous, "cheesy," yellow-green-tan secretions. The incubation period may be weeks to months. Over time, an infected animal may become exercise-intolerant, anorexic, and debilitated. Fever, increased respiratory rates, and pneumonia may also be common signs. Exotoxin-induced hemolytic crises may occur occasionally. Morbidity up to 15% is common, and morbid animals will often eventually succumb to the disease. Diagnosis is based on clinical lesions; ELISA serological testing is also available. Smears of the exudate or lymph nodes aspirates can be Gram-stained. Lymph node aspirates may also be sent for culturing. Epizootiology and transmission. The organism can survive for 6 months or more in the environment and enters via skin wounds, shearing, fighting, castration, and docking. Ingestion and aerosolization (leading to pulmonary abscesses) have been reported as alternative routes of entry. Necropsy findings. Disseminated superficial abscesses as well as lesions of the mediastinal and mesenteric lymph nodes will be identified. Cut surfaces of the affected lymph nodes may appear lamellated. Lungs, liver, spleen, and kidneys may also be affected. Cranioventral lung consolidation with hemorrhage, fibrin, and edema are seen histologically. Pathogenesis. Corynebacterium pseudotuberculosis produces an exotoxin (phospholipase D) that damages endothelial and blood cell membranes. This process enhances the organisms' ability to withstand phagocytosis. The infection spreads through the lymphatics to local lymph nodes. The necrotic lymph nodes seed local capillaries and hematogenously and lymphatically spread the organisms to other areas, especially the lungs. Differential diagnosis. Differentials include pathogens causing lymphadenopathy and abscessation. Treatment. Antibiotic therapy is not usually helpful. Abscesses can be surgically lanced and flushed with iodine-containing and/or hydrogen peroxide solutions. Abscessing lymph nodes can be removed entirely from valuable animals. During warmer months, an insect repellent should be applied to and around healing lesions. All materials used to treat animals should be disposed of properly. Because of the contagious nature of the disease, animals with draining and lanced lesions should be isolated from CLA-negative animals at least until healed. Commercial vaccines are available ( Piontkowski and Shivvers, 1998 ). Prevention and control. Minimizing contamination of the environment, using proper sanitation methods for facilities and instruments, segregating affected animals, and taking precautions to prevent injuries are all important. Research complications. This pathogen is a risk for animals undergoing routine management procedures or invasive research procedures, because of its persistence in the environment, its long clinical incubation period, and its poor response to antibiotics. k. Corynebacterium renale, C. cystitidis, and C. pilosum Infections (Pyelonephritis; Posthitis and Ulcerative Vulvovaginitis) Etiology. Corynebacterium renale, C. cystitidis, and C. pilosum are sometimes referred to as the C. renale group. These are piliated and nonmotile gram-positive rods and are distinguished biochemically. Corynebacterium renale causes pyelonephritis in cattle, and C. pilosum and C. cystitidis cause posthitis, also known as pizzle rot or sheath rot, in sheep and goats. In many references, all these clinical presentations are attributed to C. renale. Clinical signs and diagnosis. Acute pyelonephritis is characterized by fever, anorexia, polyuria, hematuria, pyuria, and arched back posture. Untreated infections usually become chronic, with weight loss, anorexia, and loss of production in dairy animals. Relapses are common, and some infections are severe and fatal. Diagnosis of pyelonephritis is based on urinalysis (proteinuria and hematuria) and rectal or vaginal palpation (assessing ureteral enlargement). Urine culturing may not be productive. In chronic cases, E. coli and other gram-negatives may be present. Posthitis and vulvovaginitis are characteriazed by ulcers, crusting, swelling and pain. The area may have a distinct malodor. Necrosis and scarring may be sequelae of more severe infections. Fly-strike may also be a complication. Diagnosis is based on clinical signs and on investigation of feeding regimens. Epizootiology and transmission. Ascending urinary tract infections with cystitis, ureteritis, and pyelonephritis are widespread problems, but incidence is relatively low. The vaginitis and posthitis contribute to the venereal transmission, but indirect transmission is possible because the organisms are stable in the environment and present on the wool or scabs shed from affected animals. Posthitis occurs in intact and castrated sheep and goats. Necropsy findings. Pyelonephritis, multifocal kidney abscessation, dilated and thickened ureters, cystitis, and purulent exudate in many sections of the urinary tract are common finding at gross necropsy. Pathogenesis. Corynebacterium renale is a normal inhabitant of bovine genitourinary tracts. The pilus mediates colonization. Conditions such as trauma, urinary tract obstruction, and anatomic anomalies may predispose to infection. In addition, more basic pH urine levels may block some immune defenses. Infections ascend through the urinary tract. The bacteria are urease-positive when tested in vitro, and the ammonia produced in vivo during an infection damages mucosal linings, with subsequent inflammation. Corynebacterium cystitidis and C. pilosum are normally found around the prepuce of sheep and goats. High-protein diets, resulting in higher urea excretion and more basic urine, are contributing factors. Posthitis and vulvovaginitis may develop within a week of change to the more concentrated or richer diet, such as pasture or the addition of high-protein forage. The ammonia produced irritates the preputial and vulvar skin, increasing the vulnerability to infection. Differential diagnosis. Urolithiasis is a primary consideration for these diseases. Contagious ecthyma should be considered for the crusting that is seen with posthitis and vulvovaginitis, although the lesions of contagious ecthyma are more likely to develop around the mouth. Ovine viral ulcerative dermatosis is also a differential for the lesions of posthitis and vulvovaginitis. Prevention and treatment. Because high-protein feed is often associated with posthitis and vulvovaginitis, feeding practices must be reconsidered. Clipping long wool and hair also is helpful. Treatment. Long-term (3 weeks) penicillin treatment is effective for pyelonephritis. Reduction of dietary protein, clipping and cleaning skin lesions, treating for or preventing fly-strike, and topical antibacterial treatments are effective for posthitis and vulvovaginitis; systemic therapy may be necessary for severe cases. Surgical debridement or correction of scarring may also be indicated in severe cases. l. Erysipelas Etiology. Erysipelothrix rhusiopathiae is a nonmotile, non-spore-forming, gram-positive rod that resides in alkaline soils. Clinical signs. Erysipelothrix causes sporadic but chronic polyarthritis in lambs less than 3 months of age. In older goats, erysipelas has been associated with joint infections. Epizootiology and transmission. The disease may follow wound inoculation associated with castration, docking, or improper disinfection of the umbilicus. Following wound contamination and a 1- to 5-day incubation period, the lamb exhibits a fever and stiffness and lameness in one or more limbs. Joints, especially the stifle, hock, elbow, and carpus, are tender but not greatly enlarged. Necropsy findings. Thickened articular capsules, mild increases in normal-appearing joint fluid and erosions of the articular cartilage are usually found. The joint capsule is infiltrated with mononuclear cells, but bacteria are difficult to find. Diagnosis is based on clinical signs of polyarthritis, and confirmation is made by culturing the organism from the joints. Differential diagnosis. Differential diagnoses include polyarthritis caused by chlamydia or other bacteria and stiffness caused by white muscle disease. Other bacteria causing septic joints include Areanobacterium pyogenes and Fusobacterium necrophorum. Caprine arthritis encephalitis (CAE) should also be considered. Prevention and control. Proper sanitation and prevention of wound contamination are important in preventing the infection in lambs. Screening of goat herds for CAE is recommended. Treatment. Erysipelas is sensitive to penicillin antibiotic therapy. m. Dermatophilosis (Cutaneous Streptothricosis, Lumpy Wool, Strawberry Foot Rot) Etiology. Dermatophilus congolensis is an aerobic, gram-positive, filamentous bacterium with branching hyphae. Dermatophilosis is a chronic bacterial skin disease characterized by crustiness and exudates accumulating at the base of the hair or wool fibers ( Scanlan et al., 1984 ). Clinical signs. Animals will be painful but will not be pruritic. Two forms of the disease exist in sheep: mycotic dermatitis (also known as lumpy wool) and strawberry foot rot. Mycotic dermatitis is characterized by crusts and wool matting, with exudates over the back and sides of adult animals and about the face of lambs. Strawberry foot rot is rare in the United States but is characterized by crusts and inflammation between the carpi and/or tarsi and the coronary bands. Animals will be lame. In goats and cattle, similar clinical signs of crusty, suppurative dermatitis are seen; the disease is often referred to as cutaneous streptothricosis in these species. Lesions in younger goats are seen along the tips of the ears and under the tail. Diagnosis is based on clinical signs as well as the typical microscopic appearance on stained skin scrapings, cultures, and serology. Epizootiology and transmission. The disease occurs worldwide, and the Dermatophilus organism is believed to be a saprophyte. Transmission occurs by direct or indirect contact and is aggravated by prolonged wet wool or hair associated with inclement weather. Biting insects may aid in transmission. Necropsy findings. Lymphadenopathy as well as liver and splenic changes may be observed. Histopathologically, superficial epidermal layers are necrotic and crusted with serum, white blood cells, and wool or hair. Dermal layers are hyperemic and edematous and may be infiltrated with mononuclear cells. Pathogenesis. Lesions typically begin around the muzzle and hooves and the dorsal midline. Prevention and control. Potash alum and aluminum sulfate have been used as wool dusts in sheep to prevent dermatophilosis. Minimizing moist conditions is helpful in controlling and preventing the disease. In addition, controlling external parasites or other factors that cause skin lesions is important. Lesions will resolve during dry periods. Treatment. Animals can be treated with antibiotics such as penicillin and oxytetracycline. Treating the animals with povidone-iodine shampoos or chlorhexidine solutions is also useful in clearing the disease. n. Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum Infection (Virulent Foot Rot; Contagious Foot Rot of Sheep and Goats; Foot Scald) Etiology. Two bacteria, Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum, work synergistically in causing contagious foot rot in sheep and goats. Other organisms may be involved as secondary invaders. Both Dichelobacter and Fusobacterium are nonmotile, non-spore-forming, anaerobic, gram-negative bacilli. Foot rot is a contagious, acute or chronic dermatitis involving the hoof and underlying tissues ( Bulgin, 1986 ). It is the leading cause of lameness in sheep. At least 20 serotypes of Dichelobacter are known. Arcanobacterium pyogenes may also contribute to the pathogenicity or to foot abscesses in goats. Foot scald, an interdigital dermatitis, is caused primarily by D. nodosus alone. Clinical signs. Varying degrees of lameness are observed in all ages of animals within 2–3 weeks of exposure to the organisms. Severely infected animals will show generalized signs of weight loss, decreased productivity, and anorexia associated with an inability to move. The interdigital skin and hooves will be moist, with a distinct necrotic odor. Morbidity may reach 70% in susceptible animals. Diagnosis is based on clinical signs. Smears and cultures confirm the definitive agents. Clinical signs of the milder disease, foot scald, include mild lameness, redness and swelling, and little to no odor. Epizootiology and transmission. Fusobacterium necrophorum is ubiquitous in soil and manure, in the gastrointestinal tract, and on the skin and hooves of domestic animals. In contrast, Dichelobacter contaminates the soil and manure but rarely remains in the environment for more than about 2 weeks. Some animals may be chronic carriers. Overcrowded, warm, and moist environments are key elements in transmission. Outbreaks are likely in the spring season. Shipping trailers and contaminated pens or yards should be considered also as likely sources of the bacteria. Pathogenesis. Both organisms are transmitted to the susceptible animal by direct or indirect contact. The organisms enter the hoof through injuries or through sites where Strongyloides papillosus larvae have penetrated. Fusobacterium necrophorum initiates the colonization and is followed by D. nodosus. The latter attaches and releases proteases; these cause necrosis of the epidermal layers and separation of the hoof from the underlying dermis. The pathogenicity of the serotypes of D. nodosus is correlated with the production of these proteases and numbers of pili. Additionally, F. necrophorum causes a severe, damaging inflammatory reaction. Differential diagnosis. Foot abscesses, tetanus, selenium/vitamin E deficiencies, copper deficiency, strawberry foot rot, bluetongue virus infection (manifested with myopathy and coronitis), and trauma are among the many differentials that must be considered. Treatment. Affected animals are best treated by manually trimming the necrotic debris from the hooves, followed by application of local antibiotics and foot wraps. Systemic antibiotics such as penicillin, oxytetracycline, and erythromycin may be used. Goats have improved dramatically when given a single dose of penicillin (40,000 U/kg) ( Smith and Sherman, 1994 ). Footbaths containing 10% zinc sulfate, 20% copper sulfate, or 10% formalin (not legal in all states) can be used for treatment as well as for prevention of the disease. Affected animals should be separated from the flock. Vaccination has been shown to be effective as part of the treatment regimen. Some breeds of sheep and some breeds and lines of goats are resistant to infection. Individual sheep may recover without treatment or are resistant to infection. Prevention and control. Prevention and control programs involve scrutiny of herd and flock management; quarantine of incoming animals; vaccination; segregation of affected animals; careful and regular hoof trimming; discarding trimmings from known or suspected infected hooves; maintaining animals in good body condition; avoiding muddy pens and holding areas; and culling individuals with chronic and nonresponsive infections. Dichelobacter nodosus bacterins are commercially available; cross protection between serotypes varies. Biannual vaccinination in wet areas may be essential. Some breeds may develop vaccination site lumps. Footbaths of 10% zinc sulfate, 10% formalin (where allowed by state regulations), or 10% copper sulfate are also considered very effective preventive measures. Goats are less sensitive than sheep to the copper in the footbaths. Research complications. Treating and controlling foot rot is costly in terms of time, initial handling and treatments and their follow-up, housing space, and medications. o. Fusobacterium necrophorum and Bacteroides melaninogenicus Infection (Foot Rot of Cattle, Interdigital Necrobacillosis of Cattle) Etiology. Interdigital necrobacillosis of cattle is caused by the synergistic infection of traumatized interdigital tissues by Fusobacterium necrophorum and Bacteroides melaninogenicus. Like F. necrophorum, B. melaninogenicus is a nonmotile, anaerobic, gram-negative bacterium. Dichelobacter nodosus, the agent of interdigital dermatitis, may be present in some cases. This is a common cause of lameness in cattle. Clinical signs. Clinical signs include mild to moderate lameness of sudden onset. Hindlimbs are more commonly affected, and cattle will often flex the pastern and bear weight only on the toe. The interdigital space will be swollen, as will be the coronet and bulb areas. Characteristic malodors will be noted, but there will be little purulent discharge. In more severe cases, animals will have elevated body temperature and loss of appetite. The lesions progress to fissures with necrosis until healing occurs. The diagnosis is by the odor and appearance. Anaerobic culturing confirms the organisms involved. Epizootiology and transmission. Cases may be sporadic, or epizootics may occur. Bos taurus dairy breeds and animals with wide interdigital spaces are more commonly affected. The factors here are comparable to those present in foot rot of smaller ruminants. Necropsy findings. Findings at necropsy include dermatitis and necrosis of the skin and subcutaneous tissues. Although necropsy would rarely be performed, secondary osteomyelitis may be noted in severe cases by sectioning limbs. Pathogenesis. The bacteria enter through the skin of the interdigital area after trauma to the interdigital skin, from hardened mud, or from softening of the skin due to, for example, constant wet conditions in pens. Colonization leads to cellulitis. In addition, F. necrophorum releases a leukocidal exotoxin that reduces phagocytosis and causes the necrosis, whereas the tissues and tendons are damaged by the proteases and collagenases produced by B. melaninogenicus. Zinc deficiency may play a role in the pathogenesis in some situations. Differential diagnoses. The most common differentials for sudden lameness include hairy heel warts and subsolar abcesses. Bluetongue virus should also be considered. Grain engorgement and secondary infection from cracks caused by selenium toxicosis should also be considered. The exotic foot-and-mouth disease virus would be considered in areas where that pathogen is found. Prevention and control. As with foot rot in smaller ruminants, management of the area and herd are important. Paddocks and pens should be kept dry, well drained, and free of material that will damage feet. Footbaths and chlortetracycline in the feed have been shown to control incidence. Affected animals should be segregated during treatment. Chronically affected or severely lame animals should be culled. New cattle should be quarantined and evaluated. Treatment. Successful treatment regimens that result in healing within a week include cleaning the feet and trimming necrotic tissue; parenteral antimicrobials, such as oxytetracycline or procaine penicillin, or sulfonomethazine in the drinking water or tetracyclines in feed; and footbaths (such as 10% zinc sulfate, 2.5% formalin, or 5% copper sulfate) twice a day. In severe cases, more aggressive therapy such as bandaging the feet or wiring the digits together may be needed. Animals can recover without treatment but will be lame for several weeks. Acquired immunity is reported to be poor. Research complications. Research complications are comparable to those noted for foot rot in smaller ruminants. p. Fusobacterium necrophorum infection (Foot Abscesses) Fusobacterium necrophorum is also associated with foot abscesses, the infection of the deeper structures of the foot, in sheep and goats. Only one claw of the affected hoof may be involved. The animals will be three-legged lame, and the affected hoof will be hot. Pockets of purulent material may be in the heel or toe. q. Heel Warts (Bovine Digital Dermatitis, Interdigital Papillomatosis, Papillomatous Digital Dermatitis, Foot Warts, Heel Warts, Hairy Foot Warts, Mortellaro's Disease) Etiology. Bacteria such as Fusobacterium spp., Bacteroides spp., and Dichelobacter nodosus have been isolated from bovine heel lesions. Spirochete-like organisms have also been shown in the lesions of cows with papillomatous digital dermatitis (PDD), in the United States and Europe; these have culturing requirements similar to those of Treponema species. Clinical signs. All lesions occur on the haired, digital skin. One or all feet may be affected. Most lesions occur on the plantar surface of the hindfoot (near the heel bulbs and/or extending from the interdigital space), but the palmar and dorsal aspect of the interdigital spaces may also be involved. Progression of lesions, typically over 2–3 weeks, includes erect hairs, loss of hair, and thickening skin. Moist plaques begin as red and remain red or turn gray or black. Exudate or blood may be present on the plaque. Plaques enlarge and "hairs" protrude from the roughened surface. Lesioned areas are painful when touched. The lesions may or may not be malodorous. Epizootiology and transmission. Facility conditions and herd management are considered contributing factors. The following have been examined as contributing factors: nutrition, particularly zinc deficiency; poorly drained, low-oxygen, organic material underfoot; poor ventilation; rough flooring; damp and dirty bedding areas; and overcrowding. These interdigital lesions occur commonly in young stock and in dairy facilities throughout the world. The disease is seen only in cattle. Pathogenesis. The organisms noted above, combined with poor facility and herd management, are critical in the pathogenesis. Differential diagnosis. Differentials for lameness will include sole abscesses, laminitis, and trauma. Prevention and control. Each facility and management condition noted above should be addressed in conjunction with appropriate antibiotic and/or antiseptic treatment regimens. All equipment used for hoof trimming must be cleaned and disinfected after every use. Trucks and trailers should also be sanitized between groups of animals. Treatment. Antibiotic and antiseptic regimens have been used successfully for this problem. Antibiotics include parenteral cephalosporins and pencillins, as well as topical tetracyclines with bandaging. Antiseptic or antibiotic solutions in footbaths include tetracyclines, zinc sulfate, lincomycin, spectinomycin, copper sulfate, and formalin. The footbaths must be well maintained, minimizing contamination by feces and other materials. Tandem arrangements, such as the cleaning footbaths and then the medicated footbaths, and preventing dilution from precipitation are useful. Other treatments such as surgical debridement, cryotherapy, and caustic topical solutions have been successful. Research complications. Infectious, contagious PPD is one of the major causes of lameness among heifers and dairy cattle and is a costly problem to treat. The outbreaks are generally worse in younger animals in chronically infected herds. The immune response is not well understood, and it may be temporary in older animals. r. Haemophilus somnus infection (Thromboembolic Meningoencephalitis) Etiology. Haemophilus somnus is a pleomorphic, nonencapsulated, gram-negative bacterium. Diseases caused by this organism include thromboembolic meningoencephalitis (TEME), septicemia, arthritis, and reproductive failures due to genital tract infections in males and females. Haemophilus somnus is a also major contributor to the bovine respiratory disease complex. Haemophilus spp. have been associated with respiratory disease in sheep and goats. Clinical signs. The neurologic presentation may be preceded by 1–2 weeks of dry, harsh coughing. Neurologic signs include depression, ataxia, falling, conscious proprioceptive deficits; signs such as head tilt from otitis interna or otitis media, opisthotonus, and convulsions may be seen as the brain stem is affected. High fever, extreme morbidity, and death within 36 hr may occur. Respiratory tract infections are usually part of the complex with infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza 3, Mycoplasma, and Pasteurella, and the synergism among these contributes to the signs of bovine respiratory disease complex (BRDC). In acute neurologic as well as chronic pneumonic infections, polyarthritis may develop. Abortion, vulvitis, vaginitis, endometritis, placentitis, and failure to conceive are manifestations of reproductive tract disease. In all cases, asymptomatic infections may also occur. Diagnosis based on culture findings is difficult because H. somnus is part of the normal nasopharyngeal flora. Paired serum samples are recommended; single titers in some animals seem to be high because of passive immunity, previous vaccination, or previous exposure. In cases of abortion, other causes should be eliminated from consideration. Epizootiology and transmission. Because the organism is considered part of the normal flora of cattle and can be isolated from numerous tissues, the distinction between the normal flora and the status of chronic carrier is not clear. Outbreaks are associated with younger cattle in feedlots in western United States, but stresses of travel and coinfection with other respiratory pathogens are involved in some cases. Adult cattle have also been affected. Vaccination for viral respiratory pathogens may increase susceptibility. Transmission is by respiratory and genital tract secretions. The organism does not persist in the environment. Necropsy findings. Pathognomonic central nervous system lesions include multifocal red-brown foci of necrosis and inflammation on and within the brain and the meninges. Many thrombi with bacterial colonies will be seen in these affected areas. Ocular lesions may also be seen, including conjunctivitis, retinal hemorrhages, and edema. Usually animals with neurological disease will not have respiratory tract lesions. The respiratory tract lesions include bronchopneumonia and suppurative pleuritis. When combined with Pasteurella infection, the pathology becomes more severe. Aborted fetuses will not show lesions, but necrotizing placentitis will be evident histologically. Pure cultures of H. somnus may be possible from these tissues. Pathogenesis. Inhalation of contaminated respiratory secretions from carrier animals is the primary means of transmission. The anatomical location of bacterial residence within the carriers has not been identified. After gaining access by way of the respiratory tract, the bacteria proliferate, and a bacteremia develops. The bacteria are phagocytosed by neutrophils but are not killed. The thrombosis formation is due to the adherence by the nonphagocytosed organisms to vascular endothelial cells, degeneration and desquamation of these cells, and exposure of subendothelial collagen, with subsequent initiation of the intrinsic coagulation pathway. Antigen-antibody complex formation, resulting in vasculitis, is also correlated with high levels of agglutinating antibodies. Differential diagnosis. Differentials in all ruminants include other pathogens associated with neurological disease and respiratory disease such as Pasteurella hemolytica, P. multocida, and P. aeruginosa. In smaller ruminants, Corynebacterium pseudotuberculosis should be considered. Prevention and control. Stressed animals or those exposed to known carriers can be treated prophylactically with tetracycline administered parenterally or orally (in the feed or water). The late-stage polyarthritis is resistant to antibiotic therapy, because of failure of the antibiotic to reach the site of infection. Planning vaccination programs carefully will decrease chances of outbreaks. For example, avoiding vaccinating animals for infectious bovine rhinotrachetitis and bovine viral diarrhea during times of stress to the cattle is worthwhile. Killed whole-cell bacterins are commercially available; these have been shown to be effective in controlling the respiratory disease presentation. Control of other clinical aspects of the H. somnus disease by these bacterins has not been well described. Treatment. Rapid treatment at the first signs of neurologic disease is important in an outbreak. Haemophilus somnus is susceptible to several antibiotics, such as Oxytetracycline and penicillin, and these are often used in sequence until the cattle are recovered. s. Leptospirosis Etiology. Seven different species of the spirochete genus Leptospira are now recognized, and pathogenic serovars exist within each species; previously pathogenic leptospires were all classified as members of the species L. interrogans. The serovars pomona, icterohaemorrhagiae, grippotyphosa, interrogans, and hardjo are recognized pathogens. Leptospira hardjo and L. pomona are the serovars most commonly diagnosed in cattle, with L. hardjo causing endemic infection. Leptospira hardjo is also the major sheep serovar. Goats are susceptible to several serovars. Clinical signs. Leptospirosis is a contagious but uncommon disease in sheep and goats. The disease may cause abortion, anemia, hemoglobinuria, and icterus and is often associated with a concurrent fever. After a 4- to 10-day incubation period, the organism enters the bloodstream and causes bacteremia, fever, and red-cell hemolysis. Leptospiremia may last up to 7 days. Immune stimulation is apparently rapid, and antibodies are detectable at the end of the first week of infection; cross-serovar protection does not occur. During active bacteremia, hemolysis may result in hemoglobin levels of 50% below normal. Hyperthermia, hemoglobinuria, icterus, and anemia may be observed during this phase, and ewes in late gestation may abort. Abortion usually occurs only once. Mortality rates of above 50% have been reported in infected ewes and lambs ( Jensen and Swift, 1982 ). Subclinical infection is more common in nonpregnant and nonlactating animals. Sheep infected with leptospirosis may display a hemolytic crisis associated with IgM acting as a cold-reacting hemagglutinin. Acute and chronic infections in cattle are more common than infections in sheep and goats. Acute forms in cattle display signs similar to those in sheep. Acute infection in calves may progress to meningitis and death. Lactating cows will have severe drops in production. Chronic cases may lead to abortion, with retained placenta, and weakened calves or animals that carry the infection. Infertility may also be a sequela. Epizootiology and transmission. Leptospires are a large genus, and leptospirosis is a complicated disease to prevent, treat, and control. The organism survives well in the environment, especially in moist, warm, stagnant water. Cattle, swine, and other domestic and wild animals are potential carriers of serovars common to particular regions. Wild animals often serve as maintenance hosts, but domestic livestock may be reservoirs also. Organisms are shed in urine, in uterine discharges, and through milk. Animals become carriers when they are infected with a host-adapted serovar; sporadic clinical disease is more commonly associated with exposure to a non-host-adapted serovar ( Heath and Johnson, 1994 ). Infection may occur via oral ingestion of contaminated feed and water, via placental fluids, or through the mucous membranes of the susceptible animal. Placental or venereal transmission may occur. As the organisms are cleared from the bloodstream, they chronically infect the renal convoluted tubules and the reproductive tract (and occasionally the cerebrospinal fluid or vitreous humor). Chronically infected animals may shed the organism in the urine for 60 days or longer. Necropsy. Diagnosis is confirmed by identification of leptospires in fetal tissues. The leptospires are visible in silver- or fluorescent antibody-stained sections of liver or kidney. Leptospires may also be seen under dark-field or phase-contrast microscopy of fetal stomach contents. Fetal and maternal serology, and diagnostic tests such as the microscopic agglutination test, are useful; interpretation is complicated because of cross reaction of antibodies to many serovars. Differential diagnosis. More than one serovar may cause infection in one animal, and each serovar should be considered as a separate pathogen. Because of the associated anemia, differential diagnoses should include copper toxicity and parasites, in addition to other abortifacient diseases. Prevention and control. Polyvalent vaccines, tailored to common serovars regionally, are available and effective for preventing leptospirosis in cattle. Immunity is serovar specific. Because serological titers tend to diminish rapidly (40–50 days in sheep [ Jensen and Swift, 1982 ]), frequent vaccination may be necessary. Other prevention measures such as species-specific housing, control of wild rodents, and proper sanitation should be instituted. Treatment. Antibiotic treatment is aimed at treating ill animals and trying to clear the carrier state. Treatment methods for acute leptospirosis include oxytetracycline for 3–6 days. Addition of oxytetracycline or chlortetracycline to the feed for 1 week may be helpful. These antibiotics are considered best for removal of the carrier state of some serovars. Vaccination and antibiotic therapy can be combined in an outbreak. Research complications. Leptospirosis is zoonotic and may be associated with flulike symptoms, meningitis, or hepatorenal failure in humans. t. Listeria (Circling Disease, Silage Disease) Etiology. Listeria monocytogenes is a pleomorphic, motile, non-spore-forming, β-hemolytic, gram-positive bacillus that inhabits the soil for long periods of time and has been often found in fermented feedstuffs such as spoiled silage. Of the 16 known serovars, several produce clinical signs in ruminants. Listeria ivanovii (associated with abortions in sheep) is serovar 5. Clinical signs. Listeriosis is an acute, sporadic, noncontagious disease associated with neurological signs or abortions in sheep and other ruminants. The overall case rate is low. The disease may present as an isolated case or with multiple animals affected. Three forms of disease are described: encephalitis, placentitis with abortion, and septicemia with hepatitis and pneumonia. The encephalitic form is most common in sheep; septicemic forms may occur in neonatal lambs ( Scarratt, 1987 ). Clinically, the encephalitic form begins with depression, anorexia, and mild hyperthermia after an incubation period of 2–3 weeks. As the disease progresses, animals exhibit nasal discharges and conjunctivitis and begin to walk in circles, as if disoriented. Facial paralytic lesions, including drooping of an ear or eyelid, dilation of a nostril, or strabismus occur unilaterally on the affected side as the result of dysfunction of some or all the cranial nerves V-XII. The neck will by flexed away from the affected side. Facial muscle twitching, protrusion of the tongue, dysphagia, hypersalivation, and nasal discharges may be noted. The hypersalivation may lead to metabolic acidosis in advanced cases in cattle. Anorexia, prostration, coma, and death follow. The placental form usually results in last-trimester abortions in ewes and does, which typically survive this form of the disease. The affected females may be asymptomatic or may show severe clinical signs such as fever and depression, with subsequent retained placenta or endometritis. Abortion usually occurs within 2 weeks of Listeria infection. In cattle, abortion occurs during the last 2 months of gestation and has been induced experimentally 6–8 days after exposure. Cows present with the range of clinical signs seen in smaller-ruminant dams. There is no long-term effect on the fertility of affected dams. Epizootiology and transmission. The organism is transmitted by oral ingestion of contaminated feeds and water or possibly by inhalation. By the oral route, the organism enters through breaks in the oral cavity and ascends to the brain stem by way of nerves. When severe outbreaks occur, feedstuffs should be assessed for spoilage. Listeria organisms can be shed by asymptomatic carriers, especially at the end of pregnancy and at lambing. Diagnosis and necropsy findings. Diagnosis is usually made from clinical signs. Culture confirms the diagnosis (cold enrichment at 20° C is preferable but not essential for isolation). Impression smears will show the pleomorphic gram-positive characterisitics of the pathogen. Tissue fluorescent antibody techniques may also be utilized. Gross lesions are not observed with the encephalitic form. Microscopic lesions include thrombosis, neutrophilic or mononuclear foci in areas of inflammation, and neuritis. The pons, medulla, and anterior spinal cord are primarily affected in the encephalitic form. Microabscesses of the midbrain are characteristic of Listeria encephalitis in sheep. Aborted fetuses that are intact may show fibrinous polyserositis, with excessive serous fluids; small, necrotic foci of the liver; and small abomasal erosions. Necrotic lesions of the fetal spleen and lungs may also be seen. In goats, Listeria-induced neurological lesions occur only in the brain stem. Placentitis, focal bronchopneumonia, hepatitis, splenitis, and nephritis may be seen with other forms. Pathogenesis. With the encephalitic form, the organism penetrates mucosal abrasions and enters the trigeminal or hypoglossal nerves. The Listeria organisms then migrate along the nerves and associated lymphatics to the brain stem (medulla and pons). In the septicemic form, the organism penetrates tissues of the gastrointestinal tract and enters the bloodstream, to be distributed to the liver, spleen, lungs, kidneys, and placenta. After infection, organisms are shed in all body secretions (infected milk is an important risk factor for zoonosis). A toxin produced by Listeria monocytogenes is correlated with pathogenicity, but the mechanism of the pathogenesis of this molecule has not been elucidated. Differential diagnoses. Rabies, bacterial meningitis, brain abscess, lead toxicity, and otitis media must be considered as differentials. In sheep, the differentials include organisms that cause abortion, and neurological signs, such as enterotoxemia due to Clostridium perfringens type D. In goats, the major differentials include caprine arthritis encephalitis viral infection and chlamydial and mycoplasmal infections. In both species, scrapie is a differential. In cattle, aberrant parasite migration or Hemophilus somnus infection must also be considered. Prevention and control. Affected dams should be segregated and treated. Other animals in the group may be treated with oxytetracycline as needed. Aborted tissues should be removed immediately. Proper storage of fermented feeds minimizes this source of contamination. When silage spoils, the pH increases, producing a suitable growth environment for the organism. Commercial vaccines are not available in the United States. Treatment. Affected animals can be treated aggressively with penicillin, ampicillin, oxytetracycline, or erythromycin. Exceptionally high levels of penicillin are required for treating affected cattle. Severely affected animals should receive appropriate fluid support and other nursing care. Treatment is less successful, and mortality is especially high in sheep. Recovered animals tend to resist reinfection. Research complications. In addition to the loss of fetal animals, stress to the dams, and risks to other animals, any aborted tissue by a ruminant should be regarded as a potential zoonotic risk. Listeria can cause mild to severe flulike symptoms in humans and may be a particular risk for pregnant women and for older or immune-compromised individuals. Listeriosis in humans is a reportable disease. u. Lyme Disease (Borrelia burgdorferi Infection, Borreliosis) Etiology. Lyme disease is caused by the spirochete Borrelia burgdorferi. Clinical signs and diagnosis. Reports in ruminants indicate seroconversion to B. burgdorferi, but there are few definitive correlations to the arthritis that is present. Diagnosis requires culturing from the affected joints and diagnostic elimination of other causes of lameness and arthritis. Epizootiology and transmission. The organism is present throughout much of the Northern Hemisphere and has been reported in many mammals and also in birds. Ticks of the Ixodes ricinus complex are the major vectors of the spirochete and must be attached for 24 hr for successful transmission. Pathogenesis. The Ixodes ticks have three life stages: larval, nymphal, and adult. Feeding occurs once during each stage, and wild animals are the source of blood meals. The larval stages feed from rodents, such as the white-footed deer mouse, Peromyscus leucopus, from which they acquire the spirochete. The nymphal stage is that which usually infects other animals. The adult ticks are usually found on deer. Differential diagnosis. Seroconversion to B. burgdorferi does not necessarily confirm the cause of arthritis. Other causes of arthritis and lameness in ruminants include trauma, caprine arthritis encephalitis virus, Mycoplasma spp., Chlamydia psittaci, Erysipelothrix spp., Arcanobacterium pyogenes, Brucella spp., and rickets. Prevention and control. Control of the tick vector is the most important factor in preventing the possibility of exposure or disease. Treatment. Antibiotic therapy, with tetracycline, penicillin, amoxicillin, and cephalosporins, is used for diagnosed or suspected Lyme arthritis. Research complications. Lyme disease is zoonotic, and the Ixodes ticks transmit the disease to humans. v. Mastitis i. Ovine mastitis Mastitis in ewes may be acute, subclinical, or chronic. Acute mastitis often results in anorexia, fever, abnormal milk, and swelling of the mammary gland. Pasteurella haemolytica is the most common cause of acute mastitis. Additional isolates may include, in order of prevalence, Staphylococcus aureus, Actinomyces (Corynebacterium) spp., and Histophilus ovis. Escherichia coli and Pseudomonas aeruginosa have also been found to cause acute mastitis. As many as six serotypes of Pasteurella haemolytica have been isolated from the mammary glands of mastitic ewes. Furthermore, intramammary inoculation of these organisms isolated from ovine and bovine pulmonary lesions has resulted in clinical mastitis in ewes ( Watkins and Jones, 1992 ). Subclinical mastitis is detected only indirectly, by counting somatic cells. The most common isolate from ewes with subclinical mastitis is coagulase-negative staphylococci. Other isolates include Actinomyces bovis, Streptococcus uberis, S. dysgalactiae, Micrococcus spp., Bacillus spp., and fecal streptococci. Most of these organisms are commonly found in the environment. Diffuse chronic mastitis, or hardbag, results from interstitial accumulations of lymphocytes in the udder. Both glands are usually affected, but no inflammation is present. Serological evidence suggests that diffuse chronic mastitis is caused by the retrovirus that causes ovine progressive pneumonia (OPP or maedi/visna virus). Other bacterial agents or Mycoplasma have not usually been isolated from udders with this type of mastitis. Acute mastitis occurs in approximately 5% of lactating ewes annually, and it usually occurs either soon after lambing or when lambs are 3–4 months old ( Lasgard and Vaabenoe, 1993 ). Subclinical mastitis occurs in 4–50% of lactating ewes ( Kirk and Glenn, 1996 ). Subclinical mastitis is more common in ewes from high-milk-producing breeds. Skin or teat lesions and dermatitis increase the prevalence of disease. Acute mastitis can be diagnosed in ewes with associated systemic signs of disease by physical examination of the udder and inspection of the milk. Subclinical mastitis is often suggested by somatic cell counts elevated above 1 × 10 6 cells/ml. When high somatic cell counts are identified, subclinical mastitis can be diagnosed by milk culture. The California mastitis test may also be helpful as an indicator of mastitis. Manual palpation of a hard, indurated udder as well as serological testing for the maedi/visna virus is helpful in confirming the diagnosis of diffuse chronic mastitis. Treatment for acute bacterial mastitis should include aggressive application of broad-spectrum antibiotics (intramammary and systemic) and supportive therapy such as fluids and anti-inflammatory drugs. It is may be helpful to milk out the infected udder frequently; oxytocin injections preceding milking will improve gland evacuation. Because somatic cell counting is often not routinely performed, treatment of subclinical mastitis is seldom done. There is currently no treatment available for diffuse chronic mastitis. ii. Caprine mastitis Lactating goats are subject to inflammation of mammary gland, or mastitis. The primary causative organisms are Staphylococcus epidermidis and other coagulase-negative Staphylococcus spp. Clinical signs of mastitis include abnormal coloration or composition of milk, mammary gland redness, heat and pain, enlargement of the mammary gland, discoloration of the mammary gland, and systemic signs of septicemia. Large abscesses may be present in the affected gland. Staphylococcus aureus is also associated with caprine mastitis, and toxemia may be part of the clinical picture. This organism produces a necrotizing alpha toxin that can result in gangrenous mastitis. Caprine mastitis may be clinical or subclinical, and the first indication of mastitis may be weak, depressed, or thin kids. Diagnosis is based on careful culture of mastitic milk. Treatment includes frequent stripping, intramammary antibiotics, and nonsteroidal anti-inflammatory drugs. Oxytocin (5–10 U) may help milk letdown for frequent strippings. Bovine mastitis products can be used in the goat; however, care should be taken not to insert the mastitis tube tip fully, because damage to the protective keratin layer lining the teat canal may occur. In severe acute systemic cases, steroids, fluids, and systemic antibiotics may be necessary. Other less common causes of mastitis in goats include Streptococcus spp. (S. agalactiae, S. dysgalactiae, S. uberis, and zooepidemicus). Gram-negative causes of caprine mastitis include Escherichia coli, Klebsiella pneumoniae, Pasteurella spp., Pseudomonas, and Proteus mirabilis. Corynebacterium pseudotuberculosis can cause mammary gland abscessation, whereas Mycoplasma mycoides may cause agalactia and systemic disease. "Hard udder" can be caused by caprine arthritis encephalitis virus (CAEV). Brucellosis and listeriosis can cause a subclinical interstitial mastitis ( Smith and Sherman, 1994 ). iii. Bovine mastitis Mastitis is the disease of greatest economic importance for the dairy cattle industry. The majority of the impact will be on the production and overall health of the cows, but low-incidence herds also diminish the risk of calves' ingesting or being exposed to pathogens. The most common bovine mastitis pathogens include Staphylococcus aureus and Streptococcus agalactiae, S. dysgalactiae, and S. uberis; coliform agents such as Escherichia coli, Enterobacter aerogenes, Serratia marcescens, and Klebsiella pneumoniae; mycoplasmal species such as Mycoplasma bovis, M. bovigenitalium, M. californicum, M. canadensis, and M. alkalescens; and Salmonella spp. such as S. typhimurium, S. newport, S. enteritidis, S. dublin, and S. muenster. Many of these agents such as Staphylococcus spp., Salmonella spp., and the coliforms can cause both acute and chronic mastitis, as well as severe systemic disease, including fever and anorexia. These must be regarded as herd and environmental pathogens in terms of treatment and prevention. The pathogenesis of staphylococcal infections is comparable to that in goats. Staphylococcus agalactiae can be cleared from udders because it does not invade other tissues, is an obligate resident of the glands, and is susceptible to penicillin. In contrast, S. uberis and S. dysgalactiae are environmental organisms and can be highly resistant to pencillin. Mycoplasma bovis is the more common of the mycoplasmal pathogens and can cause severe infections. Transmission of the mycoplasmas is not well defined but may be related to their presence in other organ systems. Treatments for mycoplasmal mastitis are not successful; culling is recommended. There are many interrelated factors associated with prevention and control of mastitis in a herd, including herd health and dry cow management, order of animals milked, milking procedures, milking equipment, condition of the teats, and the condition of the environment. Management of the overall herd includes aspects such as vaccination programs, nutrition, isolation of incoming animals, and quarantine and treatment of or culling diseased individuals. Culturing or testing newly freshened cows and monitoring the bulk milk tank serve as indicators of subclinical mastitis. Herd management will diminish teat lesions. Bacterin vaccines are available for preventing and controlling coliform mastitis and S. aureus mastitis. At the time of dry-off, all cows must be treated by intramammary route. Some infections can be successfully cleared during this time. Younger, disease-free animals should be milked first; any animals with diagnosed problems should be milked after the rest of the herd and/or segregated during treatment. Milkers' hands easily serve as a means of pathogen transmission, and wearing rubber gloves is recommended. Teat and udder cleaning practices include washing and drying with single-service paper or cloth towels or pre-and postmilking dipping. Milking equipment must be maintained to provide proper vacuum levels and pumping rates, and liners should be the appropriate size. Facilities that provide clean and dry areas for the animals to rest, feed, and move will diminish teat injuries and reduce exposures to mastitis pathogens. In that regard, inorganic bedding such as clean sand harbors few pathogens in contrast to shavings and sawdust. w. Moraxella bovis Infection (Infectious Bovine Keratoconjunctivitis, Pinkeye) Etiology. Moraxella bovis, a gram-negative coccobacillus, is the most common cause of infectious bovine keratoconjunctivitis (IBK) in cattle. This organism is not a cause of keratoconjunctivitis in sheep and goats. The disease includes conjunctivitis and ulcerative keratitis. The pathogenic M. bovis strain is piliated, and at least seven serotypes exist. Clinical signs. Lacrimation, photophobia, and blepharospasm are seen initially. Conjunctival injection and chemosis develop within a day of exposure, and then keratitis with corneal edema and ulcers. Anterior uveitis may be a sequela within a few days, and thicker mucopurulent ocular discharge may be seen. Corneal vascularization begins by 10 days after onset. Reepithelialization of the corneal ulcers occurs by 2–3 weeks after onset. Diagnosis is usually based on clinical signs, but culturing is helpful and fluorescein staining is useful for demonstrating corneal ulceration. Epizootiology and transmission. The disease is more severe in younger cattle. The clinical signs of IBK tend to be more severe in cattle that are also infected with infectious bovine rhinotracheitis (IBR) virus or those that have been vaccinated recently with modified live IBR vaccine. The bacteria are shed in nasal secretions and cattle with no clinical symptoms may be carriers. Transmission is by fomites, flies, aerosols, and direct contact. Incidence in winter months is very low. Nonhemolytic strains are associated with the winter epidemics, and hemolytic strains are associated with summer epidemics. Necropsy findings. Necropsy is not typically performed on these cases. Corneal edema, ulceration, hypopyon, and uveitis would be noted, depending on the stage of infection. Pathogenesis. The pili of M. bovis bind to receptors of corneal epithelium. The virulent strains of the bacteria then release the enzymes that damage the corneal epithelial cells. Other factors contributing to infection include ultraviolet light and trauma from dust and plant materials. Differential diagnoses. Infectious bovine rhinotrachetitis virus causes conjunctivitis, but the central corneal ulceration that is characteristic of IBK is not seen with M. bovis infections. Mycoplasma, Listeria, Branhamella (Neisseria), and adenovirus may be cultured from affected bovine eyes but none has been shown to produce the corneal lesions when inoculated into susceptible animals. Prevention and control. Cattle should not be immunized intranasally with modified live infectious bovine rhinotracheitis vaccine during IBK outbreaks; this will likely exacerbate the infection. New animals should be quarantined and treated prophylactically before introduction to herds. The available vaccines, containing. M. bovis pili or killed M. bovis, help decrease incidence and severity of disease; these preparations are not completely effective, because the M. bovis strain may not be homologous to that used for the vaccine preparation. Other preventive measures include 10% permethrin-impregnated bilateral ear tags, pour-on avermectins, or dust bags or face rubbers containing insecticide (such as 5% coumaphos) to control flies throughout the season and premises; mowing of high pasture grass to minimize ocular trauma; provision of shade; control of dust and sources of other mechanical trauma; and segregation of animals by age. Treatment. Cattle can recover without treatment, but younger animals should be treated as soon as the infection is detected. Antibiotic treatments include topical, subconjunctival administration and intramuscular dosing. Several standard topical antibiotics have been shown to be effective, including oxytetracycline, gentamicin, and triple antibiotic combinations. These should be administered twice per day. Subconjunctival injections of antibiotics, such as penicillin G, provide higher corneal levels of drug; these are typically administered only once or twice in severe cases. Intramuscular doses of long-acting oxytetracycline, given on alternate days, are effective in larger herds, and 2 doses 72 hr apart eliminate carriers. Third-eyelid flaps, temporary tarsorrhaphy, or eye patches may be useful in certain cases. Research complications. This pathogen does present a complication due to the carrier status of some animals, the likelihood of herd outbreaks, the severity of disease in younger animals, and the morbidity, possible progression to uveitis, and time and treatment costs associated with infections. The overall condition of the cattle will be affected for several weeks, and permanent visual impairment or loss, as well as ocular disfigurement, may occur. x. Mycobacterial Diseases Mycobacterium bovis Infection (Tuberculosis) Etiology. Mycobacteria are aerobic, nonmotile, non-spore-forming, acid-fast pleomorphic bacteria. Most cases of tuberculosis in sheep are related to Mycobacterium bovis or M. avium. Cases in goats have been attributed to M. bovis, M. avium, or M. tuberculosis. Mycobacterium bovis, or the bovine tubercle bacillus, is the cause in cattle but has been isolated from many domestic and wild mammals. Other agents of mammalian tuberculosis include M. microti and M. africanum. Clinical signs. Tuberculosis is a sporadic, chronic, contagious disease of ruminants and is zoonotic. The infection is often asymptomatic later in the illness, and it may be diagnosed only at necropsy. The respiratory system (M. bovis) or the digestive system (M. avium) is the primary site of infection; other tissues such as mammary tissue and reproductive tract may be infrequently involved. Locations of the characteristic tubercles will determine whether clinical signs are seen. Respiratory signs may include dyspnea, coughing, and pneumonia. Digestive tract signs include diarrhea, bloat, or constipation; diarrhea is most common. Lymphadenopathy occurs in advanced cases. Fever and generalized disease may be seen after calving. Infected goats lose weight and develop a persistent cough. Epizootiology and transmission. Although M. bovis can be killed by sunlight, it otherwise survives a long time in the environment and in cattle feces. Animals acquire the infection from the environment or from other animals via aerosols, from contaminated feed and water, and from secretions such as milk, semen, genital discharges, urine, and feces. Clinically normal animals may serve as carriers. The bacilli stimulate an initial neutrophilic tissue response. Neutrophils become necrotic and are phagocytosed by macrophages, forming giant epithelioid cells called Langhans' giant cells. An outer lymphocytic zone is formed, and fibrotic encapsulation creates the classical caseous nodules. Vascular erosion and hematogenous migration of the organisms may lead to lesions throughout the body. Necropsy findings. Yellow primary tubercles (granulomas) with central areas of caseous necrosis and calcification are present in the lungs. Caseous nodules are also associated with gastrointestinal organs and mesenteric lymph nodes. Prevention and control. Significant progress has been made in eradication programs in the United States during the past several decades, but during the 1990s, infected animals continued to be found in domestic cattle herds and particularly in captive deer herds in hunting preserves. The intradermal tuberculin test, using purified protein derivative (PPD), is usually used as a diagnostic indicator in live animals. This test should be performed annually on bovine and caprine dairy herds (and bison herds); the official tests are the caudal fold, comparative cervical, and single cervical tests. Notification to state officials is required following identification of intradermal-positive animals. Great care must be exercised in any handling of tissue or necropsies of reactors, and state animal health officials should be consulted regarding disposal of materials and cleaning of premises following depopulation of positive animals. Treatment. No treatment is recommended, and treatment is usually not allowed, because of the zoonotic potential, chronicity of the disease, and the treatment costs. Slaughter is preferred, to prevent potential transmission to humans. Research complications. The pathogen is zoonotic. Paratuberculosis, or Johne's disease (Mycobacterium paratuberculosis) Etiology. Mycobacterium paratuberculosis, the causative agent of Johne's disease, is a fastidious, non-spore-forming, acid-fast, gram-positive rod. The organism is actually a subspecies of M. avium, but M. paratuberculosis does not produce the siderophore mycobactin (an iron-binding molecule) of M. avium. Clinical signs and diagnosis. Johne's disease is a chronic, contagious, granulomatous disease of adult ruminants and is characterized by unthriftiness, weight loss, and intermittent diarrhea. In sheep and goats, chronic wasting is usually seen, occasionally with pasty feces or diarrhea. In cattle, chronic diarrhea and rapid weight loss are the most common clinical signs of the disease. Usually older adult animals are infected, but over time in an infected herd, younger animals will become infected when sufficient doses of organisms are ingested. Although clinical signs are nonspecific, Johne's disease should be considered if the affected diarrheic animals have a good appetite and are on a good anthelmintic program. The disease is diagnosed based on clinical signs and laboratory analyses, although none of the tests is more than 50% sensitive. In addition, the sensitivity of the serological tests differs between species. The standard is the fecal culture that takes 8–12 weeks. The enzyme-linked immunosorbent assay (ELISA) is now considered the most reliable serological test, but false negatives do occur. Other serological tests such as agar gel immunodiffusion (AGID) and complement fixation are useful. Herd screening may be done using the AGID or ELISA serological tests. Identification of the organism on culture, or the presence of acid-fast organisms on mucosal or mesenteric lymph node smears or from rectal biopsies, helps confirm the diagnosis. Some animals serologically negative for Johne's disease, however, have been found to be positive on fecal culture. Commercial AGID tests approved for use in cattle may be useful in diagnosing Johne's disease in sheep ( Dubash et al., 1996 ). Serological tests cross-react with other species of Mycobacterium, especially M. avium. Epizootiology and transmission. The organism is prevalent in the environment and is transmitted to young animals by direct or indirect contact. Although vertical transmission has been reported, the organism more commonly enters the gastrointestinal tract and penetrates the mucosa of the distal small intestine, primarily the ileum. Chronic carriers may intermittently shed the organisms. Pathogenesis. Mycobacterium paratuberculosis is an obligate parasite that grows only in macrophages of infected animals. Nursing infected dams are a primary source of infection of neonates. If the organism is not cleared, it proliferates slowly in the tissue, leading to inflammatory reactions that progress through neutrophilic to mononuclear stages. The organism may penetrate the lymphatics and proliferate in mesenteric lymph nodes. After an incubation period of a year or more, some of the carriers will progress to clinical disease manifested by fibrotic and hyperplastic changes in the ileum, leading to the classic thickening in the region. Gut changes result in intermittent diarrhea, with subsequent dehydration, electrolyte imbalances, and malnutrition, although this clinical sign is more common in cattle than in sheep or goats. Necropsy and diagnosis. The ileum from infected cattle is grossly thickened; this is not seen in sheep and goats. Ileal and ileocecal lymph nodes provide the best samples for histology and acid-fast staining. Differential diagnosis. Diseases causing chronic wasting and poor coat and body condition of all ruminants should be considered. These include chronic salmonellosis, peritonitis, severe parasitism, winter dysentery, and pyelonephritis. Deer can be infected, and the lesions can be confused with those of tuberculosis. Treatment. Treatment is not worthwhile. Prevention and control. Prevention is the most effective method to manage this pathogen. Efforts should be focused on eliminating the disease through test and slaughter. Neonates should not be reared by infected dams. Some states have Johne's disease eradication programs. Facilities and pastures where animals testing positive for Johne' disease were maintained should be thoroughly cleaned and kept vacant for a year after culling. Other considerations. Mycobacterium paratuberculosis is being investigated as a factor in the development of Crohn's disease in humans. y. Navel Ill (Omphalitis, Omphalophlebitis, Omphaloarteritis, Joint Ill) Etiology. The most common organism causing infection of the umbilicus is Arcanobacterium (formerly Actinomyces, Corynebacterium) pyogenes; other bacteria may be present. Arcanobacterium spp. are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Other environmental contaminants are also associated with this disease, such as Escherichia coli, Enterococcus spp., Proteus, Streptococcus spp., and Staplylococcus spp. Clinical signs and diagnosis. Navel ill is an acute localized inflammation and infection of the external umbilicus. Animals present with fever and painful enlargement of the umbilicus. Animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, and hematuria. Other common severe sequelae include septicemia, pneumonia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, uveitis, endocarditis, and diarrhea. Epizootiology and transmission. Many cases occur in neonates, and most cases occur within the first 3 months of age. Cleanliness of the birthing and housing environment and successful transfer of passive immunity are important factors in the occurrence of the disease. Dystocia resulting in weak neonates can be a factor predisposing to the development of the disease. Navel ill is diagnosed by typical clinical signs. The presence of microabscesses and palpation of the umbilical area for firm intra-abdominal structures extending from the umbilicus are abnormal. Assessment of colostral immunoglobulin transfer may contribute to determination of the prognosis. Navel ill should always be considered for young ruminants with fever of unknown origin during the first week of life and for slightly older lambs, kids, or calves that are not thriving. Arthrocentesis of affected joints and culture of the fluid for identification of the pathogen are also diagnostic options and essential for effective antimicrobial selection. Differential diagnosis. The major differential is an umbilical hernia, which will typically not be painful or infected and can often be reduced. Mycoplasmal arthritis is a differential in kids. In the past, Erysipelothrix rhusopathiae was a common navel ill pathogen in sheep. Treatment. Omphalitis can be treated with a 10 to 14 day course of broad-spectrum antibiotics such as ampicillin, amoxicillin, penicillin, ceftiofur, florfenicol, and erythromycin. If an isolated abscess is palpable, it should be surgically opened and repeatedly flushed with iodine solutions. Surgical reduction of the infected umbilicus is indicated if intra-abdominal structures are involved. The prognosis for recovery is good if systemic involvement has not occurred. Prevention and control. The disease is best prevented and controlled by providing clean birthing environments, ensuring adequate colostral immunity, thoroughly dipping the umbilicus of newborns in tincture of iodine or strong iodine solution (Lugol's), monitoring for dystocias, and maintaining young growing animals in noncontaminated environments. Research complications. The disease can be costly to treat, and the toll taken on young animals due to the consequences of systemic infection may detract from their research value. z. Pasteurellosis (Shipping Fever, Hemorrhagic Septicemia, Enzootic Pneumonia) Etiology. Pasteurella hemolytica and P. multocida are aerobic, nonmotile, non-spore-forming, bipolar, gram-negative rods. Biotype A serotypes are associated with pneumonia and septicemia in all ruminants ( Ellis, 1984 ). Serotype 1 of P. hemolytica is considered a major cause of pulmonary lesions of bovine bronchopneumonia and fibrinous bronchopneumonia. Clinical signs. Pasteurellosis is an acute bacterial disease characterized by bronchopneumonia, septicemia, and sudden death. The organism invades the mucosa of the gastrointestinal tract or respiratory tract and causes localized areas of necrosis, hemorrhage, and thrombosis. The lungs and liver are frequent areas of formation of microabscesses. Acute rhinitis or pharyngitis often precedes the respiratory form. The organism also may invade the bloodstream, causing disseminated septicemia. Clinically, the lambs may exhibit nasal discharge of mucopurulent to hemorrhagic exudate, hyperthermia, coughing, dyspnea, anorexia, and depression. With the respiratory form, auscultation of the thorax suggests dullness and consolidation of anteroventral lobes; this will be confirmed by radiographs. The disease is diagnosed by clinical signs, blood cultures from septicemic animals, blood smears showing bipolar organisms, and history of predisposing stressors. In cultures, P. hemolytica is distinguished from P. multocida by hemolysis on blood agar; only P. multocida produces indole. Epizootiology and transmission. The organism is ubiquitous in the environment and in the respiratory tracts of these animals. Younger ruminants, between 2 and 12 months of age, are especially prone to infection during times of stress, such as weaning, transportation, dietary changes, weather changes, and overcrowding. The pneumonic form appears as a complex associated with concurrent infections such as parainfluenza 3, adenovirus type 6, respiratory syncytial virus, mycoplasmas, chlamydia, Pasteurella multocida and Bordetella parapertussis ( Martin, 1996 ; Brogden et al., 1998 ). The organism is transmitted between animals by direct and indirect contact, through inhalation or ingestion. Necropsy findings. Necropsy lesions include areas of necrosis and hemorrhage in the small intestines and multifocal 1 mm lesions distributed on the surfaces of the lungs and liver. With the pneumonic form, serofibrinous exudates fill the alveoli; ventral lung lobes are consolidated and are congested and purple-gray in color. Fibrinous pleuritis, pericarditis, and hematogenously induced arthritis also may be evident. Pathogenesis. A leukotoxin is considered to be a key factor in the pathogenesis of the P. hemolytica infection. Macrophages and neutrophils are lysed by the toxin as they arrive at the lung, and the enzymes released by the neutrophils cause additional damage to the tissue. Treatment. Treatment may include the use of antibiotics such as penicillin, ampicillin, tylosin, sulfonamides, or oxytetracycline. Newer antibiotics, such as ceftiofur, tilmicosin, spectinomycin, and florfenicol, are very effective and approved for use in cattle. In outbreaks, cultures from fresh necropsies are helpful for determining sensitivities useful for the remaining group. Prevention and control. The incidence of disease can be decreased by minimizing the degree of stress; by improving management, such as nutrition and control of parasitism; and, in cattle and sheep, by vaccinating for viral respiratory infections such as parainfluenza. Early Pasteurella hemolytica bacterin vaccines for use in cattle are not considered effective, but newer products based on immunizing against the leukotoxin and some bacterial capsule surface antigens are effective. Pasteurella multocida bacterins and live streptomycin-dependent mutant vaccines are available. In young animals, passive immunity is protective. Preventive measures also include maintaining good ventilation in enclosures and barns. New animals to the flock or herds should be quarantined for at least 2 weeks before introduction. aa. Salmonellosis Etiology. Salmonella typhimurium is a motile, aerobic to facultatively anaerobic, non-spore-forming, gram-negative bacillus and is the organism associated with enteric disease and some abortions in ruminants. It is a common inhabitant of the gastrointestinal tract of ruminants. Current nomenclature categorizes S. typhimurium as a serovar within the species S. enteritidis (the other two species are S. typhi and S. choleraesuis). Salmonella typhimurium, S. dublin, and S. newport are the common species seen in bovine cases. Salmonella typhimurium, S. dublin, S. anatum, and S. montevideo are seen in ovine and caprine cases, although a host-adapted species has not been identified in the goat. Ovine abortions due to various Salmonella species are not reported in the United States but are enzootic in other countries. Salmonella serotypes have been associated with aborted fetuses in all ruminant species. Clinical signs and diagnosis. Salmonellosis causes acute gastroenteritis, dysentery, and septicemia ( Anderson and Blanchard, 1989 ). Clinically, the animals become anorexic and hyperthermic. Diarrhea or dysentery develops; feces may contain mucus and/or blood and have a putrid odor. Animals become severely depressed and weak, losing a high percentage of their body weight. Animals may die in 1–5 days because of dehydration associated with dysenteric fluid loss, septicemia, shock, and acidosis. Morbidity may be 25%, and mortality may be high. Septicemia may result in subsequent meningitis, polyarthritis, and pneumonia. Chronically infected animals may have intermittent diarrhea. In goats, salmonellosis may be recognized as diarrhea and septicemia in neonates, as enteritis in preweaned kids and mature goats, and, rarely, as abortion. Adult cases may be sporadic, with intermittent bouts of diarrhea, subacute or even chronic. Morbidity and mortality will be highest in neonates, and some may simply be found dead. The older animals generally tend to fare better during the disease. Abdominal distension with profuse yellow feces is common. Kids become severely depressed, anorexic, febrile (with temperatures as high as 106°–107°F), dehydrated, acidotic, recumbent, and comatose. Salmonella abortions may occur throughout gestation. There may not be any other clinical signs, or abortion may be seen with diarrhea, fever, and vulvar discharges. Hemorrhage, placental necrosis, and edema will be present. Metritis and placental retention may occur. Some mortality of dams may occur. Diagnosis is based on clinical signs and can be confirmed by culturing fresh feces or at necropsy. Because of intermittent shedding of organisms, culture may be difficult; repeated cultures are recommended. Leukopenia and a degenerative shift to the left are not uncommon hematological findings. Epizootiology and transmission. Stresses associated with recent shipping, overcrowding, and inclement weather may predispose the animal to enteric infection. Birds and rodents may be natural reservoirs of Salmonella in external housing environments. Transmission is fecal-oral. After ingestion, the organisms may proliferate throughout the gastrointestinal tract and may penetrate the mucosa of the intestines, invade the Peyer's patches and lymphatics, and migrate to the spleen, liver, and other organs. Animals that survive may become chronic carriers and shedders of the organisms, and this has been demonstrated experimentally ( Arora, 1983 ). Fecal-oral transmission is also associated with Salmonella abortion; veneral transmission has not been reported. Necropsy findings and diagnosis. Animals will have noticeable perineal staining. Intestines (particularly the ileum, cecum, and colon) may contain mucoid feces with or without hemorrhages. Petechial hemorrhages and areas of necrosis may be noticed on the surface of the liver, heart, and mesenteric lymph nodes. The wall of the intestines, gallbladder, and mesenteric lymph nodes will be edematous, and a pseudodiphtheritic membrane lining the distal small intestines and colon may be observed. This membrane is not normally seen in the goat ( Smith and Sherman, 1994 ). Splenomegaly may be present. Aborted fetuses will often be autolysed. Placentitis, placental necrosis, and hemorrhage are commonly seen. Serologic evidence of recent infection can be demonstrated in the dam. Salmonella can be isolated from the aborted tissues. Pathogenesis. After ingestion, the organism proliferates in the intestine. Damage to the intestines and the resulting diarrhea are due to the bacterial production of cytoxin and endotoxin. Although the Salmonella organisms will be taken up by phagocytic cells involved in the inflammatory response, they survive and multiply further. Septicemia is a common sequela, with the bacteria localizing throughout the body. In latently infected animals, it is often shed from the gallbladder and mesenteric lymph nodes. Younger animals may be susceptible because of immature immunity and intestinal flora and higher intestinal pH. Carriers may develop clinical disease when stressed. Differential diagnoses. In young animals, differentials include other enteropathogens: Escherichia coli, rotavirus and coronavirus, clostridia, cryptosporidia, and other coccidial forms. These pathogens may also be present in the affected animals. Differentials in adults include bovine viral diarrheas and winter dysentery in cattle and parasitemia and enterotoxemia in all ruminants. Prevention and control. Affected animals should be isolated during herd outbreaks. Samples for culture should include herd-mates, water and feed sources, recently arrived livestock (other species), and area wildlife, including birds and rodents. Repeated cultures, culling of animals, intensive cleaning, and disinfection of facilities are all important during outbreaks. The bacteria survive for about a week in moist cow manure. Vaccination using the commercially available killed bacterin or autologous bacterins may be useful in outbreaks involving pregnant cattle, although the J-5 bacterin is now considered better. Treatment. Nursing care includes rehydration and correction of acid-base abnormalities. Antibiotic therapy may be useful in cases with septicemia, but it is controversial because it may induce carrier animals. Gentamicin, trimethoprim-sulfadiazine, ampicillin, enrofloxacin, and amikacin antibiotics may be successful. Research complications. Salmonellosis is zoonotic, and some serotypes of the organism have caused fatalities even in immunocompetent humans. Attempts should be made to identify and cull carrier animals. bb. Spirochete-Associated Abortion in Cattle (Epizootic Foothill Abortion) Etiology. Spirochete-like organisms are associated with this disease; it is now recognized that the agent is not a chlamydial organism. The disease has been reported only in the foothills bordering the central valley of California. Clinical signs. Cows that become infected with the causative agent before 6 months of gestation abort or give birth to weak calves without any clinical sign of infection. Cows infected after 6 months of gestation give birth to normal calves. Affected cows rarely abort in subsequent pregnancies. Epizootiology and transmission. The tick vector is Ornithodorus coriaceus. Necropsy. Fetuses show several pathological changes, including enlargement of the cervical lymph nodes, spleen, and liver. The calf's thymus will be small, and histologically there will be losses of thymic cortical lymphocytes. Histologic changes in lymph nodes and spleen include vasculitis, necrosis, and histiocytosis. Treatment. Chlortetracycline treatment has been effective in controlling this disease. cc. Tularemia Etiology. Tularemia is caused by Pasteurella (Francisella) tularensis a nonmotile, non-spore-forming, aerobic, gram-negative, rod-shaped bacterium. Type A is more virulent than type B. Clinical Signs. Although tularemia is a disease of livestock, pets, and wild animals, sheep are most commonly affected. The disease is characterized by hyperthermia, muscular stiffness, and lymphadenopathy. Infected animals move stiffly, are depressed, and are hyperthermic. Anemia and diarrhea may develop, and infected lymph nodes enlarge and may ulcerate. Mortality may reach 40%. Animals that recover will have immunity of long duration. Epizootiology and transmission. The disease is most commonly transmitted by ticks or biting flies. The wood tick, Dermacentor andersoni, is an important vector in transmitting the disease in the western United States, and, as natural hosts, wild rodents and rabbits tend to be reservoirs of the pathogen. Pathogenesis. The organisms, entering the tick bite wound, move via lymphatics to lymph nodes and subsequently to the bloodstream, where they cause septicemia. The organisms can also be transmitted orally through contaminated water. Necropsy findings. Ticks may also be present on the carcasses. Suppurative, necrotic lymph nodes are typical. Lungs will be congested and edematous. Diagnosis is confirmed by prompt culturing of the organism from lymph nodes, spleen, or liver where granulomatous lesions form; P. tularensis does not survive for long periods in carcasses. Serological findings may also be helpful. Treatment. Infected animals can be treated with oxytetracycline, aminoglycosides, or cephalosporins. Differential diagnosis. When tick infestations are heavy, P. tularensis should be suspected. Pasteurella haemolytica (sheep), Haemophilus somnus (cattle), and Mycoplasma mycoides (goats), and anthrax (all ruminant species) should be considered as differentials. Control and prevention. Eliminating the tick vectors can prevent tularemia. Animals should be provided with fresh water frequently. The organism can survive in freezing conditions and in water and mud for long periods of time. Caretakers, veterinarians, and researchers should take special precautions before handling the tissues of infected sheep, because this is a method of zoonotic spread. Research complications. The disease is zoonotic, and transmission to people may result from tick bites or from handling contaminated tissues. Although not a major disease of concern in sheep, researchers using potentially infected animals from western range states of the United States should be aware of it. The organism is antigenically related to Brucella spp. dd. Yersinia Etiology. Yersiniosis is caused by infections with Yersinia enterocolitica, a gram-negative, aerobic, and facultative anaerobe of the family Enterobacteriaceae. There are 50 serotypes reported for Y. enterocolitica. Yersinia pseudotuberculosis infections have also been seen in ruminants. Enteric infections predominate in the diseases caused by these bacteria. Clinical signs and diagnosis. Clinical disease may be seen rarely in many groups of ruminants. Goats of 1–6 months old suffer from the enteric form of the disease, which is characterized by sudden death or the acute onset of watery diarrhea lasting 1 or more days. Spontaneous abortions and weak neonates are also clinical manifestations of infection. Lactating does may have mastitis that becomes chronically hemorrhagic. Bacteremia results in internal abscesses, abortion, and acute deaths. Yersinia pseudotuberculosis has been associated with laboratory goat epizootics ( Obwolo, 1976 ). Diarrhea in pastured sheep, stressed by other factors, has also been reported. Diagnosis is based on culture and serology. Epizootiology and transmission. The bacteria are carried by wild birds and rodents, and transmission is by ingestion of contaminated feed and water. Necropsy findings. Edema of mesenteric lymph nodes is the most common postmortem finding. Liver abscesses, micro-absecesses in the intestines, and granuloma formation have also been reported. Placentas are white, with opaque white foci found on cotyledons. Histologically, suppurative placentitis and suppurative pneumonia are found in the fetal tissue. Pathogenesis. After ingestion, the bacteria cause an enteric infection, and bacteremia follows. Differential diagnoses. Other causes of abortions, including abortion storms, acute deaths, enteritis, neonatal deaths, and white foci on cotyledons, should be considered. In young animals, differentials include coccidiosis and nematode parasitism. Corynebacterium pseudotuberculosis and tuberculosis are differentials for the internal abscesses. Prevention and control. Control measure are not well defined, because the epidemiology of the disease is poorly understood ( Smith and Sherman, 1994 ). Tissues from affected goats must be handled and disposed of properly. Areas housing affected goats must be thoroughly sanitized. Treatment. In case of an abortion storm, treatment of goats with tetracycline has been useful. Other broad-spectrum antibiotics may also be useful. Research complications. Yersinia is zoonotic. The risk of severe enteric disease is considered particularly great for immunocompromised persons. ee. Mycoplasmal Diseases i. Mycoplasma bovigenitalium and M. bovis infections Etiology. Mycoplasma bovigenitalium and M. bovis are associated sporadically with bovine infertility and abortions. This pathogen has also been reported associated with similar clinical signs in sheep and goats. Clinical signs and diagnosis. Infertility is more commonly caused by M. bovigenitalium infections, and granular vulvovaginitis and endometritis will be present. Granular vulvovaginitis is characterized by raised papules on the mucous membranes and mucopurulent exudate. Abortions and mastitis are associated with M. bovis infections. Calves that are born may be weak. It is rare to have a definitive diagnosis of an abortion due to Mycoplasma. After consideration of other causes of abortion and evaluation of tissues for placentitis or fetal inflammation, diagnosis is confirmed by isolation of Mycoplasma from the genital tract or aborted tissues. Epidemiology and transmission. Mycoplasmal species are considered ubiquitous, are carried in the genital tracts of males and females, and are transmitted during natural breeding or through contaminated insemination materials. Aerosols also serve as a means of transmission. In addition, transmission occurs by passage through the birth canal, by direct contact, and by contamination from urine of infected animals. Pathophysiology. Experimental infections of M. bovis have resulted in placentitis and fetal pneumonia. Differential diagnoses. Acholeplasma, Ureaplasma, and Haemophilus somnus are differentials for granular vulvovaginitis. Treatment. Fluoroquinolone antibiotics may be useful for treating Mycoplasma-induced reproductive diseases. ii. Mycoplasma ovipneumoniae (ovine mycoplasmal pneumonia) Etiology. Mycoplasma ovipneumoniae causes acute or chronic pneumonia in lambs. Clinical signs. Mycoplasmas induce serious diseases in sheep, causing pneumonia, conjunctivitis, and genitourinary disease. The disease may be coincidental with pasteurellosis. Respiratory distress, coughing, and nasal discharge are observed in infected animals. Bronchoalveolar lavage followed by culture is the best method for diagnosis (mycoplasmas are fastidious organisms requiring special handling techniques). Mycoplasmas are isolated from the genitourinary tract of sheep. Vulvovaginitis and reproductive problems are associated conditions. Treatment. Tylosin, quinolones, oxytetracycline, and gentamicin are good choices for therapy. Prevention. No vaccine is available. iii. Mycoplasma mycoides biotype F38 (contagious caprine pleuropneumonia, caprine pneumonia, pleuritis, and pleuropneumonia) Etiology. Mycoplasma mycoides biotype F38 is the agent of contagious caprine pleuropneumonia and is found worldwide. In the United States, caprine pneumonia is also caused by M. ovipneumoniae, M. mycoides subsp. capri, and M. mycoides subsp. mycoides (large colony type). Clinical signs. Contagious caprine pleuropneumonia is characterized by severe dyspnea, nasal discharge, cough, and fever ( McMartin et al., 1980 ). Infections with other Mycoplasma species also have similar clinical signs. Septicemia without respiratory involvement may also be a presentation. Epizootiology and transmission. This disease is highly contagious, with high morbidity and mortality. Transmission is by aerosols. Mycoplasma mycoides subsp. mycoides has become a serious cause of morbidity and mortality of goat kids in the United States. Necropsy. Large amounts of pale straw-colored fluid and fibrinous pneumonia and pleurisy are typical. Some lung consolidation may be present. Meningitis, fibrinous pericarditis, and fibrinopurulent arthritis may also be found. Diagnosis is usually made at necropsy by culture of the organism from lungs and other internal organs. Differential dagnosis. In the United States, the principal differential for M. mycoides subsp. mycoides is caprine arthritis encephalitis. Treatment. Tylosin and oxytetracycline are effective. Some infections are slow to resolve. Prevention and control. Vaccines are available in some areas. Infected herds are quarantined. New goats should be quarantined before introduction to the herd. Research complications. The worldwide distribution of the F38 biotype, as well as the aerosol transmission and high morbidity and mortality characteristics of mycoplasmal infectious, make these infections economically important diseases. Considerable attention is presently given to this genus as a source of morbidity and mortality in goats. iv. Mycoplasma conjunctivae (mycoplasmal keratoconjunctivitis) Etiology. Mycoplasma conjunctivae causes infectious conjunctivitis, or pinkeye, in sheep and goats with associated hyperemia, edema, lacrimation, and corneal lesions. Mycoplasma mycoides subsp. mycoides, M. agalactiae, M. arginini, and Acholeplasma oculusi have also been associated with keratoconjunctivitis in these species. Respiratory disease and other infections, such as mastitis, may also be observed. Clinical signs and diagnosis. All ages of animals may be affected. Initially, lacrimation, conjunctival vessel injection, and then keratitis and neovascularization are seen. Sometimes uveitis is evident. Although the presentation is usually unilateral, bilateral involvement is possible. Recurring infections are common. Culturing provides the better diagnostic information, and cultures will be positive even after clinical signs have diminished. Epizootiology and transmission. The infection is passed easily between animals by direct contact. Animals can become reinfected, and carrier animals may be a factor in outbreaks. Necropsy. It is unlikely that animals would die or be euthanized and undergo necropsy for this problem. Conjunctival scrapings would include neutrophils during earlier stages and lymphocytes during later stages. Epithelial cell cytoplasm should be examined for organisms. Differential diagnosis. The primary differential in sheep and goats is Chlamydia, as well as Branhamella, Rickettsia (Colesiota) conjunctivae, and infectious bovine rhinotracheitis in goats only. It is important to consider these differentials if arthritis, pneumonia, or mastitis is present in the group or the individual. Treatment. Animals do recover spontaneously within about 10 weeks. Tetracycline ointments and powders are also used. Third-eyelid flaps may be necessary if corneal ulceration develops. Prevention and control. New animals should be quarantined and, if necessary treated, before introduction to the flock or herd. ff. Rickettsial Diseases i. Eperythrozoonosis (Eperythrozoon, Haemobartonella) Etiology. Eperythrozoonosis is a rare, sporadic, noncontagious, blood-borne disease in ruminants worldwide caused by the rickettsial agent Eperythrozoon. Host-specific species of importance are E. ovis, the causative species in sheep and goats, and E. wenyoni, E. tegnodes, and E. tuomii, the causative agents in cattle. Although the disease is of minor importance, it can cause severe anemia and debilitation in affected animals. Haemobartonella bovis is also rare, and is usually found only in association with other rickettsial diseases. Clinical signs and diagnosis. The disease is more severe in sheep. Following an incubation period of 1–3 weeks, infected animals exhibit episodic hyperthermia, weakness, and anemia. Losses may be greater in younger lambs. Cattle are usually latently infected but may have swollen and tender teats and legs. Fever, anemia, and depression will be present if the cattle are stressed by another systemic disease. Diagnosis is based on clinical evidence of anemia and is confirmed by observing the rickettsiae on the surface of red blood cells in a blood smear. Epizootiology and transmission. The rickettsial organisms are transmitted typically to young sheep by biting insects, ticks, contaminated needles or blood-contaminated surgical instruments. Necropsy findings. Necropsy findings include splenic enlargement and tissue icterus. Pathogenesis. The organism invades and destroys red blood cells. It is believed that intravascular hemolysis and erythrophagocytosis contribute to the macrocytic anemia. As with other red blood cell parasites, splenectomy aggravates the disease. Differential diagnosis. Clontridium novyi type D, babesiosis, and leptospirosis are the primary differentials. Prevention and control. Following strict sanitation practices for surgical procedures and controlling external parasites prevent the disease. Treatment. Treatment is not usually recommended, but Oxytetracycline has been used. Sheep will develop immunity if supported nutritionally during the disease. Research complications. Splenectomized animals are the experimental models used to study these diseases. ii. Q fever, or query fever (Coxiella burnetii) Etiology. Coxiella burnetii is a small, gram-negative, obligate intracellular rickettsial organism that causes query fever and is regarded as a major cause of late abortion in sheep. Clinical signs. Infection of ruminants with C. burnetii is usually asymptomatic. Experimental inoculation in other mammals has resulted in transient hyperthermia, mild respiratory disease, and mastitis. Abortions, stillbirths, and births of weak lambs are also seen. Epizootiology and transmission. Coxiella burnetii is extremely resistant to environmental changes as well as to disinfectants; persistence in the environment for a year or longer is possible. The organism is associated with either a free-living or an arthropod-borne cycle. Coxiella burnetii is found in a variety of tick species, such as ixodid or argasid, where it replicates and is excreted in the feces. Once introduced into a mammal, Coxiella may be maintained without a tick intermediate. The organism is especially concentrated in placental tissues, replicates in trophoblasts, and will be in reproductive fluids. Additionally, the organism is shed in milk, urine, feces, and oronasal secretions. Necropsy findings. No specific lesion will be seen in aborted or stillborn fetuses, but necrotizing placentitis will be a finding in cases of abortion. The placenta will contain white chalky plaques and a red-brown exudate. The disease can be diagnosed by identifying the rickettsial organisms in smears of placental secretions. The organism has been found in the placentas of clinically normal animals. The organism stains red with modified Ziehl-Neelsen and Macchiavello stains and purple with Giemsa stain. Differential diagnosis. Because of the organisms' similarity to Chlamydia, confirmation must be made by culture techniques, immunofluorescent procedures, ELISA, and complement fixation tests. Treatment. Coxiella can be treated with oxytetracyclines. A vaccine is not commercially available. Prevention and control. Any aborting animals should be segregated from other animals, and other pregnant animals should be treated prophylactically with tetracycline. Serologic screening of ruminant sources should be performed routinely. Barrier housing, a review of ventilation exhaust, and defined handling procedures are often required. All placentas and all aborted tissues should be handled and disposed of carefully. Q fever has been reported in many mammalian species, including cats. Research complications. Coxiella burnetii–hee animals are particularly important in studies involving fetuses and placentation. Because of its zoonotic potential, C. burnetii presents a unique problem in the animal research facility environment. A single organism has been shown to cause disease. Some of the greatest concerns are the risk to immunocompromised individuals, pregnant women, and other animals, and the presence of carrier animals or those that may shed the organism in placentas, for example. 2. Viral Diseases a. Adenovirus Infections Etiology. The ruminant adenoviruses are DNA viruses that cause respiratory and reproductive tract diseases. Nine antigenic types of the bovine adenovirus have been identified, with type 3 associated with respiratory disease. Two of the ovine and two of the caprine antigenic types have been identified. Clinical Signs. Signs of infection range from subclinical to severe, including pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, weak calf syndrome, and abortion. Respiratory tract and intestinal tract diseases may be concurrent. Infections caused by this virus are often found associated with other viral and bacterial infections. Epizootiology and transmission. The virus is believed to be widespread, but prevalence and characteristics of infection have not been characterized. Transmission of adenoviruses in other species (e.g., canine) is by aerosols or fecal-oral routes. Necropsy findings. Lesions found after experimental infections include atelectasis, edema, and consolidation of the lungs. b. Bluetongue Virus Infection (Reoviridae) Etiology. The bluetongue virus is an RNA virus in the Orbivirus genus and Reoviridae family. Five serotypes (2, 10, 11, 13, and 17) have been identified in the United States, where it is seen mostly in western states. Bluetongue is an acute arthropod-borne viral disease of ruminants, characterized by stomatitis, depression, coronary band lesions, and congenital abnormalities ( Bulgin, 1986 ). Clinical signs and diagnosis. Sheep are the most likely to show clinical signs. Clinical disease is less common in goats and cattle. Early in the infection, animals will spike a fever and will develop hyperemia and congestion of tissues of the mouth, lips, and ears. The virus name, bluetongue, is associated with the typical cyanotic membranes. The fever may subside, but tissue lesions erode, causing ulcers. Increased salivary discharges and anorexia are often related to ulcers of the dental pad, lips, gums, and tongue, although salivation and lacrimation may precede apparent ulceration. Chorioretinitis and conjunctivitis are also common signs in cattle and sheep. Lameness may be observed associated with coronitis and is evident in the rear legs. Skin lesions such as drying and cracking of the nose, alopecia, and mammary glands are also observed. Secondary bacterial pneumonia may also occur. Animals may also develop severe diarrhea and become recumbent. Sudden deaths due to cardiomyopathy may occur at any time during the disease. Hematologically, animals will be leukopenic. The course of the disease is about 2 weeks, and mortality may reach 80%. If animals are pregnant, the virus crosses the placenta and causes central nervous system lesions. Abortions may occur at any stage of gestation in cattle. Prolonged gestation may result from cerebellar hypoplasia and lack of normal sequence to induce parturition. Cerebellar hypoplasia will also be present in young born of the infected dams, as well as hydrocephalus, cataracts, gingival hyperplasia, or arthrogryposis. Diagnosis is suspected with the characteristic clinical signs and exposure to viral vectors. Virus isolation is the best diagnostic approach if blood is collected during the febrile stage of the disease or brains from aborted fetuses. Fluorescent antibody tests, ELISA, virus neutralization tests, PCR, and agar gel immunodiffusion (AGID) tests are also used to confirm the diagnosis. Epizootiology and transmission. Severe outbreaks have occurred in other countries during this century. Screening for this disease has limited the strains present in the United States. The disease is most common in outdoor-housed animals primarily in the western United States. The virus is primarily transmitted by biting midges, Culicoides. Culicoides variipennis is the most common vector in the United States. A combination of factors associated with viral strain, available and susceptible hosts, environmental conditions (such as damp areas where flies breed), and vector presence are factors in the severity of outbreaks. The disease is rarely transmitted by animal-to-animal contact or by infected animal products. Virus-contaminated semen, transplacental transfer, and carriage on transferred embyros are other possible means of transmission. Necropsy findings. At necropsy, erosive lesions may be observed around the mouth, tongue, palate, esophagus, and pillars of the rumen. Ulceration or hyperemia of the coronary bands may also be seen. Many of the internal organs will contain petechial and ecchymotic hemorrhages of the surfaces, and hemorrhage may be seen at the base of the pulmonary artery. Pathogenesis. The virus multiplies in the hemocoel and salivary glands of the fly and is excreted in transmissible form in the insect's saliva. After entering the host, the virus causes prolonged viremia. The incubation period is 6–14 days. The virus migrates to and attacks the vascular endothelium. The resulting vasculitis accounts for the lesions of the skin, mouth, tongue, esophagus, and rumen and the edema often found in many tissues. Ballooning degeneration of affected tissues, followed by necrosis and ulceration, occurs. The effects on fetuses appear to be due to generalized infections of developing organs. Differential diagnosis. Differentials include other infectious vesicular diseases such as foot-and-mouth disease, contagious ecthyma, bovine viral diarrhea virus-mucosal disease, infectious bovine rhinotracheitis, bovine papular stomatitis, and malignant catarrhal fever. Rinderpest is a differential in countries where it is endemic. Photosensitization should be considered. Foot rot is a differential for the lameness and coronitis. Differentials for the manifestations such as arthrogryposis include border disease virus and genetic predispositions of some breeds such as Charolais cattle and Merino sheep. Prevention and control. Cellular and humoral immunity are necessary for protection from infection. The bluetongue virus is insidious because the genome is capable of reassortment, and some vaccines will not have the antigenic components represented in the local infection. In addition, there is little to no cross protection between strains. Modified live vaccines are available in some parts of the United States but should not be used in pregnant animals. Vaccinating lambs and rams in an outbreak is worthwhile, for example, but vaccinating late-gestation ewes may cause birth defects or abortions. Congenital defects are more common from vaccine use than from naturally occurring infection. Minimizing exposure to the vector in endemic areas will decrease the incidence of the disease. Treatment. Supportive care and nursing care are helpful, including gruels or softer feeds, easily accessed water, and shaded resting places. Nonsteroidal anti-inflammatory drugs are often administered. For the cases of secondary bacterial pneumonia and some cases of bluetongue conjunctivitis, antibiotics may be administered. Research complications. This is a reportable disease because clinical signs resemble foot-and-mouth disease and other exotic vesicular diseases. c. Bovine Lymphosarcoma (Bovine Leukemia Virus Infection, Bovine Leukosis) Etiology. Bovine lymphosarcoma refers to lymphoproliferative diseases in young cattle that are not associated with bovine leukemia virus (BLV) infection, and those in older cattle that are associated with BLV. BLV is a B lymphocyte-associated retrovirus ( Johnson and Kaneene, 1993 a,b,c). Clinical signs. Forms of bovine lymphosarcoma that are not associated with BLV infection are calf, or juvenile; thymic, or adolescent (animals 6 months to 2 years old); and cutaneous (any age). The calf form is rare and characterized by generalized lymphadenopathy. Onset may be sudden, and the disease is usually fatal within a few weeks. Signs include lymphadenopathy, anemia, weight loss, and weakness. Some animals may be paralyzed because of spinal cord compression from subperiosteal infiltration of neoplastic cells. The adolescent form is also rare, the course rapid, and the prognosis poor. The disease is seen most often in beef breeds such as Hereford cattle and is characterized by space-occupying masses in the neck or thorax. These masses are also often present in the brisket. Secondary effects of the masses are loss of condition, dysphagia, rumen tympany, and fatal bloat. The cutaneous presentation has a longer course and may wax and wane. The masses are found at the anus, vulva, escutcheon, shoulder, and flank; they are painful when palpated, raised, and often ulcerated. The animals are anemic, and neoplastic involvement may affect cardiac function. Generalized or limited lymphadenopathy may be apparent. Only the adult, or enzootic, form of bovine lymphosarcoma is associated with BLV infection. Many animals do not develop any malignancies or clinical signs of infection and simply remain permanently infected. Some cows manifest disease only during the periparturient period. Malignant lymphoma is the more common, whereas leukosis, due to B-lymphocyte proliferation, is rare. Clinical signs are loss of condition and a drop in production of dairy cattle, anorexia, diarrhea, ataxia, paresis, and other signs dependent on the location of the neoplastic tissue. Tumors are associated with lymphoid tissues. Common sites also include the abomasum, spinal canal, and uterus. Cardiac tumors develop at the right atrial or left ventricular myocardium, and associated beat and rate abnormalities may be auscultated. The common ocular manifestation of the disease is exophthalmos due to retrobulbar masses. Many internal organs may be involved, and tumors may be palpable per rectum. Secondary infections will be due to immunosuppression and the weakened state of the animal. Sheep have acquired BLV infection naturally and have been used as experimental models; in both situations, this species is susceptible to tumor and leukemia development. Goats seroconvert but do not develop the clinical syndromes. Diagnosis is based on the animal's age, clinical signs, serology, hematology findings according to the form, aspirates or biopsies of masses, and necropsy findings. Kits are available for running AGID, for which the BLV antigens gp-51 and gp-24 are used; antibodies may be detected within weeks after exposure and may also help in predicting disease in clinically normal cattle. ELISA and PCR diagnostic aids will also be helpful. Epizootiology and transmission. This disease is present worldwide. It is estimated that at least 50% of the cattle in the United States are infected with BLV. As few as 1% of these animals develop lymphosarcoma, but the adult form of the disease described here is the most common bovine neoplastic disease in the United States. Larger herds tend to have higher rates. Genetic predisposition may be involved; in addition to the presence of BLV, the type of bovine lymphocyte antigen (BoLA) may be correlated to resistance or susceptibility and to the course of the disease. Transmission is believed to be by inhalation of BLV in secretions; in colostrum; horizontally by contaminated equipment not sanitized between cattle; and by rectum (e.g., mucosal irritation during per-rectum exams or procedures). Natural-service bulls may transmit the infection to cows. Cows infected with BLV may transmit the infection to their calves in utero. Tabanid and other flies also serve as vectors, but these represent a minor means of transmission. Necropsy findings. Neoplastic infiltration of many organs and tissues are found in the calf form and the cutaneous forms. Tumors may be local or widely distributed in the enzootic form. Definitive diagnosis of neoplastic tissue specimens is by histology. Pathogenesis. As with other retroviruses, the BLV integrates viral DNA into host target cell DNA by means of the reverse transcriptase enzyme, creating a provirus. Prevention and control. There is no vaccine for this disease. Development and maintenance of a BLV-free herd, or controlling infection within a herd, requires financial and programmatic commitments: BLV-positive and BLV-negative animals maintained separately; serologic testing (such as at least every 6 months) and separating positive animals; and washing and then disinfecting instruments, needles (or using sterile single-use products), and equipment for ear tagging and dehorning and other such equipment between animals. A fresh rectal exam sleeve and lubricant should be used for each animal examined. Otherwise serologically positive cows may have undetectable antibodies during the periparturient period. Embryo transfer recipients should be negative, and the virus will not be transferred by the embryonic stage. Calves should be fed colostrum from serologically negative cows. Treatment. Treatment regimens of corticosteroids and cancer chemotherapeutic agents provide only short-term improvement. In cases where ova, embryos, or semen need to be collected, supportive care for the affected animals is essential. Research complications. The United States and several countries, some in Europe, have official programs for eradication of enzootic bovine leukosis. d. Bovine Herpes Mammillitis (Bovine Herpesvirus 2 Bovine Ulcerative Mammillitis) Etiology. Bovine herpesvirus 2 causes bovine herpes mammillitis, a widespread disease characterized by teat and udder lesions, as well as oral and skin lesions. Clinical signs and diagnosis. Lesions begin suddenly with teat swelling; the tissue will be edematous and tender when touched. The udder lesions may extend to the perineum. The lesions progress to vesicles, then to ulcers; these may take 10 weeks to heal. Lesions rarely may also develop focally around the mouth and generally on the skin of the udder. Secondary mastitis may occur, because of bacteria associated with the scabs. Diagnosis is by clinical signs and serologically. Epizootiology and transmission. The virus is reported to be widespread. Occurrence is often seasonal, and biting insects may be vectors. Transmission with successful infection requires deep penetration of the skin. Transmission may be by contaminated milkers' hands, contaminated equipment, and other fomites. Differential diagnosis. Differential diagnoses include other diseases that cause lesions on teats such as pseudocowpox, papillomatosis, and vesicular stomatitis. Other vesicular diseases may be considered, but other more severe clinical signs might be associated with those. Prevention and control. Established milking hygiene practices are important control measures: having milkers wash their hands with germicidal solutions or wear gloves, cleaning equipment between animals, and separating affected animals. Treatment. There is no treatment, and affected animals should be separated from the herd and milked last. Lesions can be cleaned and treated with topical antibacterials. e. Bovine Viral Diarrhea Virus Etiology. The bovine viral diarrhea virus (BVDV) is a pestivirus of the Flaviviridae family. The Flaviviridae include hog cholera virus and border disease virus of sheep. The virus contains a single strand of positive-sense RNA. A broad range of disease and immune effects is produced by BVDV only in cattle. In addition, this virus is important in the etiology of bovine pneumonias. Bovine viral diarrhea/mucosal disease (BVD/MD) is one of the most important viral diseases and one of the most complex diseases of cattle. Strains of BVDV are characterized as cytopathic (CP) and noncytopathic (NCP), based on cell-culture growth characteristics. The virus has also been categorized as type 1 and type 2 isolates. Heterologous strains exist that may confound even sound vaccination programs. Clinical signs and diagnosis. Signs of BVDV infections may be subclinical but also include abortions, congenital abnormalities, reduced fertility, persistent infection (PI) with gradual debilitation, and acute and fatal disease. The presence of antibodies, whether from passive transfer or immunizations, does not necessarily guarantee protection from the various forms of the disease. An acute form of the disease, caused by type 2 BVDV, occurs in cattle without sufficient immunity. After an incubation period of 5–7 days, clinical signs include fever, anorexia, oculonasal discharge, oral erosions (including on the hard palate), diarrhea, and decreased milk production. The disease course may be shorter with hemorrhagic syndrome and death within 2 days. Clinical signs of BVDV in calves also include severe enteritis and pneumonia. When susceptible cows are infected in utero from gestational days 50–100, or gestational cows are vaccinated with a modified live vaccine, abortion or stillbirth result. Congenital defects caused by BVDV during gestational days 90–170 include impaired immunity (thymic atrophy), cerebellar hypoplasia, ocular defects, alopecia or hypotrichosis, dysmyelinogenesis, hydranencephaly, hydrocephalus, and intrauterine growth retardation. Typical signs of cerebellar dysfunction will be evident in calves, such as wide-based stance, weakness, opisthotonus, hyperflexion, hypermetria, nystagmus, or strabismus. Some severely affected calves will not be able to stand. Ophthalmic effects include retinal degeneration and microphthalmia. Fetuses can also be infected in utero, normal at birth, immunotolerant to the virus, and persistently infected (PI). The term mucosal disease is commonly associated with this form of the infection. Many PI animals do not survive to maturity, however, and many have weakened immune systems. The PI animals are important because they shed virus and will probably show the clinical signs of mucosal disease (MD) caused by a CP BVDV strain derived from an NCP BVDV strain. These MD clinical signs include fever, anorexia, and profuse diarrhea that may include blood and fibrin casts, and oral and pharyngeal erosions, as well as erosion at the interdigital spaces and on the teats and vulva. Many other associated clinical signs include anemia, bloat, lameness, or corneal opacities and discharges. Secondary effects of hemorrhage and dehydration also contribute to the morbidity and mortality. Animals that do not succumb to the disease will be chronically unthrifty, debilitated, and infection-prone. Diagnosis in affected calves is based on herd health history, clinical signs, and antibodies to BVDV in precolostral serum. Viral culturing from blood may be useful. In older animals, oral lesions, serology, detection of viral antigen, and virus isolation contribute to the diagnosis. Leukopenia, and especially lymphopenia, are seen. Serology must be interpreted with the awareness of the possibility of PI immunotolerant animals. Vaccination against the disease carries its own set of side effects and potential problems, especially when using modified live vaccines, whether against CP or NCP strains. The condition of the animals is also a variable. Epizootiology and transmission. BVDV is present throughout the world. Transmission occurs easily by direct contact between cattle, from feed contaminated with secretions or feces, and by aborted fetuses and placentas. PI females transmit the virus to their fetuses. Semen also is a source of virus. Necropsy findings. In affected calves, histopathologic findings include necrosis of external germinal cells, focal hemorrhages, and folial edema. Later in the disease, large cavities develop in the cerebellum, and atrophy of the cerebellar folia and thin neuropil are evident. Older calves may have areas of intestinal necrosis. In cases where oral erosions occur, erosions will be found extending throughout the gastrointestinal tract to the cecum. The respiratory tract lesions will often be complicated by secondary bacterial pneumonia. When the hemorrhagic syndrome develops, petechiation and mucosal bleeding will be present. Pathogenesis. The CP and NCP strains are thought to be related mutations of the BVDV; the CP short-lived isolates are believed to arise from the NCP strains. The NCP strains are those present in the PI animals, and the strains are maintained in cattle populations. CP and NCP isolates vary in virulence, and classification of these types is based on viral surface proteins. Considerable antigenic variation also exists between strains and types. Other viral infections, such as bovine respiratory syncytial virus and infectious bovine rhinotracheitis, may also be present in the same animals. The pathology caused by BVDV is due to its ability to infect epithelial cells and impair the functioning of immune cell populations through out the bovine system. In type 2 BVDV hemorrhagic syndrome, death results from viral-induced thrombocytopenia. In fetuses, the virus infects developing germinal cells of the cerebellum. The Purkinje's cells in the granular layer are killed, and necrosis and inflammation follow. The immune effects are the result of the virus's interfering with neutrophil and macrophage functions and of lymphocyte blastogenesis. All of these predispose the affected animals to bacterial infections with Pasteurella haemolytica. BVDV damages dividing cells in fetal organ systems, resulting in abortions and congenital effects. Differential diagnosis. Many differentials must be considered for the clinical manifestations of BVDV infections. Differentials for enteritis of calves include viral infections, Cryptosporidia, Escherichia coli, Salmonella, and Coccidia. Salmonella, winter dysentery, Johne's disease, intestinal parasites, malignant catarrhal fever (MCF), and copper deficiency are differentials for the diarrhea seen in the disease in adult animals. Respiratory tract pathogens such as bovine respiratory syncytial virus, Pasteurella, Haemophilus, and Mycoplasma must be considered for the respiratory tract manifestations. Oral lesions are also produced by MCF, vesicular stomatitis, bluetongue, and papular stomatitis. Infectious bovine herpesvirus 1, leptospirosis, brucellosis, trichomoniasis, and mycosis should be considered in cases of abortion. Prevention and control. Combined with sound management in a typical cattle herd, vaccination is the best way to prevent BVDV and should be integrated into the herd health program, timed appropriately preceding breeding, gestation, or stressful events. Vaccine preparations for BVDV are modified live virus (MLV) or killed virus. Each has advantages and disadvantages. The former induces rapid immunity (within 1 week) after a single dose, provides longer duration of immunity against several strains, and induces serum neutralizing antibodies. MLV vaccines are not recommended for use in pregnant cattle, may induce mucosal disease, and may be immunosuppressive at the time of vaccination. The immunosuppression is detrimental if cattle are concurrently exposed to field-strain virus because it will facilitate infection and possible clinical disease. The MLV strains may cross the placenta, resulting in fetal infections. The killed vaccines are safer in pregnant animals but require booster doses after the initial immunization, may need to be given 2–3 times per year, and do not induce cell-mediated immunity. Passive immunity may protect most calves for up to 6–8 months of age. Subsequent vaccination with MLV may provide lifelong immunity, but this is not guaranteed. Annual boosters are recommended to protect against vaccine breaks. The virus persists in the environment for 2 weeks and is susceptible to the disfectants chlorhexidine, hypochlorite, iodophors, and aldehydes. Maintenance of a closed herd to prevent any possibility of the introduction of the virus is difficult. Isolation of new animals, avoidance of the purchase of pregnant cows, scrutiny of records from source farms, use of semen tested bulls, minimization of stress, testing of embryo-recipient cows, and maintainenance of populations of ruminants (smaller or wild species) separately on the premises will minimize viral exposure. Other management strategies may require a program for testing and culling PI cattle. This can be expensive but may be a worthwhile investment to remove the virus shedders from a herd. Treatment. No specific treatment is available. Supportive care and treatment with antibiotics to prevent secondary infection are recommended. Animals that survive the infection should be evaluated a month after recovery to determine their status as PI or virus-free. f. Cache Valley Virus Etiology. Cache Valley virus (CVV), of the arbovirus genus of the Bunyaviridae family, is a cause of congenital defects in lambs. Clinical signs and diagnosis. Teratogenic effects of in utero CVV infection in fetal and newborn lambs include arthrogryposis, microencephaly, hydranencephaly, porencephaly, cerebellar hypoplasia, and micromyelia. Stillbirths and mummified fetuses are seen. Lambs will be born weak and will act abnormally. Diagnosis is by evidence of seroconversion in precolostral blood samples or fetal fluids, as the result of in utero infection. Epizootiology and transmission. The virus is present in the western United States, although it has been isolated in a few Midwestern states. Although considered a disease of sheep, virus has been isolated from cattle and from wild ruminants and antibodies found in white-tailed deer. Transmission is by arthropods during the first trimester of pregnancy. g. Caprine Arthritis Encephalitis Virus Etiology. Caprine arthritis encephalitis virus (CAEV) occurs worldwide, with a high prevalence in the United States. Caprine arthritis encephalitis (CAE) is considered the most important viral disease of goats. The CAEV is in the Lentivirus genus of the Retroviridae family. It causes chronic arthritis in adults and encephalitis in young. CAEV is in the same viral genus as the ovine progressive pneumonia virus (OPPV). Clinical signs and diagnosis. The most common presentation in goats is an insidious, progressive arthritis in animals 6 months of age and older. Animals become stiff, have difficulty getting up, and may be clinically lame in one or both forelimbs. Carpal joints are so swollen and painful that the animal prefers to eat, drink, and walk on its "knees." In dairy goats, milk production decreases, and udders may become firmer. This retrovirus also causes neurological clinical signs in young kids 2–6 months old. Kids may be bright and alert, afebrile, and able to eat normally even when recumbent. Some kids may initially show unilateral weakness in a rear limb, which progresses to hemiplegia or tetraplegia. Mild to severe lower motor neuron deficits may be noted, but spinal reflexes are intact. Clinical signs may also include head tilt, blindness, ataxia, and facial nerve paralysis. Older animals in the group may experience interstitial pneumonia or chronic arthritis. The pneumonia is similar to the pneumonia in sheep caused by OPPV; the course is gradual but progressive, and animals will eventually lose weight and have respiratory distress. Some animals in a herd may not develop any clinical signs. Diagnosis is based on clinical signs, postmortem lesions, and positive serology for viral antibodies to CAEV. An agar gel immunodiffusion (AGID) test identifies antibodies to the virus and is used for diagnosis. Kids acquire an anti-CAEV antibody in colostrum, and this passive immunity may be interpreted as indicative of infection with the virus. The antibody does not prevent viral transmission. Epizootiology and transmission. The virus is prevalent in most industrialized countries. The common means of transmission, from adults to kids, is in the colostrum and milk in spite of the presence of anti-CAEV antibody in the colostrum. Transmission may occur among adult goats by contact. Intrauterine transmission is believed to be rare. Transmission to sheep has occurred only experimentally; there is no documented case of natural transmission. Necropsy findings. Necropsy and histopathology reveal a striking synovial hyperplasia of the joints with infiltrates of lymphocytes, macrophages, and plasma cells. Other histologic lesions include demyelination in the brain and spinal cord, with multifocal invasion of lymphocytes, macrophages, and plasma cells. In severe cases of mastitis, the udder may appear to be composed of lymphoid tissue. Pathogenesis. The virus infects cells of the mononuclear system, resulting in the formation of non-neutralizing antibody to viral core proteins and envelope proteins. Immune complex formation in synovial, mammary gland, and neurological tissue is thought to result in the clinical changes observed. Most commonly, the carpal joint is affected, followed by the stifle, hock, and hip. The infection is lifelong. Differential diagnosis. The differential diagnosis for the neurologic form of CAEV should include copper deficiency, enzootic pneumonia, white muscle disease, listeriosis, and spinal cord disease or injury. The differential diagnosis for CAEV arthritis should include chlamydia and mycoplasma. Prevention and control. Herds can be screened for CAE by testing serologically, using an AGID or an enzyme-linked immunosorbent assay (ELISA) test. The ELISA is purported to be more sensitive, whereas the AGID is more specific. Individual animals show great variation in development of antibody. Because CAE is highly prevalent in the United States, and because seronegative animals can shed organisms in the milk, retesting herds at least annually may be necessary. Recently, an immuno-precipitation test for CAE has been developed that has high sensitivity and specificity. Control measures include management practices such as test and cull, prevention of milk transmission, and isolation of affected animals. Parturition must be monitored, and kids must be removed immediately and fed heat-treated colostrum (56° C for 1 hr). CAEV-negative goats should be separated from CAEV-positive goats. Treatment. There is no treatment for CAEV. h. Infectious Bovine Rhinotracheitis Virus (Infectious Bovine Rhinotracheitis-Infectious Pustular Vulvovaginitis) Etiology. The infectious bovine rhinotracheitis virus (IBRV) is also referred to as bovine herpesvirus 1 (BHV-1) and is an alphaherpesvirus. IBRV causes or contributes to several bovine syndromes, including respiratory and reproductive tract diseases. It is one of the primary pathogens in the bovine respiratory disease complex. Strains include BHV-1.1 (associated with respiratory disease), BHV 1.2 (associated with respiratory and genital diseases), and BHV 1.4 (associated with neurological diseases), which has been reclassified as bovine herpesvirus 5. Clinical signs and diagnosis. Diseases caused by the virus include conjunctivitis, rhinotracheitis, pustular vulvovaginitis, balanoposthitis, abortion, encephalomyelitis, and mastitis. The respiratory form is known as infectious bovine rhinotracheitis, and clinical signs may range from mild to severe, the latter particularly when there are additional respiratory viral infections or secondary bacterial infections. The mortality rate in more mature cattle is low, however, unless there is secondary bacterial pneumonia. Fever, anorexia, restlessness, hyperemia of the muzzle, gray pustules on the muzzle (that later form plaques), nasal discharge (that may progress from serous to mucopurulent), hyperpnea, coughing, salivation, conjunctivitis with excessive epiphora, and decreased production in dairy animals are typical signs. Open-mouth breathing may be seen if the larynx or nasopharygneal areas are blocked by mucopurulent discharges. Neonatal calves may develop respiratory as well as general systemic disease. In these cases, in addition to the symptoms already noted, the soft palate may become necrotic, and gastrointestinal tract ulceration occurs. Young calves are most susceptible to the encephalitic form; signs include dull attitude, head pressing, vocalizations, nystagmus, head tilt, blindness, convulsions, and coma, as well as some signs, such as discharges, seen with respiratory tract presentations. This form is usually fatal within 5 days. Abortion may occur simultaneously with the conjunctival or respiratory tract diseases, when the respiratory infection appears to be mild, or may be delayed by as much as 3 months after the respiratory tract disease signs. Infectious pustular vulvovaginitis is most commonly seen in dairy cows, and clinical signs may be mild and not noticed. Otherwise, signs are fever, depression, anorexia, swelling of the vulvar labia, vulvar discharge, and vestibular mucosa reddened by pustules. The cow will often carry her tail elevated away from these lesions. These soon coalesce, and a fibrous membrane covers the ulcerated area. If uncomplicated, the infection lasts about 4–5 days, and lesions heal in 2 weeks. Younger infected bulls may develop balanoposthitis with edema, swelling, and pain such that the animals will not service cows. Epizootiology and transmission. IBRV is widely distributed throughout the world, and adult animals are the reservoirs of infection. The disease is more common in intensive calf-rearing situations and in grouped or stressed cattle. Transmission is primarily by secretions, such as nasal, during and after clinical signs of disease. Modified live vaccines are capable of causing latent infections. Necropsy findings. Fibrinonecrotic rhinotracheitis is considered pathognomic for IBRV respiratory tract infections. There will be adherent necrotic lesions in the respiratory, ocular, and reproductive mucosa. When there are secondary bacterial infections, such as Pasteurella bronchopneumonia, findings will include congested tracheal mucosa and petechial and ecchymotic hemorrhages in that tissue. Lesions from the encephalitic form include lymphocytic meningoencephalitis and will be found throughout the gray matter (neuronal degeneration, perivascular cuffing) and white matter (myelitis, demyelination). Intranuclear inclusion bodies are not a common finding with this herpesvirus. Pathogenesis. In the encephalitic form, the virus first grows in nasal mucosa and produces plaques. These resolve within 11 days, and the encephalitis develops after the virus spreads centripetally to the brain stem by the trigeminal nerve dendrites. Latent infections are also established in neural tissue. Differential diagnosis. The severe oral erosions seen with BVDV infections are rare with infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV). The conjunctivitis of IBR may initially be mistaken for that of a Moraxella bovis (pinkeye) infection; the IBR will be peripheral, and there will not be corneal ulceration. Bovine viral diarrhea virus and IBRV are the most common viral causes of bovine abortion. Differentials for balanoposthitis include trauma from service. Prevention and control. Vaccination options include inactivated, attenuated, modified live, and genetically altered preparations. Some are in combination with parainfluenza 3 (PI-3) virus. The MLV preparations are administered intranasally; these are advantageous in calves for inducing mucosal immunity even when serologic passive immunity is already present and adequate. Some newer vaccines, with gene deletion, allow for serologic differentiation between antibody responses from infection or immunization. Bulls with the venereal form of the infection will transmit the virus in semen; intranasal vaccine may be used to provide some immunity. Treatment. Uncomplicated mild infections will resolve over a few weeks; palliative treatments, such as cleaning ocular discharges and supplying softened food, are helpful in recovery. Antibiotics are usually administered because of the high likelihood of secondary bacterial pneumonia. The encephalitic animals may need to be treated with anticonvulsants. i. Parainfluenza 3 (PI-3) Etiology. Parainfluenza 3, an RNA virus of the family Paramyxoviridae, causes mild respiratory disease of ruminants when it is the sole pathogen. The viral infection often predisposes the respiratory system to severe disease associated with concurrent viral or bacterial pathogens. Viral strains are reported to vary in virulence. Serotypes seen in the smaller ruminants are distinct from those isolated from cattle. Clinical signs and diagnosis. Infections ranging from asymptomatic to mild signs of upper respiratory tract disease are associated with this virus by itself; infections are almost never fatal. Clinical signs include ocular and nasal discharges, cough, fever, and increased respiratory rate and breath sounds. In pregnant animals, exposure to PI-3 can result in abortions. Clinical signs become apparent or more severe when additional viral pathogens are present, such as bovine viral diarrhea virus, or a secondary bacterial infection, such as Pasteurella haemolytica infection, is involved. Greater morbidity and mortality will be sequelae of the bacterial infections. Viral isolation or direct immunofluorescence antibody (IFA) from nasal swabs can be used for definitive diagnosis. Epizootiology and transmission. The virus is considered ubiquitous in cattle and is a common infection in sheep. Presently it is assumed that the virus is widespread in goats, but firm evidence is lacking. Necropsy findings. For an infection of PI-3 only, findings will be negligible. Some congestion of respiratory mucosa, swelling of respiratory tract-associated lymph nodes, and mild pneumonitis may be noted grossly and histologically. Intranuclear and intracytoplasmic inclusion bodies may be present in the mucosal epithelial cells. Findings will be similar but not as severe as those caused by bovine respiratory syncytial virus. Immunohistochemistry may also be used. Pathogenesis. PI-3 infects the epithelial mucosa of the respiratory tract; however, the disease is often asymptomatic when uncomplicated. Differential diagnosis. Differentials, particularly in cattle, include infections with other respiratory tract viruses of ruminants: IBRV, BVDV, bovine respiratory syncytial virus, and type 3 bovine adenovirus. Prevention and control. Immunization, management, and nutrition are important for this respiratory pathogen, as for others. In cattle, modified live vaccines for intramuscular (IM), subcutaneous (SC), or intranasal (IN) administration are available. The IM and SC routes provide immune protection within 1 week after administration but will not provide protection in the presence of passively acquired antibodies. It is contraindicated for pregnant animals because it will cause abortion. The IN route immunizes in the presence of passively acquired antibodies, provides immunity within 3 days of administration, and stimulates the production of interferon. Other vaccine formulations, about which less information is reported, include inactivated or chemically altered live-virus preparations; both are administered IM, and followup immunizations are needed within 4 weeks. Booster vaccinations are recommended for all preparations within 2–6 months after the initial immunization. All presently marketed vaccine products come in combination with other bovine respiratory viruses as multivaccine products. The humoral immunity protects against PI-3 abortions. There is no approved PI-3 vaccine for sheep and goats. The use of the cattle formulation in these smaller ruminants is not recommended. Sound management of housing, sanitation, nutrition, and preventive medicine programs are all equally important components in prevention and control. Treatment. Uncomplicated disease is not treated. j. Respiratory Syncytial Viruses of Ruminants Etiology. The respiratory syncytial viruses are pneumoviruses of the Paramyxoviridae family and are common causes of severe disease in ruminants, especially calves and yearling cattle. Two serotypes of the bovine respiratory syncytial virus (BRSV) have been described for cattle; these may be similar or identical to the virus seen in sheep and goats. Clinical findings and diagnosis. Infections may be subclinical or develop into severe illness. Clinical signs include fever, hyperpnea, spontaneous or easily induced cough, nasal discharge, and conjunctivitis. Interstitial pneumonia usually develops, and harsh respiratory sounds are evident on auscultation. Development of emphysema indicates a poor prognosis, and death may occur in the severe cases of the viral infection. Secondary bacterial pneumonia, especially with Pasteurella haemolytica, with morbidity and mortality, is also a common sequela. Abortions have been assciated with BRSV outbreaks. Diagnosis is based on virus isolation and serology (acute and convalescent). Nasal swabs for virus isolation should be taken when animals have fever and before onset of respiratory disease. Epizootiology and transmission. These viruses are considered ubiquitous in domestic cattle and are transmitted by aerosols. Necropsy findings. Gross lesions include consolidation of anteroventral lung lobes. Edema and emphysema are present. As the name indicates, syncytia, which may have inclusions, form in areas of the lungs infected with the virus. Necrotizing bronchiolitis, bronchiolitis obliterans, and hyaline membrane formation will be evident microscopically. Pathogenesis. The severe form of the disease, which often follows a mild preliminary infection, is thought to be caused by immune-mediated factors during the process of infection in the lung. Virulence may vary greatly among viral strains. Differential diagnosis. Differentials should include other ruminant respiratory tract viruses. Prevention and control. Vaccination should be part of the standard health program, and all animals should be vaccinated regularly. Vaccinations should be administered within 1–2 months of stressful events, such as weaning, shipping, and introduction to new surroundings. Currently available vaccines include an inactivated preparation and a modified live virus preparation administered intramuscularly or subcutaneously; immunity develops well in yearling animals, and colostral antibodies develop when cows are vaccinated during late gestation. Passive immunity from colostrum provides at least partial protection to calves in herds where disease is prevalent. But this immunity suppresses the mucosal IgA response and serum antibody responses. The basis for successful immune protection is the mucosal memory IgA, but this is difficult to achieve with present vaccine formulations. The virus is easily inactivated in the environment. Preventive measures in preweaning animals should include preconditioning to minimize weaning stress. Treatment. Recovery can be spontaneous; however, antibiotics and supportive therapy are useful to prevent or control secondary bacterial pneumonia. In severe cases, antihistamines and corticosteriods may also be necessary. Use of vaccine during natural infection is not productive and may result in severe disease. k. Ulcerative Dermatosis (Ovine Venereal Disease, Balanoposthitis) Etiology. Ulcerative dermatosis is a contagious disease of sheep only. It is caused by a poxvirus similar to but distinct from the causative agent of contagious ecthyma ( "Current Veterinary Therapy," 1993 ). Clinical signs and diagnosis. Lesions include ulcers and crusts associated with the skin and mucous membranes of the genitalia, face, and feet ( Bulgin, 1986 ). Genital lesions are much more common than the facial or coronal lesions. Discomfort may be associated with the lesions. Paraphimosis occasionally occurs. These lesions are painful; during breeding season, animals will avoid coitus. Morbidity is low to moderate, and mortality negligible if the flock is otherwise healthy. Diagnosis is based on clinical signs. Epizootiology and transmission. Endemic to the western United States, ulcerative dermatosis is transmitted through direct contact with abraded skin of the prepuce, vulva, face, and feet. Necropsy findings. Necropsy would rarely be necessary to diagnose an outbreak in a healthy flock. Findings will be similar to those described for contagious ecthyma. Pathogenesis. Following an incubation period of 2–5 days, the virus replicates in the epidermal cells and leads to necrosis and pustule formation. Pustules rapidly break, forming weeping ulcers. The ulcers scab over and eventually form a fibrotic scar. The disease usually resolves in 2–6 weeks. Rarely, the disease will persist for many months to more than a year. Differential diagnosis. The main differential is contagious ecthyma, which is grossly and histopathologically associated with epithelial hyperplasia. This is also a feature of ulcerative dermatosis. Prevention and control. No vaccine is available. Affected animals, especially males, should not be used for breeding. Treatment. Affected animals should be separated from the rest of the flock. Treatment is supportive, including antiseptic ointments and astringents. Research complications. Breeding and maintenance of the flocks' condition, because of the pain associated with eating, will be compromised during an outbreak. l. Border Disease Etiology. Border disease, also known as hairy shaker disease (or "fuzzies" in the southwestern United States), is a disease of sheep caused by a virus closely related to the bovine viral diarrhea virus (BVDV), a pestivirus of the Togaviridae family. Goats are also affected. The virus causes few pathogenic effects in cattle. Clinical signs and diagnosis. Border disease in ewes causes early embryonic death, abortion of macerated or mummified fetuses, or birth of lambs with developmental abnormalities. Lambs infected in utero that survive until parturition may be born weak and often exhibit a number of congenital defects such as tremor, hirsutism (sometimes darkly pigmented over the shoulders and head), hypothyroidism, central nervous system defects, and joint abnormalities, including arthrogryposis. Later, survivors may be more susceptible to diseases and may develop persistent, sometimes fatal, diarrhea. The virus infection produces similar clinical manifestations in goats, except that the hair changes are not seen. Diagnosis includes the typical signs described above, as well as serological evidence of viral infection. Virus isolation confirms the diagnosis. Epizootiology and transmission. The virus is present worldwide, and reports of disease are sporadic. Disease has occurred when no contact with cattle has occurred. Persistently infected animals, such as lambs, are shedding reservoirs of the virus in urine, feces, and saliva throughout their lives. Necropsy findings. Lesions include placentitis, and characteristic joint and hair-coat changes in the fetus. Histologically, axonal swelling, neuronal vacuolation, dysmyelination, and focal microgliosis are observed in central nervous system structures. Pathogenesis. The virus entering the ewe via the gastrointestinal or respiratory tracts penetrates the mucous membranes and causes maternal and fetal viremia. Infection during the first 45 days of gestation causes embryonic death. In lambs infected between 45 and 80 days, the virus activates follicular development, diminishes the myelination of neurons, and causes dysfunction of the thyroid gland. Infection after 80 days of gestation results in lambs that are born persistently infected. Infected lambs have high perinatal mortality; survivors have diminished signs over time but, as noted, continue to shed the virus. Prevention and control. Border disease can be prevented by vaccinating breeding ewes with killed-BVDV vaccine. Congenitally affected lambs should be maintained separately and disposed of as soon as humanely possible. New animals to the flock should be screened serologically. If cattle are housed nearby, vaccination programs for BVDV should be maintained. Treatment. There is no treatment other than supportive care for affected animals. m. Contagious Ecthyma (Contagious Pustular Dermatitis, Sore Mouth, Orf) Etiology. Contagious ecthyma, also known as contagious pustular dermatitis, sore mouth, or orf, is an acute dermatitis of sheep and goats caused by a parapoxvirus. This disease occurs worldwide and is zoonotic. Naturally occurring disease has also been reported in other species such as musk ox and reindeer. Other parapoxviruses infect the mucous membranes and skin of cattle, causing the diseases bovine pustular dermatitis and pseudocowpox. Clinical signs and diagnosis. The disease is characterized by the presence of papules, vesicles, or pustules and subsequently scabs of the skin of the face, genitals of both sexes, and coronary bands of the feet. Lesions develop most frequently at mucocutaneous junctions and are found most commonly at the commissures of the mouth. Orf is usually found in young animals less than 1 year of age. Younger lambs and kids will have difficulty nursing and become weak. Lesions may also develop on udders of nursing dams, which may resist suckling by offspring to nurse, leading to secondary mastitis. The scabs may appear nodular and raised above the surface of the surrounding skin. Morbidity in a susceptible group of animals may exceed 90%. Mortality is low, but the course of the disease may last up to 6 weeks. Diagnosis is based on characteristic lesions. Biopsies may reveal eosinophilic cytoplasmic inclusions and proliferative lesions under the skin. Electron microscopy will reveal the virus itself. Disease is confirmed by virus isolation. Epizootiology and transmission. All ages of sheep and goats are susceptible. Seasonal occurrences immediately after lambing and after entry into a feedlot are common; stress likely plays a role in susceptibility to this viral disease. Older animals develop immunity that usually prevents reinfection for at least 1 or more years. Resistant animals may be present in some flocks or herds. The virus is very resistant to environmental conditions and may contaminate small-ruminant facilities, pens, feedlots, and the like for many years as the result of scabs that have been shed from infected animals. Transmission occurs through superficial lesions such as punctures from grass awns, scrapes, shearing, and other common injuries. Necropsy findings. Necropsy findings include ballooning degeneration of epidermal and dermal layers, edema, granulomatous inflammation, vesiculation, and cellular hyperplasia. Secondary bacterial infection may also be evident. Pathogenesis. The virus is typical of the Poxviridae, resembling sheep poxvirus (not found in the United States) and vaccinia virus and replicating in the cytoplasm of epithelial cells. Following an incubation period of 2–14 days, papules and vesicles develop around the margins of the lips, nostrils, eyelids, gums, tongue, or teats; skin of the genitalia; or coronary band of the feet. The vesicles form pustules that rupture and finally scab over. Differential diagnosis. Ulcerative dermatosis and bluetongue virus should be considered in both sheep and goats. An important differential in goats is staphylococcal dermatitis. Prevention and control. Individuals handling infected animals should be advised of precautions beforehand, should wear gloves, and should separate work clothing and other personal protective equipment. Clippers, ear tagging devices, and other similar equipment should always be cleaned and disinfected after each use. Colostral antibodies may not be protective. Vaccinating lambs and kids with commercial vaccine best prevents the disease. Dried scabs from previous outbreaks may also be used by rubbing the material into scarified skin on the inner thigh or axilla. Animals newly introduced to infected premises should be vaccinated upon arrival. Precautions must be taken when vaccinating animals, because the vaccine may induce orf in the animal handlers; it is not recommended to vaccinate animals in flocks already free of the disease. Affected dairy goats should be milked last, using disposable towels for cleaning teat ends. Treatment. Affected animals should be isolated and provided supportive care, especially tube feeding for young animals whose mouths are too sore to nurse. Treatment should also address secondary bacterial infections of the orf lesions, including systemic antibiotics for more severe infections. Treatment for myiasis may also be necessary. The viral infection is self-limiting, with recovery in about 4 weeks. Research complications. Carrier animals may be a factor in flock or herd outbreaks. Contagious ecthyma is a zoonotic disease, and human-to-human transmission can also occur. The virus typically enters through abrasions on the hands and results in a large (several centimeters) nodule that is described as being extremely painful and lasting for as many as 6 weeks. Lesions heal without scarring. n. Foot-and-Mouth Disease Etiology. Foot-and-mouth disease (FMD) is caused by the foot-and-mouth disease virus, a Picornavirus in the Aphthovirus genus. The disease is also referred to as aftosa or aphthous fever. Seven immunologically distinct types of the virus have been identified, with 60 subtypes within those 7. Epidemics of the disease have occurred worldwide. North and Central America have been free of the virus since the mid-1950s. This is a reportable disease in the United States; clinical signs are very similar to other vesicular diseases. Cattle (and swine) are primarily affected, but disease can occur in sheep and is usually subclinical in goats. Clinical signs and diagnosis. In addition to vesicle formation around and in the mouth, hooves, and teats, fever, anorexia, weakness, and salivation occur. Vesicles may be as large as 10 cm, rupture after 2 days, and subsequently erode. Secondary bacterial infections often occur at the erosions. Anorexia is likely due to the pain associated with the oral lesions. High morbidity and low mortality, except for the high mortality in young cattle, are typical. Diagnosis must be based on ELISA, virus neutralization, fluorescent antibody tests, and complement fixation. Epizootiology and transmission. Domestic and wild ruminants and several other species, such as swine, rats, bears, and llamas are hosts. Asymptomatic goats can serve as virus reservoirs for more susceptible cohoused species such as cattle. Greater mortality occurs in younger animals. The United States, Great Britain, Canada, Japan, New Zealand, and Australia are FMD-free, whereas the disease is endemic in most of South America, parts of Europe, and throughout Asia and Africa. The virus is very contagious and is spread primarily by the inhalation of aerosols, which can be carried over long distances. Transmission may also occur by fomites, such as shoes, clothing, and equipment. Human hands, soiled bedding, and animal products such as frozen or partially cooked meat and meat products, hides, semen, and pasteurized milk also serve as sources of virus. Necropsy findings. Vesicles, erosions, and ulcers are present in the oral cavity as well as on the rumen pillars and mammary alveolar epithelium. Myocardial and skeletal muscle degeneration (Zenker's) is most common (and accounts for the greater mortality) in younger animals. Histological findings include lack of inclusion bodies. Vesicular lesions include intracellular and extracellular edema, cellular degeneration, and separation of the basal epithelium. Pathogenesis. The incubation period is 2–8 days. The virus replicates in the pharynx and digestive tract in the cells of the stratum spinosum, and viremia and spread of virus to many tissues occur before clinical signs develop. Virus shedding begins about 24 hr before clinical signs are apparent. Vesicles result from the separation of the superficial epithelium from the basal epithelium. Fluid fills the basal epithelium, and erosions develop when the epithelium sloughs. Persistent infection also occurs, and virus can be found for months or years in the pharnyx; the mechanisms for the persistence are not known. Differential diagnosis. Vesicular stomatitis is the principal differential. Other differentials include contagious ecthyma (orf), rinderpest, bluetongue, malignant catarrhal fever, bovine papular stomatitis, bovine herpes mammillitis, and infectious bovine rhinotracheitis virus infection. Prevention and control. Movement of animals and animal products from endemic areas is regulated. Quarantine and slaughter are practiced in outbreaks in endemic areas. Quarantine and vaccination are also used in endemic areas, but vaccines must be type-specific and repeated 2 or 3 times per year to be effective and will provide only partial protection. Autogenous vaccines are best in an outbreak. Passive immunity protects calves for up to 5 months after birth. The virus is inactivated by extremes of pH, sunlight, high temperatures, sodium hydroxide, sodium carbonate, and acetic acid. Treatment. Nursing care and antibiotic therapy to minimize secondary reactions help with recovery. Humoral immunity is considered the more important immune mechanism, with cell-mediated immunity of less importance. Research complications. Rare cases in humans have been reported. Importation into the United States of animal products from endemic areas is prohibited. o. Malignant Catarrhal Fever Etiology. Malignant catarrhal fever (MCF) is a severe disease primarily of cattle. The agents of MCF are viruses of the Gammaherpesvirinae subfamily. Alcelaphine herpesvirus 1 and 2 and ovine herpesvirus 2 are known strains. The alcelaphine strains are seen in Africa. The ovine strain is seen in North America. The alcelaphine and ovine strains differ in incubation times and duration of illness. Disease may occur sporadically or as outbreaks. Clinical signs and diagnosis. Signs range from subclinical to recrudescing latent infections to the lethal disease seen in susceptible species, such as cattle. Sudden death may also occur in cattle. Presentations of the disease may be categorized as alimentary, encephalitis, or skin forms; all three may occur in an animal. Corneal edema starting at the limbus and progressing centripetally is a nearly pathognomonic sign; photophobia, severe keratoconjunctivitis, and ocular involvement may follow. Other signs include prolonged fever, oral mucosal erosions, salivation, lacrimation, purulent nasal discharge, encephalitis, and pronounced lymphadenopathy. As the disease progresses, cattle may shed horns and hooves. In North America, cattle will also have severe diarrhea. The course of the disease may extend to 1 week. Recovery is usually prolonged, and some permanent debilitation may occur. The disease is fatal in severely affected individuals. History of exposure, as well as the clinical signs and lesions, contributes to the diagnosis. Serology, PCR-based assays, viral isolation, and cell-culture assays, such as cytopathic effects on thyroid cell cultures, are also used. Because of the susceptibility of rabbits, inoculation of this species may be used. In less severe outbreaks or individual animal disease, definitive diagnosis may never be made. Epizootiology and transmission. Most ruminant species are susceptible to MCF. Sheep are sources of infection for cattle, which are dead-end hosts. Other ruminants, including goats, may harbor the virus. Both the African and North American strains are transmissible to rabbits; these animals develop a fatal lymphoproliferative disease. The virus is shed from the nasopharynx. Infection of lambs is horizontal from direct contact. Other sources of the virus include water troughs, placental tissues, contaminated fomites, aerosols, birds, and caretakers. Necropsy. Gross findings at necropsy include necrotic and ulcerated nasal and oral mucosa; thickened, edematous, ulcerated, and hemorrhagic areas of the intestinal tract; swollen, friable, and hemorrhagic lymph nodes and other lymphatic tissues; and erosion of affected mucosal surfaces. Lymph nodes should be submitted for histological examination. Histological findings include nonsuppurative vasculitis and encephalitis; large numbers of lymphocytes and lymphoblasts will be present without evidence of virus. Pathogenesis. The incubation period may be up to 3 months. Vascular endothelium and all epithelial surfaces will be affected. The virus is believed to cause proliferation of cytotoxic T lymphocytes with natural killer cell activities, and the resulting lesions are due to an autoimmune type of phenomenon. Differential diagnoses. The differentials for this disease are bovine viral diarrhea/mucosal disease, bovine respiratory disease complex, infectious bovine rhinotracheitis, bluetongue, vesicular stomatitis, and foot-and-mouth disease. Causes of encephalitis, such as bovine spongiform encephalopathy and rabies, should be considered. In Africa, rinderpest is also a differential. Other differentials are arsenic toxicity and chlorinated naphthalene toxicity. Prevention and control. No vaccine is available at this time. In North America, sheep, as well as cattle that have been either exposed or that have survived the disease, are reservoirs for outbreaks in other cattle. If there is concern regarding presence of the virus, animals should be screened serologically; once an animal has been infected, it remains infected indefinitely. Lambs can be free of the infection if removed from the flock at weaning. The virus is very fragile outside of host's cells and will not survive in the environment for more than a few hours. Treatment. Affected and any exposed animals should be isolated from healthy animals. There is no specific treatment for MCF; supportive treatment may improve recovery rates. Corticosteroids may be useful. p. Ovine Progressive Pneumonia (Maedi/Visna) Etiology. An RNA virus in the lentivirus group of the Retroviridae family causes ovine progressive pneumonia (OPP), or maedi/visna. Maedi refers to the progressive pneumonia presentation of the disease; visna refers to the central nervous system disease, which is reported predominantly in Iceland. Visna has been reported in goats but may have been due to caprine arthritis encephalitis infection. Clinical signs and diagnosis. OPP is a viral disease of adult sheep characterized by weakness, unthriftiness, weight loss, and pneumonia ( Pepin et al., 1998 ; de la Concha Bermejillo, 1997 ). Clinically, animals exhibit signs of progressive pulmonary disease after an extremely long incubation period of up to 2 years. Respiratory rate and dyspnea gradually increase as the disease progresses. The animal continues to eat throughout the disease; however, animals progressively lose weight and become weak. Additionally, mastitis is a common clinical feature. Thoracic auscultation reveals consolidation of ventral lung lobes; and hematological findings indicate anemia and leukocytosis. The rare neurological signs include flexion of fetlock and pastern joints, tremors of facial muscles, progressive paresis and paralysis, depression, and prostration. Death occurs in weeks to months. The disease can be serologically diagnosed with agar gel immunodiffusion (AGID) tests, virus isolation, serum neutralization, complement fixation, and enzyme-linked immunosorbent assay (ELISA) tests. Epizootiology and transmission. Sixty-eight percent of sheep in some states have been infected with the virus ( Radostits et al., 1994 ). It is transmitted horizontally via inhalation of aerosolized virus particles and vertically between the infected dam and fetus. In addition, transmission through the milk or colostrum is considered common ( Knowles, 1997 ). Necropsy findings. Lesions are observed in lungs, mammary glands, joints, and the brain. Pulmonary adhesions, ventral lung lobe consolidation, bronchial lymph node enlargement, mastitis, and degenerative arthritis are visualized grossly. Meningeal edema, thickening of the choroid plexus, and foci of leukoencephalomalacia are seen in the central nervous system (CNS). Histologically, interalveolar septal thickening, lymphoid hyperplasia, histiocyte and fibrocyte proliferation, and squamous epithelial changes are seen in the lungs. Meningitis, lymphoid hyperplasia, demyelination, and glial fibrosis are seen in the CNS. Pathogenesis. The virus has a predilection for the lungs, mediastinal lymph nodes, udder, spleen, joints, and rarely the brain. After initial infection, the virus integrates into the DNA of mature monocytes and persists as a provirus. Later in the animal's life, infected monocytes mature as lung (and other tissue) macrophages and establish active infection. The virus induces lymphoproliferative disease, histiocyte and fibrocyte proliferation in the alveolar septa, and squamous metaplasia. Pulmonary alveolar and vascular changes impinge on oxygen and carbon dioxide exchange and lead to serious hypoxia and pulmonary hypertension. Secondary bacterial pneumonia may contribute to the animal's death. Differential diagnosis. Pulmonary adenomatosis is the differential diagnosis. Prevention and control. Isolating or removing infected animals can prevent the disease. Facilities and equipment should also be disinfected. Treatment. Treatment is unsuccessful. q. Poxviruses of Ruminants i. Ovine viral dermatosis. Ovine viral dermatosis is a venereal disease of sheep caused by a parapoxvirus distinct from contagious ecthyma. The disease resolves within 2 weeks in healthy animals, but lesions are painful and resemble those of Corynebacterium renale posthitis/vulvovaginitis. Symptomatic treatment may be necessary in some cases. There is no vaccine. Animals should not be used for breeding while clinical signs are present. ii. Proliferative stomatitis (bovine papular stomatitis) Etiology. A parapoxvirus is the causative agent of bovine papular stomatitis. This virus is considered to be closely related to the parapoxvirus that causes contagious ecthyma and pseudocowpox. It is also a zoonotic disease. The disease is not considered of major consequence, but high morbidity and mortality may be seen in severe outbreaks. In addition, lesions are comparable in appearance to those seen with vesicular stomatitis, bovine viral diarrhea virus, and foot-and-mouth disease. The disease occurs worldwide. Clinical signs and diagnosis. Raised red papules or erosions or shallow ulcers on the muzzle, nose, oral mucosa (including the hard palate), esophagus, and rumen of younger cattle are the most common findings. In some outbreaks, the papules will be associated with ulcerative esophagitis, salivation, diarrhea, and subsequent weight loss. Lesions persist or may come and go over a span of several months. Morbidity among herds may be 100%. Mortalities are rare. Bovine papular stomatitis is associated with "rat tail" in feedlot cattle. Animals continue to eat and usually do not show a fever. No lesion is seen on the feet. The infection may also be asymptomatic. Diagnosis is based on clinical signs, histological findings, and viral isolation. Epizootiology and transmission. Cattle less than 1 year of age are most commonly affected, and disease is rare in older cattle. Transmission is by animal-to-animal contact. Necropsy findings. Raised papules may be found around the muzzle and mouth and involve the mucosa of the esophagus and rumen. Histologically, epithelial cells will show hydropic degeneration and hyperplasia of the lamina propria. Eosinophilic inclusions will be in the cytoplasm of infected epithelial cells. Pathogenesis. Following exposure to the virus, erythematous macules most commonly appear on the nares, followed by the mouth. These become raised papules within a day, regressing after days to weeks; the lesions that remain will be persistent yellow, red, or brown spots. Some infections may recur or persist, with animals showing lesions intermittently or continuously over several months. Differential diagnosis. Pseudocowpox, vesicular stomatitis, foot-and-mouth disease, and bovine viral diarrhea virus infection are the differentials for this disease. The differential for the "rat tail" clinical sign is Sarcocystis infection. Prevention and control. There is no vaccine available for bovine papular stomatitis. Because of the similarity of this virus to the parapoxvirus of contagious ecthyma, it is important to be aware of the persistence in the environment and susceptibility of younger cattle. Vaccination using the local strain, and the skin scarification technique for orf, have been protective. Handlers should wear gloves and protective clothing. Treatment. Cattle usually will not require extensive nursing care, but lesions with secondary bacterial infections should be treated with antibiotics. Research complications. Handlers may develop lesions on their hands at sites of contact with lesions of cattle. iii. Pseudocowpox Etiology. Pseudocowpox is a worldwide cattle disease caused by a parapoxvirus related to the causative agents of contagious ecthyma and bovine papular stomatitis (see Sections III,A,2,m and III,A,2,q,ii). Lesions are confined to the teats. This is also a zoonotic disease. Clinical signs and diagnosis. Minor lesions are usually confined to the teats. These are distinctive because of the ring- or horseshoe-shaped scab that develops after 10 days. Additional lesions sometimes develop on the udder, the medial aspect of the thighs, and the scrotum. The teat lesions may predispose to mastitis. Pathogenesis. The virus is spread by contaminated hands, equipment, and fomites. Differential diagnosis. Differentials include bovine herpes mammillitis and papillomatosis. Prevention and control. Milking hygiene is helpful in control. Treatment. Lesions should be treated symptomatically, and affected animals milked last. Research complications. Like other related poxviruses, this virus causes nodular lesions on humans. r. Pulmonary Adenomatosis (Jaagsiekte) Etiology. Pulmonary adenomatosis is a rare but progressive wasting disease of sheep, with worldwide distribution. Pulmonary adenomatosis is caused by a type D retrovirus antigenically related to the Mason-Pfizer monkey virus. Jaagsiekte was the designation when the disease was described originally in South Africa. Clinical signs and diagnosis. Typical clinical signs include progressive respiratory signs such as dyspnea, rapid respiration, and wasting. The disease is diagnosed by these chronic clinical signs and histology. Epizootiology and transmission. The disease is transmitted by aerosols. Body fluids of viremic animals, such as milk, blood, saliva, tears, semen, and bronchial secretions, will contain the virus or cells carrying the virus. Necropsy. The adenomas and adenocarcinomas will be small firm lesions distributed throughout the lungs. The adenocarcinomas metastasize to regional lymph nodes. Pathogenesis. As with ovine progressive pneumonia (OPP), the incubation period is up to 2 years long. Adenocarcinomatous lesions arising from type II alveolar epithelial cells may be discrete or confluent and involve all lung lobes. Differential diagnosis. This disease occurs coincidentally with or is a differential diagnosis for OPP. Treatment. No treatment is effective. s. Papillomatosis (Warts, Verrucae) Etiology. Cutaneous papillomatosis is a very common disease in cattle and is much less common among sheep and goats. The disease is a viral-induced proliferation of the epithelium of the neck, face, back, and legs. These tumors are caused by a papillomavirus (DNA virus) of the Papovaviridae family, and the viruses are host-specific and often body site-specific. Most are benign, although some forms in cattle and one form in goats can become malignant. In cattle, the site specificity of the papillomavirus strains are particularly well recognized. Designations of the currently recognized bovine papillomavirus (BPV) types are BPV-1 through BPV-5. Clinical signs and diagnosis. The papillomas may last up to 12 months and are seen more frequently in younger animals. Lesions have typical wart appearances and may be single or multiple, small (1 mm) or very large (500 mm). The infections will generally be benign, but pain will be evident when warts develop on occlusal surfaces or within the gastrointestinal tract. In addition, when infections are severe, weight loss may occur. When warts occur on teats, secondary mastitis may develop. In cattle, BPV-1 and BPV-2 cause fibropapillomas on teats and penises or on head, neck, and dewlap, respectively. BPV-3 causes flat warts that occur in all body locations, BPV-4 causes warts in the gastrointestinal tract, and BPV-5 causes small white warts (called rice-grain warts) on teats. Warts caused by BPV-3 and BPV-5 do not regress spontaneously. Prognosis in cattle is poor only when papillomatosis involves more than 20% of the body surface. In sheep, warts are the verrucous type. The disease is of little consequence unless the warts develop in an area that causes discomfort or incapacitation such as between the digits, on the lips, or over the joints. In adult sheep, warts may transform to squamous cell carcinoma. In goats, the disease is rare, and the warts are also of the verrucous type and occasionally may develop into squamous cell carcinoma. Warts on goat udders tend to be persistent. Diagnosis is made by observing the typical proliferative lesions. Epizootiology and transmission. Older animals are less sensitive to papillomatosis than young animals, although immunosu-pressed animals of any age may develop warts as the result of harbored latent infections. The virus is transmitted by direct and indirect (fomite) contact, entering through surface wounds and sites such as tattoos. Pathogenesis. The incubation period ranges from 1 to 6 months. The virus induces epidermal and fibrous tissue proliferation, often described as cauliflower-like skin tumors. The disease is generally self-limiting. Differential diagnosis. In sheep and goats, differentials include contagious ecthyma, ulcerative dermatosis, strawberry foot rot, and sheep and goat pox. Prevention and control. Commercial vaccines (available only for cattle) or autogenous vaccines must be used with a recognition that papovavirus strains are host-specific and that immunity from infection or vaccination is viral-type-specific. Autogenous vaccines are generally considered more effective. Some vaccine preparations are effective at prevention but not treatment of outbreaks. Viricidal products are recommended for disinfection of contaminated environments. Minimizing cutaneous injuries and sanitizing equipment (tattoo devices, dehorners, ear taggers, etc.) in a virucidal solution between uses are also recommended preventive and control measures. Halters, brushes, and other items may also be sources of virus. Treatment. Warts will often spontaneously resolve as immunity develops. In severe cases or with flockwide or herdwide problems, affected animals should be isolated from nonaffected animals, and premises disinfected. Warts can be surgically excised and autogenous vaccines can be made and administered to help prevent disease spread. Cryosurgery with liquid nitrogen or dry ice has also proven to be successful for wart removal. Topical agents such as podophyllin (various formulations) and dimethyl sulfoxide may be applied to individual lesions once daily until regression. t. Pseudorabies (Mad Itch, Aujeszky's Disease) Etiology. Pseudorabies is an acute encephalitic disease caused by a neurotropic alphaherpesvirus, the porcine herpesvirus 1. One serotype is recognized, but strain differences exist. The disease has worldwide distribution. It is a primarily a clinical disease of cattle, with less frequent reports (but no less severe clinical manifestations) in sheep and goats. Clinical signs and diagnosis. A range of clinical signs is seen during the rapid course of this usually fatal disease. At the site of virus inoculation or in other locations, abrasions, swelling, intense pruritus, and alopecia are seen. Pruritus will not be asymmetric. Animals will also become hyperthermic and will vocalize frantically. Other neurological signs range from hoof stamping, kicking at the pruritic area, salivation, tongue chewing, head pressing and circling, to paresthesia or hyperesthesia, ataxia, and conscious proprioceptive deficits. Nystagmus and strabismus are also seen. Animals will be fearful or depressed, and aggression is sometimes seen. Recumbency and coma precede death. Diagnostic evidence includes clinical findings; virus isolation from nasal or pharyngeal secretions or postmortem tissues; and histological findings at necropsy. Serology of affected animals is not productive, because of the rapid course. If swine are housed nearby, or if swine were transported in the same vehicles as affected animals, serological evaluations are worthwhile from those animals. Epizootiology and transmission. Swine are the primary hosts for pseudorabies virus, but they are usually asymptomatic and serve as reservoirs for the virus. The infection can remain latent in the trigeminal ganglion of pigs and recrudesce during stressful conditions. Other animals are dead-end hosts. The unprotected virus will survive only a few weeks in the environment but may remain viable in meat (including carcasses) or saliva and will survive outside the host, in favorable conditions, in the summer for several weeks and the winter for several months. Transmission is by oral, intranasal, intradermal, or subcutaneous introduction of the virus. When the virus is inhaled, the clinical signs of pruritus are less likely to be seen. Transmission can also be by inadvertent exposure (e.g., contaminated syringes) of ruminants to the modified live vaccines developed for use in swine. Spread between infected ruminants is a less likely means of transmission, because of the relatively short period of virus shedding. Transport vehicles used for swine may also be sources of the virus. Raccoons are believed to be vectors of the virus. Horses are resistant to infection. Necropsy findings. There is no pathognomonic gross lesion. Definitive histologic findings include severe, focal, nonsuppurative encephalitis and myelitis. Eosinophilic intranuclear inclusion bodies (Cowdry type A) may be present in some affected neurons. Methods such as immunofluorescence and immunoperoxidase staining can be used to show presence of the porcine herpesvirus 1. Pathogenesis. The incubation period is 90–156 hr and duration of the illness is 8–72 hr. The longest duration is seen in animals with pruritus around the head. Differential diagnoses. Differentials for the neurologic signs of pseudorabies infection include rabies, polioencephalomalacia, salt poisoning, meningitis, lead poisoning, hypomagnesemia, and enterotoxemia. Those for the intense pruritus include psoroptic mange and scrapie in sheep, sarcoptic mange, and pediculosis. Prevention and control. Pseudorabies is a reportable disease in the United States, where a nationwide eradication program exists; states are rated regarding status. Effective disinfectants include sodium hypochlorite (10% solution), formalin, peracetic acid, tamed iodines, and quaternary ammonium compounds. Five minutes of contact time is required, and then surfaces must be rinsed. Other disinfectant methods for viral killing include 6 hr of formaldehyde fumigation, or 360 min of ultraviolet light. Transport vehicles should be cleaned and disinfected between species. Serological screening for pseudorabies of swine housed near ruminants is essential. Treatment. There is no treatment, and most affected animals die. Research complications. Swine housed close to research ruminants should be serologically screened prior to purchase, and all transport vehicles should be cleaned and disinfected between loads of large animals. Humans have been reported to seroconvert. The porcine herpesvirus 1 shares antigens with the infectious bovine rhinotracheitis virus. u. Rabies (Hydrophobia) Etiology. Rabies is a sporadic but fatal, acute viral disease affecting the central nervous system. The rabies virus is a neurotropic RNA virus of the Lyssavirus genus and the Rhabdoviridae family. Sheep, goats, and cattle are susceptible. The zoonotic potential of this virus must be kept in mind at all times when handling moribund animals with neurological signs characteristic of the disease. Rabies is endemic in many areas of the world and within areas of the Unites States. This is a reportable disease in North America. Clinical findings and diagnosis. Animals generally progress through three phases: prodromal, excitatory, and paralytic. Many signs in the different species during these stages are nonspecific, and forms of the disease are also referred to as dumb or furious. During the short prodromal phase, animals are hyperthermic and apprehensive. Animals progress to the excitatory phase, during which they refuse to eat or drink and are active and aggressive. Repeated vocalizations, tenesmus, sexual excitement, and salivation occur during this phase. The final paralytic stage, with recumbency and death, occurs over several hours to days. This paralytic stage is common in cattle, and animals may simply be found dead. The clinical course is usually 1–4 days. Diagnosis is based on clinical signs, with a progressive and fatal course. Confirmation presently is made with the fluorescent antibody technique on brain tissue. Epizootiology and transmission. The rabies virus is transmitted via a bite wound inflicted by a rabid animal. Cats, dogs, raccoons, skunks, foxes, wild canids, and bats are the common disease vectors in North America. Virus is also transmitted in milk and aerosols. Necropsy findings. Few lesions are seen at necropsy. Many secondary lesions from manic behaviors during the course of disease may be evident. Histological findings will include nonsuppurative encephalitis. Negri bodies in the cytoplasm of neurons of the hippocampus and in Purkinje's cells are pathognomonic histologic findings. Pathogenesis. After exposure, the incubation period is variable, from 2 weeks to several months, depending on the distance that the virus has to travel to reach the central nervous system. The rabies virus proliferates locally, gains access to neurons by attaching to acetylcholine receptors, via a viral surface glycoprotein, migrates along sensory nerves to the spinal cord and brain, and then descends via cranial nerves (trigeminal, facial, olfactory, glossopharyngeal) to oral and nasal cavity structures (i.e., salivary glands). The fatal outcome is currently believed to be multifactorial, related to anorexia, respiratory paralysis, and effects on the pituitary. Differential diagnosis. Rabies should be included on the differential list when clinical signs of neurologic disease are evident. Other differentials for ruminants include herpesvirus encephalitis, thromboemobolic meningoencephalitis, nervous ketosis, grass tetany, and nervous cocciodiosis. Prevention and control. Vaccines approved for use cattle and sheep are commercially available and contain inactivated virus; there is not one available in the United States for goats. Ruminants in endemic areas, such as the East Coast of the United States, should be routinely vaccinated. Any animals housed outside that may be exposed to rabid animals should be vaccinated. Vaccination programs generally begin at 3 months of age, with a booster at 1 year of age and then annual or triennial boosters. Awareness of the current rabies case reports for the region and wildlife reservoirs, however, is important. Monitoring for and exclusion of wildlife from large-animal facilities are worthwhile preventive measures. The virus is fragile and unstable outside of a host animal. Research complications. Aerosolized virus is infective. Personal protective equipment, including gloves, face mask, and eye shields, must be worn by individuals handling animals that are manifesting neurological disease signs. v. Transmissible Spongiform Encephalopathies i. Bovine spongiform encephalopathy (mad cow disease). Bovine spongiform encephalopathy, a transmissible spongiform encephalopathy (TSE), is not known to occur in the United States, where since 1989 it has been listed as a reportable disease. The profound impact of this disease on the cattle industry in Great Britain during the past two decades is well known. The disease may be caused by a scrapielike (prion) agent. It is believed that the source of infection for cattle was feedstuff derived from sheep meat and bonemeal that had been inadequately treated during processing. The incubation period of years, the lack of detectable host immune response, the debilitating and progressive neurological illness, and the pathology localized to the central nervous system are characteristics of the disease, and are is comparable to the characteristics of other TSE diseases such as scrapie, which affects sheep and goats. In addition, the infectious agent is extremely resistant to dessication and disinfectants. Confirmation of disease is by histological examination of brain tissue collected at necropsy; the vacuolation that occurs during the disease will be symmetrical and in the gray matter of the brain stem. Molecular biology techniques, such as Western blots and immunohistochemistry, may also be used to identify the presence of the prion protein. Differentials include many infectious or toxic agents that affect the bovine nervous and musculoskeletal systems, such as rabies, listeriosis, and lead poisoning. Metabolic disorders such as ketosis, milk fever, and grass tetany are also differentials. There is no vaccine or treatment. Prevention focuses on import regulations and not feeding ruminant protein to ruminants; recent USDA regulations prohibit feeding any mammalian proteins to ruminants. ii. Scrapie Etiology. Scrapie is a sporadic, slow, neurodegenerative disease caused by a prion. Scrapie is a reportable disease. It is much more common in sheep than in goats. The disease is similar to transmissible mink encephalopathy, kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy (mad cow disease). Prions are nonantigenic, replicating protein agents. Clinical signs and diagnosis. During early clinical stages, animals are excitable and hard to control. Tremors of head and neck muscles, as well as uncoordinated movements and unusual "bunny-hopping" gaits are observed. In advanced stages of the disease, animals experience severe pruritus and will self-mutilate while rubbing on fences, trees, and other objects. Blindness and abortion may also be seen. Morbidity may reach 50% within a flock. Most animals invariably die within 4–6 weeks; some animals may survive 6 months. In goats, the disease is also fatal. Pruritus is generally less severe but may be localized. A wide range of clinical signs have also been noted in goats, including listlessness, stiffness or restlessness, or behavioral changes such as irritability, hunched posture, twitching, and erect tail and ears. As with sheep, the disease gradually progresses to anorexia and debilitation. Diagnosis can be made by clinical signs and histopathological lesions. A newer diagnostic test in live animals is based on sampling from the third eyelid. Tests for genetic resistance or susceptibility require a tube of EDTA blood and are reasonably priced. Epizootiology and transmission. The Suffolk breed of sheep tends to be especially susceptible. Scrapie has also been reported in several other breeds, including Cheviot, Dorset, Hampshire, Corriedale, Shropshire, Merino, and Rambouillet. It is believed that there is hereditary susceptibility in these breeds. Targhees tend to be resistant. Genomic research indicates there are two chromosomsal sites governing this trait; these sites are referred to codons 171 (Q, R, or H genes can be present) and 136 (A or V genes can be present). Of the five genes, R genes appear to confer immunity to clinical scrapie in Suffolks in the United States. Affected Suffolks in the United States that have been tested have been AA QQ. The disease is also enzootic is many other countries. The disease tends to affect newborns and young animals; however, because the incubation period tends to range from 2 to 5 years, adult animals display signs of the disease. Scrapie is transmitted horizontally by direct or indirect contact; nasal secretions or placentas serve as sources of the infectious agent. Vertical transmission is questioned, and transplacental transmission is considered unlikely. Necropsy findings. At necropsy, no gross lesion is observed. Histopathologically, neuronal vacuolization, astrogliosis, and spongiform degeneration are visualized in the brain stem, the spinal cord, and especially the thalamus. Inflammatory lesions are not seen. Pathogenesis. Replication of the prions probably occurs first in lymphoid tissues throughout the host's body and then progresses to neural tissue. Differential diagnosis. In sheep and goats, depending on the speed of onset, differentials for the pruritus include ectoparasites, pseudorabies, and photosensitization. Prevention and control. If the disease diagnosed in a flock, quarantine and slaughter, followed by strict sanitation, are usually required. The U.S. Department of Agriculture has approved the use of 2% sodium hydroxide as the only disinfectant for sanitation of scrapie-infected premises. Prions are highly resistant to physicochemical means of disinfection. Artificial insemination or embryo transfer has been shown to decrease the spread of scrapie ( Linnabary et al., 1991 ). Treatment. No vaccine or treatment is available. Research complications. As noted, this is a reportable disease. Stringent regulations exist in the United States regarding importation of small ruminants from scrapie-infected countries. w. Vesicular Stomatitis Virus Etiology. Vesicular stomatitis (VS) is caused by the vesicular stomatitis virus (VSV), a member of the Rhabdoviridae. Three serotypes are recognized: New Jersey, Indiana, and Isfahan. The New Jersey and Indiana strains cause sporadic disease in cattle in the United States. The disease is rare in sheep. Clinical signs and diagnosis. Adult cattle are most likely to develop VS. Fever and development of vesicles on the oral mucous membranes are the initial clinical signs. Lesions on the teats and interdigital spaces also develop. The vesicles progress quickly to ulcers and erosions. The animal's tongue may be severely involved. Anorexia and salivation are common. Weight loss and decreased milk production are noticeable. Morbidity will be high in an outbreak, but mortality will be low to nonexistent. Diagnostic work should be initiated as soon as possible to distinguish this from foot-and-mouth disease. Diagnosis is based on analysis of fluid, serum, or membranes associated with the vesicles. Virus isolation, enzyme-linked immunosorbent assay (ELISA), competitive ELISA (CELISA), complement fixation, and serum neutralization are used for diagnosis. Epizootiology and transmission. This disease occurs in several other mammalian species, including swine, horses, and wild ruminants. VSV is an enveloped virus and survives well in different environmental conditions, including in soil, extremes of pH, and low temperatures. Outbreaks of VS occur sporadically in the United States, but it is not understood how or in what species the virus survives between these outbreaks. Incidence of disease decreases during colder seasons. Equipment, such as milking machines, contaminated by secretions is a mechanical vector, as are human hands. Transmission may also be from contaminated water and feed. Transmission is also believed to occur by insects (blackflies, sand flies, and Culicoides) that may simply be mechanical vectors. It is believed that carrier animals do not occur in this disease. Necropsy. It is rare for animals to be necropsied as the result of this disease. Typical vesicular lesion histology is seen, with ballooning degeneration and edema. There is no inclusion body formation. Pathogenesis. Lesions often begin within 24 hr after exposure. The virus invades oral epithelium. Injuries or trauma in any area typically affected, such as mouth, teats, or interdigital areas, will increase the likelihood of lesions developing there. Animals will develop a long-term immunity; this immunity can be overwhelmed, however, by a large dose of the virus. Differential diagnosis. Foot-and-mouth disease lesions are identical to VS lesions. Other differentials in cattle include bovine viral diarrhea, malignant catarrhal fever, contagious ecthyma, photosensitization, trauma, and caustic agents. Prevention and control. Quarantine and restrictions on shipping infected animals or animals from the premises housing affected animals are required in an outbreak. Vaccines are available for use in outbreaks and have decreased the severity of lesions. Phenolics, quaternaries, and halogens are effective for inactivating and disinfecting equipment and facilities. Treatment. Affected animals should be segregated from the rest of the herd and provided with separate water and softened feed. These animals should be cared for after unaffected animals. Any feed or water contaminated by these animals should not be used for other animals; contaminated equipment should be disinfected. Topical or systemic antibiotics control secondary bacterial infections. Cases of mastitis secondary to teat lesions must be treated as necessary. Any abrasive materials that could cause further trauma to the animals should be removed. Research complications. Animals developing vesicular lesions must be reported promptly to eliminate the possibility of an outbreak of foot-and-mouth disease. Personal protective equipment, especially gloves, should be worn when handling any animals with vesicular lesions. VSV causes a flulike illness in humans. x. Viral Diarrhea Diseases i. Ovine. Rotavirus, of the family Reoviridae, induces an acute, transient diarrhea in lambs within the first few weeks of life. Four antigenic groups (A-D) have been identified by differences in capsid antigens VP3 and VP7. Primarily group A, but also groups B and C, have been isolated from sheep. The disease is characterized by yellow, semifluid to watery diarrhea occurring 1–4 days after infection. The disease can progress to dehydration, anorexia and weight loss, acidosis, depression, and occasionally death. The virus is ingested with contaminated feed and water and selectively infects and destroys the enterocytes at the tips of the small intestinal villi. The villi are replaced with immature cells that lack sufficient digestive enzymes; osmotic diarrhea results. Virus may remain in the environment for several months. The disease is diagnosed by virus isolation, electron microscopy of feces, fecal fluorescent antibody, fecal ELISA tests (marketed tests generally detect group A rotavirus), and fecal latex agglutination tests. Rotavirus diarrhea is treated by supportive therapy, including maintaining hydration, electrolyte, and acid-base balance. A rotavirus vaccine is available for cattle; because of cross-species immunity, oral administration of high-quality bovine colostrum from vaccinated cows to infected sheep may be helpful ( "Current Veterinary Therapy," 1993 ). Coronavirus, of the family Coronaviridae, produces a more severe, long-lasting disease when compared with rotavirus. Clinical signs are similar to above, although the incubation period tends to be shorter (20–36 hr), and animals exhibit less anorexia than those with rotavirus. Additionally, mild respiratory disease may be noted ( Janke, 1989 ). Like rotavirus, coronavirus also destroys enterocytes of the villus tips. The virus can be visualized with electron microscopy. Treatment is supportive; close consideration of hydration and acid-base status is essential. Bovine vaccines are available. ii. Caprine. Rotavirus, coronavirus, and adenoviruses affect neonatal goats; however, little has been documented on the pathology and significance of these agents in this age group. It appears that bacteria play a more important role in neonatal kid diarrheal diseases then in neonatal calf diarrheas. iii. Bovine. Rotaviruses, coronaviruses, parvoviruses, and bovine viral diarrhea virus (BVDV) are associated with diarrheal disease in calves. Each pathogen multiplies within and destroys the intestinal epithelial cells, resulting in villous atrophy and clinical signs of diarrhea (soft to watery feces), dehydration, and abdominal pain. These viral infections may be complicated by parasitic infections (e.g., Cryptosporidium, Eimeria) or bacterial infections (e.g., Escherichia coli, Salmonella, Campylobacter). Treatment is aimed at correcting dehydration, electrolyte imbalances, and acidosis; cessation of milk replacers and administration of fluid therapy intravenously and by stomach tube may be necessary, depending on the presence of suckle reflex and the condition of the animals. Diagnosis is by immunoassays available for some viruses, viral culture, exclusion or identification of presence of other pathogens (by culture or fecal exams), and microscopic examination of necropsy specimens. Prevention focuses on calves suckling good-quality colostrum; other recommendations for calf care are in Section II,B,5. Combination vaccine products are available for immunizing dams against rotavirus, coronavirus, and enterotoxigenic E. coli. Additional supportive care for calves includes providing calves with sufficient energy and vitamins until milk intake can resume. Rotaviruses of serogroup A are the most common type in neonatal calves; 4- to 14-day old calves are typically affected, but younger and older animals may also be affected. The small intestine is the site of infection. Antirotavirus antibody is present in colostrum, and onset of rotavirus diarrhea coincides with the decline of this local protection. Transmission is likely from other affected calves and asymptomatic adult carriers. The diarrhea is typically a distinctive yellow. Colitis with tenesmus, mucus, and blood may be seen. This virus may be zoonotic. Coronaviruses are commonly associated with disease in calves during the first month of life, and they infect small- and large-intestinal epithelial cells. The virus infection may extend to mild pneumonia. Transmission is by infected calves and also by asymptomatic adult cattle, including dams excreting virus at the time of parturition. Calves that appear to have recovered continue to shed virus for several weeks. Parvovirus infections are usually associated with neonatal calves. BVDV infections also are seen in neonates and also affect many systems and produce other clinical signs and syndromes that are described in Section III,A,2,e. iv. Winter Dysentery. Winter dysentery is an acute, winter-seasonal, epizootic diarrheal disease of adult cattle, although it has been reported in 4-month-old calves. The etiology has not yet been defined, but a viral pathogen is suspected. Coronavirus-like viral particles have been isolated from cattle feces, either the same as or similar to the coronavirus of calf diarrhea. Outbreaks typically last a few weeks, and first-lactation or younger cattle are affected first, with waves of illness moving through a herd. Individual cows are ill for only a few days. The incubation period is estimated at 2–8 days. The outbreaks of disease are often seen in herds throughout the local area. Clinical signs include explosive diarrhea, anorexia, depression, and decreased production. The diarrhea has a distinctive musty, sweet odor and is light brown and bubbly, but some blood streaks or clots may be mixed in with the feces. Animals will become dehydrated quickly but are thirsty. Respiratory symptoms such as nasolacrimal discharges and coughing may develop. Recovery is generally spontaneous. Mortalities are rare. Diagnosis is based on characteristic patterns of clinical signs, and elimination of diarrheas caused by parasites such as coccidia, bacterial organisms such as Salmonella or Mycobacterium paratuberculosis, and viruses such as BVDV. Pathology is present in the colonic mucosa, and necrosis is present in the crypts. 3. Chlamydial Diseases a. Enzootic Abortion of Ewes (Chlamydial Abortion) Etiology. Chlamydia psittaci is a nonmotile, obligate, intracytoplasmic, gram-negative bacterium. Clinical signs. Enzootic abortion in sheep and goats is a contagious disease characterized by hyperthermia and late abortion or by birth of stillborn or weak lambs or kids ( Rodolakis et al., 1998 ). The only presenting clinical sign may be serosanguineous vulvar discharges. Other animals may present with arthritis or pneumonia. Infection of animals prior to about 120 days of gestation results in abortion, stillbirths, or birth of weak lambs. Infection after 120 days results in potentially normal births, but the dams or offspring may be latently infected. Latently infected animals that were infected during their dry period may abort during the next pregnancy. Ewes or does generally only abort once, and thus recovered animals will be immune to future infections. Epizootiology and transmission. Chlamydia possess group and specific antigens associated with the cell surface. The group antigen is common among all Chlamydia; the specific antigen is common to related subgroups. Two subgroups are recognized, one that causes EAE and one that causes polyarthritis and conjunctivitis. The disease is transmitted by direct contact with infectious secretions such as placental, fetal, and uterine fluids or by indirect contact with contaminated feed and water. Necropsy. Placental lesions include intercotyledonary plaques and necrosis and cotyledonary hemorrhages. Histopathological evidence of leukocytic infiltration, edema, and necrosis is found throughout the placentome. Fetal lesions include giant-cell accumulation in mesenteric lymph nodes and lymphohistiocytic proliferations around the blood vessels within the liver. Diagnosis is based on clinical signs and laboratory (serological or histopathological) identification of the organism. Impression smears in placental tissues stained with Giemsa, Gimenez, or modified Ziehl-Neelsen can provide preliminary indications of the causative agent. Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) methods also aid in diagnosis. Differential diagnosis. Q fever will be the major differential for late-term abortion and necrotizing placentitis. Campylobacter and Toxoplasma should also be considered for late-term abortion. Treatment. Animals may respond to treatment with oxytetra cycline. Abortions are prevented through administration of a commercial vaccine, but the vaccine will not eliminate infections. This is a sheep vaccine and should be administered before breeding and annually to at least the young females entering the breeding herd or flock. Research complications. In addition to losses or compromise of research animals, pregnant women should not handle aborted tissues. b. Chlamydial Polyarthritis of Sheep Etiology. Chlamydia psittaci is a nonmotile, obligate intracellular, gram-negative bacterium. Chlamydial polyarthritis is an acute, contagious disease characterized by fever, lameness ( Bulgin, 1986 ), and conjunctivitis (see Section III,A,3,c) in growing and nursing lambs. Clinical signs. Clinically, animals will appear lame on one or all legs and in major joints, including the scapulohumeral, humeroradioulnar, coxofemoral, femorotibial, and tibiotarsal joints. Lambs may be anorexic and febrile. Animals frequently also exhibit concurrent conjunctivitis. The disease usually resolves in approximately 4 weeks. Joint inflammation usually resolves without causing chronic articular changes. Epizootiology and transmission. The disease is transmitted to susceptible animals by direct contact as well as by contaminated feed and water. The organism penetrates the gastrointestinal tract and migrates to joints and synovial membranes as well as to the conjunctiva. The organism causes acute inflammation and associated fibrinopurulent exudates. Necropsy findings. Lesions are found in joints, tendon sheaths, conjunctiva, and lungs. Pathological sites will be edematous and hyperemic, with fibrinous exudates but without articular changes. Lesions will be infiltrated with mononuclear cells. Lung lesions include atelectasis and alveolar inspissation. Diagnosis is based on clinical signs. Synovial taps and subsequent smears may allow the identification of chlamydial inclusion bodies. Treatment. Animals respond to treatment with parenteral oxytetracycline. c. Chlamydial Conjunctivitis (Infectious Keratoconjunctivitis, Pinkeye) Etiology. Chlamydia psittaci, a nonmotile, obligate intracellular, gram-negative bacterium, is the most common cause of infectious keratoconjunctivitis in sheep. Chlamydia and Mycoplasma are considered to be the most common causes of this disease in goats. Chlamydial conjunctivitis is not a disease of cattle. Clinical signs. Infectious keratoconjunctivitis is an acute, contagious disease characterized in earlier stages by conjunctival hyperemia, epiphora, and edema and in later stages by, corneal edema, ulceration, and opacity. Perforation may result from the ulceration. Animals will be photophobic. In less severe cases, corneal healing associated with fibrosis and neovascularization occurs in 3–4 days. Lymphoid tissues associated with the conjunctiva and nictitating membrane may enlarge and prolapse the eyelids. Morbidity may reach 80–90%. Bilateral and symmetrical infections characterize most outbreaks. Relapses may occur. Other concurrent systemic infections may be seen, such as polyarthritis or abortion in sheep and polyarthritis, mastitis, and uterine infections in goats. Epizootiology and transmission. Direct contact, and mechanical vectors such as flies easily spread the organism. Necropsy. If the chlamydial or mycoplasmal agents are suspected, diagnostic laboratories should be contacted for recommendations regarding sampling. Conjunctival smears are also useful. Pathogenesis. The pathogen penetrates the conjunctival epithelium and replicates in the cytoplasm by forming initial and elementary bodies. The infection moves from cell to cell and causes an acute inflammation and resultant purulent exudate. The chlamydial organism may penetrate the bloodstream and migrate to the opposite eye or joints, leading to arthritis. Diagnosis is suggested by the clinical signs. Cytoplasmic inclusions observed on conjunctival scrapings and immunofluorescent techniques help confirm the diagnosis. Differential diagnosis. Nonchlamydial keratoconjunctivitis also occurs in sheep and goats. The primary agents involved include Mycoplasma conjunctiva, M. agalactiae in goats, and Branhamella (Neisseria) ovis. A less common differential for sheep and cattle is Listeria monocytogenes. Other differentials include eye worms, trauma, and foreign bodies such as windblown materials (pollen, dust) and poor-quality hay; these latter irritants and stress may predispose the animals' eyes to the infectious agents. Prevention and control. Source of mechanical irritation should be minimized whenever possible. Quarantine of new animals and treatment, if necessary, before introduction into the flock or herd are important measures. Shade should be provided for all animals. Treatment. The infections can be self-limiting in 2–3 weeks without treatment. Treatment consists of topical application of tetracycline ophthalmic ointments. Systemic or oral oxytetracycline treatments have been used with the topical treatment. Atropine may be added to the treatment regimen when uveitis is present. Shade should be provided. 4. Parasitic Diseases a. Protozoa i. Anaplasmosis Etiology. Anaplasmosis is an infectious, hemolytic, noncontagious, transmissible disease of cattle caused by the protozoan Anaplasma marginale. Anaplasma is a member of the Anaplas-matacae family within the order Rickettsiales. In sheep and goats, the disease is caused by A. ovis and is an uncommon cause of hemolytic disease. Anaplasmosis has not been reported in goats in the United States. Some controversy exists regarding the classification. Most recently it is classified as a protozoal disease because of similarities to babesiosis. It has also been classified as a rickettsial pathogen. This summary addresses the disease in cattle with limited reference to A. ovis infections, but there are many similarities to the disease in cattle. Clinical signs and diagnosis. Acute anemia is the predominant sign in anaplasmosis, and fever coincides with parasitemia. Weakness, pallor, lethargy, dehydration, and anorexia are the result of the anemia. Four disease stages—incubation, developmental, convalescent, and carrier—are recognized. The incubation stage may be long, 3–8 weeks, and is characterized by a rise in body temperature as the infection moves to the next stage. Most clinical signs occur during the 4- to 9-day developmental stage, with hemolytic anemia being common. Death is most likely to occur at this stage or at the beginning of the convalescent stage. Death may also occur from anoxia, because of the animal's inability to handle any exertion or stress, especially if treatment is initiated when severe anemia exists. Reticulocytosis characterizes the convalescent stage, which may continue for many weeks. Morbidity is high, and mortality is low. The carrier stage is defined as the time in the convalescent stage when the animal host becomes a reservoir of the disease, and Anaplasma organisms and any parasitemia are not discernible. Common serologic tests are the complement fixation test and the rapid card test. These become positive after the incubation phase and do not distinguish between the later three stages of disease. Definitive diagnosis is made by clinical and necropsy findings. Staining of thin blood smears with Wright's or Giemsa stain allows detection of basophilic, spherical A. marginale bodies near the red blood cell peripheries. Evidence will most likely be found before a hemolytic episode. A negative finding should not eliminate the pathogen from consideration. Epizootiology and transmission. The disease is common in cattle in the southern and western United States. Anaplasma organisms are spread biologically or mechanically. Mechanical transmission occurs when infected red blood cells are passed from one host to another on the mouthparts of seasonal biting flies. Sometimes mosquitoes or instruments such as dehorners or hypodermic needles may facilitate transfer of infected red cells from one animal to another. Biological transmission occurs when the tick stage of the organism is passed by Dermacentor andersoni and D. occidentalis ticks. The carrier stage covers the time when discernible Anaplasma organisms can be found on host blood smears. Recovered animals serve as immune carriers and disease reservoirs. Necropsy. Pale tissues and watery, thin blood are typical findings. Splenomegaly, hepatomegaly, and gallbladder distension are common findings. Pathogenesis. The parasites infect the host's red blood cells, and acute hemolysis occurs during the parasites' developmental stage. The four stages of the parasite's life cycle are described above because these are closely linked to the clinical stages. Differential diagnosis. The clinical disease closely resembles the protozoal disease babesiosis. Prevention and control. Offspring of immune carriers resist infection up to 6 months of age because of passive immunity. Vector control and attention to hygiene are essential, such as between-animal rinsing in disinfectant of mechanical vectors such as dehorners. There is no entirely effective means, however, to prevent and control the disease. Vaccination (killed whole organism) programs are not entirely effective, and vaccine should not be administered to pregnant cows. Neonatal isoerythrolysis may occur because of the antierythrocyte antibodies stimulated by one vaccine product. Vaccinated animals can still become infected and become carriers. The cattle vaccine has shown no efficacy in smaller ruminants, and there is no A. ovis vaccine. Identifying carriers serologically and treating with tetracycline during and/or after vector seasons may be an option. Removing carriers to a separate herd is also an approach. Interstate movement of infected animals is regulated. Treatment. Oxytetracycline, administered once, helps reduce the severity of the infection during the developmental stage. Other tetracycline treatment programs have been described to help control carriers. ii. Babesiosis (red water, Texas cattle fever, cattle tick fever) Etiology. Babesia bovis and Ba. bigemina are protozoa that cause subclinical infections or disease in cattle. These are intraerythrocytic parasites. Babesia bovis is regarded as the more virulent of the two organisms. This disease is not seen in the smaller ruminants in the United States. Clinical signs and diagnosis. The more common presentation is liver and kidney failure due to hemolysis with icterus, hemoglobinuria, and fever. Hemoglobinuria indicates a poor prognosis. Acute encephalitis is a less common presentation and begins acutely with fever, ataxia, depression, deficits in conscious proprioception, mania, convulsions, and coma. The encephalitic form generally also has a poor prognosis. Sudden death may occur. Thin blood smears stained with Giemsa will show Babesia trophozoites at some stages of the disease, but lack of these cannot be interpreted as a negative. The trophozoites occur in a variety of shapes, such as piriform, round, or rod. Complement fixation, immunofluorescent antibody, and enzyme immunoassay are the most favored of the available serologic tests. Epizootiology and transmission. Babesiosis is present on several continents, including the Americas. In addition to domestic cattle, some wild ruminants, such as white-tailed deer and American buffalo, are also susceptible. Bos indicus breeds have resistance to the disease and the tick vectors. Innate resistance factors have been found in all calves. If infected, these animals will not show many signs of disease during the first year of life and will become carriers. Stress can cause disease development. Necropsy findings. Signs of acute hemolytic crisis are the most common findings, including hepatomegaly, splenomegaly, dark and distended gallbladder, pale tissues, thin blood, scattered hemorrhages, and petechiation. Animals dying after a longer course of disease will be emaciated and icteric, with thin blood, pale kidneys, and enlarged liver. Pathogenesis. The protozoon is transmitted by the cattle fever ticks Boophilus annulatus, B. microplus, and B. decoloratus; these one-host ticks acquire the protozoon from infected animals. It is passed transovarially, and both nymph and adult ticks may transmit to other cattle. Only B. ovis is transmitted by the larval stage. Clinical signs develop about 2 weeks after tick infestations or mechanical transmission but may develop sooner with the mechanical transmission. Hemolysis is due to intracellular reproduction of the parasites and occurs intra- and extravascularly. In addition to the release of merozoites, proteolytic enzymes are also released, and these contribute to the clinical metabolic acidosis and anoxia. The development of the encephalitis form is believed to be the result of direct invasion of the central nervous system, disseminated intravascular coagulation, capillary thrombosis by the parasites and infarction, and/or tissue anoxia. Differential diagnosis. In addition to anaplasmosis, other differentials for the hemolytic form of the disease are leptospirosis, chronic copper toxicity, and bacillary hemoglobinuria. Several differentials in the United States for the encephalitic presentation include rabies, nervous system coccidiosis, polioencephalomalacia, lead poisoning, infectious bovine rhinotracheitis, salt poisoning, and chlorinated hydrocarbon toxicity. Prevention and control. Control or eradication of ticks and cleaning of equipment to prevent mechanical transmission, as noted in Section III,A,3,a,i, are important preventive measures. Some vaccination approaches have been effective, but a commercial product is not available. Treatment. Supportive care is indicated, including blood transfusions, fluids, and antibiotics. Medications such as diminazene diaceturate, phenamidine diisethionate, imidocarb diprionate, or amicarbalide diisethionate are most commonly used. Treatment outcomes will be either elimination of the parasite or development of a chronic carrier state immune to further disease. Research complications. This is a reportable disease in the United States. iii. Coccidiosis Etiology. Coccidiosis is an important acute and chronic protozoal disease of ruminants. In young ruminants, it is characterized primarily by hemorrhagic diarrhea. Adult ruminants may carry and shed the protozoa, but they rarely display clinical signs. Intensive rearing and housing conditions and stress increase the severity of the disease in all age groups. Coccidia are protozoal organisms of the phylum Apicomplexa, members of which are obligatory intracellular parasites. There are at least 11 reported species of coccidia in sheep, of which several are considered pathogenic: Eimeria ashata, E. crandallis, and E. ovinoidalis ( Schillhorn van Veen, 1986 ). At least 9 species of Eimeria have been recognized in the goat ( Foreyt, 1990 ). Eimeria ninakohlyakimovae, E. arloingi, and E. christenseni are regarded as the most pathogenic. Eimeria bovis and E. zuernii (highly pathogenic), and E. auburnensis and E. alabamensis (moderately pathogenic), are among the 13 species known to infect cattle. Eimeria zuernii is more commonly seen in older cattle and is the agent of "winter coccidiosis." Clinical signs and diagnosis. Hemorrhagic diarrhea develops 10 days to 3 weeks after infection. Fecal staining of the tail and perineum will be present. Animals will frequently display tenesmus; rectal prolapses may also develop. Anorexia, weight loss, dehydration, anemia, fever (infrequently), depression, and weakness may also be seen in all ruminants. The diarrhea is watery and malodorous and will contain variable amounts of blood and fibrinous, necrotic tissues. The intestinal hemorrhage may subsequently lead to anemia and hypoproteinemia. Depending on the predilection of the coccidial species for small and/or large intestines, malabsorption of nutrients or water may occur, and electrolyte imbalances may be severe. Concurrent disease with other enteropathogens may also be part of the clinical picture. In sheep, secondary bacterial infection with organisms such as Fusobacterium necrophorum may ensue. Young goats may die peracutely or suffer severe anemia from blood loss into the bowel. Older goats may lose the pelleted form of feces. Cattle may have explosive diarrhea and develop anal paralysis. The disease is usually diagnosed by history and clinical signs. Numerous oocysts will frequently be observed in fresh fecal flotation (salt or sugar solution) samples as the diarrhea begins. Laboratory results are usually reported as number of oocysts per gram of feces. Coccidia seen on routine fecal evaluations reflect shedding, possibly of nonpathogenic species, without necessarily being indicative of impending or resolving mild disease. Epizootiology and transmission. As noted, coccidiosis is a common disease in young ruminants. In goats, young animals aged 3 weeks to 5 months are primarily affected, but isolated outbreaks in adults may occur after stressful conditions such as transportation or diet changes. Coccidia are host-specific and also host cell-specific. The disease is transmitted via ingestion of sporulated oocysts. Coccidial oocysts remain viable for long periods of time when in moist, shady conditions. Necropsy. Necropsies provide information on specific locations and severity of lesions that correlate with the species involved. Ileitis, typhlitis, and colitis with associated necrosis and hemorrhage will be observed. Mucosal scrapings will frequently yield oocysts. Various coccidial stages associated with schizogony or gametogony may be observed in histopathological sections of the intestines. Fibrin and cellular infiltrates will be found in the lamina propria. Pathogenesis. This parasite has a complex life cycle in which sexual and asexual reproduction occurs in gastrointestinal enterocytes ( Speer, 1996 ). The severity of the disease is correlated primarily with the number of ingested oocysts. Specifics of life cycles vary with the species, and those characteristics contribute to the pathogenicity. In most cases, the disease is well established by the time clinical signs are seen. Oocysts must undergo sporulation over a 3- to 10-day period in the environment. After ingestion of the sporulated oocysts, sporozoites are released and penetrate the intestinal mucosa and form schizonts. Schizonts initially undergo replication by fission to form merozoites and eventually undergo sexual reproduction, forming new oocysts. The organisms cause edema and hyperemia; penetration into the lamina propria may lead to necrosis of capillaries and hemorrhage. Differential diagnosis. Differential diagnoses include the many enteropathogens associated with acute diarrhea in young ruminants: cryptosporidia, colibacilli, salmonella, enterotoxins, Yersinia, viruses, and other intestinal parasites such as helminths. In cattle, for example, bovine viral diarrhea virus and helminthiasis caused by Ostergia must be considered. Management factors, such as dietary-induced diarrheas, are also differentials. In older animals, differentials in addition to stress are malnutrition, grain engorgement, and other intestinal parasitisms. Prevention and control. Good management practices will help prevent the disease. Oocysts are resistant to disinfectants but are susceptible to dry or freezing conditions. Proper sanitation of animal housing and minimizing overcrowding are essential. Coccidiostats added to the feed and water are helpful in preventing the disease in areas of high exposure. Treatment. Affected animals should be isolated. On an individual basis, treatment should also include provision of a dry, warm environment, fluids, electrolytes (orally or intravenously), antibiotics (to prevent bacterial invasion and septicemia), and administration of coccidiostats. Coccidiostats are preferred to coccidiocidals because the former allow immunity to develop. Although many coccidial infections tend to be self-limiting, sulfonamides and amprolium may be used to aid in the treatment of disease. Other anticoccidial drugs include decoquinate, lasalocid, and monensin; labels should be checked for specific approval in a species or specific indications. Animals treated with amprolium should be monitored for development of secondary polioencephalomalacia. Pen mates of affected animals should be considered exposed and should be treated to control early stages of infection. Mechanisms of immunity have not been well defined but appear to be correlated with the particular coccidial species and their characteristics (for example, the extent of intracellular penetration). Immunity may result when low numbers are ingested and there is only mild disease. Immunity also may develop after more severe infections. iv. Cryptosporidiosis Etiology. Cryptosporidium organisms are a very common cause of diarrhea in young ruminants. Four Cryptosporidium species have been described in vertebrates: C. baileyi and C. meleagridis in birds and C. parvum and C. muris in mammals. Cryptosporidium parvum is the species affecting sheep ( Rings and Rings, 1996 ). Debate continues regarding whether there are definite host-specific variants. Clinical signs and diagnosis. Cryptosporidiosis is characterized by protracted, watery diarrhea and debilitation. The diarrhea may last only 6–10 days or may be persistent and fatal. The diarrhea is watery and yellow, and blood, mucus, bile, and undigested milk may also be present. Infected animals will display tenesmus, anorexia and weight loss, dehydration, and depression. In relapsing cases, animals become cachectic. Overall, morbidity will be high, and mortality variable. Mucosal scrapings or fixed stained tissue sections may be useful in diagnosis. The disease is also diagnosed by detecting the oocysts in iodine-stained feces or in tissues stained with periodic acid-Schiff stain or methenamine silver. Cryptosporidium also stains red on acid-fast stains such as Kinyoun or Ziehl-Neelsen. Fecal flotations should be performed without sugar solutions or with sugar solutions at specific gravity of 1.27 (Foryet, 1990). Fecal immunofluorescent antibody (IFA) techniques have also been described. Epizootiology and transmission. Younger ruminants are commonly affected: lambs, kids (especially kids between the ages of 5 and 10 days old), and calves less than 30 days old. Like other coccidians, Cryptosporidium is transmitted via the fecal-oral route. In addition to local contamination, water supplies have also been sources of the infecting oocysts. The oocysts are extremely resistant to desiccation in the environment and may survive in the soil and manure for many months. Necropsy findings. The lesions caused by Cryptosporidium are nonspecific. Animals will be emaciated. Moderate enteritis and hyperplasia of the crypt epithelial cells with villous atrophy as well as villous fusion, primarily in the lower small intestines, will be present. Cecal and colonic mucosae may sometimes be involved. Gastrointestinal smears may be made at necropsy and stained as described above. Pathogenesis. Although Cryptosporidium infections are clinically similar to Eimeria infections ( Moore, 1989 ), Cryptosporidium, in contrast to Eimeria, invades just under the surface but does not invade the cytoplasm of enterocytes. There is no intermediate host. The oocysts are half the size of Eimeria oocysts and are shed sporulated; they are, therefore, immediately infective. Within 2–7 days of exposure, diarrhea and oocyst shedding occur. The diarrhea is the result of malabsorption and, in younger animals, intraluminal milk fermentation. Autoinfection within the lumen of the intestines may also occur and result in persistent infections. In addition, several other pathogens may be involved, such as concurrent coronavirus and rotavirus infections in calves. Environmental stressors such as cold weather increase mortality. Intensive housing arrangements increase morbidity and mortality. Differential diagnosis. Other causes of diarrhea in younger ruminants include rotavirus, coronavirus, and other enteric viral infections; enterotoxigenic Escherichia coli; Clostridium; other coccidial pathogens; and dietary causes (inappropriate use of milk replacers). In addition, these other agents may also be causing illness in the affected animals and may complicate the diagnosis and the treatment picture. Eimeria is more likely to cause diarrhea in calves and lambs at 3–4 weeks of age. Giardia organisms may be seen in fecal preparations from young ruminants but are not considered to play a significant role in enteric disease. Prevention and control. Precautions should be taken when handling infected animals. Affected animals must be removed and isolated as soon as possible. Animal housing areas should be disinfected with undiluted commercial bleach or 5% ammonia. Formalin (10%) fumigation has proven successful (Foryet, 1990). After being cleaned, areas should be allowed to dry thoroughly and should remain unpopulated for a period of time. Because enteric disease often is multifactorial, other pathogens should also be considered, and management and husbandry should be examined. Treatment. No known drug treatment is available. The disease is generally self-limiting, so symptomatic, supportive therapy aimed at rehydrating, correcting electrolyte and acid-base balance, and providing energy is often effective. Supplementation with vitamin A may be helpful. Age resistance begins to develop when the animals are about 1 month old. Research complications. Cryptosporidiosis is a zoonotic disease. It is easily spread from calves to humans, for example, even as the result of simply handling clothing soiled by calf diarrhea. Adult immunocompetent humans are reported to experience watery diarrhea, cramping, flatulence, and headache. The disease can be life-threatening in immunocompromised individuals. v. Giardiasis Etiology. Giardia lamblia (also called G. intestinalis and G. duodenalis) is a flagellate protozoon. Giardiasis is a worldwide protozoal-induced diarrheal disease of mammals and some birds ( Kirkpatrick, 1989 ), but it not considered to be a significant pathogen in ruminants. Clinical signs and diagnosis. Diarrhea may be continuous or intermittent, is pasty to watery, is yellow, and may contain blood. Animals exhibit fever, dehydration, and depression. Chronic cases may result in a "poor doer" syndrome with weight loss and unthriftiness. Giardia can be diagnosed by identifying the motile piriform trophozoites in fresh fecal mounts. Oval cysts can be floated with zinc sulfate solution (33%). Standard solutions tend to be too hyperosmotic and to distort the cysts. Newer enzyme-linked immunosorbent assay (ELISA) and IFA tests are sensitive and specific. Epizootiology and transmission. Giardia infection may occur at any age, but young animals are predisposed. Chronic oocyst shedding is common. Transmission of the cyst stage is fecal-oral. Wild animals may serve as reservoirs. Necropsy findings. Gross lesions may not be evident. Villous atrophy and cuboidal enterocytes may be evident histologically. Pathogenesis. Following ingestion, each Giardia cyst releases four trophozoites, which attach to the enterocytes of the duodenum and proximal jejunum and subsequently divide by binary fission or encyst. The organism causes little intestinal pathology, and the cause of diarrhea is unknown but is thought to be related to disruption of digestive enzyme function, leading to malabsorption. Disturbances in intestinal motility may also occur ( Rings and Rings, 1996 ). Prevention and control. Intensive housing and warm environments should be minimized. Cysts can survive in the environment for long periods of time but are susceptible to desiccation. Effective disinfectants include quaternary ammonium compounds, bleach-water solution (1:16 or 1:32), steam, or boiling water. After cleaning, areas should be left empty and allowed to dry completely. Treatment. Giardia has been successfully treated with oral metronidazole. Benzimidazole anthelmintics are also effective, but these are not approved for use in animals for this purpose. Research complications. Giardia is zoonotic. Precautions should be taken when handling infected animals. vi. Neosporosis Etiology. Neosporosis is a common, worldwide cause of bovine abortion caused by the protozoal species Neospora caninum. Abortions have also been reported in sheep and goats. Neonatal disease is seen in lambs, kids, and calves. Until 1988, these infections were misdiagnosed as caused by Toxoplasma gondii. Some similarities exist between the life cycles and pathogeneses of both organisms. Clinical signs and diagnosis. Abortion is the only clinical sign seen in adult cattle and occurs sporadically, endemically, or as abortion storms. Bovine abortions occur between the third and seventh month of gestation; fetal age at abortion correlates with the parity of the dam as well as with pattern of abortion in the herd. Although cows that abort tend to be culled after the first or second abortion, repeated N. caninum- caused abortions will occur progressively later in gestation (up to about 6 months) and within a shorter time frame in the same cow ( Thurmond and Hietala, 1997 ). Although infections in adults are asymptomatic other than the abortions, decreased milk production has been noted in congenitally infected cows. Many Neospo ra-infected calves will be born asymptomatic. Weakness will be evident in some infected calves, but this resolves. Rare clinical signs include exophthalmos or asymmetric eyes, weight loss, ataxia, hyperflexion or hyperextension of all limbs, decreased patellar reflexes, and loss of conscious proprioception. Some fetal deaths will occur, and resorption, mummification, autolysis, or stillbirth will follow. Immunohistochemistry and histopathology of fetal tissue are the most efficient and reliable means of establishing a postmortem diagnosis. Serology (IFA and ELISA) is useful, including precolostral levels in weak neonates, but this indicates only exposure. Titers of dams will not be elevated at the time of abortion; fetal serology is influenced by the stage of gestation and course of infection. Earlier and rapid infections are less likely to yield antibodies against Neospora. None of the currently available tests is predictive of disease. Epizootiology and transmission. The parasite is now acknowledged to be widespread in dairy and cattle herds. The life cycle of N. caninum is complex, and many aspects remain to be clarified. The definitive host is the dog ( McAllister et al., 1998 ). Placental or aborted tissues are the most likely sources of infection for the definitive host and play a minor role in transmission to the intermediate hosts. The many intermediate hosts include ruminants, deer, and horses. Transplacental transmission is the major mode of transmission in dairy cattle and is the means by which a herd's infection is perpetuated. A less significant mode of transmission is by ingestion of oocysts, which sporulate in the environment or in the intermediate host's body. Reactivation in a chronically infected animal's body is the result of rupture of tissue cysts in neural tissue. Seropositive immunity does not protect a cow from future abortions. Many seropositive cows and calves will never abort or show clinical signs, respectively. Some immunological cross-reactivity may exist among Neospora, Cryptosporidia, and Coccidium. Necropsy findings. Aborted fetuses will usually be autolysed. In those from which tissue can be recovered, tissue cysts are most commonly found in the brain. Spinal cord is also useful. Histological lesions include mild to moderate gliosis, nonsuppurative encephalitis, and perivascular infiltration by mixed mononuclear cells. Pathogenesis. As with Toxoplasma, cell death is the result of intracellular multiplication of Neospora tachyzoites. Neospora undergoes sexual replication in the dog's intestinal tract, and oocysts are shed in the feces. The intermediate hosts develop nonclinical systemic infections, with tachyzoites in several organs, and parasites then localize and become encysted in particular tissues, especially the brain. Infections of this type are latent and lifelong. Except when immunocompromised, most cattle do not usually develop clinical signs and do not have fetal loss. Fetuses become infected, leading to fetal death, mid-gestation abortions, or live calves with latent infections or congenital brain disease. It usually takes 2–4 weeks for a fetus to die and to be expelled. Many aspects of the role of the maternal immune response and pregnancy-associated immunodeficiency in the patterns of Neospora abortions remain to be elucidated. Differential diagnosis. Even when there is a herd history of confirmed Neospora abortions, leptospirosis, bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), salmonellosis, and campylobacteriosis should be considered. BVDV in particular should be considered for abortion storms. Differentials for weak calves are BVDV, perinatal hypoxia following dystocia (immediate postpartum time), bluetongue virus, Toxoplasma, exposure to teratogens, or congenital defects. Prevention and control. The primary preventive measure is preventing contact with contaminated feces. Oocysts will not survive dry environments or extremes of temperature. Dog populations should be controlled, and dogs and other canids should not have access to placentas or aborted fetuses. Dogs should also be restricted from feed bunks and other feed storage areas. Preventive culling is not economically practical for most producers. A vaccine recently became available. If embryo transfer is practiced, recipients should be screened serologically before use. Treatment. There is no known treatment or immunoprophylaxis. vii. Sarcocystosis Etiology. Sarcocystosis is the disease caused by the cyst-forming sporozoon Sarcocystis. Sarcocystis capricanus, S. ovi-canus, and S. tenella are the species that infect sheep and goats. Sarcocystis cruzi, S. hirsuta, and S. hominis are the species that infect cattle. Definitive hosts are carnivores, and all ruminant species are intermediate hosts. Clinical signs and diagnosis. Clinical signs of sarcocystosis infection are seen in cattle during the stage when the parasite encysts in soft tissues. Often the infections are asymptomatic. Fever, anemia, ataxia, symmetric lameness, tremors, tail-switch hair loss, excessive salivation, diarrhea, and weight loss are clinical signs. Abortions in cattle occur during the second trimester and in smaller ruminants 28 days after ingestion of the sporulated oocysts. Definitive diagnosis is based on finding merozoites and meronts in neural tissue lesions. Clinical hematology results include decreased hematocrit, decreased serum protein, and prolonged prothrombin times. Sarcocystis-specific IgG will increase dramatically by 5–6 weeks after infection. There is no cross-reaction between Sarcocystis and Toxoplasma. Epizootiology and transmission. Infection rates among cattle in the United States are estimated to be very high. Transmission is by ingestion of feed and water contaminated by feces of the definitive hosts. Dogs are the definitive hosts for the species that infect the smaller ruminants. Cats, dogs, and primates (including humans when S. hominis is involved) are the definitive hosts for the species that infect cattle. Necropsy. Aborted fetuses may be autolysed. Lesions in neural tissues, including meningoencephalomyelitis, focal malacia, perivascular cuffing, neuronal degeneration, and gliosis, are most marked in the cerebellum and midbrain. Lesions may be found in other tissues, such as lymphadenopathy, and hemorrhages may be found in muscles and on serous surfaces. Cysts in cardiac and skeletal muscles are common incidental findings during necropsies. Pathogenesis. Ingestion of muscle flesh from an infected ruminant results in Sarcocystis cysts' being broken down in the carnivore's digestive system, release of bradyzoites, infection of intestinal mucosal cells by the bradyzoites, differentiation into sexual stages, fusion of the male and female gametes to form oocysts, and shedding as sporocysts by the definitive hosts. The sporocysts are eaten by the ruminant and penetrate the bowel walls; several stages of development occur in endothelial cells of arteries. Merozoites are the form that enters soft tissues, such as muscle, and subsequently encysts. Prevention and control. Feed supplies of ruminants must be protected from fecal contamination by domestic and wild carnivores. These animals should be controlled and must also not have access to carcasses. In larger production situations, monensin may be fed as a prophylactic measure. Treatment. Monensin fed during incubation is prophylactic, but the efficacy in clinically affected cattle is not known. viii. Toxoplasmosis Etiology. Toxoplasmosis is caused by the obligate intracellular protozoon Toxoplasma gondii, a coccidial parasite of the family Eimeridae. Cats are the only definitive hosts, and several warm-blooded animals, including ruminants, have been shown to be intermediate hosts. The disease is a major cause of abortion in sheep and goats and less common in cattle. Clinical signs and diagnosis. Clinical signs depend on the organ or tissue parasitized. Toxoplasmosis is typically associated with placentitis, abortion, stillbirths, or birth of weak young ( Underwood and Rook, 1992 ; Buxton, 1998 ). It has also been shown to cause pneumonia and nonsuppurative encephalitis. The enteritis at the early stage of infection may be fatal in some hosts. Hydrocephalus does not occur in animals as it does in human fetal Toxoplasma infections. Rare clinical presentations in ruminants include retinitis and chorioretinitis; these are usually asymptomatic. Infection of the ewe during the first trimester usually leads to fetal resorption, during the second trimester leads to abortion, and during the third trimester leads to birth of weak to normal lambs with subsequent high perinatal mortality. Congenitally infected lambs may display encephalitic signs of circling, incoordination, muscular paresis, and prostration. In sheep, weak young will develop normally if they survive the first week after birth. Infected adult sheep show no systemic illness. Infected adult goats, however, may die. Diagnosis may be difficult, and biological, serological, and histological methods are helpful. Serological tests are the most readily available. Complement fixation and the Sabin-Feldman antibody test may assist in diagnosis. Antibodies found in fetuses are indicative of congenital infection and are typically detectable 35 days after infection; fetal thoracic fluid is especially useful in demonstrating serological evidence of exposure. Biological methods, such as tissue culture or inoculation of mice with maternal body fluids, or with postmortem or necropsy tissues, are more time-consuming and expensive. Epizootiology and transmission. This protozoon is considered ubiquitous. Fifty percent (50%) of adult western sheep and 20% of feedlot lambs have positive hemagglutination titers (1:64 or higher) ( Jensen and Swift, 1982 ). Transmission among the definitive host is by ingestion of tissue cysts. Necropsy findings. At necropsy, placental cotyledons contain multiple small white areas that are sites of necrosis, edema, and calcification. Fetal brains may show nonspecific lesions such as coagulative necrosis, nonsuppurative encephalomyelitis, pneumonia, myocarditis, and hepatitis. Histologically, granulomas with Toxoplasma organisms may be seen in the retina, myocardium, liver, kidney, brain, and other tissues. Impression smears of these tissues, stained appropriately (e.g., with Giemsa), provide a rapid means of diagnosis. Identification of the organism in tissue sections (especially of the heart and the brain) also confirms the findings. Toxoplasma gondii is crescent-shaped, with a clearly visible nuclei, and will be found within macrophages. Pathogenesis. The protozoon has three infectious stages: the tachyzoite, the bradyzoite, and the sporozoite within the oocyst. The definitive hosts, felids, become infected by ingesting cyst stages in mammalian tissues, by ingesting oocysts in feces, and by transplacental transfer. Ingested zoites invade epithelial cells and eventually undergo sexual reproduction, resulting in new oocysts, which the cats will shed in the feces. Cats rarely show clinical signs of infection. One cat can shed millions of oocysts in 1 gm of feces, but the asymptomatic shedding takes place for only a few weeks in its life. Oocysts sporulate in cat feces after 1 day. Ruminants are intermediate hosts of toxoplasmosis and become infected by ingesting sporulated oocyst-contaminated water or feed. As in the definitive host, the ingested sporozoite invades epithelial cells within the intestine but also further invades the bloodstream and is transported throughout the host. The organism migrates to tissues such as the brain, liver, muscles, and placenta. Placental infection develops about 14 days after ingestion of the oocysts. The damage caused by an infection is due to multiplication within cells. Toxoplasma does not produce any toxin. Differential diagnosis. Differentials for abortion include Campylobacter, Chlamydia, and Q fever. Prevention and control. Feline populations on source farms should be controlled. Eliminating contamination of feed and water with cat feces is the best preventive measure. Sporulated oocysts can survive in soil and other places for long periods of time and are resistant to desiccation and freezing. Vaccines for abortion prevention in sheep are available in New Zealand and Europe. Treatment. Toxoplasmosis treatment is ineffective, although feeding monensin during pregnancy may be helpful ( Underwood and Rook, 1992 ). (Monensin is not approved for this use in the Unites States.) Weak lambs that survive the first week after birth will mature normally and will not deliver Toxoplasma- infected young. Research complications. Because toxoplasmosis is zoonotic, precautions must be taken when handling tissues from any abortions or neurological cases. Infections in immunocompromised humans have been fatal. ix. Trichomoniasis Etiology. Trichomoniasis is an insidious venereal disease of cattle caused by Tritrichomonas (also referred to as Trichomonas) fetus, a large, pear-shaped, flagellated protozoon. The organism is an obligate parasite of the reproductive tract, and it requires a microaerophilic environment to establish chronic infections. In the United States, it is now primarily a disease seen in western beef herds. There are many similarities between trichomoniasis and campylobacteriosis; both diseases cause herd infertility problems. Clinical signs and diagnosis. Clinical signs include infertility manifested by high nonpregnancy rates as well as periodic pyometras and abortions during the first half of gestation. Often the problem is not recognized until herd pregnancy checks indicate many "open," delayed-estrus, late-bred cows, or cows with postcoital pyometras. The abortion rate varies from 5% to 30%, and placentas will be expelled or retained. Tritrichomonas fetus also causes mild salpingitis but this does not result in permanent damage. Other than these manifestations, infection with T. fetus causes no systemic signs. Diagnosis is based on patterns of infertility and pyometras. For example, pyometras in postcoital heifers or cows are suggestive of this pathogen. Diagnostic methods include identifying or culturing the trichomonads from preputial smegma, cervicovaginal mucus, uterine exudates, placental fluids, or abomasal contents of aborted fetuses. Other nonpathogenic protozoa from fecal contamination may be present in the sample. The trichomonad has three anterior flagellae, one posterior flagella, and an undulating membrane; it travels in fluids with a characteristic jerky movement. Culturing must be done on specific media, such as Diamond's or modified Pastridge. Epizootiology and transmission. All transmission is by venereal exposure from breeding bulls or cows or, in some cases, contaminated breeding equipment. Necropsy findings. Nonspecific lesions, such as pyogranulomatous bronchopneumonia of fetuses and placentitis, may be seen in aborted material; some cases will have no gross lesions. Histologically, trichomonads may be visible in the fetal lung lesions and the placenta; those tissues are also the most useful for culturing. Pathogenesis. Tritrichomonas fetus colonizes the female reproductive tract, and subsequent clinical manifestations may be related to the size of the initial infecting dose. Tritrichomonas fetus does not interfere with conception. Embryonic death occurs within the first 2 months of infection. Affected cows will clear the infection over a span of months and maintain immunity for about 6 months. Infections in younger bulls are transient; apparently organisms are cleared by the bulls' immune systems and are dependent on exposure to infected females. Older bulls become chronic carriers, probably because of the ability of T. fetus to colonize deeper epithelial crypts of the prepuce and penis. Differential diagnosis. Campylobacteriosis is the other primary differential for reduced reproductive efficiency of a herd. Other venereal diseases should be considered when infertility problems are noted in a herd: brucellosis, mycoplasmosis, ureaplasmosis, and infectious pustular vulvovaginitis. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. A bacterin vaccine is available. Heifers, cows, and breeding bulls are vaccinated subcutaneously twice at 2 to 4 week intervals, with the booster dose administered 4 weeks before breeding season starts. Similar timing is recommended for administration of the annual booster; a long, anamnestic response does not occur. Bulls used for artificial insemination (AI) are screened routinely for T. fetus (and Campylobacter). AI reduces but does not eliminate the disease. The use of younger, vaccinated bulls is recommended in all circumstances. New animals should be tested before introduction to the herd. Control measures also include culling affected cows or else removing them from the breeding herd for 3 months to rest and clear the infection. Culling chronically infected bulls is strongly recommended. Treatment. Imidazole compounds have been effective, but the use of these is not permitted in food animals in the United States. Therapeutic immunizations are worthwhile when a positive diagnosis has been made. These will not curtail fetal losses but will shorten the convalescence of the affected cows and improve immunity of breeding bulls. Research complications. Trichomoniasis should be considered whenever natural service is used and fertility problems are encountered. b. Nematodes Nematodes are important ruminant pathogens that cause acute, chronic, subclinical, and clinical disease in adults and adolescents. The major helminths may cause gastroenteritis associated with intestinal hemorrhage and malnutrition. Nematodiasis is associated with grazing exposure to infective larvae; animals procured for research may have had exposure to these helminths. Mixed infections of these parasites are common. Generally, older animals develop resistance to some of the species; thus, animals between about 2 months and 2 years of age are most susceptible to infection. Because of the parasites' effects on the animals' physiology, infection in these younger animals is a major contributor to a cycle of poor nutrition and digestion, compromised immune responses, and impaired growth and development. Diagnosis is primarily based on fecal flotation techniques; however, because many of these nematodes have similar-appearing ova, hatching the ova and identifying the larvae are often required (Baermann technique). A number of anthelmintics can be used to interrupt nematode life cycles. See Zajac and Moore (1993) and Pugh et al. (1998 ) for comprehensive reviews of treatment and control of nematodiasis. i. Haemonchus contortus, H. placei (barber's pole worm, large stomach worm). Haemonchus contortus is the most important internal parasite of sheep and goats, and the brief description here focuses on the disease in the smaller ruminants. Haemonchus contortus and H. placei infections do occur in younger cattle and are similar to the disease in sheep. Haemonchus is extremely pathogenic, and the adults feed by sucking blood from the mucosa of the abomasum. Severe anemia may lead to death. Weight loss, decreased milk production, poor wool growth, and intermandibular and cervical edema due to hypoproteinemia ("bottle jaw") are also common clinical signs. Diarrhea is not seen in all cases but may sometimes be severe or chronic. The life cycle is direct. Under optimal conditions, a complete life cycle, from ingestion of larvae to eggs passed in the feces, occurs in 3 weeks. Embryonated eggs may develop into infective larvae within a week. Hypobiotic (arrested) larvae may exist for several months in animal tissues, serving as a reservoir for future pasture contamination. Periparturient increases in egg shedding by ewes contribute to large numbers of eggs spread on spring pastures ("spring rise"). Resistance to common anthelmintics has developed; currently ivermectin or benzimidazole products are used, with a minimum of 2 dosings given 2–3 weeks apart. Levamisole is also used. In severe cases, animals may benefit from blood transfusions and iron supplementation. Because animals may easily acquire infective larvae from ingestion of contaminated feed and from contaminated pastures, general facility sanitation and pasture management and rotation are important preventive and control measures. Haemonchus contortus is susceptible to destruction by freezing temperatures and dry conditions. ii. Ostertagia (Teladorsagia) circumcincta (medium stomach worm). Ostertagia circumcincta is also highly pathogenic for sheep and goats and, like Haemonchus, attaches to the abomasal mucosa and ingests blood. The life cycle is comparable to that of Haemonchus, including the phenomenon of hypobiosis. Larvae are especially resistant to cool temperatures, however, and will overwinter on pastures. Larvae-induced hyperplasia of abomasal epithelial glands results in a change of gastric pH from about 2.0 to near 7.0, leading to decreased digestive enzyme activity and malnutrition. Clinical syndromes are categorized as type 1 or type 2. The former type is associated with infections acquired in fall or spring and is seen in younger animals. The latter type is associated with emergence of the arrested larvae during spring or fall. Clinical signs include anemia, weight loss, decreased milk production, and unthriftiness. Diarrhea is usually seen in type 1 only; the symptoms of type 2 are comparable to those of Haemonchus infections. Anthelmintic drug therapy is comparable to that for Haemonchus, and drug resistance is also a problem with Ostertagia. iii. Ostertagia ostertagi (cattle stomach worm). Ostertagia ostertagi is the most pathogenic and most costly of the cattle nematodes. Ostertagia leptospicularis and O. bisonis also cause disease. The life cycle is direct, and egg shedding by the cattle may occur within 3–4 weeks of ingestion of infective larvae. Hypobiosis is also a characteristic of O. ostertagi. In the initial steps of infection, the normal processes of the abomasum are profoundly disrupted and cells are destroyed as the larvae develop within and emerge from the glands. Moroccan leather appearance is the term to describe the result of cellular hyperplasia and loss of cell differentiation. Cycles of infection and morbidity depend on geographic location, climate, and production cycles. Type 1 cattle ostertagiasis is associated with ingestion of large numbers of infective larvae, occurs in animals less than 2 years old, and causes diarrhea and anorexia. Type 2 ostertagiasis occurs in cattle 2–4 years old and older adults, is the result of the emergence and development of hypobiotic larvae, and in addition to signs seen with type 1, hypoproteinemia with development of submandibular edema, fever, and anemia is a clinical sign. Treatment options include ivermectin, fenbendazole, and levamisole; all are effective against the arrested larvae. Ostertagia is susceptible to desiccation but is resistant to freezing. iv. Trichostrongylus vitrinus, T. axei, T. colubriformis (hair worms). Trichostrongylus species favor cooler conditions, and some larvae may overwinter. Although the different species may affect different segments of the gastrointestinal tract, the nematode attaches to the mucosa and affects secretion and/or absorption. Trichostrongylus vitrinus and T. colubriformis infect the small intestine of sheep and goats. Trichostrongylus axei infects the abomasum of cattle, sheep, and goats and causes increases in abomasal pH similar to those seen with Ostertagia. Mucosal hyperplasia is not seen. The prepatent period is about 3 weeks. Affected animals display unthriftiness, anorexia, decreased milk production, weight loss, diarrhea, and dehydration. These worms show intermediate resistance to freezing temperatures and dry conditions. v. Nematodirus spathiger, N. battus (thread-necked worms). Nematodirus has lower pathogenicity compared with other gastrointestinal nematodes. The larvae cause small-intestinal necrosis and inflammation. The larvae are especially resistant to desiccation and freezing. Clinical signs include depression, weight loss, anorexia, and diarrhea. vi. Cooperia (small intestinal worms). Cooperia primarily affects younger animals less than 1 year of age. Cooperia curticei infects the small intestine of sheep and goats; C. punctata and C. oncophora infect the small intestines of cattle, sheep, and goats. Cooperia pectinata infects the stomach of cattle. Large numbers lead to clinical infection, and the prepatent period is about 3 weeks. Cooperia and Osteragia infections, like infections of some other nematode species, may act synergistically. Because these nematodes suck blood, clinical signs include anemia, gastrointestinal hemorrhage, and malnutrition. Animals exhibit weight loss, diarrhea, and depression. Cooperia species are intermediate to resistant to the effects of cold temperatures. vii. Strongyloides papillosus. Strongyloides papillosus is a small-intestinal parasite of sheep and cattle. Strongyloides has a different life cycle from that of many nematodes. The eggs, expelled in the feces, are larvated, and when they hatch, they form both free-living males and females or parasitic females only. The parasitic females may enter the gastrointestinal tract through oral ingestion, such as in milk during nursing, or through direct penetration of the skin. Penetrating larvae enter the bloodstream and are transported to the lungs, where they penetrate the alveoli, are coughed up, and then swallowed to ultimately enter the gastrointestinal tract. Adult females may reproduce in the small intestines by parthenogenesis. Clinical signs associated with Strongyloides include weight loss, diarrhea, unthriftiness, and dermatitis in cases where large numbers migrate through the skin. The current broad-spectrum anthelmintics are effective against Strongyloides. viii. Bunostomum trigonocephalum (hookworm). Bunostomum trigonocephalum is a hookworm that occasionally infects sheep in locales in the southwestern United States. Like Strongyloides, Bunostomum infection may involve oral ingestion or direct penetration of the skin (followed by tracheal migration and swallowing). The larvae mature in the small intestines and suck blood. Larvae are susceptible to desiccation and freezing. Heavy infection with Bunostomum may result in anemia, diarrhea, intestinal hemorrhage, edema, and weight loss. ix. Oesophagostomum columbianum, O. venulosum (nodule worms). Oesophagostomum spp. primarily infect the large intestine and occasionally the distal small intestine, causing nodule worm disease, or simply gut. Oesophagostomum columbianum and O. venulosum infect sheep and cattle. These nematodes may affect sheep from 3 months to 2 years of age, and the prepatent period is about 6 weeks. Larvae are highly sensitive to freezing and desiccation and rarely overwinter. Larvae penetrate the large-intestinal mucosa but occasionally move into the deeper areas of the intestinal wall near the serosa. The resultant inflammatory reaction may lead to the formation of a caseous nodule that may mineralize over time. Intestinal lesions may accelerate peristalsis, leading to diarrhea, or may inhibit peristalsis (later stages), resulting in constipation. Clinical signs include weakness, unthriftiness, alternating episodes of diarrhea and constipation, and severe weight loss. Nodular lesions are typical at necropsy. x. Chabertia ovis (large-mouth bowel worm). Chabertia ovis is a minor colon parasite of sheep, goats, and cattle and is seen primarily in sheep. Signs of infection are not usually seen in cattle. Prepatent periods are up to 50 days. Heavy infection, which may result from as few as 100 worms located at the proximal end of the colon, may lead to hemorrhagic mucoid diarrhea, weight loss, weakness, colitis, and mild anemia. xi. Trichuris (whipworms). Trichuris spp. are mildly pathogenic nematodes and are usually attached to the cecal mucosa. Trichuris has a rather long prepatent period, extending from 1 to 3 months. The oval eggs are double-operculated and survive well in pasture environmental extremes. The adult worms also have a characterisitic morphology, with one thicker end appearing as a whip handle. The nematodes cause a minor cecitis and will feed on blood. Clinical infection is rare and results in diarrhea with mucus and blood. Treatment and prevention methods are similar to those for other nematodes. xii. Dictyocaulus (lungworms). Dictyocaulus spp., or lungworms, are nematodes that cause varying clinical signs in ruminants. In sheep, Dictyocaulus filaria, Protostrongylus rufescens, and Muellerius capillaris cause disease; Dictyocaulus is the most pathogenic. Goats are infected by the same species as sheep, but infections are uncommon. Dictyocaulus viviparus is the only lungworm found in cattle, causing "fog fever." Infections with these parasites in the United States tend to be associated with cooler, moister climates. Lungworms induce a severe parasitic bronchitis (known as husk, or verminous pneumonia) in sheep between approximately 2 and 18 months of age. Sheep infected with any of the lungworm species may display coughing, dyspnea, nasal discharge, weight loss, unthriftiness, and occasionally fever. Coughing and dyspnea are symptoms in goats. Diagnosis is suggested by persistent coughing and nasal discharge and is confirmed by identifying larvae in the feces or adults in pathological samples. The Baermann technique, involving prompt examination of room-temperature feces, is usually used; zinc sulfate flotation is also used. Dictyocaulus has a direct life cycle. The adult worms reside in the large bronchi. Dictyocaulus produces embryonated eggs that are coughed up and swallowed; the eggs then hatch in the intestines, and larvae are expelled in the feces. The expelled larvae are infectious in about 7–10 days and, after ingestion, penetrate the intestinal mucosa and move through the lymphatics and blood into the lungs, where they develop into adults in about 5 weeks. Dictyocaulus filaria causes an especially severe bronchitis in sheep. Protostrongylus inhabits smaller bronchioles. Muellerius is of minor pathogenicity. Protostrongylus and Muellerius require the snail or slug as an intermediate host. Infection occurs through ingestion of infected snails; infections are less likely than those caused by the direct ingestion of Dictyocaulus larvae. Immunity wanes over a year. Viral and bacterial respiratory tract infections may be associated with the parasitic infection. Dictyocaulus viviparus causes the obvious signs in cattle. More severe illness is seen after infections with Cooperia and Ostertagia, because of a synergism between the nematodes even if the cattle are not currently infected with those parasites. Hypobiosis (arrested development of immature worms in lung tissue) is associated with Dictyocaulus infections; cattle will be silent carriers, showing no clinical signs and serving as a means for the infection to survive over winter or a dry season. Pastures can be heavily contaminated during the next grazing season. Necropsy lesions include bronchiolitis and bronchitis, atelectasis, and hyperplasia of peribronchiolar lymphoid tissue. Nematodes frequently reside in the bronchi of the diaphragmatic lung lobes and are frequently enmeshed with frothy exudate. Prevention and control of the disease involve appropriate pasture management. Elimination of intermediate hosts is important in sheep and goat pastures. In a laboratory setting, animals may be procured that are already harboring the disease. Infected animals can be treated with anthelmintics such as ivermectin or levamisole. Muellerius tends to be resistant to levamisole. There is no anthelmintic currently approved for goats, but fenbendazole, administered 2 weeks apart, has been effective for all three nematodes. Treating D. viviparus depends on the type and stage of life of the cattle; label directions must be followed. There is no vaccine for D. viviparus in the United States. Even if infections are not severe and do resolve with treatment, permanent lesions may be inflicted on the lung tissue. c. Cestodes (Tapeworms) i. Moniezia expansa and Thysanosoma actinoides infections. Tapeworms are rarely of clinical or economic importance. In younger animals, heavy infections result in potbellies, constipation or mild diarrhea, poor growth, rough coat, and anemia. Moniezia expansa, and less commonly Moniezia benedini, inhabit the small intestines of grazing ruminants. Moniezia expansa has the widest distribution of the tapeworm species in North America. Soil mites (Galumna spp. and Oribatula spp.) contribute to the life cycle as intermediate hosts, a period that lasts up to 16 weeks. Cysticercoids released from the mites are grazed, pass into the small intestines, and mature. No clinical or pathological sign is usually observed with Moniezia infection; diagnosis is made by observing the characteristic triangular-shaped eggs in fecal flotation examinations. Infection is treated with cestocides. Thysanosoma actinoides, or the fringed tapeworm, is a cestode that resides in the duodenum, bile duct, and pancreatic duct of sheep and cattle raised primarily west of the Mississippi River in the United States. Thysanosoma is of the family Anoplocephalidae. The life cycle is indirect, and the intermediate host is the psocid louse. Larval forms, or cysticercoids, are ingested by grazing animals, and the prepatent period is several months. Typically, no clinical signs are observed with Thysanosoma infection; nonetheless, liver damage, resulting in liver condemnation at slaughter, occurs. Necropsy lesions include bile and/or ductal hyperplasia and fibrosis. Thysanosoma is diagnosed premortem by identifying the gravid segments in the feces. ii. Abdominal or visceral cysticercosis. Abdominal or visceral cysticercosis is an occasional finding at slaughter. The so-called bladder worms typically affect the liver or peritoneal cavity and are the larval form of Taenia hydatigena, the common tapeworm of the dog family. Taenia hydatigena resides in the small intestines of canids, and its gravid segments, oncospheres, contaminate feed and water sources. After ingestion, the larvae penetrate the intestinal mucosa, are transported via the bloodstream to the liver, and cause migration tracts throughout the liver parenchyma. The larvae may leave the liver and migrate into the peritoneal cavity, where they attach and develop over the next 1–9 months into small fluid-filled bladders. The life cycle is completed only after these bladders are ingested by a carnivore, thus completing the maturation of the adult tapeworms. Although larval migration may cause nonspecific signs such as anorexia, hyperthermia, and weight loss, affected animals are usually asymptomatic. At necropsy, the bladder worms will be observed attached to the peritoneal or organ surfaces. Migration tracts may result in fibrosis and inflammation. Diagnosis is usually made at necropsy. Because of the migration through the liver, Fasciola hepatica is a differential diagnosis. Minimizing exposure to canine feces-contaminated feeds and water effectively interrupts the life cycle. Research animals may have been exposed prior to purchase. iii. Echinococcosis (hydatidosis, hydatid cyst disease). Echinococcosis, like cysticercosis, is an occasional finding at slaughter or necropsy. The hydatid cyst is the larval intermediate of the adult tapeworm Echinococcus granulosus, which resides in the small intestines of dogs and wild canids. Embryonated ova are expelled in the feces of the primary host and are ingested by herbivores, swine, and potentially humans. The eggs hatch in the gastrointestinal tract, and the oncospheres penetrate the mucosal lining, enter the bloodstream, and are transported to various organs such as the liver and lungs. The cystic structure develops and potentially ruptures, forming new cystic structures. Clinically, echinococcosis presents minimal clinical signs; unthriftiness or pneumonic lesions may be associated with infected organs. Cysts are typically observed at necropsy. Prevention should be aimed at decreasing fecal contamination of feed and water by canids. Additionally, tapeworm-infected dogs can be treated with standard tapeworm therapies. Treatment of infected ruminants is uncommon. iv. Gid. Coenuris cerebralis, the larval form of the canid tapeworm Taenia (Multiceps) multiceps, is the causative agent of the rare condition called gid. The disease occurs in ruminants as well as many other mammalian species. The larval parasite, ingested from fecal-contaminated food and water, invades the brain and spinal cord and develops as a bladder worm that causes pressure necrosis of the nervous tissues. The resultant signs of hyperesthesia, meningitis, paresis, paralysis, ataxia, and convulsions are observed. Diagnosis is usually made at necropsy. Eliminating transfer from the canid hosts prevents the disease. d. Trematodes i. Fascioliasis (liver fluke disease). Liver flukes are an important cause of acute and chronic disease in grazing sheep and cattle. There are three common species of flukes in ruminants of the continental United States: Fasciola hepatica, Fascioloides magna, and Dicrocoelium dendriticum. Fasciola hepática infections are primarily seen in Gulf Coast and western states. Fascioloides magna infections are typically seen in Gulf, Great Lake, and northwestern states, where ruminants share pasture with deer, elk, and moose. Dicrocoelium dendriticum infections occur only in New York State. Liver fluke eggs are passed in the bile and feces and hatch in 2–3 weeks to form the free-swimming miracidia. It is important to note that each fluke egg represents the source of eventually thousands of cercariae or metacercariae. The miracidia penetrate the body of an intermediate host (usually freshwater snails) and develop through sporocyst and redia stages, finally forming cercariae. (Dicrocoelium is unique because it utilizes a land snail that expels slime balls, each containing several hundred cercariae. These are eaten by a second intermediate host, the ant Formica fusca.) The cercariae leave the intermediate host, swim to grassy vegetation, lose their tail, and become a cystlike metacercaria. The metacercariae may remain in a dormant stage on the grass for 6 months or longer until ingested by a ruminant. The ingested metacercariae penetrate the small-intestinal wall and migrate through the abdominal cavity to the liver. There they locate in a bile duct, mature, and remain for up to 4 years. Acute liver fluke disease is related to the damage caused by the migration of immature flukes. Migratory flukes may lead to liver inflammation, hemorrhage, necrosis, and fibrosis. Fascioloides magna infections in sheep and goats can be fatal as the result of just one fluke tunneling through hepatic tissue. In cattle, infections are often asymptomatic because of the host's encapsulation of the parasite. Liver fluke damage may predispose to invasion by anaerobic Clostridium species such as C. novyi that could lead to fatal black disease or bacillary hemoglobinuria. Chronic disease may result from fluke-induced physical damage to the bile ducts and cholangiohepatitis. Blood loss into the bile may lead to anemia and hypoproteinemia. Liver damage also is evidenced by increases in liver enzymes such as γ-glutamyl transpeptidase (GGT). Persistent eosinophilia is also seen with liver fluke disease. Other clinical signs of liver fluke disease include anorexia, weight loss, unthriftiness, edema, and ascites. At necropsy, livers will be pale and friable and may have distinct migration tunnels along the serosal surfaces. Bile ducts will be enlarged, and areas of fibrosis will be evident. Diagnosis can be made from clinical signs and postmortem analyses. Blood chemistries suggestive of liver disease and eosinophilia support the diagnosis. Liver fluke control involves removal of the intermediate hosts. In a laboratory setting, liver fluke infection is unlikely. Nonetheless, incoming animals from pasture environments may be infected. Liver flukes can be treated by using the anthelmintic albendazole. ii. Rumen fluke infections (paramphistomosis). Paramphistomosis is an uncommon disease found in sheep and cattle in southern states. Paramphistomum microbothrioides and P. cervi inhabit the duodenum and rumen of affected sheep. Eggs are passed in the feces and hatch in approximately 1 month, and the miracidia penetrate the intermediate snail hosts. Cercariae develop in the snail over the next month, emerge, and encyst on grasses as metacercariae. When eaten, the metacercariae develop into adult flukes and attach to the mucosal lining. The life cycle is complete in approximately 100 days. The flukes cause localized injury to the mucosa and, by interfering with digestive processes, cause diarrhea and protein loss. Clinically, animals may experience anorexia, dehydration, weight loss, and diarrhea with or without blood. Mortality may reach 25%. Diagnosis is based on clinical findings as well as the identification of flukes or eggs in the feces. Animals can be treated with fluki-cides. Eliminating the intermediate host prevents the disease. e. Mites (Mange) Mites cause a chronic dermatitis. The principal symptom of these infections is intense pruritus. In addition, papules, crusts, alopecia, and secondary dermatitis are seen. Anemia, disruption of reproductive cycles, and increased susceptibility to other diseases may also occur. Mites are rare in ruminants in the United States, but infections of Sarcoptes and Psorergates mange must be reported to animal health officials. Ruminants in poorly managed facilities are generally the most susceptible to infection, and infections are more frequent during winter months. Diagnosis is based on signs, examination of skin scrapings, and response to therapy. No effective treatment for demodectic mange in large animals has been found. The differential for mite infestations is pediculosis. Several genera of mites may affect sheep. These have been eradicated from flocks in the United States or are very rare and include Psoroptes ovis (common scabies), Sarcoptes scabiei (head scabies, barn itch), Psorergates ovis (sheep itch mite), Chorioptes ovis (foot scabies, tail mange), and Demodex ovis (follicular mange). Goats can also be infected by sarcoptic, chorioptic, and psoroptic mange. The scabies mite Sarcoptes rupicaprae invades epidermal tissue and causes focal pruritic areas around the head and neck. The chorioptic mite, either Chorioptes bovis or C. caprae, does not invade epidermal tissue but rather feeds on dead skin tissue. The chorioptic mite prefers distal limbs, the udder, and the scrotum and can be a significant cause of pruritus. The psoroptic mite Psoroptes cuniculi commonly occurs in the ear canal and causes head shaking and scratching. Repeated treatments of lime sulfur, amitraz, or ivermectin may be effective ( Smith and Sherman, 1994 ). Goats are also susceptible to demodectic mange caused by Demodex caprae. Adult mites invade hair follicles and sebaceous glands. Pustules may develop with secondary bacterial infection. Psoroptes bovis continues to be present in cattle in the United States, although it has been eradicated from sheep. Chorioptes bovis typically infects lower hindlimbs, perineum, tail, and scrotum but can become generalized. The sarcoptic mange mite S. scabei can survive off the host, so fomite transmission is a factor. The mange usually begins around the head but then spreads. This parasite can be transmitted to humans. Demodex bovis infects cattle; nodules on the face and neck are typical. Demodex bovis infections may resolve without treatment. Lindane, coumaphos, malathion, and lime sulfur are used to treat Psoroptes and Psorergates. Ivermectin is effective against Sarcoptes and is approved for use in cattle. f. Lice (Pediculosis) Lice that infect ruminants are of the orders Mallophaga, biting or chewing lice, and Anoplura, sucking lice. These are wingless insects. Members of the Mallophaga are colored yellow to red; members of the Anoplura are blue gray. Lice produce a seasonal (winter-to-spring), chronic dermatitis. In sheep, biting lice include Damalinia (Bovicola) ovis (sheep body louse). Sucking lice that infect sheep include Linognathus ovillus (blue body louse) and L. pedalis (sheep foot louse). In goats, biting lice infection are caused by D. caprae (goat biting louse), D. limbatus (Angora goat biting louse), and D. crassipes. Sucking louse infections in goats are caused by L. stenopis and L. africanus. Damalinia bovis is the cattle biting louse. Sucking lice include L. vituli, Solenopotes capillatus, Haematopinus eurysternus, and H. quadripertusus. Pruritus is the most common sign and often results in alopecia and excoriation. The host's rubbing and grooming may not correlate with the extent of infestation. Hairballs can result from overgrooming in cattle. In severe cases, the organisms can lead to anemia, weight loss, and damaged wool in sheep and damaged pelts in other ruminants. Young animals with severe infestations of sucking lice may become anemic or even die. Pregnant animals with heavy infestations may abort. In sheep infected with the foot louse, lameness may result. Lice are generally species-specific. Those infecting ruminants are usually smaller than 5 mm. Goats may serve as a source of infection for sheep by harboring Damalinia ovis. Transmission is primarily by direct contact between animals. Transmission can also occur by attachment to flies or by fomites. Some animals are identified as carriers and seem to be particularly susceptible to infestations. Biting or chewing lice inhabit the host's face, lower legs, and flanks and feed on epidermal debris and sebaceous secretions. Sucking lice inhabit the host's neck, back, and body region and feed on blood. Lice eggs or nits are attached to hairs near the skin. Three nymphal stages, or instars, occur between egg and adult, and the growth cycle takes about 1 month for all species. Lice cannot survive for more than a few days off the host. All ruminant mite infestations are differentials for the clinical signs seen with pediculosis. Animals that are carriers should be culled, because these individuals may perpetuate the infection in the group. Lice are effectively treated with a variety of insecticides, including coumaphos, dichlorvos, crotoxyphos, avermectin, and pyrethroids. Label directions should be read and adhered to, including withdrawal times. Products should not be used on female dairy animals. Treatments must be repeated at least twice at intervals appropriate for nit hatches (about every 16 days) because nits will not be killed. Fall treatments are useful in managing the infections. Systemic treatments in cattle are contraindicated when there may be concurrent larvae of cattle grubs (Hypoderma lineatum and H. bovis). Back rubbers with insecticides, capitalizing on self-treatment, are useful for cattle. Sustained-release insecticide-containing ear tags are approved for use in cattle. g. Ticks Etiology. Ruminants are susceptible to many species of Ixodidae (hard-shell ticks) and Argasidae (softshell ticks). Many diseases, including anaplasmosis, babesiosis, and Q fever are transmitted by ticks. Clinical signs and diagnosis. Tick infestations are associated with decreased productivity, loss of blood and blood proteins, transmission of diseases, debilitation, and even death. Feeding sites on the host vary with the tick species. Ticks are associated with an acute paralytic syndrome called tick paralysis. This disease is characterized by ascending paralysis and may lead to death if the tick is not removed before the paralysis reaches the respiratory muscles. Diagnosis is based on identification of the species. Epizootiology and transmission. Ticks are not as host-specific as lice. Ticks are classified as one-host, two-host, or three-host; this refers to whether they drop off the host between larval and nymphal stages to molt. Pathogenesis of tick infestations. Patterns of feeding on the host differ between Argasidae and Ixodidae. The former feed repeatedly, whereas the latter feed once during each life stage. Pathogenesis of tick paralysis. Following a tick-feeding period of 4–6 days, the tick salivary toxin travels hematogenously to the myoneural junctions and spinal cord and inhibits nerve transmission. Removal of the ticks reverses the syndrome unless paralysis has migrated anteriorly to the respiratory centers of the medulla. In these cases, death due to respiratory failure occurs. Treatment. Ticks can be treated using systemic or topical insecticides. h. Other Parasites i. Nasal bots (nasal myiasis, head grubs). Nasal myiasis causes a chronic rhinitis and sinusitis. The disease is caused by the larval forms of the botfly Oestrus ovis. The botfly deposits eggs around the nostrils of sheep. The ova hatch, and the larvae migrate throughout the nasal cavity and sinuses, feeding on mucus and debris. In 2–10 months, the larvae complete their growing phase, migrate back to the nasal cavity, and are sneezed out. The mature larvae penetrate the soil and pupate for 1–1.5 months and emerge as botflies. Clinically, early in the disease course, animals display unique behaviors such as stamping, snorting, sneezing, and rubbing their noses against each other or objects. Hypersensitivity to the larvae occurs ( Dorchies et al., 1998 ). Later, mucopurulent nasal discharges associated with the larval-induced inflammation of mucosal linings will be observed. At necropsy, larvae will be observed in the nasal cavity or sinuses. Mild inflammatory reactions, mucosal thickening, and exudates will accompany the larvae. The disease is diagnosed by observing the behaviors or identifying organisms at necropsy. Up to 80% of a flock will potentially be infected; treatment should be employed on the rest of the flock. Ivermectins and other insecticides will eliminate the larvae; but treatment should be done in the early fall, when larvae are small. Fly repellents may be helpful at preventing additional infections. ii. Screwworm flies. Cochliomyia hominivorax (Callitroga americana) is the the screwworm that causes occasional disease in the southwestern United States along the Mexico border. Eradication programs have been pursued, and the disease is reportable. Large greenish flies lay large numbers of white eggs as shinglelike layers at the edges of open wounds (including docking and castration sites), soiled skin, or abrasions. Eggs hatch within 24 hr. Larvae are obligate parasites of living tissue, and the cycle is perpetuated because the increasingly large wound continues to be attractive to the next generation of flies. Larvae eventually drop off, pupate best in hot climates, and hatch in 3 weeks. Large cavities in parasitized tissue are formed, and lesions are characterized by malodor, large volumes of brown exudate, and necrosis. Single animals or entire herds may be affected. Treatment is intensive, with dressings and larvicidal applications. If there is no intervention, the host succumbs to secondary infections and fluid loss. Effective current control regimens include subcutaneous injection of ivermectin and programs that release sterile male flies. iii. Sheep keds ("sheep ticks"). In sheep and goats, sheep keds produce a chronic irritation and dermatitis with associated pruritus. The disease is caused by Melophagus ovinus, which is a flat, brown, blood-sucking, wingless fly; the term sheep tick is incorrectly used. The adult fly lives entirely on the skin of sheep. Females mate and produce 10–15 larvae following a gestation of about 10–12 days. The larvae attach to the wool or hair and then pupate for about 3 weeks. The adult female feeds on blood and lives for 4–5 months; the life cycle is completed in about 5–6 weeks. Infection is highest in fall and winter. Pruritus develops around the neck, sides, abdomen, and rump. In severe cases, anemia may occur. Keds can transmit bluetongue virus. Keds are diagnosed by gross or microscopic identification. Ivermectin or other insecticides are useful treatment agents. 5. Fungal Disease: Dermatophytes (Ringworm) Etiology. Dermatophytosis, or infection of the keratinized layers of skin, is caused mostly by species of the genera Trichophyton and Microsporum. The primary causes in sheep are T. mentagrophytes and T. verrucosum. In goats, the agents are T. mentagrophytes, M. canis, M. gypseum, T. verrucosum, T. schoenleinii, and Epidermophyton floccosum. In cattle, T. verrucosum is the primary causative agent. Dermatophytosis is a common fungal infection of the epidermis of cattle and is less common in sheep and goats. Clinical signs and diagnosis. Multiple, gray, crusty, circumscribed, hyperkeratotic lesions are characteristic of infection. Lesions will vary in size. In all ruminants, lesions will be around the head, neck, and ears. In goats and cattle, lesions will extend down the neck, and in cattle, lesions develop particularly around the eyes and on the thorax. Cattle lesions are unique in the marked crustiness, which progressively appears wartlike. Hair shafts become brittle and break off. Intense pruritus is often associated with the alopecic lesions. The disease can be diagnosed by microscopic identification of hyphae and conidia on the hairs following skin scraping and 20% potassium hydroxide digestion. Dermatophyte test media (DTM) cultures are the most reliable means to diagnose the fungus. Broken hairs from the periphery of the lesion are the best sources of the fungus. Epizootiology and transmission. Younger animals are more susceptible, and factors such as crowding, indoor housing, warm and humid conditions, and poor nutrition are also important. Transmission is by direct contact or by contact with contaminated fomites, such as equipment, fencing, or feed bunks. Pathogenesis. Incubation can be as long as 6 weeks. The organisms invade and multiply in hair shafts. Treatment. Spontaneous recovery occurs in all species in 1–4 months. Although cell-mediated immunity is considered important, other immune mechanisms are not well understood. Immunity may not be of long duration. Recovery is enhanced by correcting nutritional deficiencies and improving housing and ventilation problems. A number of topical treatments, such as 2–5% lime-sulfur solution, 3% captan, iodophors, thiabendazole, and 0.5% sodium hypochlorite, can be used. In severe cases, systemic therapy with griseofulvin may be successful. Prevention and control. The animals' environment and overall physical condition should be reassessed with particular attention to ventilation, crowding, sanitation, and nutrition. Pens should be thoroughly cleaned and disinfected. Research complications. Ringworm is a zoonotic disease. B. Genetic, Metabolic, Nutritional, and Management-Related Diseases 1. Genetic Diseases a. Entropion Inverted eyelids are a common inherited disorder of lambs and kids of most breeds. Generally, the lower eyelid is affected and turns inward, causing various degrees of trauma to the conjunctiva and cornea. Young animals will display tearing, blepharospasm, and photophobia initially. If the disorder is left uncorrected, corneal ulcers, perforating ulcers, uveitis, and blindness may occur. Placing a suture or a surgical staple in the lower eyelid and the cheek, effectively anchoring the lid in an everted position, successfully treats the condition. The procedure likely results in the formation of some degree of scar tissue within the lower lid, because when the suture eventually is removed, the condition rarely returns. Other treatments include the injection of a "bleb" of penicillin in the lid, regular manual correction over a 2-day period early in the animal's life, and application of ophthalmic ointments, powders, and solutions. Boric acid or 10% Argyrol solutions have been used as treatments. Because of the genetic predisposition, prevention of the condition requires removal of maternal or paternal carriers. b. β-Mannosidosis of Goats β-Mannosidosis is an autosomal recessive lysosomal storage disease of goats. The disease affects kids of the Nubian breed and is identified by intention tremors and difficulty or inability of newborns to stand. Cells of affected animals are vacuolated because of a lack of lysosomal hydroxylase, which results in accumulation of oligosaccharides. Newborn kids are unable to rise, and they have characteristic flexion of the carpal joint and hyperextension of the pastern joint. Kids are born deaf and with musculoskeletal deformities such as domed skull, small narrow muzzle, small palpebral fissures, enophthalmos, and depressed nasal bridge ( Smith and Sherman, 1994 ). Carrier adults can be identified by plasma measurements of β-mannosidase activity. c. Congenital Myotonia of Goats Caprine congenital myotonia is an inherited autosomal dominant disease that affects voluntary striated skeletal muscles. Goats with this disease are commonly known as fainting goats. "Fainting" is actually transient spasms of skeletal musculature brought about by visual, tactile, or auditory stimuli ( Smith and Sherman, 1994 ). Muscle fiber membranes appear to have fewer chloride channels than normal, resulting in decreased chloride conduction across the membrane, with subsequent increased membrane excitability and repetitive firing ( Smith and Sherman, 1994 ). Contractions of skeletal muscle are sustained for up to 1 min. Kids exhibit the condition by 6 weeks of age, and males appear to exhibit more severe clinical signs than females ( Smith and Sherman, 1994 ). Electromyographic studies produce an audible "dive-bomber" sound characteristic of hyper-excitable cell membranes ( Smith and Sherman, 1994 ). d. Inherited Conditions of Cattle i. Congenital erythropoietic porphyria. Congenital erythropoietic porphyria (CEP) is an autosomal recessive disease of cattle seen primarily in Holsteins, Herefords, and Shorthorns. The disease also occurs in Limousin cattle, humans, and some other species. In the homozygous recessive animal, symptoms of the disease may vary from mild to severe and occur at different times of the year and in different ages of animals. A reddish brown discoloration of teeth and bones is a characteristic of the disease, as is discolored urine, general weakness and failure to thrive, photosensitization, and photophobia. Bones are more fragile compared with bones of normal animals. A regenerative anemia occurs as the result of the shortened life span of erythrocytes, due to accumulations of porphyrins. The genetic defect is associated with low activity of an essential enzyme, uroporphyrinogen III synthase, in the porphyrin–heme synthesis pathway in erythrocytic tissue. The ranges in the presentation of the disease are believed to be related to varying cycles of porphyrin synthesis. Porphyrins are excreted in varying amounts in the urine and the discoloration fluoresces under a Wood's lamp. Diagnosis is based on these clinical and visible signs of porphyria; skin biopsy provides definitive diagnosis. Heterozygotes may have milder symptoms. Many other genetic defects, in all major organ systems, have been described in numerous breeds of cattle and are described in detail elsewhere ( "Large Animal Internal Medicine," 1996 ). In many cases, the genetic basis has been clarified, and associated defects also noted. Many defects are reported in particular breeds, but as crossbreeding increases and new breeds are developed, these traits are appearing in these animals. The bovine genome continues to be further characterized, and more linkage maps and gene locations are forthcoming ( Womack, 1998 ). Some bovine genetic defects are also regarded as models of genetic disease, such as leukocyte adhesion deficiency of Holstein cattle. Some of the more commonly reported defects include syndactyly in Holsteins and other breeds and Polydactyly in Simmentals; lysosomal storage diseases such as α-mannosidosis in some beef breeds; enzyme deficiencies such as citrulline-mia in Holsteins; and progressive degenerative myeloencephalopathy ("weaver") in Brown Swiss. ii. Goiter of sheep. A defect in the synthesis of thyroid hormone has been identified in Merino sheep ( Radostits et al., 1994 ). Lambs born with the defect have enlargement of the thyroid gland, a silky appearance to the wool, and a high degree of mortality. Edema, bowing of the legs, and facial abnormalities have also been noted in animals with this disorder. Immaturity of the lungs at birth causes neonatal respiratory distress and results in dyspnea and respiratory failure. iii. Spider lamb syndrome (hereditary chondrodysplasia). Spider lamb syndrome is an inherited, often lethal, musculoskeletal disorder primarily occurring in Suffolk and Hampshire breeds. Severely affected lambs die shortly after birth. Animals that survive the perinatal period develop angular limb deformities, scoliosis, and facial deformities. With time, affected animals become debilitated, exhibit joint pain, and develop neurological problems associated with the spinal abnormalities. Radiologically, secondary ossification centers—especially the physis, subchondral areas, and cuboidal bones—are affected. Abnormal endochondral ossification leads to excess cartilage formation, notably apparent in the elbows. Lambs will typically display abnormally long limbs, medial deviation of the carpus and tarsus, flattening of the sternum, scoliosis/kyphosis of the vertebrae, and a rounded nose. Muscle atrophy is common. Diagnosis can be based on typical clinical signs, which are similar to those seen with Marfan syndrome in humans ( Rook et al., 1986 ). Long-term survival is rare; treatment is unsuccessful. 2. Metabolic Diseases a. Abomasal Disorders i. Abomasal and duodenal ulcers. Abomasal and duodenal ulcers occur more frequently in calves and adult cattle than in sheep and goats. Like rumenitis, abomasal and duodenal ulcers may be associated with lactic acidosis. Concurrent disease, such as salmonellosis, bluetongue, or overuse of anti-inflammatory drugs, or recent shipping or environmental stresses may also lead to ulcer formation. Copper deficiency, dietary changes, mycotic infections, Clostridium perfringens abomasitis, and abomasal bezoars are associated with this disease in calves. In older adult cattle, abomasal lymphosarcoma may be the underlying condition. Gastric acid hypersecretion in conjunction with insufficient gastric mucous secretion will physically destroy the gastric epithelium. Deep ulceration may cause serious hemorrhage and/or perforation with peritonitis. Chronic hemorrhage may lead to anemia. Although ulcers are often asymptomatic in calves, perforation with peritonitis is more common than hemorrhage. Dark feces or melena and abdominal pain may be observed. Arched back, restlessness, kicking at the abdomen, bruxism, and anorexia are common signs of abdominal pain. Fecal occult blood is as an easy diagnostic test. Treatment includes gastrointestinal protectants and histamine antagonists. Anemia may be symptomatically treated with parenteral iron injections and anabolic steroids. Preventive measures in cattle herds include ensuring optimal passive immunity for calves, minimizing stress to calves, and striving for a herd free of bovine leukosis virus. ii. Abomasal emptying defect. Abomasal emptying defect of sheep is a sporadic syndrome associated with abomasal distension and weight loss. Suffolks tend to be especially predisposed, although the disease has been diagnosed in Hampshires, Columbias, and Corriedales. The mechanism of the disease is unknown. Affected animals will exhibit a gradual weight loss with a history of normal appetites. Feces will continue to be normal. Ventral abdominal distension associated with abomasal accumulation of feedstuffs will be apparent in many of the animals. Diagnosis is primarily based on history and clinical signs. Elevations in rumen chloride concentrations (>15 mEq/liter) are commonly found. Radiography or ultrasonography may be helpful at identifying the distended abomasum. Abomasal emptying defect is usually eventually fatal. Medical treatment with metoclopramide and mineral oil may be helpful in early disease. iii. Abomasal displacement. Displaced abomasum (DA) is a sporadic disorder usually associated with multiparous 4- to 7-year-old dairy cows in early lactation, but the condition can occur even in young calves. Displacement to the right (RDA) may be further complicated by torsion (RTA), a surgical emergency. Left displacement (LDA) is more common than RDA. Clinical signs include anorexia, lack of cud chewing, decreased frequency of ruminal contractions, shallow respirations, increased heart rate, treading, and decreased milk production. Diagnosis is based on characteristic areas of tympanic resonance during auscultation-percussion of the lateral to lateral-ventral abdomen ("pings"), ruminal displacement palpated per rectum, and clinical signs. Cow-side clinical chemistry findings include hypoglycemia and ketonuria; more extensive evaluations will often indicate moderate to severe electrolyte and acid-base abnormalities. DA occurs because of gas accumulation within the viscus, and the abomasum "floats" up from its normal ventral location to the lateral abdominal wall. No exact cause of DA has been identified, but it is commonly associated with stress; high levels of concentrate in the diet, leading to forestomach atony; and many disorders, including lack of regular exercise, mastitis, hypocalcemia, retained placenta, metritis, or twins. Factors such as body size and conformation indicate the possibility of genetic predisposition. Treatments include surgical and nonsurgical techniques for LDA; the former has a better chance of permanent correction. Emergency surgery is necessary for RTA; the disorder is fatal within 72 hr. Recurrence is rare after surgical correction. Electrolyte and acid-base imbalances are likely in severe cases and especially with RTA. Prevention includes reducing stress, taking greater care in the introduction and feeding of concentrates, and reducing incidence of predisposing diseases noted above ( Rohrbach et al., 1999 ). b. Fat Cow Syndrome, Hepatic Lipidosis Fat cow syndrome is seen in peri- or postparturient overconditioned or obese multiparous dairy cows. Factors in the development of the condition include negative energy balance related to the normal decreased dry matter intake as parturition approaches; hormonal changes associated with parturition; and concurrent diseases of parturition that decrease feed intake and increase energy needs. The possible concurrent diseases include metritis, retained fetal membranes, mastitis, parturient paresis, and displaced abomasum. Signs are nonspecific and include depression, anorexia, and weakness. Prognosis is usually guarded. Diagnosis is based on herd management, the animal's condition, ketonuria, and clinical signs. In prepartum cattle and in lactating cows, blood levels of nonesterified fatty acids (NEFA) greater than 1000 μEq/liter and 325–400 μEq/liter, respectively, are abnormal ( Gerloff and Herdt, 1999 ). Triglyceride analysis of liver biposy specimens are useful. In affected cows, body fat is mobilized, in the form of NEFA in response to the energy demands. Hepatic lipidosis occurs rapidly as the NEFA are converted into hepatic triglycerides. The ability of the liver to extract the albumin-bound NEFA from the blood is better than that of other tissues that need and can also use NEFA as an energy source. Treatment for any concurrent diseases must be pursued aggressively, as well as measures to increase and stabilize blood glucose, decrease NEFA production, and increase forestomach digestion to improve production of normally metabolized volatile fatty acids. Therapeutic measures include intravenous glucose drips, insulin (NPH or Lente) injections every 12 hr, and transfaunation of ruminal fluid from a normal cow. Prevention includes minimizing stress to late-gestation cows. Dry and lactating cows should be maintained separately; their energy, protein, and dry matter requirements are very different. Cows with prolonged lactation or delayed breeding should be managed to prevent weight gain. c. Rumen and Reticulum Disorders i. Bloat. Bloat or tympanites refers to an excessive accumulation of gas in the rumen. The condition most frequently occurs in animals that have been recently fed abundant quantities of succulent forages or grains. Bloat is classified into two broad categories: frothy bloat and free-gas bloat. Frothy bloat is associated with ingestion of feeds that produce a stable froth that is not easily expelled from the rumen. Fermentation gases such as CO 2 , CH 4 , and minor gases such as N 2 , O 2 , H 2 , and H 2 S incorporate into the froth, overdistend the rumen, and eventually compromise respiration by limiting diaphragm movement. The froth is often derived from a combination of salivary mucoproteins, protozoal or bacterial proteins, and proteins, pectins, saponins, or hemicellulose associated with ingested leaves or grain. Typical foodstuffs that cause frothy bloat include green legumes, leguminous hay (alfalfa, clover), or grain (especially barley, corn, and soybean meal). Free-gas bloat is less related to feeds ingested; rather, it is caused by rumen atony or by physical or pathological problems that prevent normal gas eructation. Some examples of causes of free-gas bloat are esophageal obstructions (foreign bodies, tumors, abscesses, and enlarged cervical or thoracic lymph nodes), vagal nerve paralysis or injury, and central nervous system conditions that affect eructation reflexes. Clinically, the animal will exhibit rumen distension, and tympany will be observed in the left paralumbar fossa. Additional signs may include colic-like pain of the abdomen and dyspnea. Passage of a stomach tube helps to differentiate between free-gas bloat and frothy bloat; and with free-gas bloat, expulsion of gas through the stomach tube aids in treatment of the disorder. Once rumen distension is alleviated with free-gas bloat, the underlying cause must be investigated to prevent recurrence. Frothy bloat is more difficult to treat, because the foam blocks the stomach tube. Addition of mineral oil, household detergents, or antifermentative compounds via the tube may help break down the surface tension, allowing the gas to be expelled. In acute, life-threatening cases of bloat, treatment should be aimed at alleviating rumen distension by placing a trocar or surgical rumenotomy into the rumen via the paralumbar fossa. Limiting the consumption of feedstuffs prone to induce bloat can prevent the disease. Additionally, poloxalene or monensin will decrease the incidence of frothy bloat. ii. Lactic acidosis. Lactic acidosis, or rumen acidosis, is an acute metabolic disease caused by engorgement of grains or other highly fermentable carbohydrate sources. The disease is most frequently related to a rapid change in diet from one containing high roughage to one containing excessive carbohydrates. Diet components that predispose to acidosis include common feed grains; feedstuffs such as sugar beets, molasses, and potatoes; by-products such as brewer's grains; and bakery products. Biochemically, ingestion of large amounts of the carbohydrate-rich diet causes the normally gram-negative rumen bacterial populations to shift to gram-positive Streptococcus and Lactobacillus species. The gram-positive organisms efficiently convert the starches to lactic acid. The lactic acid acidifies the rumen contents, leading to rumen mucosal inflammation, and increases the osmolality of rumen fluids, leading to sequestration of fluids and osmotic attraction of plasma and tissue fluid to the rumen. Lactic acid-induced rumenitis predisposes the animal to ulcers, to liver abscesses from "absorbed" bacterial pathogens, to laminitis from absorbed toxins, and to polioencephalomalacia from the inability of the new rumen bacterial populations to produce sufficient thiamine needed to maintain normal nervous system function. Clinically, animals will become anorexic, depressed, and weak within 1–3 days after the initial insult. Incoordination, ataxia, dehydration, hemoconcentration, rapid pulse and respiration, diarrhea, abdominal pain, and lameness will also be noted. Rumen distension and an acetone-like odor to the breath, milk, or urine may also be observed. Diagnosis is based on history and clinical signs. Blood, urine, or milk ketones can be detected ( Moore and Ishler, 1997 ). Additionally, rumen pH, which is normally above 6.0, will drop to less than 5.0 and in severe cases may achieve levels as low as 3.8. Similarly, urine pH will become acidic, blood pH will drop below 7.4, and hematocrit will appear to increase due to the relative hemoconcentration. Necropsy findings will be determined by secondary conditions. The primary lactic acidosis will cause swelling and necrosis of rumen papillae and abomasal hemorrhages and ulcers. Treatment must be applied early in the syndrome. In early hours of severe carbohydrate engorgement, rumenotomy and evacuation of the contents are appropriate. The patient should be given mineral oil and antifermentatives to prevent the continued conversion of starches to acids and the absorption of metabolic products. Bicarbonate or other antacids like magnesium carbonate or magnesium hydroxide introduced into the rumen will aid in adjusting rumen pH. Furthermore, animals can be given oral tetracycline or penicillin, which will decrease the gram-positive bacterial population. iii. Rumen parakeratosis. Parakeratosis is a degenerative condition of the rumen mucosa that leads to keratinization of the papillary epithelium. Excessive and continuous feeding of diets low in roughage causes the mucosal changes. Generally, this condition is seen in feedlot lambs and steers that are fed an all-grain diet. Clinically, animals may exhibit only poor rates of gain, due to changes in the absorptive capacity of the injured mucosa. At necropsy, papillae will be thickened and rough. They will frequently be dark in color, and multiple papillae will clump together. Abscessation may be observed. Histopathologically, papilla surfaces will have hyperkeratinization of the squamous epithelium. Chronic laminitis may be observed. However, diagnosis of parakeratosis is generally made at necropsy. Feeding adequate roughage, such as stemmy hay, will prevent the disease. Antibiotics may be administered to prevent secondary liver abscess formation. iv. Rumenitis. Rumenitis is an acute or chronic inflammation of the rumen, which occurs most commonly as a sequela to lactic acidosis. In addition to concentrate feeding, inadequate roughage in the diet is also associated with this disorder. Rumenitis may occur with contagious ecthyma infection or following ingestion of poisons or other irritants. Because rumenitis is often associated with lactic acidosis, it tends to occur in feedlot animals. The inflamed ruminal epithelium becomes necrotic and sloughs, creating ulcers. Endogenous rumen bacteria such as Fusobacterium necrophorum may invade the ulcers, penetrate the circulatory system, and induce abscesses of the liver. Clinically, the animals will appear depressed and anorexic. Rumen motility will be decreased, and animals will lose weight. The disease may resolve in a week to 10 days; mortality may reach 20%. Necropsy lesions include rumen inflammation and ulcers in the anteroventral sac. Granulation tissue and scarring may be observed following healing. Rumenitis is not typically diagnosed clinically; thus, specific treatment is not commonly done. The disease can be prevented by minimizing the incidence of lactic acidosis. d. Traumatic Reticulitis-Reticuloperitonitis (Hardware Disease) Etiology. Traumatic reticulitis–reticuloperitonitis is a disease of cattle related to their exploratory tendencies and ingestion of many different, nonvegetative materials. The disease is rarely seen in smaller ruminants. Clinical signs. Clinical signs range from asymptomatic to severe, depending on the penetration and damage by the foreign object after settling in the animal's forestomach. Many signs during the early, acute stages will be nonspecific, ranging from arched back, listlessness, anorexia, fever, decrease in production, ketosis, regurgitation, decrease or cessation of ruminal contractions, bloat, tachypnea, tachycardia, and grunts when urinating, defecating, or being forced to move. The prognosis is poor when peritonitis becomes diffuse. Sudden death can occur if the heart, coronary vessels, or other large vessels are punctured by the migrating object. Epizootiology and transmission. This is a noncontagious disease. The occurrence is directly related to sharp or metallic indigestible items in the feed or environment that the cattle mouth and swallow. Necropsy findings. In severe cases, necropsy findings include extensive inflammation throughout the cranial abdomen, malodorous peritoneal fluid accumulations, and lesions at the reticular sites of migration of the foreign objects. Cardiac puncture will be present in those animals succumbing to sudden death. Pathogenesis. Consumed objects initially settle in the rumen but are dumped into the reticulum during the digestive process, and normal contraction may eventually lead to puncture of the reticular wall. This sets off a localized inflammation or a localized or more generalized peritonitis. The inflammation may also temporarily or permanently affect innervation of local tissues and organs. Further damage may result from migration and penetration of the diaphragm, pericardium, and heart. Diagnosis is based on clinical signs, knowledge of herd management techniques in terms of placement of forestomach magnets, and reflection of acute or chronic infection on the hemogram. Radiographs and abdominocentesis may be useful. Differential diagnosis. Differentials include abomasal ulcers, hepatic ulcers, neoplasia (such as lymphosarcoma, usually in older animals, or intestinal carcinoma), laminitis, and cor pulmonale. Infectious diseases that are differentials include systemic leptospirosis and internal parasitism. Diseases causing sudden death may need to be considered. Prevention and control. This problem can be prevented entirely by elimination of sharp objects in cattle feed and in the housing and pasture environments. Adequately sized magnets placed in feed handling equipment and forestomach magnets (placed per os with a balling gun in young stock at 6–8 months of age) are also significant prevention measures. Treatment. Provision of a forestomach magnet, confinement, and nursing care, including antibiotics, are the initial treatments. In severe cases, rumenotomy may be considered. e. Pregnancy Toxemia (Ketosis), Protein Energy Malnutrition Etiology. Pregnancy toxemia is a primary metabolic disease of ewes and does in advanced pregnancy. Beef heifers are susceptible to protein energy malnutrition (PEM) syndrome, which is also referred to as pregnancy toxemia. Clinical signs. In sheep, this disease is characterized by hypoglycemia, ketonemia, ketonuria, weakness, and blindness. Hypoglycemic and ketotic ewes begin to wander aimlessly and to move away from the flock. They become anorexic and act uncoordinated, frequently leaning against objects. Advanced signs may include blindness, muscle tremors, teeth grinding, convulsions, and coma. Body temperature, heart rate, respiratory rate, and rumen motility continue normally. Up to 80% of infected ewes may die from the disease. The course of the disease may last up to a week. In goats, the disease usually occurs in the last 6 weeks of gestation, especially in does carrying triplets. Pregnancy toxemia should be considered with any goat showing signs of illness in late gestation. The doe may separate herself from the herd, stagger, or circle and may appear blind. Appetite is poor, and tremors may be evident. A rapid metabolic acidosis results in subsequent recumbency. Urinalysis will readily reveal ketonuria. If fetal death occurs, acute toxemia and death of the doe may result. In beef heifers, weight loss and thin body condition, weakness and inability to stand, and depression are clinical signs. Some cows develop diarrhea. Because the catabolic state is often so advanced, most affected heifers die even if treated. Pregnancy toxemia is diagnosed by evidence of typical clinical signs. Sodium nitroprusside tablets or ketosis dipsticks may be used to identify ketones in the urine or plasma of ewes and does. Blood glucose levels found to be below 25 mg/dl and ketonuria are good diagnostic indicators. In cattle, ketonuria is not a typical finding; hypocalcemia and anemia may be present. Epizootiology. Pregnancy toxemia occurs primarily in ewes that are obese or bearing twins or triplets. The disease develops during the last 6 weeks of pregnancy. PEM most frequently occurs in heifers during the final trimester of pregnancy. Necropsy findings. At necropsy, affected ewes will often have multiple fetuses, which may have died and decomposed. The liver will be enlarged, yellow, and friable, with fatty degeneration. The adrenal gland may also be enlarged. In cattle, heifers will be very thin, and in addition to a fatty liver, signs of concurrent diseases may be present. Pathogenesis. Rapid fetal growth, a decline in maternal nutrition, and a voluntary decrease in food intake in overfat ewes result in an inadequate supply of glucose needed for both maternal and fetal tissues. The ewe develops a severe hypoglycemia in early stages of the disease. The ruminant absorbs little dietary glucose; rather, it produces and absorbs volatile fatty acids (acetic, propionic, and butyric acids) from consumed feedstuffs. Propionic acid is absorbed and selectively converted to glucose through gluconeogenesis. When the animal is in a state of negative energy balance, it hydrolyzes fats to glycerol and fatty acids. Glycerol is converted to glucose while the fatty acids are metabolized for energy. The oxidation of fatty acids in the face of declining oxaloacetate levels (required for normal Krebs cycle function) results in the formation of ketone bodies (acetone, acetoacetic acid, and β-hydroxybutyric acid), thus causing the condition ketoacidosis. Heifer cattle have high energy requirements for completing normal body growth and supporting a pregnancy. Additional energy requirements are needed during pregnancy for winter conditions and during concurrent diseases. Marginal diets and poor-quality forage will place the cows in a negative energy balance. Differential diagnosis. Hypocalcemia is a common differential diagnosis. In cattle, differentials include chronic or untreated diseases such as Johne's disease, lymphosarcoma, parasitism, and chronic respiratory diseases. Prevention and control. Pregnancy toxemia can be prevented by providing adequate nutrition during late gestation and by maintaining animals in appropriate nonfat condition during pregnancy. In late pregnancy, the dietary energy and protein should be increased 1.5–2 times the maintenance level. PEM can be prevented by maintaining appropriate body condition earlier in pregnancy and supplying good-quality forage for the last trimester. Treatment. In sheep, because the morbidity may be as high as 20%, treatment should be directed at the flock rather than the individual. Treating the individual is usually unsuccessful. Oral administration of 200 ml of propylene glycol or 50% glucose twice a day, anabolic steroids, and high doses of adrenocorti-costeroids may be helpful. If ewes are still responsive and not severely acidotic or in renal failure, cesarean section may be successful by rapidly removing the fetus, which is the dietary drain for the ewe. In goats, pregnancy toxemia is best treated by removal of the fetuses either by cesarean section or induction of parturition. Parturition can be induced in does by either dexamethasone (10 mg) or PGF 2a (10 pg). In addition, goats may be treated with 10% dextrose (100 to 200 ml iv) or propylene glycol (60 ml per os 2 or 3 times a day). Adjunctive therapy includes normalizing acid base and hydration status, administration of vitamin B 12 and transfaunation. Heifers may be force-fed alfalfa gruels, given propylene glycol per os, placed on IV 50% glucose drips, and treated for concurrent disease. Research complications. In research requiring pregnant ewes in late stages of gestation, for example, this disease should be considered if the animals are likely to bear twins and will be transported or stressed in other ways during that time. f. Hypocalcemia (Parturient Paresis, Milk Fever) Etiology. Hypocalcemia is an acute metabolic disease of ruminants that requires emergency treatment; the presentation is slightly different in ewes, does, and cows. Clinical signs and diagnosis. In sheep, the disease is seen in ewes during the last 6 weeks of pregnancy and is characterized by muscle tetany, incoordination, paralysis, and finally coma. As calcium levels drop, ewes begin to show early signs such as stiffness and incoordination of movements, especially in the hindlimbs. Later, muscular tremors, muscular weakness, and recumbency will ensue. Animals will frequently be found breathing rapidly despite a normal body temperature. Morbidity may approach 30%, and mortality may reach as high as 90% in untreated animals. Affected does become bloated, weak, unsteady, and eventually recumbent. Cows are affected within 24–48 hr before or after parturition. Cows initially are weak and show evidence of muscle tremors, then deteriorate to sternal recumbency, with the head usually tucked to the abdomen, and an inability to stand. Tachycardia, dilated pupils, anorexia, hypothermia, depression, ruminal stasis, bloat, uterine inertia, and loss of anal tone are also seen at this stage. The terminal stage of disease is a rapid progression from coma to death. Heart rates will be high, but pulse may not be detectable. Hypocalcemia is diagnosed based on the pregnancy stage of the female and on clinical signs. It is later confirmed by laboratory findings of low serum calcium. With hypocalcemia in ewes, the plasma concentrations of calcium drop from normal values of 8–12 mg/dl to values of 3–6 mg/dl. In cattle, plasma levels below 7.5 mg/dl are hypocalcemic; at the terminal stages levels may be 2 mg/dl. Epizootiology. Hypocalcemia occurs primarily in overweight ewes during the last 6 weeks of pregnancy or during the first few weeks of lactation. The disease is not as common in the dairy goat as in the dairy cow. High-producing, older, multiparous dairy cows are the most susceptible, and the Jersey breed is considered susceptible. Cows that have survived one episode are prone to recurrence. In addition, dry cows must be managed carefully regarding limiting dietary calcium. The disease is not common in beef cattle unless there is an overall poor nutrition program. Necropsy findings. There is no pathognomonic or typical finding at necropsy. Pathogenesis. During the periparturient period, calcium requirements for fetal skeletal growth exceed calcium absorbed from the diet and from bone metabolism. Additionally, dietary calcium intake is thought to be compromised because, in advanced pregnancy, animals may not be able to eat enough to sustain adequate nutrient levels, and intestinal absorption capabilities do not respond as quickly as needed. After parturition, calcium needs increase dramatically because of calcium levels in colostrum and milk. Recent information suggests that legume and grass forages, high in potassium and low in magnesium, create a slight physiological alkalosis (at least in cattle), which antagonizes normal calcium regulation ( Rings et al., 1997 ). Thus, bone resorption, renal resorption, and gastrointestinal absorption of calcium are less than maximal. Prevention and control. Maintaining appropriate nutrition during the last trimester is helpful in preventing the disease. In cows and does, for example, limiting calcium intake by removing alfalfa from the diet is helpful. Treatment. Hypocalcemia must be treated quickly based on clinical signs; pretreatment blood samples can be saved for later confirmation. Twenty percent calcium borogluconate solution should be administered by slow intravenous infusion. Response will often be rapid, with the resolution of the animal's dull mentation. Less severely affected animals will often try to stand in a short time. Relapses are common, however, in sheep and cattle. Hypermagnesemia and hypophosphatemia often coincide with hypocalcemia. These imbalances should be considered when animals appear to be unresponsive to treatment. Hypocalcemia in the goat can be treated with 50–100 ml of calcium borogluconate. Heart rate should be monitored closely throughout calcium administration. If an irregular or rapid heart rate is detected, then calcium treatment should be slowed or discontinued. Calcium gels and boluses are also available for treatment ( Rings et al., 1997 ). Prognosis is generally good if the animal is treated early in the disease, but the prognosis will often be poor when treatment is initiated in later stages of the disease. g. Urinary Calculi (Obstructive Urolithiasis, Water Belly) Etiology. Urolithiasis is a metabolic disease of intact and castrated male sheep, goats, and cattle that is characterized by the formation of bladder and urethral crystals, urethral blockage, and anuria (Murray, 1985). The disease occurs rarely in female ruminants. Clinical signs and diagnosis. Affected animals will vocalize and begin to show signs of uneasiness, such as treading, straining postures, arched backs, raised tails, and squatting while attempting to urinate. These postures may be mistaken for tenesmus. Male cattle may develop swelling along the ventral perineal area. Affected animals will not stay with the herd or flock. Small amounts of urine may be discharged, and crystal deposits may be visible attached to the preputial hairs. Additionally, in smaller ruminants, the filiform urethral appendage (pizzle) often becomes dark purple to black in color. The pulsing pelvic urethra may be detected by manual or digital rectal palpation, and bladder distention may be noticeable in cattle by the same means. As the disease progresses to complete urethral blockage, the animal will become anorexic and show signs of abdominal pain, such as kicking at the belly. The abdomen will swell as the bladder enlarges, and rupture can occur within 36 hr after development of clinical signs. Bladder or urethral rupture may cause a short-lived period of apparent pain relief; subsequent development of uremia will eventually lead to death. The disease may progress over a period of 1–2 weeks, and the mortality is high unless the blockages are reversed. Diagnosis is made by the typical clinical signs. Abdominal taps may yield urine. Calculi are usually composed of calcium phosphate or ammonium phosphate matrices. Epizootiology and transmission. Clinical disease is usually seen in growing intact or castrated males. The disease may be sporadic or there may be clusters of cases in the flock or herd. Necropsy findings. Necropsy findings include urine in the abdomen with or without bladder or urethral rupture. Renal hydronephrosis may be evident. Calculi or struvite crystal sediment will be observed in the bladder and urethra. Histologically, trauma to the urethra and ureters will be present. Pathogenesis. Urolithiasis is multifactorial and involves dietary, anatomical, hormonal, and environmental factors. Male sheep and goats have a urethral process that predisposes them to entrapment of calculi. In cattle, the urethra narrows at the sigmoid flexure, and calculi lodge there most frequently. Additionally, the removal of testosterone by early castration is thought to result in hypoplasia of the urethra and penis. This physical reduction in the size of the excretory tube may predispose to the precipitation of and blockage by the struvite minerals. Grains fed to growing animals tend to be high in phosphorus and magnesium content. These calculogenic diets lead to the formation of struvite (magnesium ammonium phosphate) crystals. Other minerals associated with urolithiasis include silica (range grasses), carbonates (some grasses and clover pastures), calcium (exclusively alfalfa hay), and oxalates (fescue grasses). Differential diagnosis. Grain engorgement colic, gastrointestinal blockage, and causes of tenemus, such as enteritis or trauma, are differentials. Trauma to the urethral process should be considered. Urinary tract infections are uncommon in ruminants. Prevention and control. One case often is indicative of a potential problem in the group. Urolithiasis can be minimized by monitoring the calcium:phosphorus ratio in the diet. The normal ratio should be 2:1. Additionally, increasing the amount of dietary roughage will help balance the mineral intake. Increasing the amount of salt (sodium chloride, 2–4%) in the diet to increase water consumption, or adding ammonium chloride to the diet, at 10 gm/head/day or 2% of the ration, to acidify the urine, will aid in the prevention of this disease. Palatability of and accessibility to water should be assessed as well as functioning of automatic watering equipment. Treatment. Treatment is primarily surgical (Van Metre et al. 1996). Initially, amputation of the filiform urethral appendage may alleviate the disease since urethral blockage often begins here. As the disease progresses, urethral blockage in the sigmoid flexure as well as throughout the urethra may occur. In more advanced stages, perineal urethrostomy may yield good results. The prognosis is poor when the condition becomes chronic, reoccurs, or surgery is required. Research complications. Young castrated and intact male ruminants used in the laboratory setting will be the susceptible age group for this disorder. h. Rickets Rickets is a disease of young, growing animals but rarely occurs in goats. It is a metabolic disease characterized by a failure of bone matrix mineralization at the epiphysis of long bones due to lack of phosphorus. The condition can occur as an absolute deficiency in vitamin D 2 , an inadequate dietary supply of phosphorus, or a long-term dietary imbalance of calcium and phosphorus. The syndrome must be differentiated from epiphisitis (unequal growth of the epiphyses of long bones in young, rapidly growing kids fed diets with excess calcium). Clinical signs include poor growth, enlarged costochondral junctions, narrow chests, painful joints, and reluctance to move. Spontaneous fractures of long bones may occur. Animals will recover when dietary phosphorus is provided and if joint damage is not severe. 3. Nutritional Diseases a. Copper Deficiency (Enzootic Ataxia, Swayback) Etiology. Chronic copper deficiency in pregnant ewes and does may produce a metabolic disorder in their lambs and kids called enzootic ataxia. In goats, this deficiency also causes swayback in the fetuses. Clinical signs and diagnosis. This disease results in a progressive hindlimb ataxia and apparent blindness in lambs up to about 3 months of age. Additionally, because copper is essential for osteogenesis, hematopoiesis, myelination, and pigmentation of wool and hair, ewes may appear unthrifty, may be anemic, and may have poor, depigmented wool with a decrease in wool crimp. Affected kids are born weak, tremble, and have a characteristic concavity to the spinal cord, leading to the name swayback. When the deficiency occurs later during gestation, demyelination is limited to the spinal cord and brain stem. Kids are born normally but develop a progressive ataxia, leading to paralysis, muscle atrophy, and depressed spinal reflexes with lower motor neuron signs. Diagnosis is based on low copper levels found in feedstuffs and tissues at necropsy. Diagnosis is based on clinical signs, feed analysis, and pathological findings. Epizootiology and transmission. Enzootic ataxia is rarely seen in western states; most North American diets have sufficient copper levels to prevent this disease. Copper antagonists in the feed or forage at sufficient levels, such as molybdenum, sulfate, and cadmium, however, may predispose to copper deficiencies. Pathogenesis. The maternal copper deficiency leads to a disturbance early in the embryonic development of myelination in the central nervous system and the spinal cord. Copper is part of the cytochrome oxidase system and other enzyme complexes and is important in myelination, osteogenesis, hematopoiesis (iron absorption and hemoglobin formation), immune system development, and maintenance and normal growth ( Smith and Sherman, 1994 ). Differential diagnosis. The differential diagnosis for newborns includes β-mannosidosis, hypoglycemia, and hypothermia. For older animals the differential should include caprine arthritis encephalitis (goats), enzootic muscular dystrophy, listeriosis, spinal trauma or abscessation, and cerebrospinal nematodiasis. Prevention and control. Copper deficiency can be prevented by providing balanced nutrition for pregnant animals. Necropsy findings. Gross encephalomalacia has been noted. Histopathologically, white matter of the brain and spinal cord displays gelatinization and cavitation. Extensive nerve demyelination and necrosis are evident. Postmortem lesions include extensive demyelination and neuronal degeneration. Treatment. Because the condition is developmental, supplemental copper may improve clinical signs but not eliminate them. b. Copper Toxicosis Etiology: Acute or chronic copper ingestion or liver injury often causes a severe, acute hemolytic anemia in weanling to adult sheep and in calves and adult dairy cattle. Growing lambs may be the most susceptible. Copper toxicosis is rare in goats. Clinical signs and diagnosis. The clinical course in sheep can be as short as 1–4 days, and mortality may reach 75%. Hemolysis, anemia, hemoglobinuria, and icterus characterize the acute hemolytic crisis, associated with copper released from the overloaded liver. Some clinical signs are related to direct irritation to the gastrointestinal tract mucosa. Weakness, vomiting, abdominal pain, bruxism, diarrhea, respiratory difficulty, and circulatory collapse are followed by recumbency and death. Hepatic biopsy is currently considered the best diagnostic approach; serum or plasma levels of copper and hepatic enzymes such as aspartate aminotransferase (AST) and γ-glutamyltransferase (GGT) may provide some information, but it is generally believed that these will not accurately reflect total copper load or hepatic damage. Epizootiology and transmission. A single toxic dose for sheep and goats is the range of 20–100 mg/kg, and for cattle it is 220–880 mg/kg. Chronic poisoning in sheep may occur when 3.5 mg/kg is ingested. Copper-containing pesticides, soil additives, therapeutics, and improperly formulated feeds may potentially lead to copper toxicity. Phytogenous sources include certain pastures such as subterranean clover. Feed low in molybdenum, zinc, or calcium may lead to increased uptake of copper from properly balanced rations. A common cause of the disease in sheep is feeding concentrates balanced for cattle; cattle feeds and mineral blocks contain much higher quantities of copper than are required for sheep. Chronic ingestion of these feedstuffs leads to copper accumulation and toxicity. Copper toxicosis has been reported in calves given regular oral or parenteral copper supplements, and in adult dairy cattle given copper supplements to compensate for copper-deficient pasture. Pregnant dairy cattle may be more susceptible to copper toxicity. Rare sources of copper ingestion may include copper sulfate footbaths. Necropsy findings. Common findings at necropsy include icterus; a soft, dark, friable, enlarged spleen; an enlarged, yellow-brown friable liver; and "gun-barrel" black kidneys. Hemoglobin-stained urine will be visible in the bladder. Copper accumulations in the liver reaching 1000–3000 ppm are toxic. Pathogenesis. Hemolysis occurs when sufficient amounts of copper are ingested or released suddenly from the liver and is believed to be due direct interaction of the copper with red-cell surface molecules. Stresses such as transportation, lactation, and poor nutrition or exercise may precipitate the hemolysis. Differential diagnosis. Other causes of hemolytic disease include babesiosis, trypanosomiasis, and plant poisonings such as kale. Arsenic ingestion, organophosphate toxicity, and cyanide or nitrate poisoning should also be considered as the source of poisoning. Urethral obstruction and gastrointestinal emergencies should be considered for the abdominal pain. Control and prevention. The disease is prevented by carefully monitoring copper access in sheep and copper supplementation in cattle. Sheep and goats should not be fed feedstuffs formulated for cattle, and dairy calf milk replacer should not be used for lambs and kids. Molybdenum may be administered to animals considered at high risk. Molybdenum-deficient pastures may be treated with molybdenum superphosphate. Herd copper supplementation should be undertaken with the knowledge of existing hepatic copper levels, and existing copper and molybdenum levels, in the feedstuffs. Treatment. Oral treatment for sheep consists of ammonium or sodium molybdenate (50–100 mg/day), and sodium thiosulfate (0.5–1.0 mg/day) for 3 weeks aids in excretion of copper. Oral D-penicillamine daily for 6 days (50 mg/kg) has also been shown to increase copper excretion in sheep. Ammonium molybdenate has been administered intravenously to goats at 1.7 mg/kg for 3 treatments on alternate days. Cattle have been treated orally with sodium molybdenate (3 gm/day) or sodium thiosulfate (5 gm/day). Treatment for anemia and nephrosis may be necessary in severe cases. Research complications. Some breeds of sheep, such as Merino crosses and the British breeds, may be more susceptible to copper toxicosis caused by phytogenous sources. c. Selenium/Vitamin E Deficiency (Nutritional Muscular Dystrophy, Nutritional Myodegeneration, White Muscle Disease, Stiff Lamb Disease) Etiology. White muscle disease, also known as stiff lamb disease, is a nutritional muscular dystrophy caused by a deficiency of selenium or vitamin E. Clinical signs and diagnosis. Clinically two forms of the disease have been identified: cardiac and skeletal. The cardiac form occurs most commonly in neonates. In these, respiratory difficulty will be a manifestation of damage to cardiac, diaphragmatic, and intercostal muscles. Young will be able to nurse when assisted. In slightly older animals, the disease is characterized by locomotor disturbances and/or circulatory failure. Clinically, animals may display paresis, stiffness or inability to stand, rapid but weak pulse, and acute death. Mortality may reach 70% ( Jensen and Swift, 1982 ). Paresis and sudden death in neonates with associated pathological signs are frequently diagnostic. With the skeletal form, affected animals are stiff and reluctant to move, and muscles of affected animals are painful. Young will be reluctant to get up but will readily nurse when assisted. Peracute to acute myocardial degeneration may occur in the cardiac form, and animals may simply be found dead. Serum selenium levels are usually below 50 ppb (normal is 158–160 ppb) ( Nelson, 1983 ). Diagnosis may also include determination of antemortem whole blood levels of selenium and plasma levels of vitamin E. Glutathione peroxidase levels in red blood cells can be measured as an indirect test. Clinical biochemistry findings of significant elevations of aspartate aminotransferase (AST) in creatinine kinase (CK) are also supportive of the diagnosis. Epizootiology and transmission. Selenium deficiency has been associated with formulated diets deficient in selenium, forages grown on selenium-deficient soils in certain geographic regions, and forages such as alfalfa and clover that have an inability to efficiently extract available selenium from the soils. Rumen bacterial reduction of selenium compounds to unavailable elemental selenium may also contribute to the disease. Necropsy findings. Necropsy lesions include petechial hemorrhages and muscle edema. Hallmarks are pale white streaking of affected skeletal and cardiac muscle. These are due to coagulation necrosis. Pale striated muscles of the limb, diaphragm, and tongue are also seen. Pathogenesis. Selenium and vitamin E function together as antioxidants that protect lipid membranes from oxidative destruction. Selenium is a cofactor for glutathione peroxidase, which converts hydrogen peroxide to water and other nontoxic compounds. Lack of one or both results in loss of membrane integrity. Differential diagnosis. In neonatal ruminants presenting with respiratory and cardiac dysfunction, differentials include congenital cardiac anomalies. Differentials generally for weak neonates or sudden or peracute neonatal deaths should include septicemia, pneumonia, toxicity, diarrhea, and dehydration. Prevention and control. Awareness of regional selenium deficiencies is important. Control involves providing goodquality roughage, vitamin E and selenium supplementation, and parenteral injections prior to parturition and weaning. Treatment. Affected animals may be treated by administering vitamin E or selenium injections. Administering vitamin E or selenium to ewes in late pregnancy can prevent white muscle disease ( Kott et al., 1998 ). The label dose for selenium is 2.5–3 mg/45 kg of body weight. Combination products are available and can be used in goats at the sheep dose ( Smith and Sherman, 1994 ). Proper mineral balance in the diet is critical. d. Selenium Toxicity Selenium toxicity occurs most frequently as the result of excessive dosing to prevent or correct selenium deficiency or as the result of ingestion of selenium-converting plants. The main preventive measure for the former is the use of the appropriate product for the species. Secondarily, the concentration of the available product should be double-checked. In the United States, ruminants in the Midwest and western areas may be subject to selenium toxicity when pastured in areas containing selenium-converting plants. Signs of overdosing include weakness, dyspnea, bloating, and diarrhea. Shock, paresis, and death may occur. Initial clinical signs of excessive selenium intake from plants are observed in the distal limb, with cracked hoof walls and subsequent infection and irregular hoof growth. e. Thiamin Deficiency (Polioencephalomalacia) Etiology. Polioencephalomalacia (PEM) is a noninfectious, noncontagious disease characterized by neurological signs. Growing and adult ruminants on high-concentrate diets are typically affected. Animals exposed to toxic plants or moldy feed containing thiaminases, feed high in sulfates, or unusually high doses of some medications are also at risk. Clinical signs and diagnosis. An early sign may be mild diarrhea. Acute clinical signs include bruxism, hyperesthesia, involuntary muscle contractions, depression, partial or complete opisthotonus, nystagmus, dorsomedial strabismus, seizures, and death. In subacute cases of the disease, animals may appear to walk aimlessly as if blind or may display head-pressing postures. Hypersalivation may be present, but body temperatures and ocular reflexes are normal. Morbidity and mortality may be high, especially in younger animals. Diagnosis is suggestive from clinical signs and from response to intensive parental thiamine hydrochloride. Epizootiology and transmission. PEM is caused by a thiamin deficiency. The disease tends to be seen more frequently in cattle and sheep feedlots where the concentrates fed are high in fermentable carbohydrates. Pastured animals are also vulnerable if grain is feed. Thiaminase-containing plants, such as bracken fern, are often unpalatable so will less likely be a contributing factor. Recent studies have also indicated that high levels of sulfate in the diet, such as in the fermentable, low-fiber concentrates, may play an important role. Medications such as as amprolium, levamisole, and thiabendazole have thiaminantagonizing activity when given in excessive doses. Necropsy signs. Cerebral lesions characterized by softening and discoloration are grossly observed in the gray matter. Microscopically, neurons will exhibit edema, chromatolysis, and shrinkage. Gliosis and cerebral capillary proliferation may be observed. Pathogenesis. A lack of thiamin results in inappropriate carbohydrate metabolism and accumulation of pyruvate and other intermediaries that lead to cerebral edema and neuronal degeneration. Differential diagnosis. Several important differentials include acute lead poisoning, nitrofuran toxicity, hypomagnesemia, vitamin A deficiency, listeriosis, pregnancy toxemia, infectious thromboembolic meningoencephalitis, and type D clostridial enterotoxemia. Prevention and control. The disease can be prevented by monitoring the diet and by providing adequate roughage necessary to prevent overgrowth of thiaminase-producing ruminal flora and to maximize ruminal production of B vitamins. If excess sulfur is the primary factor, immediate removal of the source is critical. Treatment. Early aggressive treatment is essential to save animals. The disease is treated by frequent parenteral administration of thiamine hydrochloride, the first dose being administered intravenously. Dexamethasone, B vitamins, and diazepam may also be required. Treatment is less successful when sulfur plays a prominent role in the etiology. Research complications. This disease is preventable. Although the disease is less likely to occur in smaller groups of confined ruminants, the risks of feeding concentrates or moldy feed, for example, with minimal good-quality roughage, should be kept in mind. f. Vitamin D Toxicity Vitamin D toxicity can result either from iatrogenic overadministration or ingestion of the plant Trisetum flavescens. Serum calcium levels may be high enough that blood in EDTA tubes will clot. g. Nutritional Deficiencies In goats, nutritional deficiencies often manifest as a generalized poor coat that is dry, scaly, thin, and erectile. Zinc-responsive dermatitis has been reported in goats ( Smith and Sherman, 1994 ). Vitamin A deficiencies associated with hyperkeratosis have been reported, as well as vitamin E-responsive and selenium-responsive dermatitis. 4. Management-Related Diseases a. Failure of Passive Transfer Neonatal ruminants are born without immunoglobulins and must receive colostrum by 24 hr after birth. The morbidity and mortality associated with failure of or inadequate passive transfer, such as enteric and respiratory illnesses, can be severe. Measures to assure passive immunity for neonatal ruminants are covered in Section II,B,5, and clinical signs of illness associated with lack of immunity are addressed in the discussions of bacterial diseases (e.g., Escherichia coli infections) and, of viral diseases (e.g., diarrheas) in Section III,A,1 and III,A,2. Generally, transfer of less than 600 mg/dl of immunoglobulins in the serum is classified as failure of transfer, 600–1600 mg/dl is partial, and above 1600 mg/dl is complete transfer. Methods to determine success of transfer should be performed within a week of birth and include single radial immunodiffusion (quantitates immunogloblin classes); zinc sulfate turbidity (semiquantitative); sodium sulfite precipitation (semiquantitative); glutaraldehyde coagulation (coagulates above specific level); and, γ-glutamyltransferase (assays enzyme in high concentration in colostrum and absorbed simultaneously with colostrum). b. Laminitis Laminitis is common in ruminants and can be caused by sudden changes in diet, excess dietary energy, and grain overload (or overeating). Laminitis is also associated with mastitis and metritis. Facility conditions, such as concrete flooring, poor manure management, and inadequate resting areas may also contribute to the pathogenesis of the disease. The complete pathogenesis of laminitis is poorly understood; however, it is thought that changes in the diet cause changes in rumen microbial populations, resulting in acidosis and endotoxemia. Dramatic changes in the vascular endothelium result in chronic inflammation of the sensitive laminae of the hoof, separation of corium and hoof wall, and rotation of the third phalanx. Affected animals may be reluctant to get up or walk, will shift their weight frequently, and will grind teeth or walk on carpi. Chronically, the hoof wall takes on a "slipper" appearance. Treatment consists of identifying the underlying cause, administering antiinflammatories (phenylbutazone, flunixin meglumin), feeding good-quality forages only, and regular foot trimming. c. Nutritional Diarrhea Otherwise normal, well-managed lambs, kids, and calves can develop loose, pasty feces due to a nutritional imbalance caused by overfeeding and/or improper mixing of milk replacers. Only milk replacer formulated for the particular species should be used. Once nutritional imbalances are corrected, the feces readily return to normal. Sudden changes in diet can also result in loose feces. d. Photosensitization (Bighead) Photosensitization is an acute dermatitis associated with an interaction between photosensitive chemicals and sunlight. The photosensitive chemicals are usually ingested, but in some cases exposure may be by contact. Animals with a lack of pigment are more susceptible to the disease. Three types of photosensitization occur: primary; secondary, or hepatogenous; and aberrant. Primary photosensitization is related to uncommon plant pigments or to drugs such as phenothiazine, sulfonamides, or tetracyclines. Secondary photosensitization is more common in large animals and is specifically related to the plant pigment phylloerythrin. Phylloerythrin, a porphyrin compound, is a degradation product of chlorophyll released by rumen microbial digestion. Liver disease or injury, which prevents normal conjugation of phylloerythrin and excretion through the biliary system, predisposes to photosensitization. The only example of aberrant photosensitization is congenital porphyria of cattle (see Section III,B,1). Pathologically, the photosensitive chemical is deposited in the skin and is activated by absorbed sunlight. The activated pigments transfer their energy to local proteins and amino acids, which, in the presence of oxygen, are converted to vasoactive substances. The vasoactive substances increase the permeability of capillaries, leading to fluid and plasma protein losses and eventually to local tissue necrosis. Photosensitization can occur within hours to days after sun exposure and produces lesions of the face, vulva, and coronary bands; lesions are most likely to occur on white-haired areas. Initially, edema of the lips, corneas, eyelids, nasal planum, face, vulva, or coronary bands occurs. The facial edema, nostril constriction, and swollen lips potentially lead to difficulty in breathing. With secondary photosensitization, icterus is also common. Necrosis and gangrene may occur. Diagnosis is based on clinical lesions and exposure to the photosensitive chemicals and sunlight. Treatment is symptomatic. The prognosis for hepatogenous type may be guarded if hepatic disease is severe. e. Reproductive Prolapses (Vaginal, Uterine) Vaginal and uterine prolapses occur in ewes, does, and cows. The conditions are not common in does. Vaginal prolapses usually occur during late gestation and may be related to relaxation of the pelvic ligaments in response to hormone levels. In sheep, these are most common in overconditioned ewes that are also carrying twins or triplets. Overconsumption of roughages, which distends the rumen, and lack of exercise leading to intra-abdominal fat may predispose an animal to vaginal prolapse by increasing intra-abdominal pressure. The condition may result from excessive straining associated with dysuria from the pressure of the fetuses and/or abdominal contents on the bladder. If the prolapse obstructs subsequent urination, rupture of the bladder may occur. The vaginal prolapse can be reduced and repaired if discovered early, and techniques in small and large ruminants are comparable. The animal should be restrained, and the prolapsed tissue should be cleansed with disinfectants. Best done under epidural anesthesia, the vagina is replaced into the pelvic canal and the vulvar or vestibular opening is sutured closed (Buhner suture). Alternatively, a commercial device called a bearing retainer (or truss) can be placed into the reduced vagina and tied to the wool, thereby holding the vagina in proper orientation without interfering with subsequent lambing. Vaginal prolapses may have a hereditary basis in ewes and cows and may prolapse the following year. These animals should be culled. Vaginal prolapses may occur in nonpregnant animals that graze estrogenic plants or as a sequela to docking the tail too close to the body ( Ross, 1989 ). Uterine prolapses occur sporadically in postpartum ewes and cattle. The gravid horn invaginates after delivery and protrudes from the vulva. The cause is unknown, but excessive traction utilized to correct dystocia or retained placenta, uterine atony, hypocalcemia, and overconditioning or lack of exercise have been implicated. In cattle, the uterine prolapses usually develop within 1 week of calving, are more common in dairy cows than in beef cows, and are often associated with dystocia or hypocalcemia. Cows may also have concurrent parturient paresis. Initially, the tissue will appear normal, but edema and environmental contamination or injuries of the tissue develop quickly. Clinical signs will include increased pulse and respiratory rates, straining, restlessness, and anorexia. If identified early, the uterus can be replaced as for vaginal prolapses. Electrolyte imbalances should be corrected if present. Additional supportive therapy, including the use of antibiotics should always be considered. Tetanus prophylaxis should be included. Oxytocin should be administered to induce uterine reduction. Vaginal closures are less successful at retaining uterine prolapses. Preventive and control measures include regular exercise for breeding animals, and management of prepartum nutrition and body condition. f. Rectal Prolapse Rectal prolapse is common in growing, weaned lambs and in cattle from 6 months to 2 years old. The physical eversion of the rectum through the anal sphincter is usually secondary to other diseases or management-related circumstances. Rectal prolapses may occur secondary to gastrointestinal infection or inflammation, especially when the colon is involved. Diseases that cause tenesmus, such as coccidiosis, salmonellosis, and intestinal worms, may result in prolapse. Urolithiasis may result in prolapses as the animal strains to urinate. Any form of cystitis or urethritis, vaginal irritation, or vaginal prolapse and some forms of hepatic disease may lead to rectal prolapse. Abdominal enlargement related to advanced stages of pregnancy, excessive rumen filling or bloat, and overconditioning may cause prolapse. Finally, excessive coughing during respiratory tract infections, improper tail docking (too short), growth implants, prolonged recumbency, or overcrowded housing with animal piling may lead to prolapses. Diagnosis is based on clinical signs. Early prolapses may be corrected by holding the animal with the head down, while a colleague places a pursestring suture around the anus. The mucosa and underlying tissue of prolapses that have been present for longer periods of time will often become necrotic, dry, friable, and devitalized and will require surgical amputation or the placement of prolapse rings to remove the tissue. Rectal prolapse may also be accompanied by intestinal intussusceptions that will further complicate the treatment and increase mortality. Occasionally, acute rectal prolapse with evisceration will result in shock and prompt death of the animal. Prognosis depends on the cause and extent of the prolapse as well as the timeliness of intervention. In all cases of treatment, determination and elimination of the underlying cause are essential. g. Trichobezoars Gastrointestinal accumulations or obstructions of hair (and/or sometimes very coarse roughage, forming bezoars) occur in cattle and sheep. Cattle that are maintained on a low-roughage diet, that lick their coats frequently, that have long hair coats from outdoor housing, or that have heavy lice or mite infestations and associated pruritus will often develop bezoars. In addition, younger calves with abomasal ulcers have been found to be more likely to have abomasal trichobezoars as well. Clinical signs may be mild or severe according to size, number, and location. Ruminal trichobezoars rarely result in clinical signs. Obstruction will be accompanied by signs of pain, development of bloat, and decreased fecal production. Serum profiles will show hypochloridemia; other imbalances depend on the duration of the problem. Diagnosis is also based on abdominal auscultation, rectal palpation, and ultrasound (useful in calves and smaller ruminants). Treatment is surgical, such as paracostal laparotomy (for abomasal), paralumbar celiotomy with manual breakdown, or enterotomy. Supportive care should be administered as necessary to correct electrolyte imbalances and to prevent inflammation and sepsis. Prognosis is generally good if the condition is diagnosed and treated before dehydration and imbalances become severe and peritonitis develops. Prevention includes providing good-quality roughage and treating lice and mange infestations. C. Traumatic Disorders (Wounds, Bites, and Entrapped Foreign Bodies) Wounds may be sustained from poorly constructed pens or fences, or from skirmishes among animals. Predators will usually be sources of bite wounds. Standard veterinary wound assessment and care are essential for wounds or bites. Tetanus antitoxin may be indicated. Use of approved antibiotics may be appropriate. The lesion should be cleaned with disinfectants and repaired with primary closure if it is clean and uncontaminated. Thorough cleaning, regular monitoring, and healing by second intention are recommended for older wounds. Abscesses may also occur in the soft tissues of the hooves (sole abscesses; see Section III,C,3) because of entrapped foreign bodies or hoof cracks that fill with dirt. Preventive measures include improvement of housing facilities, pens, and pastures; monitoring hierarchies among animals penned together; and implementing predator control measures, such as sound fencing, flock guard dogs, or donkeys, in pasture situations. D. Iatrogenic Diseases 1. Anaphylactic Reactions Acute anaphylatic reactions in sheep, goats, and cattle are often clinically referable to the respiratory system. Anaphylactic vaccine reactions cause acute lung edema; lungs are the primary site of lesions if collapse and death are sequelae. The animals will also be anxious and shivering and will become hyperthermic. Salivation, diarrhea, and bloat also occur. Immediate therapy must include epinephrine by intravenous infusion at (1 ml of 1:1000 per 50 kg of body weight for goats and 1:10,000 (0.1 mg/ml) or 0.01 mg/kg (about 5 ml) for adult cows.) Furosemide (5 mg/kg) may be beneficial to reduce edema. Prognosis is usually guarded. Recovery can occur within 2 hr. 2. Catheter Sites and Experimental Surgeries In a research environment, catheter sites or experimental surgeries may be sources of iatrogenic infection. Traumatic injuries to peripheral nerves can cause acute lameness. Improper administration of therapeutics can easily cause this type of lameness. Injections given in gluteals or between the semimembranosus and semitendinosus can cause irritation to the sciatic nerve and subsequent lameness. Contraction of the quadriceps results in the limb being pulled forward. Injections in the caudal thigh can damage the peroneal nerve and cause knuckling at the fetlock. Traumatic injury to the radial nerve can result in a "dropped elbow" ( Nelson, 1983 ). Husbandry procedures such as tail docking, castration, dehorning, dosing with a balling gun, and shearing may result in superficial lesions, dermal infections, or cases of tetanus. Balling-gun injuries to the pharynx may lead to cellulitis with coughing, decreased appetite, and sensitivity to palpation. Standard veterinary assessment and care are essential for these cases. Local and systemic antibiotics with supportive care may be indicated. Swelling around peripheral nerves caused by inoculations may be reduced by diuretics and anti-inflammatories. Mild cases of peripheral nerve damage may recover in 7–14 days. Personnel training, including review of relevant anatomy, preprocedure preparation, appropriate technique, careful surgical site preparation, rigorous instrument sanitation, and sterile technique will minimize the incidence of potential complications from surgical procedures. E. Neoplastic Diseases Neoplasia and tumors are relatively rare in ruminants. Lymphosarcoma/leukemia in sheep has been shown to result from infection by a virus related (or identical) to the bovine leukemia virus. Pulmonary carcinoma (pulmonary adenomatosis) and hepatic tumors are found in sheep. Virus-induced papillomatosis (warts), discussed in Section III,A,2,s, and squamous cell carcinomas have also been reported in sheep. In goats, thymoma is one of the two most common neoplasias reported, although no distinct clinical syndrome has been described. Cutaneous papillomas are the most common skin and udder tumor of goats, and although outbreaks involve multiple animals, no wart virus has been identified. Persistent udder papillomas may progress to squamous cell carcinoma. Lymphosarcoma is reported rarely in goats. Although adrenocortical adenomas have been reported frequently and almost exclusively in older wethers, no clinical condition has been described. Lymphosarcoma of various organ systems and "cancer eye" (bovine ocular squamous cell carcinoma, or OSCC) are the most commonly reported cancers in cattle. Lymphosarcoma is described in Section III,A,2,c. Lack of periocular pigmentation and the amount and intensity of exposure to solar ultraviolet light are considered important factors in OSCC. Genetic factors may also play a role. Many cases occur in Herefords. This is a disease of older cattle; no case has been reported in animals less than 4 years of age. The cancer metastasizes through the lymph system to major organs. Treatment in either lymphosarcoma or OSCC is recommended only as a palliative measure. The extent of ocular neoplastic involvement is a significant criterion for carcass condemnation. Papillomatosis (warts) are common in cattle (see Section III,A,2,s). F. Miscellaneous 1. Amyloidosis Amyloidosis in adult cattle is due to accumulations of amyloid protein in the kidney, liver, adrenal glands, and gastrointestinal tract. The disease has been classified as AA type, or associated with chronic inflammatory disease, although other unknown factors are believed to be involved in some cases. Clinical signs include chronic diarrhea, weight loss, decreased production, nonpainful renomegaly, and generalized edema. The loss of protein in the urine contributes to abnormal plasma albumin values and foaming urine. The proteinuria also distinguishes amyloidosis (and glomerulonephritis) from other causes of weight loss and diarrhea in cattle such as Johne's disease, parasitism, copper deficiency, salmonellosis, and bovine viral diarrhea virus infection. Prognosis is poor, and no treatment is reported. 2. Dental Wear Dental wear is seen most commonly in sheep. As sheep age, excessive dental wear may lead to an inability to properly masticate feed, manifesting as weight loss and unthriftiness. Several factors predisposing to dental wear should be considered. The diet should be properly balanced for minerals, especially calcium and phosphorus, because primary or secondary calcium deficiency during teeth development results in softening of the enamel and dentin. Dietary contamination with silica (i.e., hays and grains harvested in sandy regions) will lead to mechanical wear on the teeth. Likewise, animals grazing or being fed in sandy environments will have excessive tooth wear. Sheep older than about 5 years of age are especially prone to tooth wear and should be checked frequently, especially if signs of weight loss or malnutrition are evident. Managing the content and consistency of the diets can best prevent the disease. 3. Sole abscesses Of the ruminants, cows are the most frequently affected by subsolar absesses. Dirt becomes packed into cracks in the horny layer of the sole of the hoof, and contamination eventually extends into the sensitive areas of the hoof, with lameness and infection resulting. Animals maintained in very soiled or muddy conditions, combined with poor hoof care, are more likely affected. Fusobacterium necrophorum is often the pathogen involved. Separation of the animal, supportive care, surgical drainage, and antibiotic treatment are indicated. A. Infectious Diseases 1. Bacterial, Mycoplasmal, and Rickettsial Diseases a. Actinobacillosis ("Wooden Tongue") Etiology. Actinobacillus lignieresii is an aerobic, nonmotile, non-spore-forming, gram-negative rod that is widespread in soil and manure and is found as normal flora of the respiratory, gastrointestinal, and reproductive tracts of ruminants. In sheep and cattle, A. lignieresii causes sporadic, noncontagious, and potentially chronic disease characterized by diffuse abscess and granuloma formation in tissues of the head and occasionally other body organs. This disease, called wooden tongue, has not been documented in goats. Clinical signs. Skin lesions are common. Tongue lesions are more common in cattle than in sheep. Lip lesions are more common in sheep. Soft-tissue or lymph node swelling accompanied by draining tracts is observed in the head and neck regions, as well as other areas. Animals may have difficulty prehending food; may be anorexic, weak, unthrifty and depressed; and may salivate excessively. Diagnosis is made based on clinical signs and is confirmed by culture. Epizootiology and transmission. The organism penetrates wounds of the skin, mouth, nose, gastrointestinal tract, testicles, and mammary gland. Rough feed material and foreign bodies may play a role in causing abrasions. Actino bacillus lignieresii then enters into deeper tissues, where it causes chronic inflammation and abscess formation. Lymphatic spread may occur, leading to abscessation of lymph nodes or infection of other organs. Necropsy findings. Purulent discharges of white-green exudate drain from the tracts that often extend from the area of colonization to the skin surface. Exudates will also contain characteristic small white-gray (sulfurlike) granules. The pus is usually nonodorous. Differential diagnosis. Contagious ecthyma and caseous lymphadenitis are the primary differentials. Diseases or injuries causing oral pain and discomfort, such as dental infections, foreign bodies, and trauma, should be considered. Treatment. Animals should be fed softer feeds. Antibiotics such as sulfonamides, tetracyclines, and ampicillin are effective, although high doses and long durations of therapy are required. Penicillin is not effective. Weekly systemic administration of sodium iodide for several weeks is not as effective as antibiotic therapy. Surgical excision and drainage are not recommended. Prevention and control. Because the organism enters through tissue wounds, especially those associated with oral trauma, feedstuffs should be closely monitored for coarse material and foreign bodies. b. Arcanobacterium Infection (Formerly actinomycosis, or "Lumpy Jaw ") Etiology. Arcanobacterium (formerly known as Actinomyces or Corynebacterium) pyogenes and A. bovis are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Arcanobacterium bovis is a normal part of the ruminant oral microflora and is the organism associated with "lumpy jaw" in cattle; this syndrome is rarely seen in sheep and goats. This organism has also been associated with pharyngitis and mastitis in cattle. Clinical signs and diagnosis. Arcanobacterium bovis causes mandibular lesions primarily. The mass will be firm, non-painful, and immovable. Draining tracts may develop over time. If teeth roots become involved, painful eating and weight loss are evident. Radiographic studies are helpful for determining fistulas. Diagnosis is based on clinical signs, and culture is required to confirm Arcanobacterium. The prognosis is poor for lumpy jaw. Epizootiology and transmission. These organisms are normal flora of the gastrointestinal tracts of ruminants and gain entrance into the tissues through abrasions and penetrating wounds. Necropsy. Draining lesions with sulfurlike granules (as with actinobacillosis) are frequently observed. Pathogenesis. Arcanobacterium pyogenes is known to produce an exotoxin, which may be involved in the pathogenesis. Differential diagnosis. Actinobacillus lignieresii and caseous lymphadenitis are important differentials for draining tracts. A major differential for omphalophlebitis is an umbilical hernia, which will typically not be painful or infected. There are many differentials for septic joints and polyarthritis: Chlamydia spp., Mycoplasma spp., streptococci, coliforms, Erysipelothrix rhusiopathiae, Fusobacterium necrophorum, and Salmonella spp. Tumors, trauma to the affected area, such as the mandible, and dental disease or oral foreign body should also be considered. Prevention and control. Arcanobacterium bovis lesions can be prevented or minimized by feeds without coarse or sharp materials. Treatment. Penicillin or derivatives such as ampicillin or amoxicillin are treatments of choice. Sodium iodides (intravenous) and potassium iodides (orally) have been utilized also. Extended antibiotic therapy may be necessary. Surgical excision is an option. In addition to medications noted above, isoniazid is somewhat effective for A. bovis infections in nonpregnant cattle. Research complications. The possibility of long-term infection and long therapy are factors that will diminish the value of affected research animals. c. Actinomycosis Omphalophlebitis, omphaloarteritis, omphalitis, and navel ill are terms referring to infection of the umbilicus in young animals. Arcanobacterium pyogenes is the most common organism causing omphalophlebitis, an acute localized inflammation and infection of the external umbilicus. Most cases occur within the first 3 months of age, and animals are presented with a painful enlargement of the umbilicus. Animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, hematuria, and so on. Severe sequelae may include septicemia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, and endocarditis. Research complications. Young stock affected by omphalophlebitis may be inappropriate subjects because of growth setbacks and physiologic stresses from the infection. Affected adult animals will not thrive and, even with therapy, may not be appropriate research subjects. d. Anthrax Etiology. Bacillus anthracis is a nonmotile, capsulated, spore-forming, aerobic, gram-positive bacillus that is found in alkaline soil, contaminated feeds (such as bonemeal), and water. Common names for the disease anthrax include woolsorters' disease, splenic fever, charbon, and milzbrand. Clinical signs and diagnosis. Anthrax is a sporadic but very serious infectious disease of cattle, sheep, and goats characterized by septicemia, hyperthermia, anorexia, depression, listlessness, depression, and tremors. Subacute and chronic cases may occur also and are characterized by swelling around the shoulders, ventral neck, and thorax. The incubation period is 1 day to 2 weeks. Bloody secretions such as hematuria and bloody diarrhea often occur. Abortion and blood-tinged milk may also be noted. The disease is usually fatal, especially in sheep and goats, after 1–3 days. Death is the result of shock, renal failure, and anoxia. Diagnosis is based on the clinical signs of peracute deaths and hemorrhage. Stained blood smears may show short, single to chained bacilli. Blood may be collected from a superficial vein and submitted for culture. Epizootiology and transmission. Cattle and sheep tend to be affected more commonly than goats, because of grazing habits. Older animals are more vulnerable than younger, and bulls are more vulnerable than cows. Although the disease occurs worldwide, and even in cold climates, most cases in the United States occur in the central and western states, and outbreaks usually occur as the result of spore release after abrupt climatic changes such as heavy rainfall after droughts or during warmer, dryer months. Spores survive very well in the environment. The anthrax organisms (primarily spores) are generally ingested, sporulate, and replicate in the local tissues. Abrasive forages may play a role in infection. Transmission via insect bites or through skin abrasions rarely occurs. Necropsy. Necropsies should not be done around animal pens or pastures, and definitive diagnoses may be made without opening the animals. Incomplete rigor mortis, rapid putrefaction, and dark, uncoagulated blood exuding from all body orifices are common findings. Blood collected carefully and promptly from peripheral veins of freshly dead animals can be used diagnostically. Splenomegaly, cyanosis, epicardial and subcutaneous hemorrhages, and lymphadenopathy are characterisitic of the disease. Pathogenesis. The rapidly multiplying organisms enter the lymphatics and bloodstream and result in a severe septicemia and neurotoxicosis. Encapsulation protects the organisms from phagocytosis. Liberated toxins cause local edema. Differential diagnosis. Although anthrax should always be considered when an animal healthy the previous day dies acutely, other causes of acute death in ruminants should be considered, e.g., bloat, poisoning, enterotoxemia, malignant edema, blackleg, and black disease. Prevention and control. Outbreaks must be reported to state officials. Anthrax is of particular concern as a bioterrorism agent. Any vaccination programs should also be reviewed with regulatory personnel. Herds in endemic areas and along waterways are usually vaccinated routinely with the Sterne-strain spore vaccine (virulent, nonencapsulated, live). Careful hygiene and quarantine practices are crucial during outbreaks. Dead animals and contaminated materials should be incinerated or buried deeply. Biting insects should be controlled. The disease is zoonotic and a serious public health risk. Treatment. Treatment of animals in early stages with penicillin and anthrax antitoxin (hyperimmune serum, if available) may be helpful. Amoxicillin, erythromycin, oxytetracycline, gentamicin, and fluoroquinolones are also good therapeutic agents. During epidemics, animals should be vaccinated with the Sterne vaccine. Research complications. Natural and experimental anthrax infections are a risk to research personnel; the pathogen may be present in many body fluids and can penetrate intact skin. The organism sporulates when exposed to air, and spores may be inhaled during postmortem examinations. e. Brucellosis Etiology. Brucella is a nonmotile, non-spore-forming, nonencapsulated, gram-negative coccobacillus. Brucella abortus is one of several Brucella species that infects domestic animals but cross-species infections occur rarely. Brucella abortus or B. melitensis may cause brucellosis in sheep, cattle, and goats. Brucella melitensis (biovar 1, 2, or 3) is the primary cause of sheep disease ( Garin-Bastuji et al., 1998 ). Brucella ovis is more commonly associated with ovine epididymitis or orchitis than abortion. In the United States, clusters of brucellosis are still found in western areas contiguous to Yellowstone National Park. Bang's disease is the common name given to the disease in ruminants. Clinical signs and diagnosis. Brucella melitensis in the adult ewe is generally asymptomatic and self-limiting within about 3 months. However, because the organism may enter and cause necrosis of the chorionic villi and fetal organs, abortion or stillbirths may occur. Abortion usually occurs in the third trimester, after which the ewe will appear to recover. It has been reported that up to 20% of infected ewes may abort more than once. Rams will also be infected and may develop orchitis or pneumonia. The disease caused by B. ovis is manifested by clinical or subclinical infection of the epididymis, leading to epididymal enlargement and testicular atrophy. Brucella ovis causes decreased fertility. Brucella melitensis is the more common cause of brucellosis in goats. Brucella abortus has been shown to infect goats in natural and experimental infections, and B. ovis has also been shown to infect goats experimentally. Does infected with B. melitensis will also abort during the third trimester. Infections with B. abortus in cattle produce few clinical signs. There may be a brief septicemia during which organisms are phagocytosed by neutrophils and fixed macrophages in lymph nodes. In cows, the organism localizes in supramammary lymph nodes and udders and in the endometrium and placenta of pregnant cows. Infection may cause abortions after the fifth month, with resulting retained placentas. Permanent infection of the udder is common and results in shedding of organisms in milk. In bulls, the organism may cause unilateral orchitis and epidydimitis and involvement of the secondary sex organs. Organisms may be in the semen. In infected herds, lameness may also be a clinical sign. Diagnosis of brucellosis can be made by bacterial isolation of the Brucella organism from necropsy samples (especially the fetal stomach contents), as well as by supportive serological evidence. Many serological tests are available, such as the tube and plate agglutination tests, the card or rose bengal test, the rivanol precipitation test, complement fixation, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and others. Test selection is often dependent on state requirements in the United States. Epizootiology and transmission. The primary route of transmission of B . abortus is ingestion of the organism from infected tissues and fluids (milk, vaginal and uterine discharges) during and for a few weeks after abortion or parturition; contaminated semen is considered to be a minor source of infection. Exposure to the organism may occur via the gastrointestinal tract (contaminated feed or water), the respiratory tract (droplet infection), or the reproductive tract (contaminated semen) and through other mucous membranes such as the conjunctiva. Brucella ovis is transmitted in the semen, as well as orally or nasally through contaminated feed and bedding. Necropsy findings. A sheep fetus aborted due to Brucella will exhibit generalized edema. The liver and spleen will be swollen, and serosal surfaces will be covered with petecchial hemorrhages. Peritoneal and pleural cavities often contain serofibrinous exudates. The placenta will be leathery. Pathogenesis. Ruminants are considered especially susceptible to Brucella infection, because of higher levels of erythritol (a sugar alcohol), which is a growth stimulant for the organism. Brucella utilizes erythritol preferentially over glucose as an energy source. Placentas and male genitalia also contain high levels of erythritol. Brucella organisms also evade lysis when phagocytosed by macrophages and neutrophils and survive intracellularly in phagosomes. Abortion is the result of placentitis, typically during the third trimester of gestation. Brucella ovis enters the host through the mucous membranes, then passes into the lymphatics, causes hyperplasia of reticuloendothelial cells, and is spread to various organs via the blood. The organism localizes in the epididymides, the seminal vesicles, the bulbourethral glands, and the ampullae. Orchitis may be a sequelae of the disease. Epididymitis can be diagnosed by identifying gross lesions by palpation of the epididymides, by serological evidence of antibodies to B. ovis, and by semen cultures. Differential diagnosis. Differential diagnoses include all other abortion-causing diseases. Many other agents, such as Actinobacillus spp., Arcanobacterium (Actinomyces) pyogenes, Eschericia coli, Pseudomonas spp., Proteus mirabilis, Chlamydia, Mycoplasma, and others may be associated with ovine epididymitis and orchitis. A clinically and pathologically similar agent, Actinobacillus seminis, has been isolated from virgin rams. This organism has morphological and staining characteristics similar to those of B. ovis and complicates the diagnosis ( Genetzky, 1995 ). Prevention and control. The Rev 1 vaccine has been recommended for vaccination of ewe lambs in endemic areas, but this vaccine is not used in the United States. Separating young rams from potentially infected older males, sanitizing facilities, and vaccinating them with B. ovis bacterin can prevent the disease. Over the past 20 years, aggressive federal and state regulatory and cattle herd health programs in the United States have provided control and prevention mechanisms for this pathogen through a combination of serological monitoring of herds, slaughter of diseased animals, herd management, vaccination programs, and monitoring of transported animals. Most states are considered brucellosis-free in the cattle populations; thus, procurement of ruminants that have been exposed to this infectious agent will be unlikely. Cattle vaccination programs can be very successful when conducted on a herd basis to reduce likelihood of exposure. Strain 19 and the recently validated attentuated strain RB51 are live vaccines and can be used in healthy heifer calves 4–12 months old. Vaccination for older animals may be done under certain circumstances. Vaccination of bull calves is not recommended, because of low likelihood of spread through semen and possibility of vaccination-induced orchitis. The strain 19 vaccine induces long-term cell-mediated immunity, protects a herd from abortions, and protects the majority of a herd from reactors during a screening and culling program. The vaccine will not, however, protect the animals from becoming infected with B. abortus. Strain 19 vaccine induces an antibody response in cattle. The RB51 vaccine does not result in antibody titers and therefore is advantageous because infection with Brucella can be determined serologically. The RB51 vaccine has been designated as the official calfhood bovine brucellosis vaccine in the United States by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS) ( Stevens et al., 1997 ). Brucella vaccine should be administered to unstressed, healthy cattle, with attention to particular side effects of the vaccination material and to prevention of compounding stresses associated with weaning, regrouping, other management changes, and shipping. The RB51 is regarded as less pathogenic and abortigenic in cattle. Treatment. Definite confirmation of Brucella infection is important from the standpoint of public and herd health. Culling is considered the treatment of choice in cattle herds. Rams infected with B. ovis should be isolated and treated with tetracyclines. Research complications. Brucellosis represents a research complication as a cause of abortions and of infections in male ruminants. Impairment of the infected host's immune system, especially alteration of phagocytic cells where the bacteria stay in membrane-bound vesicles, should be considered. The potential complications of needle sticks by large-animal veterinarians with the strain 19 vaccine and the public health risks (undulant fever) are well known. Less is known presently regarding the RB51 vaccine effects in humans. Epidemiologic and diagnostic methodologies are being developed to track and monitor these cases. There is also a risk of human infection from handling infected materials during a dystocia or postmortem. Worldwide, B. melitensis is the leading cause of human brucellosis. f. Campylobacteriosis (Vibriosis) i. Campylobacter fetus subsp. intestinalis; C. jejuni infection (ovine vibriosis) Etiology. Campylobacter (Vibrio) fetus subsp. intestinalis, a pleomorphic curved to coccoid, motile, non-spore-forming, gram-negative bacterium, causes campylobacteriosis, the most important cause of ovine abortion in the United States. There are few reports of campylobacteriosis in goats in the United States. Vibriosis is derived from the name formerly given to the genus; the term is still frequently used. Clinical signs and diagnosis. Ovine vibriosis is a contagious disease that causes abortion, stillbirths, and weak lambs. The organism inhabits the intestines and gallbladder in subclinical carriers. Abortion generally occurs in the last trimester, and abortion storms may occur as more susceptible animals, such as maiden ewes, become exposed to the infectious tissues. It is reported that 20–25% of the flock may become infected and up to 5% of the ewes will die ( Jensen and Swift, 1982 ). Some lambs may be born alive but will be weak, and dams will not be able to produce milk. Diagnosis is achieved by microscopic identification or isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharges. Tentative identification of the organism can be made by observing curved ("gull-wing") rods in Giemsa-stained or Ziehl–Neelsen–stained smears from fetal stomach contents, placentomes, or maternal uterine fluids. Epizootiology and transmission. Campylobacteriosis occurs worldwide. Campylobacter spp., such as C. jejuni, normally inhabit ovine gastrointestinal tracts. Transmission of the disease occurs through the gastrointestinal tract, followed by shedding, especially associated with aborted tissues and fluids. In abortion storms, considerable contamination of the environment will occur due to placenta, fetuses, and uterine fluids. Ewes may have active Campylobacter organisms in uterine discharges for several months after abortion. The bacteria will also be shed in feces, and feed and water contamination serve as another source. There is no venereal transmission in the ovine. Necropsy. Aborted fetuses will be edematous, with accumulation of serosanguinous fluids within the subcutis and muscle tissue fascia. The liver may contain 2–3 cm pale foci. Placental tissues will be thickened and edematous and will contain serous fluids similar to those of the fetus. The placental cotyledons may appear gray. Pathogenesis. The organism enters the bloodstream and causes a short-term bacteremia (1–2 weeks) prior to the localizing of the bacteria in the chorionic epithelial cells and finally passing into the fetus. Differential diagnosis. Toxoplasma, Chlamydia, and Listeria should be considered in late gestation ovine abortions. Prevention and control. A bacterin is available to prevent the disease. Carrier states have been cleared by treating with a combination of antibiotics, including penicillin and oral Chlortetracycline. Aborting ewes should be isolated immediately from the rest of the flock. After an outbreak, ewes will develop immunity lasting 2–3 years. Treatment. Infected animals should be isolated and provided with supportive therapy. Prompt decontamination of the area and disposal of the aborted tissues and discharges are important. Research complications. Losses from abortion may be considerable. Campylobacter ssp. are zoonotic agents, and C. fetus subsp. intestinalis may be the cause of "shepherd's scours." ii. Campylobacter fetus subsp. venerealis infection (bovine vibriosis) Etiology. Campylobacter fetus subsp. venerealis is the main cause of bovine campylobacteriosis abortions. It does not cause disease in other ruminant species. Clinical signs and diagnosis. Preliminary signs of a problem in the herd will be a high percentage of cows returning to estrus after breeding and temporary infertility. This will be particularly apparent in virgin heifers that may return to estrus by 40 days after breeding. Long interestrous intervals also serve an indication of a problem. Spontaneous abortions will occur in some cases, typically during the fourth to eighth months of gestation. Severe endometritis may lead to salpingitis and permanent infertility. Demonstration or isolation of the organism, a curved rod with corkscrew motility, is the basis for diagnosis. The vaginal mucous agglutination test is used to survey herds for campylobacteriosis. Serology will not be worthwhile, because the infection does not trigger a sufficient antibody response. Culture from breeding animals may be difficult because Campylobacter will be overgrown by faster-growing species also present in the specimens. Epizootiology and transmission. The bacteria is an obligate, ubiquitous organism of the genital tract. Transmission is from infected bulls to heifers. Older cows develop effective immunity. Necropsy findings. Necrotizing placentitis, dehydration, and fibrinous serositis will be found grossly. In addition, bronchopneumonia and hepatitis will be seen histologically. Pathogenesis. Campylobacter organisms grow readily in the genital tract, and infection is established within days of exposure. The resulting endometritis prevents conception or causes embyronic death. Differential diagnosis. The primary differential diagnosis for campylobacteriosis is trichomoniasis. Other venereal diseases should be considered when infertility problems are noted in a herd. These include brucellosis, mycoplasmosis, ureaplasmosis, infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV), and bovine virus diarrhea (BVD). Leptospirosis should also be considered. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. Killed bacterin vaccines are available, either as oil adjuvant or as aluminum hydroxide adsorbed. The former is preferred because of duration of immunity but causes granulomas. That vaccine also has specific recommendations regarding administration several months before the breeding season. The latter product is administered closer to the breeding season, and the duration of immunity is not as prolonged. In both cases, boosters should be given after the initial immunization and as part of the regular prebreeding regimen. Only one bacterin product is approved for use in bulls. Many combination vaccine products contain only the aluminum hydroxide adsorbed product. Artificial insemination (AI) is particularly useful at controlling the disease, but bulls used for AI must be part of a screening program for this and other venereal diseases such as trichomoniasis. Treatment. Cows will usually recover from the infection, and treatment with antibiotics such as penicillin, administered as an intrauterine infusion, improve the chances of returning to breeding condition. g. Caprine Staphylococcal Dermatitis Etiology. The most common caprine bacterial skin infection is caused by Staphylococcus intermedius or S. aureus and is known as staphylococcal dermatitis ( Smith and Sherman, 1994 ). The Staphylococcus organisms are cocci and are categorized as primary pathogens or ubiquitous skin commensals of humans and animals. Staphylococcus aureus and S. intermedius are classified as primary pathogens and produce coagulase, a virulence factor. Clinical signs and diagnosis. Small pustular lesions, caused by bacterial infection and inflammation of the hair follicle, occur around the teats and perineum. Occasionally, the infection may involve the flanks, underbelly, axilla, inner thigh, and neck. Staphylococcal dermatitis may occur secondary to other skin lesions. Diagnosis is based on lesions. Culture will distinguish S. aureus. Pathogenesis. Simple boredom may cause rubbing, followed by staphylococcal infection of damaged epidermis. Differential diagnosis. The presence of scabs makes contagious ecthyma a differential diagnosis, along with fungal skin infections and nutritional causes of skin disease. Treatment. Severe infections should be treated with antibiotics based on culture and sensitivity. Severe lesions and lesions localized to the underbelly, thighs, and udder benefit by periodic cleaning with an iodophor shampoo and spraying with an antibiotic and an astringent ( Smith and Sherman, 1994 ). h. Clostridial Diseases i. Clostridium perfringens type C infection (enterotoxemia and struck) Etiology. Clostridium perfringens is an anaerobic, gram-positive, nonmotile, spore-forming bacterium that lives in the soil, in contaminated feed, and in gastrointestinal tracts of ruminants. The bacteria is categorized by toxin production. Toxins include alpha (hemolytic), beta (necrotizing), delta (cytotoxic and hemoltyic), epsilon, and iota. Types of C. perfingens are A, B, C, D, and E. This is a common and economically significant disease of sheep, goats, and cattle. Clinical signs and diagnosis. The beta toxin associated with overgrowth of this bacterium results in a fatal hemorrhagic enterocolitis within the first 72 hr of a young ruminant's life. Many animals may be found dead, with no clinical presentation. Affected animals are acutely anemic, dehydrated, anorexic, restless, and depressed and may display tremors or convulsions as well as abdominal pain. Feces may range from loose gray-brown to dark red and malodorous. Morbidity and mortality may be nearly 100%. A similar noncontagious but acutely fatal form of enterotoxemia in adult sheep, called struck, occurs in yearlings and adults. Struck is rare in the United States. The disease is also caused by the beta toxin of C. perfringens type C and is often associated with rapid dietary changes or shearing stresses in sheep. Although affected animals are usually found dead, clinical signs include uneasiness, depression, and convulsions. Mortality is usually less than 15%. Diagnosis is usually based on necropsy findings, although confirmation can be made by culture of the organism. Identification of the beta toxin in intestinal contents may be difficult because of instability of the toxin. Epizootiology and transmission. Clostridial organisms are ubiquitous in the environment as well as in the gastrointestinal tract and contaminated feeds. Confinement and poor sanitation predisposes to infection with C. perfringens. Transmission is by ingestion of contaminated material. Necropsy findings. Necropsy findings include a milk-filled abomasum, and hemorrhage in the distal small intestine and throughout the large intestine. Petechial hemorrhages of the serosal surfaces of many organs, especially the thymus, heart, and gastrointestinal tract, will be visible. Hydropericardium, hydroperitoneum, and hemorrhagic mesenteric lymph nodes will also be present. Pulmonary and brain edema may also be seen. Histologically, the gram-positive C. perfringens organisms may be visible in excess numbers along the mucosal surface of the swollen, congested, necrotic intestines. In cases of struck, necropsy findings include congestion and erosions of the mucosa of the gastrointestinal tract, serosal hemorrhages, and serous peritoneal and pericardial fluids. In late stages of the disease and especially if prompt necropsy is not performed, the organism will infiltrate the muscle fascial layers and produce serohemorrhagic and gaseous infiltration of perimysial and epimysial spaces. Pathogenesis. Hemorrhagic enterotoxemia is an acute, sporadic disease caused by the beta toxin of Clostridium perfringens type C. Neonates ingest the organism, which then proliferates and attaches to the gastrointestinal microvilli and elaborates primarily the beta toxins. The trypsin inhibitors present in colostrum prevent inactivation of the beta toxin. The toxins injure intestinal epithelial cells and then enter the blood, leading to acute toxemia. The intestinal injury may result in diarrhea, with small amounts of hemorrhage. Associated electrolyte and water loss result in dehydration, acidosis, and shock. Differential diagnosis. Differential diagnoses include other clostridial diseases such as blackleg and black disease, as well as coccidiosis, salmonellosis, anthrax, and acute poisoning. Prevention and control. A commercial toxoid is available and should be administered to the pregnant animals prior to parturition. An alternative includes administration of an antitoxin to the newborn lambs. The disease may become endemic once it is on the premises. Treatment. Treatment is difficult and usually unsuccessful. Antitoxin may be useful in milder cases, and the antitoxin and toxoid can also be administered during an outbreak. Research complications. This disease can be costly in losses of neonates and younger animals. ii. Clostridium perfringens type D infection (pulpy kidney disease) Etiology. Clostridium perfringens type D releases epsilon toxin that is proteolytically activated by trypsin. This disease caused by C. perfringens tends to be associated with sheep and is of less importance in goats and cattle. Clinical signs. The peracute condition in younger animals is characterized by sudden deaths, which are occasionally preceded by neurological signs such as incoordination, opisthotonus, and convulsions. Because the disease progresses so rapidly to death (within 1–2 hr), clinical signs are rarely observed. Hypersalivation, rapid respirations, hyperthermia, convulsions, and opisthotonus have been noted. In acute cases, hyperglycemia and glucosuria are considered almost pathognomonic. Clinical signs in chronic cases in older animals, such as adult goats, include soft stools, weight loss, anorexia, depression, and severe diarrhea, sometimes with mucus and blood. Mature affected sheep may be blind and anorectic and may head-press. Necropsy findings. Necropsy findings are similar to those seen with C. perfringens type C. Additionally, extremely necrotic, soft kidneys ("pulpy kidneys") are usually observed immediately following death. (This phenomenon is in contrast to what is normally associated with later stages of postmortem autolysis.) Focal encephalomalacia, and petechial hemorrhages on serosal surfaces of the brain, diaphragm, gastrointestinal tract, and heart are common findings. Diagnosis can be made from the typical clinical signs and necropsy findings as well as the observation of glucose in the urine at necropsy. Pathogenesis. The epsilon toxin causes neuronal death and shock, probably through vascular damage. The noncontagious, peracute form of enterotoxemia occurs in suckling, fast-growing animals, either nursing from their dams or on high-protein, high-energy concentrates. The largest, fastest-growing animals generally are predisposed to this condition; for example, lambs, fat ewe lambs, and usually singleton lambs tend to be most susceptible. The hyperglycemia and glucosuria seen in acute cases are due to epsilon toxin effects on liver glycogen metabolism. Differential diagnosis. Tetanus, enterotoxigenic E. coli, botulism, polioencephalomalacia, grain overload, and listeriosis are differentials. Prevention and control. Vaccination prevents the disease. Maternal antibodies last approximately 5 weeks postpartum; thus young animals should be vaccinated at about this time. Feeding regimens to young, fast-growing animals and feeding of concentrates to adults should be evaluated carefully. Treatment. Treatment consists of support (fluids, warmth), antitoxin administration, oral antibiotics, and diet adjustment. iii. Clostridium tetani infection (tetanus, lockjaw) Etiology. Clostridium tetani is a strictly anaerobic, motile, spore-forming, gram-positive rod that persists in soils and manure and within the gastrointestinal tract. At least 10 serotypes of C. tetani exist. Clinical signs. Infection by C. tetani is characterized by a sporadic, acute, and fatal neuropathy. After an incubation period of 4 days to 3 weeks, the animal exhibits bloat; muscular spasticity; prolapse of the third eyelid; rigidity and extension of the limbs, leading to a stiff gate; an inability to chew; and hyperthermia. Erect or drooped ears, retracted lips, drooling, hypersensitivity to external stimuli, and a "sawhorse" stance are frequent signs. The animal may convulse. Death occurs within 3–10 days, and mortality is nearly 100%, primarily from respiratory failure. Diagnosis is based on clinical signs. Muscle-related serum enzymes such as aspartate aminotransferase (AST), creatinine kinase (CK), and lactate dehydrogenase (LDH) might be elevated. ( Jensen and Swift, 1982 ). Serum cortisol may also be elevated, and stress hyperglycemia may be evident. Permanent lameness may result in survivors. Epizootiology and transmission. Clostridium tetani is a soil contaminant and is often found as part of the gut microflora of herbivores. The organisms sporulate and persist in the environment. All species of livestock are susceptible, but sheep and goats are more susceptible than cattle. Individual cases may occur, or herd outbreaks may follow castration, tail docking, ear tagging, or dehorning. Mouth wounds may also be sites of entry. Pathogenesis. Tetanus, or lockjaw, is caused by the toxins of C. tetani. All serovars produce the same exotoxin, which is a multiunit protein composed of tetanospasmin, which is neurotoxic, and tetanolysin, which is hemolytic. A nonspasmogenic toxin is also produced. Contamination of wounds results in anaerobic proliferation of the bacterium and liberation of the tetanospasmin, which diffuses through motor neurons in a retrograde direction to the spinal cord. The toxin inhibits the release of glycine and γ-aminobutyric acid from Renshaw cells; this results in hypertonia and muscular spasms. Proliferation of C. tetani in the gut of affected animals may also serve as a source and may produce clinical signs. The uterus is the most common site of infection in postparturient dairy cattle with retained placentas. Differential diagnoses. Early in the course of the infection, differential diagnoses include bloat, rabies, hypomagnesemic tetany, polioencephalomalacia, white muscle disease, enterotoxemia in lambs, and lead poisoning. Polyarthritis of cattle is a differential for the gait changes in that species. Necropsy findings. Findings are nonspecific except for the inflammatory reaction associated with the wound. Because of the low number of organisms necessary to cause neurotoxicosis, isolation of C. tetani from the wound may be difficult. Treatment. Treatment consists of cleaning the infected wound; administering tetanus antitoxin (e.g., at least 500 IU in an adult sheep or goat); vaccinating with tetanus toxoid; administering of antibiotics (penicillin, both parenterally [potassium penicillin intravenously and procaine penicillin intramuscularly] and flushed into the cleaned wound), a sedative or tranquilizer (e.g., acepromazine or chlorpromazine) and a muscle relaxant; and keeping the animal in a dark, quiet environment. Supportive fluids and glucose must be administered until the animal is capable of feeding. If the animal survives, revaccination should be done 14 days after the previous dose. Prevention and control. Like other ubiquitous clostridial diseases, tetanus is impossible to eradicate. The disease can be controlled and prevented by following good sanitation measures, aseptic surgical procedures, and vaccination programs. Tetanus toxoid vaccine is available and very effective for stimulating long-term immunity. Tetanus antitoxin can be administered (200 IU in lambs) as a preventive or in the face of disease as an adjunct to therapy. Both the toxoid and the antitoxin can be administered to an animal at the same time, but they should not be mixed in the syringe, and each should be administered at different sites, with a second toxoid dose administered 4 weeks later. Animals should be vaccinated 2 or 3 times during the first year of life. Does and ewes should receive booster vaccinations within 2 months of parturition to ensure colostral antibodies. Research complications. Unprotected, younger ruminants may be affected following routine flock or herd management procedures. Contaminated or inadequately managed open wounds or lesions in older animals may provide anaerobic incubation sites. iv. Clostridium novyi infection (bighead; black disease; bacillary hemoglobinuria, or red water) and C. chauvoei infection (blackleg) Etiology. Clostridium novyi, an anaerobic, motile, spore-forming, gram-positive bacteria, is the agent of bighead and black disease. Clostridium novyi type D (C. hemolyticum) is the cause of bacillary hemoglobinuria, or "red water." Clostridium chauvoei is the causative agent of blackleg. Clinical signs. Bighead is a disease of rams characterized by edema of the head and neck. The edema may migrate to ventral regions such as the throat. Additional clinical signs include swelling of the eyelids and nostrils. Most animals will die within 48–72 hours. Black disease, or infectious necrotic hepatitis, is a peracute, fatal disease associated with C. novyi. It is more common in cattle and sheep but may be seen in goats. The clinical course is 1–2 days in cattle and slightly shorter in sheep. Otherwise healthy-appearing adult animals are often affected. Clinical signs are rarely seen, because of the peracute nature of the disease. Occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency are observed prior to death. Bacillary hemoglobinuria is an acute disease seen primarily in cattle and characterized by fever and anorexia, in addition to the hemoglobinemia and hemoglobinuria indicated by the name. Animals that survive a few days will develop icterus. Mortality may be high. Blackleg, a disease similar to bighead, causes necrosis and emphysema of muscle masses, serohemorrhagic fluid accumulation around the infected area, and edema ( Jackson et al., 1995 ). Blackleg is more common in cattle than in sheep. The incubation period is 2–5 days and is followed by hyperthermia, muscular stiffness and pain, anorexia, and gangrenous myositis. The clinical course is short, 24–48 hr, and untreated animals invariably die. Blackleg in cattle can be associated with subcutaneous edema or crepitation; these do not usually occur in sheep. Most lesions are associated with muscles of the face, neck, perineum, thigh, and back. Epizootiology and transmission. Bighead is caused by the toxins of C. novyi, which enters through wounds often associated with horn injuries during fighting. The C. novyi type B organisms produce alpha and beta toxins, and the alpha toxins are mostly responsible for toxemia, tissue necrosis, and subsequent death. Clostridium novyi type D is endemic in the western United States. It is hypothesized that the C. chauvoei organisms enter through the gastrointestinal tract. Black disease and bacillary hemoglobinuria are associated with concurrent liver disease, often associated with Fasciola infections (liver flukes); it is sometimes seen as a sequela to liver biopsies. The diseases are more common in summer months, and fecal contamination of pastures, flooding, and infected carcasses are sources of the organism. Birds and wild animals may be vectors of the pathogen. Ingested spores are believed to develop in hepatic tissue damaged and anoxic from the fluke migrations. Necropsy. Diagnosis of black disease is usually based on postmortem lesions. Subcutaneous vessels will be engorged with blood, resulting in dried skin with a dark appearance. Carcasses putrefy quickly. In addition, hepatomegaly and endocardial hemorrhages are common, and hepatic damage from flukes may be so severe that diagnosis is difficult. Blood coagulates slowly in affected animals. Pathogenesis. The propagation of the clostridial organisms is self-promoted by the damage caused by the toxins and the increased local anaerobic environment created. Clostridium novyi proliferates in the soft tissues of the head and neck, and the resultant clostridial toxin causes increased capillary permeability and the liberation of serous fluids into the tissues. Mixed infections with related clostridial organisms may lead to increasing hemorrhage and necrosis in the affected tissues. Diagnosis is based on clinical signs. In black disease and bacillary hemoglobinuria disease, the ingested clostridial spores are absorbed, enter the liver, and cause hepatic necrosis. Associated toxemia causes subcutaneous vascular dilatation; increased pericardial, pleural, and peritoneal fluid; and endocardial hemorrhages. The toxins produced by C. novyi, identified as beta, eta, and theta, and each having enzymatic or lytic properties or both, also contribute to the hemolytic disease. Clostridium chauvoei spores proliferate in traumatized muscle areas damaged by transportation, rough handling, or injury. Differential diagnosis. Differential diagnoses include other clostridial diseases as well as photosensitization. Hemolytic diseases such as babesiosis, leptospirosis, and hemobartonellosis should be included as differentials. Treatment. For C. chauvoei infection (blackleg), early treatment with penicillin or tetracycline may be helpful. Treatment for black disease is not rewarding even if the animal is found before death. Carcasses from bacillary hemoglobinuria losses should be burned, buried deeply, or removed from the premises. Prevention and control. Vaccinating animals with multivalent clostridial vaccines can prevent these diseases. Subcutaneous administration of vaccine material is recommended over intramuscular. Vaccinations may be useful in an outbreak. Careful handling of ruminants during shipping and transfers will contribute to fewer muscular injuries. For bighead, mature rams penned together should be monitored for lesions, especially during breeding season. Control of fascioliasis is very important in prevention and control of black disease and in the optimal timing of vaccinations. v. Clostridium septicum infection (malignant edema) Etiology. Clostridium septicum is the species usually associated with malignant edema, but mixed infections involving other clostridial species such as C. chauvoei, C. novyi, C. sordellii, and C. perfringens may occur. Clostridium spp. are motile (C. chauvoei, C. septicum) or nonmotile, anaerobic, spore-forming, gram-positive rods. Clinicial signs. Malignant edema, or gas gangrene, is an acute and often fatal bacterial disease caused by Clostridium spp. The incubation period is approximately 2–4 days. The affected area will be warm and will contain gaseous accumulations that can be palpated as crepitation of the subcutaneous tissue around the infected area. Regional lymphadenopathy and fever may occur. The animal becomes anorexic, severely depressed, and possibly hyperthermic. Edema and crepitation may be noted around the wound; death occurs within 12 hr to 2 days. Epizootiology and transmission. The organisms are ubiquitous in the environment and may survive in the soil for years. The disease is especially prevalent in animals that have had recent wounds such as those that have undergone castration, docking, ear notching, shearing, or dystocia. Necropsy findings. The tissue necrosis and hemorrhagic serous fluid accumulations resemble those of other clostridial diseases. Pathogenesis. In most cases, the clostridial organisms cause a spreading infection through the fascial planes around the area of the injury; vegetative organisms then produce potent exotoxins, which result in necrosis (alpha toxin) and/or hemolysis (beta toxin). Furthermore, the toxins enter the bloodstream and central nervous system, resulting in systemic collapse and high mortality. Necropsy. Spreading, crepitant lesions around wounds are suggestive of malignant edema. Affected tissues are inflamed and necrotic. Gas and serosanguineous fluids with foul odors infiltrate the tissue planes. Large rod-shaped bacteria may be observed on histopathology; confirmation is made through culture and identification. Intramuscular inoculation of guinea pigs causes a necrotizing myositis and death. Organisms can be cultured from guinea pig tissues. Treatment. Infected animals can be treated with large doses of penicillin and fenestration of the wound is recommended. Prevention and control. Proper preparation of surgical sites, correct sanitation of instruments and the housing environment, and attention to postoperative wounds will help prevent this disease. Multivalent clostridial vaccines are available. Research complications. Morbidity or loss of animals from lack of or unsuccessful vaccination and from contaminated surgical sites or wounds may be consequences of this disease. i. Colibacillosis Etiology. Escherichia coli is a motile, aerobic, gram-negative, non-spore-forming coccobacillus commonly found in the environment and gastrointestinal tracts of ruminants. Escherichia coli organisms have three areas of surface antigenic complexes (O, somatic; K, envelope or pili; and H, flagellar), which are used to "group" or classify the serotypes. Colibacillosis is the common term for infections in younger animals caused by this bacteria. Clinical signs. Presentation of E. coli infections vary with the animal's age and the type of E. coli involved. Enterotoxigenic E. coli infection causes gastroenteritis and/or septicemia in lambs and calves. Colibacillosis generally develops within the first 72 hr of life when newborn animals are exposed to the organism. The enteric infection causes a semifluid, yellow to gray diarrhea. Occasionally blood streaking of the feces may be observed. The animal may demonstrate abdominal pain, evidenced by arching of the back and extension of the tail, classically described as "tucked up." Hyperthermia is rare. Severe acidosis, depression, and recumbancy ensue, and mortality may be as high as 75%. The septicemic form generally occurs between 2 and 6 weeks of age. Animals display an elevated body temperature and show signs suggestive of nervous system involvement such as incoordination, head pressing, circling, and the appearance of blindness. Opisthotonos, depression, and death follow. Occasionally, swollen, painful joints may be observed with septicemic colibacillosis. Blood cultures may be helpful in identifying the septicemic form. In ruminants, E. coli is is a less common cause of cystitis and pyelonephritis. The cystitis is characterized by dysuria and pollakiuria; gross hematuria and pyuria may be present. The infection may or may not be restricted to the bladder; in the later presentation, and in cases of pyelonephritis, a cow will be acutely depressed, have a fever and ruminal stasis, and be anorexic. In chronic cases, animals will be polyuric and undergo weight loss. Escherichia coli may also cause in utero disease in cattle, resulting in abortion or weakened offspring. Epizootiology and transmission. Escherichia coli is one of the most common gram-negative pathogens isolated from ruminant neonates. Zeman et al. (1989 ) classify E. coli infections into four groups: enterotoxigenic, enterohemorrhagic, enteropathogenic, and enteroinvasive. Enterotoxigenic E. coli (ETEC) attach to the enterocytes via pili, produce enterotoxins, and are the primary cause of colibacillosis in animals and humans. Fimbrial (pili) antigens associated with ovine disease include K99 and F41. Enterohemorrhagic E. coli (EHEC) attach and efface the microvillus, produce verotoxins, and occasionally cause disease in humans and animals. Enteropathogenic E. coli (EPEC) colonize and efface the microvillus but do not produce verotoxins. EPEC are associated with disease in humans and rabbits and cause a secretory diarrhea. Enteroinvasive E. coli (EIEC) invade the enterocytes of humans and cause a shigella-like disease. Overcrowding and poor sanitation contribute significantly to the development of this disease in young animals. The organism will be endemic in a contaminated environment and present on dams' udders. The bacteria rapidly proliferate in the neonates' small intestines. The bacteria and associated toxins cause a secretory diarrhea, resulting in the loss of water and electrolytes. If the bacteria infiltrate the intestinal barrier and enter the blood, septicemia results. Diagnosis of the enteric form can be made by observation of clinical signs, including diarrhea and staining of the tail and wool. Necropsy findings. Swollen, yellow to gray, fluid-filled small and large intestines, swollen and hemorrhagic mesenteric lymph nodes, and generalized tissue dehydration are common. Septicemic lambs may have serofibrinous fluid in the peritoneal, thoracic, and pericardial cavities; enlarged joints containing fibrinopurulent exudates; and congested and inflamed meninges. Isolation and serotyping of E. coli confirm the diagnosis. ELISA and latex agglutination tests are available diagnostic tools. Differential diagnosis. Differential diagnoses include the enterotoxemias caused by C. perfringens type A, B, or C; Campylobacter jejuni; Coccidia, rotavirus, coronavirus, Salmonella, and Cryptosporidia. Other contributing causes of abomasal tympany in young ruminants, such as dietary changes, copper deficiency, excessive intervals between feedings of milk replacer, or feeding large volumes should be considered. Prevention and control. The best preventive measures are maintenance of proper housing conditions, limiting overcrowding, and frequently sanitizing lambing areas. Attention to colostrum feeding techniques and colostral quality are important means of preventing disease. Treatment must include intravenous fluid hydration and reestablishment of acid-base and electrolyte abnormalities. Treatment. Antibiotics such as trimethoprim-sulfadiazine, enrofloxacin, cephalothin, amikacin, and apramycin may be helpful; oral antibiotics are not recommended. Vaccines are available for prevention of colibacillosis in cattle. j. Corynebacterium pseudotuberculosis Infection (Caseous Lymphadenitis) Etiology. Corynebacterium pseudotuberculosis (previously C. ovis) are nonmotile, non-spore-forming, aerobic, short and curved, gram-positive coccobacilli. Caseous lymphadenitis (CLA) is such a common, chronic contagious disease of sheep and goats that any presentation of abscessing and draining lymph nodes should be presumed to be this disease until proven otherwise. The disease has been reported occasionally in cattle. Clinical signs and diagnosis. Abscessation of superficial lymph nodes, such as the superficial cervical, retropharyngeal, subiliacs (prefemoral), mammary, superficial inguinals, and popliteal nodes, and of deep nodes, such as mediastinal and mesenteric lymph nodes, is typical. Radiographs may be helpful in identifying affected central nodes. Peripheral lymph nodes may erode and drain caseous, "cheesy," yellow-green-tan secretions. The incubation period may be weeks to months. Over time, an infected animal may become exercise-intolerant, anorexic, and debilitated. Fever, increased respiratory rates, and pneumonia may also be common signs. Exotoxin-induced hemolytic crises may occur occasionally. Morbidity up to 15% is common, and morbid animals will often eventually succumb to the disease. Diagnosis is based on clinical lesions; ELISA serological testing is also available. Smears of the exudate or lymph nodes aspirates can be Gram-stained. Lymph node aspirates may also be sent for culturing. Epizootiology and transmission. The organism can survive for 6 months or more in the environment and enters via skin wounds, shearing, fighting, castration, and docking. Ingestion and aerosolization (leading to pulmonary abscesses) have been reported as alternative routes of entry. Necropsy findings. Disseminated superficial abscesses as well as lesions of the mediastinal and mesenteric lymph nodes will be identified. Cut surfaces of the affected lymph nodes may appear lamellated. Lungs, liver, spleen, and kidneys may also be affected. Cranioventral lung consolidation with hemorrhage, fibrin, and edema are seen histologically. Pathogenesis. Corynebacterium pseudotuberculosis produces an exotoxin (phospholipase D) that damages endothelial and blood cell membranes. This process enhances the organisms' ability to withstand phagocytosis. The infection spreads through the lymphatics to local lymph nodes. The necrotic lymph nodes seed local capillaries and hematogenously and lymphatically spread the organisms to other areas, especially the lungs. Differential diagnosis. Differentials include pathogens causing lymphadenopathy and abscessation. Treatment. Antibiotic therapy is not usually helpful. Abscesses can be surgically lanced and flushed with iodine-containing and/or hydrogen peroxide solutions. Abscessing lymph nodes can be removed entirely from valuable animals. During warmer months, an insect repellent should be applied to and around healing lesions. All materials used to treat animals should be disposed of properly. Because of the contagious nature of the disease, animals with draining and lanced lesions should be isolated from CLA-negative animals at least until healed. Commercial vaccines are available ( Piontkowski and Shivvers, 1998 ). Prevention and control. Minimizing contamination of the environment, using proper sanitation methods for facilities and instruments, segregating affected animals, and taking precautions to prevent injuries are all important. Research complications. This pathogen is a risk for animals undergoing routine management procedures or invasive research procedures, because of its persistence in the environment, its long clinical incubation period, and its poor response to antibiotics. k. Corynebacterium renale, C. cystitidis, and C. pilosum Infections (Pyelonephritis; Posthitis and Ulcerative Vulvovaginitis) Etiology. Corynebacterium renale, C. cystitidis, and C. pilosum are sometimes referred to as the C. renale group. These are piliated and nonmotile gram-positive rods and are distinguished biochemically. Corynebacterium renale causes pyelonephritis in cattle, and C. pilosum and C. cystitidis cause posthitis, also known as pizzle rot or sheath rot, in sheep and goats. In many references, all these clinical presentations are attributed to C. renale. Clinical signs and diagnosis. Acute pyelonephritis is characterized by fever, anorexia, polyuria, hematuria, pyuria, and arched back posture. Untreated infections usually become chronic, with weight loss, anorexia, and loss of production in dairy animals. Relapses are common, and some infections are severe and fatal. Diagnosis of pyelonephritis is based on urinalysis (proteinuria and hematuria) and rectal or vaginal palpation (assessing ureteral enlargement). Urine culturing may not be productive. In chronic cases, E. coli and other gram-negatives may be present. Posthitis and vulvovaginitis are characteriazed by ulcers, crusting, swelling and pain. The area may have a distinct malodor. Necrosis and scarring may be sequelae of more severe infections. Fly-strike may also be a complication. Diagnosis is based on clinical signs and on investigation of feeding regimens. Epizootiology and transmission. Ascending urinary tract infections with cystitis, ureteritis, and pyelonephritis are widespread problems, but incidence is relatively low. The vaginitis and posthitis contribute to the venereal transmission, but indirect transmission is possible because the organisms are stable in the environment and present on the wool or scabs shed from affected animals. Posthitis occurs in intact and castrated sheep and goats. Necropsy findings. Pyelonephritis, multifocal kidney abscessation, dilated and thickened ureters, cystitis, and purulent exudate in many sections of the urinary tract are common finding at gross necropsy. Pathogenesis. Corynebacterium renale is a normal inhabitant of bovine genitourinary tracts. The pilus mediates colonization. Conditions such as trauma, urinary tract obstruction, and anatomic anomalies may predispose to infection. In addition, more basic pH urine levels may block some immune defenses. Infections ascend through the urinary tract. The bacteria are urease-positive when tested in vitro, and the ammonia produced in vivo during an infection damages mucosal linings, with subsequent inflammation. Corynebacterium cystitidis and C. pilosum are normally found around the prepuce of sheep and goats. High-protein diets, resulting in higher urea excretion and more basic urine, are contributing factors. Posthitis and vulvovaginitis may develop within a week of change to the more concentrated or richer diet, such as pasture or the addition of high-protein forage. The ammonia produced irritates the preputial and vulvar skin, increasing the vulnerability to infection. Differential diagnosis. Urolithiasis is a primary consideration for these diseases. Contagious ecthyma should be considered for the crusting that is seen with posthitis and vulvovaginitis, although the lesions of contagious ecthyma are more likely to develop around the mouth. Ovine viral ulcerative dermatosis is also a differential for the lesions of posthitis and vulvovaginitis. Prevention and treatment. Because high-protein feed is often associated with posthitis and vulvovaginitis, feeding practices must be reconsidered. Clipping long wool and hair also is helpful. Treatment. Long-term (3 weeks) penicillin treatment is effective for pyelonephritis. Reduction of dietary protein, clipping and cleaning skin lesions, treating for or preventing fly-strike, and topical antibacterial treatments are effective for posthitis and vulvovaginitis; systemic therapy may be necessary for severe cases. Surgical debridement or correction of scarring may also be indicated in severe cases. l. Erysipelas Etiology. Erysipelothrix rhusiopathiae is a nonmotile, non-spore-forming, gram-positive rod that resides in alkaline soils. Clinical signs. Erysipelothrix causes sporadic but chronic polyarthritis in lambs less than 3 months of age. In older goats, erysipelas has been associated with joint infections. Epizootiology and transmission. The disease may follow wound inoculation associated with castration, docking, or improper disinfection of the umbilicus. Following wound contamination and a 1- to 5-day incubation period, the lamb exhibits a fever and stiffness and lameness in one or more limbs. Joints, especially the stifle, hock, elbow, and carpus, are tender but not greatly enlarged. Necropsy findings. Thickened articular capsules, mild increases in normal-appearing joint fluid and erosions of the articular cartilage are usually found. The joint capsule is infiltrated with mononuclear cells, but bacteria are difficult to find. Diagnosis is based on clinical signs of polyarthritis, and confirmation is made by culturing the organism from the joints. Differential diagnosis. Differential diagnoses include polyarthritis caused by chlamydia or other bacteria and stiffness caused by white muscle disease. Other bacteria causing septic joints include Areanobacterium pyogenes and Fusobacterium necrophorum. Caprine arthritis encephalitis (CAE) should also be considered. Prevention and control. Proper sanitation and prevention of wound contamination are important in preventing the infection in lambs. Screening of goat herds for CAE is recommended. Treatment. Erysipelas is sensitive to penicillin antibiotic therapy. m. Dermatophilosis (Cutaneous Streptothricosis, Lumpy Wool, Strawberry Foot Rot) Etiology. Dermatophilus congolensis is an aerobic, gram-positive, filamentous bacterium with branching hyphae. Dermatophilosis is a chronic bacterial skin disease characterized by crustiness and exudates accumulating at the base of the hair or wool fibers ( Scanlan et al., 1984 ). Clinical signs. Animals will be painful but will not be pruritic. Two forms of the disease exist in sheep: mycotic dermatitis (also known as lumpy wool) and strawberry foot rot. Mycotic dermatitis is characterized by crusts and wool matting, with exudates over the back and sides of adult animals and about the face of lambs. Strawberry foot rot is rare in the United States but is characterized by crusts and inflammation between the carpi and/or tarsi and the coronary bands. Animals will be lame. In goats and cattle, similar clinical signs of crusty, suppurative dermatitis are seen; the disease is often referred to as cutaneous streptothricosis in these species. Lesions in younger goats are seen along the tips of the ears and under the tail. Diagnosis is based on clinical signs as well as the typical microscopic appearance on stained skin scrapings, cultures, and serology. Epizootiology and transmission. The disease occurs worldwide, and the Dermatophilus organism is believed to be a saprophyte. Transmission occurs by direct or indirect contact and is aggravated by prolonged wet wool or hair associated with inclement weather. Biting insects may aid in transmission. Necropsy findings. Lymphadenopathy as well as liver and splenic changes may be observed. Histopathologically, superficial epidermal layers are necrotic and crusted with serum, white blood cells, and wool or hair. Dermal layers are hyperemic and edematous and may be infiltrated with mononuclear cells. Pathogenesis. Lesions typically begin around the muzzle and hooves and the dorsal midline. Prevention and control. Potash alum and aluminum sulfate have been used as wool dusts in sheep to prevent dermatophilosis. Minimizing moist conditions is helpful in controlling and preventing the disease. In addition, controlling external parasites or other factors that cause skin lesions is important. Lesions will resolve during dry periods. Treatment. Animals can be treated with antibiotics such as penicillin and oxytetracycline. Treating the animals with povidone-iodine shampoos or chlorhexidine solutions is also useful in clearing the disease. n. Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum Infection (Virulent Foot Rot; Contagious Foot Rot of Sheep and Goats; Foot Scald) Etiology. Two bacteria, Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum, work synergistically in causing contagious foot rot in sheep and goats. Other organisms may be involved as secondary invaders. Both Dichelobacter and Fusobacterium are nonmotile, non-spore-forming, anaerobic, gram-negative bacilli. Foot rot is a contagious, acute or chronic dermatitis involving the hoof and underlying tissues ( Bulgin, 1986 ). It is the leading cause of lameness in sheep. At least 20 serotypes of Dichelobacter are known. Arcanobacterium pyogenes may also contribute to the pathogenicity or to foot abscesses in goats. Foot scald, an interdigital dermatitis, is caused primarily by D. nodosus alone. Clinical signs. Varying degrees of lameness are observed in all ages of animals within 2–3 weeks of exposure to the organisms. Severely infected animals will show generalized signs of weight loss, decreased productivity, and anorexia associated with an inability to move. The interdigital skin and hooves will be moist, with a distinct necrotic odor. Morbidity may reach 70% in susceptible animals. Diagnosis is based on clinical signs. Smears and cultures confirm the definitive agents. Clinical signs of the milder disease, foot scald, include mild lameness, redness and swelling, and little to no odor. Epizootiology and transmission. Fusobacterium necrophorum is ubiquitous in soil and manure, in the gastrointestinal tract, and on the skin and hooves of domestic animals. In contrast, Dichelobacter contaminates the soil and manure but rarely remains in the environment for more than about 2 weeks. Some animals may be chronic carriers. Overcrowded, warm, and moist environments are key elements in transmission. Outbreaks are likely in the spring season. Shipping trailers and contaminated pens or yards should be considered also as likely sources of the bacteria. Pathogenesis. Both organisms are transmitted to the susceptible animal by direct or indirect contact. The organisms enter the hoof through injuries or through sites where Strongyloides papillosus larvae have penetrated. Fusobacterium necrophorum initiates the colonization and is followed by D. nodosus. The latter attaches and releases proteases; these cause necrosis of the epidermal layers and separation of the hoof from the underlying dermis. The pathogenicity of the serotypes of D. nodosus is correlated with the production of these proteases and numbers of pili. Additionally, F. necrophorum causes a severe, damaging inflammatory reaction. Differential diagnosis. Foot abscesses, tetanus, selenium/vitamin E deficiencies, copper deficiency, strawberry foot rot, bluetongue virus infection (manifested with myopathy and coronitis), and trauma are among the many differentials that must be considered. Treatment. Affected animals are best treated by manually trimming the necrotic debris from the hooves, followed by application of local antibiotics and foot wraps. Systemic antibiotics such as penicillin, oxytetracycline, and erythromycin may be used. Goats have improved dramatically when given a single dose of penicillin (40,000 U/kg) ( Smith and Sherman, 1994 ). Footbaths containing 10% zinc sulfate, 20% copper sulfate, or 10% formalin (not legal in all states) can be used for treatment as well as for prevention of the disease. Affected animals should be separated from the flock. Vaccination has been shown to be effective as part of the treatment regimen. Some breeds of sheep and some breeds and lines of goats are resistant to infection. Individual sheep may recover without treatment or are resistant to infection. Prevention and control. Prevention and control programs involve scrutiny of herd and flock management; quarantine of incoming animals; vaccination; segregation of affected animals; careful and regular hoof trimming; discarding trimmings from known or suspected infected hooves; maintaining animals in good body condition; avoiding muddy pens and holding areas; and culling individuals with chronic and nonresponsive infections. Dichelobacter nodosus bacterins are commercially available; cross protection between serotypes varies. Biannual vaccinination in wet areas may be essential. Some breeds may develop vaccination site lumps. Footbaths of 10% zinc sulfate, 10% formalin (where allowed by state regulations), or 10% copper sulfate are also considered very effective preventive measures. Goats are less sensitive than sheep to the copper in the footbaths. Research complications. Treating and controlling foot rot is costly in terms of time, initial handling and treatments and their follow-up, housing space, and medications. o. Fusobacterium necrophorum and Bacteroides melaninogenicus Infection (Foot Rot of Cattle, Interdigital Necrobacillosis of Cattle) Etiology. Interdigital necrobacillosis of cattle is caused by the synergistic infection of traumatized interdigital tissues by Fusobacterium necrophorum and Bacteroides melaninogenicus. Like F. necrophorum, B. melaninogenicus is a nonmotile, anaerobic, gram-negative bacterium. Dichelobacter nodosus, the agent of interdigital dermatitis, may be present in some cases. This is a common cause of lameness in cattle. Clinical signs. Clinical signs include mild to moderate lameness of sudden onset. Hindlimbs are more commonly affected, and cattle will often flex the pastern and bear weight only on the toe. The interdigital space will be swollen, as will be the coronet and bulb areas. Characteristic malodors will be noted, but there will be little purulent discharge. In more severe cases, animals will have elevated body temperature and loss of appetite. The lesions progress to fissures with necrosis until healing occurs. The diagnosis is by the odor and appearance. Anaerobic culturing confirms the organisms involved. Epizootiology and transmission. Cases may be sporadic, or epizootics may occur. Bos taurus dairy breeds and animals with wide interdigital spaces are more commonly affected. The factors here are comparable to those present in foot rot of smaller ruminants. Necropsy findings. Findings at necropsy include dermatitis and necrosis of the skin and subcutaneous tissues. Although necropsy would rarely be performed, secondary osteomyelitis may be noted in severe cases by sectioning limbs. Pathogenesis. The bacteria enter through the skin of the interdigital area after trauma to the interdigital skin, from hardened mud, or from softening of the skin due to, for example, constant wet conditions in pens. Colonization leads to cellulitis. In addition, F. necrophorum releases a leukocidal exotoxin that reduces phagocytosis and causes the necrosis, whereas the tissues and tendons are damaged by the proteases and collagenases produced by B. melaninogenicus. Zinc deficiency may play a role in the pathogenesis in some situations. Differential diagnoses. The most common differentials for sudden lameness include hairy heel warts and subsolar abcesses. Bluetongue virus should also be considered. Grain engorgement and secondary infection from cracks caused by selenium toxicosis should also be considered. The exotic foot-and-mouth disease virus would be considered in areas where that pathogen is found. Prevention and control. As with foot rot in smaller ruminants, management of the area and herd are important. Paddocks and pens should be kept dry, well drained, and free of material that will damage feet. Footbaths and chlortetracycline in the feed have been shown to control incidence. Affected animals should be segregated during treatment. Chronically affected or severely lame animals should be culled. New cattle should be quarantined and evaluated. Treatment. Successful treatment regimens that result in healing within a week include cleaning the feet and trimming necrotic tissue; parenteral antimicrobials, such as oxytetracycline or procaine penicillin, or sulfonomethazine in the drinking water or tetracyclines in feed; and footbaths (such as 10% zinc sulfate, 2.5% formalin, or 5% copper sulfate) twice a day. In severe cases, more aggressive therapy such as bandaging the feet or wiring the digits together may be needed. Animals can recover without treatment but will be lame for several weeks. Acquired immunity is reported to be poor. Research complications. Research complications are comparable to those noted for foot rot in smaller ruminants. p. Fusobacterium necrophorum infection (Foot Abscesses) Fusobacterium necrophorum is also associated with foot abscesses, the infection of the deeper structures of the foot, in sheep and goats. Only one claw of the affected hoof may be involved. The animals will be three-legged lame, and the affected hoof will be hot. Pockets of purulent material may be in the heel or toe. q. Heel Warts (Bovine Digital Dermatitis, Interdigital Papillomatosis, Papillomatous Digital Dermatitis, Foot Warts, Heel Warts, Hairy Foot Warts, Mortellaro's Disease) Etiology. Bacteria such as Fusobacterium spp., Bacteroides spp., and Dichelobacter nodosus have been isolated from bovine heel lesions. Spirochete-like organisms have also been shown in the lesions of cows with papillomatous digital dermatitis (PDD), in the United States and Europe; these have culturing requirements similar to those of Treponema species. Clinical signs. All lesions occur on the haired, digital skin. One or all feet may be affected. Most lesions occur on the plantar surface of the hindfoot (near the heel bulbs and/or extending from the interdigital space), but the palmar and dorsal aspect of the interdigital spaces may also be involved. Progression of lesions, typically over 2–3 weeks, includes erect hairs, loss of hair, and thickening skin. Moist plaques begin as red and remain red or turn gray or black. Exudate or blood may be present on the plaque. Plaques enlarge and "hairs" protrude from the roughened surface. Lesioned areas are painful when touched. The lesions may or may not be malodorous. Epizootiology and transmission. Facility conditions and herd management are considered contributing factors. The following have been examined as contributing factors: nutrition, particularly zinc deficiency; poorly drained, low-oxygen, organic material underfoot; poor ventilation; rough flooring; damp and dirty bedding areas; and overcrowding. These interdigital lesions occur commonly in young stock and in dairy facilities throughout the world. The disease is seen only in cattle. Pathogenesis. The organisms noted above, combined with poor facility and herd management, are critical in the pathogenesis. Differential diagnosis. Differentials for lameness will include sole abscesses, laminitis, and trauma. Prevention and control. Each facility and management condition noted above should be addressed in conjunction with appropriate antibiotic and/or antiseptic treatment regimens. All equipment used for hoof trimming must be cleaned and disinfected after every use. Trucks and trailers should also be sanitized between groups of animals. Treatment. Antibiotic and antiseptic regimens have been used successfully for this problem. Antibiotics include parenteral cephalosporins and pencillins, as well as topical tetracyclines with bandaging. Antiseptic or antibiotic solutions in footbaths include tetracyclines, zinc sulfate, lincomycin, spectinomycin, copper sulfate, and formalin. The footbaths must be well maintained, minimizing contamination by feces and other materials. Tandem arrangements, such as the cleaning footbaths and then the medicated footbaths, and preventing dilution from precipitation are useful. Other treatments such as surgical debridement, cryotherapy, and caustic topical solutions have been successful. Research complications. Infectious, contagious PPD is one of the major causes of lameness among heifers and dairy cattle and is a costly problem to treat. The outbreaks are generally worse in younger animals in chronically infected herds. The immune response is not well understood, and it may be temporary in older animals. r. Haemophilus somnus infection (Thromboembolic Meningoencephalitis) Etiology. Haemophilus somnus is a pleomorphic, nonencapsulated, gram-negative bacterium. Diseases caused by this organism include thromboembolic meningoencephalitis (TEME), septicemia, arthritis, and reproductive failures due to genital tract infections in males and females. Haemophilus somnus is a also major contributor to the bovine respiratory disease complex. Haemophilus spp. have been associated with respiratory disease in sheep and goats. Clinical signs. The neurologic presentation may be preceded by 1–2 weeks of dry, harsh coughing. Neurologic signs include depression, ataxia, falling, conscious proprioceptive deficits; signs such as head tilt from otitis interna or otitis media, opisthotonus, and convulsions may be seen as the brain stem is affected. High fever, extreme morbidity, and death within 36 hr may occur. Respiratory tract infections are usually part of the complex with infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza 3, Mycoplasma, and Pasteurella, and the synergism among these contributes to the signs of bovine respiratory disease complex (BRDC). In acute neurologic as well as chronic pneumonic infections, polyarthritis may develop. Abortion, vulvitis, vaginitis, endometritis, placentitis, and failure to conceive are manifestations of reproductive tract disease. In all cases, asymptomatic infections may also occur. Diagnosis based on culture findings is difficult because H. somnus is part of the normal nasopharyngeal flora. Paired serum samples are recommended; single titers in some animals seem to be high because of passive immunity, previous vaccination, or previous exposure. In cases of abortion, other causes should be eliminated from consideration. Epizootiology and transmission. Because the organism is considered part of the normal flora of cattle and can be isolated from numerous tissues, the distinction between the normal flora and the status of chronic carrier is not clear. Outbreaks are associated with younger cattle in feedlots in western United States, but stresses of travel and coinfection with other respiratory pathogens are involved in some cases. Adult cattle have also been affected. Vaccination for viral respiratory pathogens may increase susceptibility. Transmission is by respiratory and genital tract secretions. The organism does not persist in the environment. Necropsy findings. Pathognomonic central nervous system lesions include multifocal red-brown foci of necrosis and inflammation on and within the brain and the meninges. Many thrombi with bacterial colonies will be seen in these affected areas. Ocular lesions may also be seen, including conjunctivitis, retinal hemorrhages, and edema. Usually animals with neurological disease will not have respiratory tract lesions. The respiratory tract lesions include bronchopneumonia and suppurative pleuritis. When combined with Pasteurella infection, the pathology becomes more severe. Aborted fetuses will not show lesions, but necrotizing placentitis will be evident histologically. Pure cultures of H. somnus may be possible from these tissues. Pathogenesis. Inhalation of contaminated respiratory secretions from carrier animals is the primary means of transmission. The anatomical location of bacterial residence within the carriers has not been identified. After gaining access by way of the respiratory tract, the bacteria proliferate, and a bacteremia develops. The bacteria are phagocytosed by neutrophils but are not killed. The thrombosis formation is due to the adherence by the nonphagocytosed organisms to vascular endothelial cells, degeneration and desquamation of these cells, and exposure of subendothelial collagen, with subsequent initiation of the intrinsic coagulation pathway. Antigen-antibody complex formation, resulting in vasculitis, is also correlated with high levels of agglutinating antibodies. Differential diagnosis. Differentials in all ruminants include other pathogens associated with neurological disease and respiratory disease such as Pasteurella hemolytica, P. multocida, and P. aeruginosa. In smaller ruminants, Corynebacterium pseudotuberculosis should be considered. Prevention and control. Stressed animals or those exposed to known carriers can be treated prophylactically with tetracycline administered parenterally or orally (in the feed or water). The late-stage polyarthritis is resistant to antibiotic therapy, because of failure of the antibiotic to reach the site of infection. Planning vaccination programs carefully will decrease chances of outbreaks. For example, avoiding vaccinating animals for infectious bovine rhinotrachetitis and bovine viral diarrhea during times of stress to the cattle is worthwhile. Killed whole-cell bacterins are commercially available; these have been shown to be effective in controlling the respiratory disease presentation. Control of other clinical aspects of the H. somnus disease by these bacterins has not been well described. Treatment. Rapid treatment at the first signs of neurologic disease is important in an outbreak. Haemophilus somnus is susceptible to several antibiotics, such as Oxytetracycline and penicillin, and these are often used in sequence until the cattle are recovered. s. Leptospirosis Etiology. Seven different species of the spirochete genus Leptospira are now recognized, and pathogenic serovars exist within each species; previously pathogenic leptospires were all classified as members of the species L. interrogans. The serovars pomona, icterohaemorrhagiae, grippotyphosa, interrogans, and hardjo are recognized pathogens. Leptospira hardjo and L. pomona are the serovars most commonly diagnosed in cattle, with L. hardjo causing endemic infection. Leptospira hardjo is also the major sheep serovar. Goats are susceptible to several serovars. Clinical signs. Leptospirosis is a contagious but uncommon disease in sheep and goats. The disease may cause abortion, anemia, hemoglobinuria, and icterus and is often associated with a concurrent fever. After a 4- to 10-day incubation period, the organism enters the bloodstream and causes bacteremia, fever, and red-cell hemolysis. Leptospiremia may last up to 7 days. Immune stimulation is apparently rapid, and antibodies are detectable at the end of the first week of infection; cross-serovar protection does not occur. During active bacteremia, hemolysis may result in hemoglobin levels of 50% below normal. Hyperthermia, hemoglobinuria, icterus, and anemia may be observed during this phase, and ewes in late gestation may abort. Abortion usually occurs only once. Mortality rates of above 50% have been reported in infected ewes and lambs ( Jensen and Swift, 1982 ). Subclinical infection is more common in nonpregnant and nonlactating animals. Sheep infected with leptospirosis may display a hemolytic crisis associated with IgM acting as a cold-reacting hemagglutinin. Acute and chronic infections in cattle are more common than infections in sheep and goats. Acute forms in cattle display signs similar to those in sheep. Acute infection in calves may progress to meningitis and death. Lactating cows will have severe drops in production. Chronic cases may lead to abortion, with retained placenta, and weakened calves or animals that carry the infection. Infertility may also be a sequela. Epizootiology and transmission. Leptospires are a large genus, and leptospirosis is a complicated disease to prevent, treat, and control. The organism survives well in the environment, especially in moist, warm, stagnant water. Cattle, swine, and other domestic and wild animals are potential carriers of serovars common to particular regions. Wild animals often serve as maintenance hosts, but domestic livestock may be reservoirs also. Organisms are shed in urine, in uterine discharges, and through milk. Animals become carriers when they are infected with a host-adapted serovar; sporadic clinical disease is more commonly associated with exposure to a non-host-adapted serovar ( Heath and Johnson, 1994 ). Infection may occur via oral ingestion of contaminated feed and water, via placental fluids, or through the mucous membranes of the susceptible animal. Placental or venereal transmission may occur. As the organisms are cleared from the bloodstream, they chronically infect the renal convoluted tubules and the reproductive tract (and occasionally the cerebrospinal fluid or vitreous humor). Chronically infected animals may shed the organism in the urine for 60 days or longer. Necropsy. Diagnosis is confirmed by identification of leptospires in fetal tissues. The leptospires are visible in silver- or fluorescent antibody-stained sections of liver or kidney. Leptospires may also be seen under dark-field or phase-contrast microscopy of fetal stomach contents. Fetal and maternal serology, and diagnostic tests such as the microscopic agglutination test, are useful; interpretation is complicated because of cross reaction of antibodies to many serovars. Differential diagnosis. More than one serovar may cause infection in one animal, and each serovar should be considered as a separate pathogen. Because of the associated anemia, differential diagnoses should include copper toxicity and parasites, in addition to other abortifacient diseases. Prevention and control. Polyvalent vaccines, tailored to common serovars regionally, are available and effective for preventing leptospirosis in cattle. Immunity is serovar specific. Because serological titers tend to diminish rapidly (40–50 days in sheep [ Jensen and Swift, 1982 ]), frequent vaccination may be necessary. Other prevention measures such as species-specific housing, control of wild rodents, and proper sanitation should be instituted. Treatment. Antibiotic treatment is aimed at treating ill animals and trying to clear the carrier state. Treatment methods for acute leptospirosis include oxytetracycline for 3–6 days. Addition of oxytetracycline or chlortetracycline to the feed for 1 week may be helpful. These antibiotics are considered best for removal of the carrier state of some serovars. Vaccination and antibiotic therapy can be combined in an outbreak. Research complications. Leptospirosis is zoonotic and may be associated with flulike symptoms, meningitis, or hepatorenal failure in humans. t. Listeria (Circling Disease, Silage Disease) Etiology. Listeria monocytogenes is a pleomorphic, motile, non-spore-forming, β-hemolytic, gram-positive bacillus that inhabits the soil for long periods of time and has been often found in fermented feedstuffs such as spoiled silage. Of the 16 known serovars, several produce clinical signs in ruminants. Listeria ivanovii (associated with abortions in sheep) is serovar 5. Clinical signs. Listeriosis is an acute, sporadic, noncontagious disease associated with neurological signs or abortions in sheep and other ruminants. The overall case rate is low. The disease may present as an isolated case or with multiple animals affected. Three forms of disease are described: encephalitis, placentitis with abortion, and septicemia with hepatitis and pneumonia. The encephalitic form is most common in sheep; septicemic forms may occur in neonatal lambs ( Scarratt, 1987 ). Clinically, the encephalitic form begins with depression, anorexia, and mild hyperthermia after an incubation period of 2–3 weeks. As the disease progresses, animals exhibit nasal discharges and conjunctivitis and begin to walk in circles, as if disoriented. Facial paralytic lesions, including drooping of an ear or eyelid, dilation of a nostril, or strabismus occur unilaterally on the affected side as the result of dysfunction of some or all the cranial nerves V-XII. The neck will by flexed away from the affected side. Facial muscle twitching, protrusion of the tongue, dysphagia, hypersalivation, and nasal discharges may be noted. The hypersalivation may lead to metabolic acidosis in advanced cases in cattle. Anorexia, prostration, coma, and death follow. The placental form usually results in last-trimester abortions in ewes and does, which typically survive this form of the disease. The affected females may be asymptomatic or may show severe clinical signs such as fever and depression, with subsequent retained placenta or endometritis. Abortion usually occurs within 2 weeks of Listeria infection. In cattle, abortion occurs during the last 2 months of gestation and has been induced experimentally 6–8 days after exposure. Cows present with the range of clinical signs seen in smaller-ruminant dams. There is no long-term effect on the fertility of affected dams. Epizootiology and transmission. The organism is transmitted by oral ingestion of contaminated feeds and water or possibly by inhalation. By the oral route, the organism enters through breaks in the oral cavity and ascends to the brain stem by way of nerves. When severe outbreaks occur, feedstuffs should be assessed for spoilage. Listeria organisms can be shed by asymptomatic carriers, especially at the end of pregnancy and at lambing. Diagnosis and necropsy findings. Diagnosis is usually made from clinical signs. Culture confirms the diagnosis (cold enrichment at 20° C is preferable but not essential for isolation). Impression smears will show the pleomorphic gram-positive characterisitics of the pathogen. Tissue fluorescent antibody techniques may also be utilized. Gross lesions are not observed with the encephalitic form. Microscopic lesions include thrombosis, neutrophilic or mononuclear foci in areas of inflammation, and neuritis. The pons, medulla, and anterior spinal cord are primarily affected in the encephalitic form. Microabscesses of the midbrain are characteristic of Listeria encephalitis in sheep. Aborted fetuses that are intact may show fibrinous polyserositis, with excessive serous fluids; small, necrotic foci of the liver; and small abomasal erosions. Necrotic lesions of the fetal spleen and lungs may also be seen. In goats, Listeria-induced neurological lesions occur only in the brain stem. Placentitis, focal bronchopneumonia, hepatitis, splenitis, and nephritis may be seen with other forms. Pathogenesis. With the encephalitic form, the organism penetrates mucosal abrasions and enters the trigeminal or hypoglossal nerves. The Listeria organisms then migrate along the nerves and associated lymphatics to the brain stem (medulla and pons). In the septicemic form, the organism penetrates tissues of the gastrointestinal tract and enters the bloodstream, to be distributed to the liver, spleen, lungs, kidneys, and placenta. After infection, organisms are shed in all body secretions (infected milk is an important risk factor for zoonosis). A toxin produced by Listeria monocytogenes is correlated with pathogenicity, but the mechanism of the pathogenesis of this molecule has not been elucidated. Differential diagnoses. Rabies, bacterial meningitis, brain abscess, lead toxicity, and otitis media must be considered as differentials. In sheep, the differentials include organisms that cause abortion, and neurological signs, such as enterotoxemia due to Clostridium perfringens type D. In goats, the major differentials include caprine arthritis encephalitis viral infection and chlamydial and mycoplasmal infections. In both species, scrapie is a differential. In cattle, aberrant parasite migration or Hemophilus somnus infection must also be considered. Prevention and control. Affected dams should be segregated and treated. Other animals in the group may be treated with oxytetracycline as needed. Aborted tissues should be removed immediately. Proper storage of fermented feeds minimizes this source of contamination. When silage spoils, the pH increases, producing a suitable growth environment for the organism. Commercial vaccines are not available in the United States. Treatment. Affected animals can be treated aggressively with penicillin, ampicillin, oxytetracycline, or erythromycin. Exceptionally high levels of penicillin are required for treating affected cattle. Severely affected animals should receive appropriate fluid support and other nursing care. Treatment is less successful, and mortality is especially high in sheep. Recovered animals tend to resist reinfection. Research complications. In addition to the loss of fetal animals, stress to the dams, and risks to other animals, any aborted tissue by a ruminant should be regarded as a potential zoonotic risk. Listeria can cause mild to severe flulike symptoms in humans and may be a particular risk for pregnant women and for older or immune-compromised individuals. Listeriosis in humans is a reportable disease. u. Lyme Disease (Borrelia burgdorferi Infection, Borreliosis) Etiology. Lyme disease is caused by the spirochete Borrelia burgdorferi. Clinical signs and diagnosis. Reports in ruminants indicate seroconversion to B. burgdorferi, but there are few definitive correlations to the arthritis that is present. Diagnosis requires culturing from the affected joints and diagnostic elimination of other causes of lameness and arthritis. Epizootiology and transmission. The organism is present throughout much of the Northern Hemisphere and has been reported in many mammals and also in birds. Ticks of the Ixodes ricinus complex are the major vectors of the spirochete and must be attached for 24 hr for successful transmission. Pathogenesis. The Ixodes ticks have three life stages: larval, nymphal, and adult. Feeding occurs once during each stage, and wild animals are the source of blood meals. The larval stages feed from rodents, such as the white-footed deer mouse, Peromyscus leucopus, from which they acquire the spirochete. The nymphal stage is that which usually infects other animals. The adult ticks are usually found on deer. Differential diagnosis. Seroconversion to B. burgdorferi does not necessarily confirm the cause of arthritis. Other causes of arthritis and lameness in ruminants include trauma, caprine arthritis encephalitis virus, Mycoplasma spp., Chlamydia psittaci, Erysipelothrix spp., Arcanobacterium pyogenes, Brucella spp., and rickets. Prevention and control. Control of the tick vector is the most important factor in preventing the possibility of exposure or disease. Treatment. Antibiotic therapy, with tetracycline, penicillin, amoxicillin, and cephalosporins, is used for diagnosed or suspected Lyme arthritis. Research complications. Lyme disease is zoonotic, and the Ixodes ticks transmit the disease to humans. v. Mastitis i. Ovine mastitis Mastitis in ewes may be acute, subclinical, or chronic. Acute mastitis often results in anorexia, fever, abnormal milk, and swelling of the mammary gland. Pasteurella haemolytica is the most common cause of acute mastitis. Additional isolates may include, in order of prevalence, Staphylococcus aureus, Actinomyces (Corynebacterium) spp., and Histophilus ovis. Escherichia coli and Pseudomonas aeruginosa have also been found to cause acute mastitis. As many as six serotypes of Pasteurella haemolytica have been isolated from the mammary glands of mastitic ewes. Furthermore, intramammary inoculation of these organisms isolated from ovine and bovine pulmonary lesions has resulted in clinical mastitis in ewes ( Watkins and Jones, 1992 ). Subclinical mastitis is detected only indirectly, by counting somatic cells. The most common isolate from ewes with subclinical mastitis is coagulase-negative staphylococci. Other isolates include Actinomyces bovis, Streptococcus uberis, S. dysgalactiae, Micrococcus spp., Bacillus spp., and fecal streptococci. Most of these organisms are commonly found in the environment. Diffuse chronic mastitis, or hardbag, results from interstitial accumulations of lymphocytes in the udder. Both glands are usually affected, but no inflammation is present. Serological evidence suggests that diffuse chronic mastitis is caused by the retrovirus that causes ovine progressive pneumonia (OPP or maedi/visna virus). Other bacterial agents or Mycoplasma have not usually been isolated from udders with this type of mastitis. Acute mastitis occurs in approximately 5% of lactating ewes annually, and it usually occurs either soon after lambing or when lambs are 3–4 months old ( Lasgard and Vaabenoe, 1993 ). Subclinical mastitis occurs in 4–50% of lactating ewes ( Kirk and Glenn, 1996 ). Subclinical mastitis is more common in ewes from high-milk-producing breeds. Skin or teat lesions and dermatitis increase the prevalence of disease. Acute mastitis can be diagnosed in ewes with associated systemic signs of disease by physical examination of the udder and inspection of the milk. Subclinical mastitis is often suggested by somatic cell counts elevated above 1 × 10 6 cells/ml. When high somatic cell counts are identified, subclinical mastitis can be diagnosed by milk culture. The California mastitis test may also be helpful as an indicator of mastitis. Manual palpation of a hard, indurated udder as well as serological testing for the maedi/visna virus is helpful in confirming the diagnosis of diffuse chronic mastitis. Treatment for acute bacterial mastitis should include aggressive application of broad-spectrum antibiotics (intramammary and systemic) and supportive therapy such as fluids and anti-inflammatory drugs. It is may be helpful to milk out the infected udder frequently; oxytocin injections preceding milking will improve gland evacuation. Because somatic cell counting is often not routinely performed, treatment of subclinical mastitis is seldom done. There is currently no treatment available for diffuse chronic mastitis. ii. Caprine mastitis Lactating goats are subject to inflammation of mammary gland, or mastitis. The primary causative organisms are Staphylococcus epidermidis and other coagulase-negative Staphylococcus spp. Clinical signs of mastitis include abnormal coloration or composition of milk, mammary gland redness, heat and pain, enlargement of the mammary gland, discoloration of the mammary gland, and systemic signs of septicemia. Large abscesses may be present in the affected gland. Staphylococcus aureus is also associated with caprine mastitis, and toxemia may be part of the clinical picture. This organism produces a necrotizing alpha toxin that can result in gangrenous mastitis. Caprine mastitis may be clinical or subclinical, and the first indication of mastitis may be weak, depressed, or thin kids. Diagnosis is based on careful culture of mastitic milk. Treatment includes frequent stripping, intramammary antibiotics, and nonsteroidal anti-inflammatory drugs. Oxytocin (5–10 U) may help milk letdown for frequent strippings. Bovine mastitis products can be used in the goat; however, care should be taken not to insert the mastitis tube tip fully, because damage to the protective keratin layer lining the teat canal may occur. In severe acute systemic cases, steroids, fluids, and systemic antibiotics may be necessary. Other less common causes of mastitis in goats include Streptococcus spp. (S. agalactiae, S. dysgalactiae, S. uberis, and zooepidemicus). Gram-negative causes of caprine mastitis include Escherichia coli, Klebsiella pneumoniae, Pasteurella spp., Pseudomonas, and Proteus mirabilis. Corynebacterium pseudotuberculosis can cause mammary gland abscessation, whereas Mycoplasma mycoides may cause agalactia and systemic disease. "Hard udder" can be caused by caprine arthritis encephalitis virus (CAEV). Brucellosis and listeriosis can cause a subclinical interstitial mastitis ( Smith and Sherman, 1994 ). iii. Bovine mastitis Mastitis is the disease of greatest economic importance for the dairy cattle industry. The majority of the impact will be on the production and overall health of the cows, but low-incidence herds also diminish the risk of calves' ingesting or being exposed to pathogens. The most common bovine mastitis pathogens include Staphylococcus aureus and Streptococcus agalactiae, S. dysgalactiae, and S. uberis; coliform agents such as Escherichia coli, Enterobacter aerogenes, Serratia marcescens, and Klebsiella pneumoniae; mycoplasmal species such as Mycoplasma bovis, M. bovigenitalium, M. californicum, M. canadensis, and M. alkalescens; and Salmonella spp. such as S. typhimurium, S. newport, S. enteritidis, S. dublin, and S. muenster. Many of these agents such as Staphylococcus spp., Salmonella spp., and the coliforms can cause both acute and chronic mastitis, as well as severe systemic disease, including fever and anorexia. These must be regarded as herd and environmental pathogens in terms of treatment and prevention. The pathogenesis of staphylococcal infections is comparable to that in goats. Staphylococcus agalactiae can be cleared from udders because it does not invade other tissues, is an obligate resident of the glands, and is susceptible to penicillin. In contrast, S. uberis and S. dysgalactiae are environmental organisms and can be highly resistant to pencillin. Mycoplasma bovis is the more common of the mycoplasmal pathogens and can cause severe infections. Transmission of the mycoplasmas is not well defined but may be related to their presence in other organ systems. Treatments for mycoplasmal mastitis are not successful; culling is recommended. There are many interrelated factors associated with prevention and control of mastitis in a herd, including herd health and dry cow management, order of animals milked, milking procedures, milking equipment, condition of the teats, and the condition of the environment. Management of the overall herd includes aspects such as vaccination programs, nutrition, isolation of incoming animals, and quarantine and treatment of or culling diseased individuals. Culturing or testing newly freshened cows and monitoring the bulk milk tank serve as indicators of subclinical mastitis. Herd management will diminish teat lesions. Bacterin vaccines are available for preventing and controlling coliform mastitis and S. aureus mastitis. At the time of dry-off, all cows must be treated by intramammary route. Some infections can be successfully cleared during this time. Younger, disease-free animals should be milked first; any animals with diagnosed problems should be milked after the rest of the herd and/or segregated during treatment. Milkers' hands easily serve as a means of pathogen transmission, and wearing rubber gloves is recommended. Teat and udder cleaning practices include washing and drying with single-service paper or cloth towels or pre-and postmilking dipping. Milking equipment must be maintained to provide proper vacuum levels and pumping rates, and liners should be the appropriate size. Facilities that provide clean and dry areas for the animals to rest, feed, and move will diminish teat injuries and reduce exposures to mastitis pathogens. In that regard, inorganic bedding such as clean sand harbors few pathogens in contrast to shavings and sawdust. w. Moraxella bovis Infection (Infectious Bovine Keratoconjunctivitis, Pinkeye) Etiology. Moraxella bovis, a gram-negative coccobacillus, is the most common cause of infectious bovine keratoconjunctivitis (IBK) in cattle. This organism is not a cause of keratoconjunctivitis in sheep and goats. The disease includes conjunctivitis and ulcerative keratitis. The pathogenic M. bovis strain is piliated, and at least seven serotypes exist. Clinical signs. Lacrimation, photophobia, and blepharospasm are seen initially. Conjunctival injection and chemosis develop within a day of exposure, and then keratitis with corneal edema and ulcers. Anterior uveitis may be a sequela within a few days, and thicker mucopurulent ocular discharge may be seen. Corneal vascularization begins by 10 days after onset. Reepithelialization of the corneal ulcers occurs by 2–3 weeks after onset. Diagnosis is usually based on clinical signs, but culturing is helpful and fluorescein staining is useful for demonstrating corneal ulceration. Epizootiology and transmission. The disease is more severe in younger cattle. The clinical signs of IBK tend to be more severe in cattle that are also infected with infectious bovine rhinotracheitis (IBR) virus or those that have been vaccinated recently with modified live IBR vaccine. The bacteria are shed in nasal secretions and cattle with no clinical symptoms may be carriers. Transmission is by fomites, flies, aerosols, and direct contact. Incidence in winter months is very low. Nonhemolytic strains are associated with the winter epidemics, and hemolytic strains are associated with summer epidemics. Necropsy findings. Necropsy is not typically performed on these cases. Corneal edema, ulceration, hypopyon, and uveitis would be noted, depending on the stage of infection. Pathogenesis. The pili of M. bovis bind to receptors of corneal epithelium. The virulent strains of the bacteria then release the enzymes that damage the corneal epithelial cells. Other factors contributing to infection include ultraviolet light and trauma from dust and plant materials. Differential diagnoses. Infectious bovine rhinotrachetitis virus causes conjunctivitis, but the central corneal ulceration that is characteristic of IBK is not seen with M. bovis infections. Mycoplasma, Listeria, Branhamella (Neisseria), and adenovirus may be cultured from affected bovine eyes but none has been shown to produce the corneal lesions when inoculated into susceptible animals. Prevention and control. Cattle should not be immunized intranasally with modified live infectious bovine rhinotracheitis vaccine during IBK outbreaks; this will likely exacerbate the infection. New animals should be quarantined and treated prophylactically before introduction to herds. The available vaccines, containing. M. bovis pili or killed M. bovis, help decrease incidence and severity of disease; these preparations are not completely effective, because the M. bovis strain may not be homologous to that used for the vaccine preparation. Other preventive measures include 10% permethrin-impregnated bilateral ear tags, pour-on avermectins, or dust bags or face rubbers containing insecticide (such as 5% coumaphos) to control flies throughout the season and premises; mowing of high pasture grass to minimize ocular trauma; provision of shade; control of dust and sources of other mechanical trauma; and segregation of animals by age. Treatment. Cattle can recover without treatment, but younger animals should be treated as soon as the infection is detected. Antibiotic treatments include topical, subconjunctival administration and intramuscular dosing. Several standard topical antibiotics have been shown to be effective, including oxytetracycline, gentamicin, and triple antibiotic combinations. These should be administered twice per day. Subconjunctival injections of antibiotics, such as penicillin G, provide higher corneal levels of drug; these are typically administered only once or twice in severe cases. Intramuscular doses of long-acting oxytetracycline, given on alternate days, are effective in larger herds, and 2 doses 72 hr apart eliminate carriers. Third-eyelid flaps, temporary tarsorrhaphy, or eye patches may be useful in certain cases. Research complications. This pathogen does present a complication due to the carrier status of some animals, the likelihood of herd outbreaks, the severity of disease in younger animals, and the morbidity, possible progression to uveitis, and time and treatment costs associated with infections. The overall condition of the cattle will be affected for several weeks, and permanent visual impairment or loss, as well as ocular disfigurement, may occur. x. Mycobacterial Diseases Mycobacterium bovis Infection (Tuberculosis) Etiology. Mycobacteria are aerobic, nonmotile, non-spore-forming, acid-fast pleomorphic bacteria. Most cases of tuberculosis in sheep are related to Mycobacterium bovis or M. avium. Cases in goats have been attributed to M. bovis, M. avium, or M. tuberculosis. Mycobacterium bovis, or the bovine tubercle bacillus, is the cause in cattle but has been isolated from many domestic and wild mammals. Other agents of mammalian tuberculosis include M. microti and M. africanum. Clinical signs. Tuberculosis is a sporadic, chronic, contagious disease of ruminants and is zoonotic. The infection is often asymptomatic later in the illness, and it may be diagnosed only at necropsy. The respiratory system (M. bovis) or the digestive system (M. avium) is the primary site of infection; other tissues such as mammary tissue and reproductive tract may be infrequently involved. Locations of the characteristic tubercles will determine whether clinical signs are seen. Respiratory signs may include dyspnea, coughing, and pneumonia. Digestive tract signs include diarrhea, bloat, or constipation; diarrhea is most common. Lymphadenopathy occurs in advanced cases. Fever and generalized disease may be seen after calving. Infected goats lose weight and develop a persistent cough. Epizootiology and transmission. Although M. bovis can be killed by sunlight, it otherwise survives a long time in the environment and in cattle feces. Animals acquire the infection from the environment or from other animals via aerosols, from contaminated feed and water, and from secretions such as milk, semen, genital discharges, urine, and feces. Clinically normal animals may serve as carriers. The bacilli stimulate an initial neutrophilic tissue response. Neutrophils become necrotic and are phagocytosed by macrophages, forming giant epithelioid cells called Langhans' giant cells. An outer lymphocytic zone is formed, and fibrotic encapsulation creates the classical caseous nodules. Vascular erosion and hematogenous migration of the organisms may lead to lesions throughout the body. Necropsy findings. Yellow primary tubercles (granulomas) with central areas of caseous necrosis and calcification are present in the lungs. Caseous nodules are also associated with gastrointestinal organs and mesenteric lymph nodes. Prevention and control. Significant progress has been made in eradication programs in the United States during the past several decades, but during the 1990s, infected animals continued to be found in domestic cattle herds and particularly in captive deer herds in hunting preserves. The intradermal tuberculin test, using purified protein derivative (PPD), is usually used as a diagnostic indicator in live animals. This test should be performed annually on bovine and caprine dairy herds (and bison herds); the official tests are the caudal fold, comparative cervical, and single cervical tests. Notification to state officials is required following identification of intradermal-positive animals. Great care must be exercised in any handling of tissue or necropsies of reactors, and state animal health officials should be consulted regarding disposal of materials and cleaning of premises following depopulation of positive animals. Treatment. No treatment is recommended, and treatment is usually not allowed, because of the zoonotic potential, chronicity of the disease, and the treatment costs. Slaughter is preferred, to prevent potential transmission to humans. Research complications. The pathogen is zoonotic. Paratuberculosis, or Johne's disease (Mycobacterium paratuberculosis) Etiology. Mycobacterium paratuberculosis, the causative agent of Johne's disease, is a fastidious, non-spore-forming, acid-fast, gram-positive rod. The organism is actually a subspecies of M. avium, but M. paratuberculosis does not produce the siderophore mycobactin (an iron-binding molecule) of M. avium. Clinical signs and diagnosis. Johne's disease is a chronic, contagious, granulomatous disease of adult ruminants and is characterized by unthriftiness, weight loss, and intermittent diarrhea. In sheep and goats, chronic wasting is usually seen, occasionally with pasty feces or diarrhea. In cattle, chronic diarrhea and rapid weight loss are the most common clinical signs of the disease. Usually older adult animals are infected, but over time in an infected herd, younger animals will become infected when sufficient doses of organisms are ingested. Although clinical signs are nonspecific, Johne's disease should be considered if the affected diarrheic animals have a good appetite and are on a good anthelmintic program. The disease is diagnosed based on clinical signs and laboratory analyses, although none of the tests is more than 50% sensitive. In addition, the sensitivity of the serological tests differs between species. The standard is the fecal culture that takes 8–12 weeks. The enzyme-linked immunosorbent assay (ELISA) is now considered the most reliable serological test, but false negatives do occur. Other serological tests such as agar gel immunodiffusion (AGID) and complement fixation are useful. Herd screening may be done using the AGID or ELISA serological tests. Identification of the organism on culture, or the presence of acid-fast organisms on mucosal or mesenteric lymph node smears or from rectal biopsies, helps confirm the diagnosis. Some animals serologically negative for Johne's disease, however, have been found to be positive on fecal culture. Commercial AGID tests approved for use in cattle may be useful in diagnosing Johne's disease in sheep ( Dubash et al., 1996 ). Serological tests cross-react with other species of Mycobacterium, especially M. avium. Epizootiology and transmission. The organism is prevalent in the environment and is transmitted to young animals by direct or indirect contact. Although vertical transmission has been reported, the organism more commonly enters the gastrointestinal tract and penetrates the mucosa of the distal small intestine, primarily the ileum. Chronic carriers may intermittently shed the organisms. Pathogenesis. Mycobacterium paratuberculosis is an obligate parasite that grows only in macrophages of infected animals. Nursing infected dams are a primary source of infection of neonates. If the organism is not cleared, it proliferates slowly in the tissue, leading to inflammatory reactions that progress through neutrophilic to mononuclear stages. The organism may penetrate the lymphatics and proliferate in mesenteric lymph nodes. After an incubation period of a year or more, some of the carriers will progress to clinical disease manifested by fibrotic and hyperplastic changes in the ileum, leading to the classic thickening in the region. Gut changes result in intermittent diarrhea, with subsequent dehydration, electrolyte imbalances, and malnutrition, although this clinical sign is more common in cattle than in sheep or goats. Necropsy and diagnosis. The ileum from infected cattle is grossly thickened; this is not seen in sheep and goats. Ileal and ileocecal lymph nodes provide the best samples for histology and acid-fast staining. Differential diagnosis. Diseases causing chronic wasting and poor coat and body condition of all ruminants should be considered. These include chronic salmonellosis, peritonitis, severe parasitism, winter dysentery, and pyelonephritis. Deer can be infected, and the lesions can be confused with those of tuberculosis. Treatment. Treatment is not worthwhile. Prevention and control. Prevention is the most effective method to manage this pathogen. Efforts should be focused on eliminating the disease through test and slaughter. Neonates should not be reared by infected dams. Some states have Johne's disease eradication programs. Facilities and pastures where animals testing positive for Johne' disease were maintained should be thoroughly cleaned and kept vacant for a year after culling. Other considerations. Mycobacterium paratuberculosis is being investigated as a factor in the development of Crohn's disease in humans. y. Navel Ill (Omphalitis, Omphalophlebitis, Omphaloarteritis, Joint Ill) Etiology. The most common organism causing infection of the umbilicus is Arcanobacterium (formerly Actinomyces, Corynebacterium) pyogenes; other bacteria may be present. Arcanobacterium spp. are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Other environmental contaminants are also associated with this disease, such as Escherichia coli, Enterococcus spp., Proteus, Streptococcus spp., and Staplylococcus spp. Clinical signs and diagnosis. Navel ill is an acute localized inflammation and infection of the external umbilicus. Animals present with fever and painful enlargement of the umbilicus. Animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, and hematuria. Other common severe sequelae include septicemia, pneumonia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, uveitis, endocarditis, and diarrhea. Epizootiology and transmission. Many cases occur in neonates, and most cases occur within the first 3 months of age. Cleanliness of the birthing and housing environment and successful transfer of passive immunity are important factors in the occurrence of the disease. Dystocia resulting in weak neonates can be a factor predisposing to the development of the disease. Navel ill is diagnosed by typical clinical signs. The presence of microabscesses and palpation of the umbilical area for firm intra-abdominal structures extending from the umbilicus are abnormal. Assessment of colostral immunoglobulin transfer may contribute to determination of the prognosis. Navel ill should always be considered for young ruminants with fever of unknown origin during the first week of life and for slightly older lambs, kids, or calves that are not thriving. Arthrocentesis of affected joints and culture of the fluid for identification of the pathogen are also diagnostic options and essential for effective antimicrobial selection. Differential diagnosis. The major differential is an umbilical hernia, which will typically not be painful or infected and can often be reduced. Mycoplasmal arthritis is a differential in kids. In the past, Erysipelothrix rhusopathiae was a common navel ill pathogen in sheep. Treatment. Omphalitis can be treated with a 10 to 14 day course of broad-spectrum antibiotics such as ampicillin, amoxicillin, penicillin, ceftiofur, florfenicol, and erythromycin. If an isolated abscess is palpable, it should be surgically opened and repeatedly flushed with iodine solutions. Surgical reduction of the infected umbilicus is indicated if intra-abdominal structures are involved. The prognosis for recovery is good if systemic involvement has not occurred. Prevention and control. The disease is best prevented and controlled by providing clean birthing environments, ensuring adequate colostral immunity, thoroughly dipping the umbilicus of newborns in tincture of iodine or strong iodine solution (Lugol's), monitoring for dystocias, and maintaining young growing animals in noncontaminated environments. Research complications. The disease can be costly to treat, and the toll taken on young animals due to the consequences of systemic infection may detract from their research value. z. Pasteurellosis (Shipping Fever, Hemorrhagic Septicemia, Enzootic Pneumonia) Etiology. Pasteurella hemolytica and P. multocida are aerobic, nonmotile, non-spore-forming, bipolar, gram-negative rods. Biotype A serotypes are associated with pneumonia and septicemia in all ruminants ( Ellis, 1984 ). Serotype 1 of P. hemolytica is considered a major cause of pulmonary lesions of bovine bronchopneumonia and fibrinous bronchopneumonia. Clinical signs. Pasteurellosis is an acute bacterial disease characterized by bronchopneumonia, septicemia, and sudden death. The organism invades the mucosa of the gastrointestinal tract or respiratory tract and causes localized areas of necrosis, hemorrhage, and thrombosis. The lungs and liver are frequent areas of formation of microabscesses. Acute rhinitis or pharyngitis often precedes the respiratory form. The organism also may invade the bloodstream, causing disseminated septicemia. Clinically, the lambs may exhibit nasal discharge of mucopurulent to hemorrhagic exudate, hyperthermia, coughing, dyspnea, anorexia, and depression. With the respiratory form, auscultation of the thorax suggests dullness and consolidation of anteroventral lobes; this will be confirmed by radiographs. The disease is diagnosed by clinical signs, blood cultures from septicemic animals, blood smears showing bipolar organisms, and history of predisposing stressors. In cultures, P. hemolytica is distinguished from P. multocida by hemolysis on blood agar; only P. multocida produces indole. Epizootiology and transmission. The organism is ubiquitous in the environment and in the respiratory tracts of these animals. Younger ruminants, between 2 and 12 months of age, are especially prone to infection during times of stress, such as weaning, transportation, dietary changes, weather changes, and overcrowding. The pneumonic form appears as a complex associated with concurrent infections such as parainfluenza 3, adenovirus type 6, respiratory syncytial virus, mycoplasmas, chlamydia, Pasteurella multocida and Bordetella parapertussis ( Martin, 1996 ; Brogden et al., 1998 ). The organism is transmitted between animals by direct and indirect contact, through inhalation or ingestion. Necropsy findings. Necropsy lesions include areas of necrosis and hemorrhage in the small intestines and multifocal 1 mm lesions distributed on the surfaces of the lungs and liver. With the pneumonic form, serofibrinous exudates fill the alveoli; ventral lung lobes are consolidated and are congested and purple-gray in color. Fibrinous pleuritis, pericarditis, and hematogenously induced arthritis also may be evident. Pathogenesis. A leukotoxin is considered to be a key factor in the pathogenesis of the P. hemolytica infection. Macrophages and neutrophils are lysed by the toxin as they arrive at the lung, and the enzymes released by the neutrophils cause additional damage to the tissue. Treatment. Treatment may include the use of antibiotics such as penicillin, ampicillin, tylosin, sulfonamides, or oxytetracycline. Newer antibiotics, such as ceftiofur, tilmicosin, spectinomycin, and florfenicol, are very effective and approved for use in cattle. In outbreaks, cultures from fresh necropsies are helpful for determining sensitivities useful for the remaining group. Prevention and control. The incidence of disease can be decreased by minimizing the degree of stress; by improving management, such as nutrition and control of parasitism; and, in cattle and sheep, by vaccinating for viral respiratory infections such as parainfluenza. Early Pasteurella hemolytica bacterin vaccines for use in cattle are not considered effective, but newer products based on immunizing against the leukotoxin and some bacterial capsule surface antigens are effective. Pasteurella multocida bacterins and live streptomycin-dependent mutant vaccines are available. In young animals, passive immunity is protective. Preventive measures also include maintaining good ventilation in enclosures and barns. New animals to the flock or herds should be quarantined for at least 2 weeks before introduction. aa. Salmonellosis Etiology. Salmonella typhimurium is a motile, aerobic to facultatively anaerobic, non-spore-forming, gram-negative bacillus and is the organism associated with enteric disease and some abortions in ruminants. It is a common inhabitant of the gastrointestinal tract of ruminants. Current nomenclature categorizes S. typhimurium as a serovar within the species S. enteritidis (the other two species are S. typhi and S. choleraesuis). Salmonella typhimurium, S. dublin, and S. newport are the common species seen in bovine cases. Salmonella typhimurium, S. dublin, S. anatum, and S. montevideo are seen in ovine and caprine cases, although a host-adapted species has not been identified in the goat. Ovine abortions due to various Salmonella species are not reported in the United States but are enzootic in other countries. Salmonella serotypes have been associated with aborted fetuses in all ruminant species. Clinical signs and diagnosis. Salmonellosis causes acute gastroenteritis, dysentery, and septicemia ( Anderson and Blanchard, 1989 ). Clinically, the animals become anorexic and hyperthermic. Diarrhea or dysentery develops; feces may contain mucus and/or blood and have a putrid odor. Animals become severely depressed and weak, losing a high percentage of their body weight. Animals may die in 1–5 days because of dehydration associated with dysenteric fluid loss, septicemia, shock, and acidosis. Morbidity may be 25%, and mortality may be high. Septicemia may result in subsequent meningitis, polyarthritis, and pneumonia. Chronically infected animals may have intermittent diarrhea. In goats, salmonellosis may be recognized as diarrhea and septicemia in neonates, as enteritis in preweaned kids and mature goats, and, rarely, as abortion. Adult cases may be sporadic, with intermittent bouts of diarrhea, subacute or even chronic. Morbidity and mortality will be highest in neonates, and some may simply be found dead. The older animals generally tend to fare better during the disease. Abdominal distension with profuse yellow feces is common. Kids become severely depressed, anorexic, febrile (with temperatures as high as 106°–107°F), dehydrated, acidotic, recumbent, and comatose. Salmonella abortions may occur throughout gestation. There may not be any other clinical signs, or abortion may be seen with diarrhea, fever, and vulvar discharges. Hemorrhage, placental necrosis, and edema will be present. Metritis and placental retention may occur. Some mortality of dams may occur. Diagnosis is based on clinical signs and can be confirmed by culturing fresh feces or at necropsy. Because of intermittent shedding of organisms, culture may be difficult; repeated cultures are recommended. Leukopenia and a degenerative shift to the left are not uncommon hematological findings. Epizootiology and transmission. Stresses associated with recent shipping, overcrowding, and inclement weather may predispose the animal to enteric infection. Birds and rodents may be natural reservoirs of Salmonella in external housing environments. Transmission is fecal-oral. After ingestion, the organisms may proliferate throughout the gastrointestinal tract and may penetrate the mucosa of the intestines, invade the Peyer's patches and lymphatics, and migrate to the spleen, liver, and other organs. Animals that survive may become chronic carriers and shedders of the organisms, and this has been demonstrated experimentally ( Arora, 1983 ). Fecal-oral transmission is also associated with Salmonella abortion; veneral transmission has not been reported. Necropsy findings and diagnosis. Animals will have noticeable perineal staining. Intestines (particularly the ileum, cecum, and colon) may contain mucoid feces with or without hemorrhages. Petechial hemorrhages and areas of necrosis may be noticed on the surface of the liver, heart, and mesenteric lymph nodes. The wall of the intestines, gallbladder, and mesenteric lymph nodes will be edematous, and a pseudodiphtheritic membrane lining the distal small intestines and colon may be observed. This membrane is not normally seen in the goat ( Smith and Sherman, 1994 ). Splenomegaly may be present. Aborted fetuses will often be autolysed. Placentitis, placental necrosis, and hemorrhage are commonly seen. Serologic evidence of recent infection can be demonstrated in the dam. Salmonella can be isolated from the aborted tissues. Pathogenesis. After ingestion, the organism proliferates in the intestine. Damage to the intestines and the resulting diarrhea are due to the bacterial production of cytoxin and endotoxin. Although the Salmonella organisms will be taken up by phagocytic cells involved in the inflammatory response, they survive and multiply further. Septicemia is a common sequela, with the bacteria localizing throughout the body. In latently infected animals, it is often shed from the gallbladder and mesenteric lymph nodes. Younger animals may be susceptible because of immature immunity and intestinal flora and higher intestinal pH. Carriers may develop clinical disease when stressed. Differential diagnoses. In young animals, differentials include other enteropathogens: Escherichia coli, rotavirus and coronavirus, clostridia, cryptosporidia, and other coccidial forms. These pathogens may also be present in the affected animals. Differentials in adults include bovine viral diarrheas and winter dysentery in cattle and parasitemia and enterotoxemia in all ruminants. Prevention and control. Affected animals should be isolated during herd outbreaks. Samples for culture should include herd-mates, water and feed sources, recently arrived livestock (other species), and area wildlife, including birds and rodents. Repeated cultures, culling of animals, intensive cleaning, and disinfection of facilities are all important during outbreaks. The bacteria survive for about a week in moist cow manure. Vaccination using the commercially available killed bacterin or autologous bacterins may be useful in outbreaks involving pregnant cattle, although the J-5 bacterin is now considered better. Treatment. Nursing care includes rehydration and correction of acid-base abnormalities. Antibiotic therapy may be useful in cases with septicemia, but it is controversial because it may induce carrier animals. Gentamicin, trimethoprim-sulfadiazine, ampicillin, enrofloxacin, and amikacin antibiotics may be successful. Research complications. Salmonellosis is zoonotic, and some serotypes of the organism have caused fatalities even in immunocompetent humans. Attempts should be made to identify and cull carrier animals. bb. Spirochete-Associated Abortion in Cattle (Epizootic Foothill Abortion) Etiology. Spirochete-like organisms are associated with this disease; it is now recognized that the agent is not a chlamydial organism. The disease has been reported only in the foothills bordering the central valley of California. Clinical signs. Cows that become infected with the causative agent before 6 months of gestation abort or give birth to weak calves without any clinical sign of infection. Cows infected after 6 months of gestation give birth to normal calves. Affected cows rarely abort in subsequent pregnancies. Epizootiology and transmission. The tick vector is Ornithodorus coriaceus. Necropsy. Fetuses show several pathological changes, including enlargement of the cervical lymph nodes, spleen, and liver. The calf's thymus will be small, and histologically there will be losses of thymic cortical lymphocytes. Histologic changes in lymph nodes and spleen include vasculitis, necrosis, and histiocytosis. Treatment. Chlortetracycline treatment has been effective in controlling this disease. cc. Tularemia Etiology. Tularemia is caused by Pasteurella (Francisella) tularensis a nonmotile, non-spore-forming, aerobic, gram-negative, rod-shaped bacterium. Type A is more virulent than type B. Clinical Signs. Although tularemia is a disease of livestock, pets, and wild animals, sheep are most commonly affected. The disease is characterized by hyperthermia, muscular stiffness, and lymphadenopathy. Infected animals move stiffly, are depressed, and are hyperthermic. Anemia and diarrhea may develop, and infected lymph nodes enlarge and may ulcerate. Mortality may reach 40%. Animals that recover will have immunity of long duration. Epizootiology and transmission. The disease is most commonly transmitted by ticks or biting flies. The wood tick, Dermacentor andersoni, is an important vector in transmitting the disease in the western United States, and, as natural hosts, wild rodents and rabbits tend to be reservoirs of the pathogen. Pathogenesis. The organisms, entering the tick bite wound, move via lymphatics to lymph nodes and subsequently to the bloodstream, where they cause septicemia. The organisms can also be transmitted orally through contaminated water. Necropsy findings. Ticks may also be present on the carcasses. Suppurative, necrotic lymph nodes are typical. Lungs will be congested and edematous. Diagnosis is confirmed by prompt culturing of the organism from lymph nodes, spleen, or liver where granulomatous lesions form; P. tularensis does not survive for long periods in carcasses. Serological findings may also be helpful. Treatment. Infected animals can be treated with oxytetracycline, aminoglycosides, or cephalosporins. Differential diagnosis. When tick infestations are heavy, P. tularensis should be suspected. Pasteurella haemolytica (sheep), Haemophilus somnus (cattle), and Mycoplasma mycoides (goats), and anthrax (all ruminant species) should be considered as differentials. Control and prevention. Eliminating the tick vectors can prevent tularemia. Animals should be provided with fresh water frequently. The organism can survive in freezing conditions and in water and mud for long periods of time. Caretakers, veterinarians, and researchers should take special precautions before handling the tissues of infected sheep, because this is a method of zoonotic spread. Research complications. The disease is zoonotic, and transmission to people may result from tick bites or from handling contaminated tissues. Although not a major disease of concern in sheep, researchers using potentially infected animals from western range states of the United States should be aware of it. The organism is antigenically related to Brucella spp. dd. Yersinia Etiology. Yersiniosis is caused by infections with Yersinia enterocolitica, a gram-negative, aerobic, and facultative anaerobe of the family Enterobacteriaceae. There are 50 serotypes reported for Y. enterocolitica. Yersinia pseudotuberculosis infections have also been seen in ruminants. Enteric infections predominate in the diseases caused by these bacteria. Clinical signs and diagnosis. Clinical disease may be seen rarely in many groups of ruminants. Goats of 1–6 months old suffer from the enteric form of the disease, which is characterized by sudden death or the acute onset of watery diarrhea lasting 1 or more days. Spontaneous abortions and weak neonates are also clinical manifestations of infection. Lactating does may have mastitis that becomes chronically hemorrhagic. Bacteremia results in internal abscesses, abortion, and acute deaths. Yersinia pseudotuberculosis has been associated with laboratory goat epizootics ( Obwolo, 1976 ). Diarrhea in pastured sheep, stressed by other factors, has also been reported. Diagnosis is based on culture and serology. Epizootiology and transmission. The bacteria are carried by wild birds and rodents, and transmission is by ingestion of contaminated feed and water. Necropsy findings. Edema of mesenteric lymph nodes is the most common postmortem finding. Liver abscesses, micro-absecesses in the intestines, and granuloma formation have also been reported. Placentas are white, with opaque white foci found on cotyledons. Histologically, suppurative placentitis and suppurative pneumonia are found in the fetal tissue. Pathogenesis. After ingestion, the bacteria cause an enteric infection, and bacteremia follows. Differential diagnoses. Other causes of abortions, including abortion storms, acute deaths, enteritis, neonatal deaths, and white foci on cotyledons, should be considered. In young animals, differentials include coccidiosis and nematode parasitism. Corynebacterium pseudotuberculosis and tuberculosis are differentials for the internal abscesses. Prevention and control. Control measure are not well defined, because the epidemiology of the disease is poorly understood ( Smith and Sherman, 1994 ). Tissues from affected goats must be handled and disposed of properly. Areas housing affected goats must be thoroughly sanitized. Treatment. In case of an abortion storm, treatment of goats with tetracycline has been useful. Other broad-spectrum antibiotics may also be useful. Research complications. Yersinia is zoonotic. The risk of severe enteric disease is considered particularly great for immunocompromised persons. ee. Mycoplasmal Diseases i. Mycoplasma bovigenitalium and M. bovis infections Etiology. Mycoplasma bovigenitalium and M. bovis are associated sporadically with bovine infertility and abortions. This pathogen has also been reported associated with similar clinical signs in sheep and goats. Clinical signs and diagnosis. Infertility is more commonly caused by M. bovigenitalium infections, and granular vulvovaginitis and endometritis will be present. Granular vulvovaginitis is characterized by raised papules on the mucous membranes and mucopurulent exudate. Abortions and mastitis are associated with M. bovis infections. Calves that are born may be weak. It is rare to have a definitive diagnosis of an abortion due to Mycoplasma. After consideration of other causes of abortion and evaluation of tissues for placentitis or fetal inflammation, diagnosis is confirmed by isolation of Mycoplasma from the genital tract or aborted tissues. Epidemiology and transmission. Mycoplasmal species are considered ubiquitous, are carried in the genital tracts of males and females, and are transmitted during natural breeding or through contaminated insemination materials. Aerosols also serve as a means of transmission. In addition, transmission occurs by passage through the birth canal, by direct contact, and by contamination from urine of infected animals. Pathophysiology. Experimental infections of M. bovis have resulted in placentitis and fetal pneumonia. Differential diagnoses. Acholeplasma, Ureaplasma, and Haemophilus somnus are differentials for granular vulvovaginitis. Treatment. Fluoroquinolone antibiotics may be useful for treating Mycoplasma-induced reproductive diseases. ii. Mycoplasma ovipneumoniae (ovine mycoplasmal pneumonia) Etiology. Mycoplasma ovipneumoniae causes acute or chronic pneumonia in lambs. Clinical signs. Mycoplasmas induce serious diseases in sheep, causing pneumonia, conjunctivitis, and genitourinary disease. The disease may be coincidental with pasteurellosis. Respiratory distress, coughing, and nasal discharge are observed in infected animals. Bronchoalveolar lavage followed by culture is the best method for diagnosis (mycoplasmas are fastidious organisms requiring special handling techniques). Mycoplasmas are isolated from the genitourinary tract of sheep. Vulvovaginitis and reproductive problems are associated conditions. Treatment. Tylosin, quinolones, oxytetracycline, and gentamicin are good choices for therapy. Prevention. No vaccine is available. iii. Mycoplasma mycoides biotype F38 (contagious caprine pleuropneumonia, caprine pneumonia, pleuritis, and pleuropneumonia) Etiology. Mycoplasma mycoides biotype F38 is the agent of contagious caprine pleuropneumonia and is found worldwide. In the United States, caprine pneumonia is also caused by M. ovipneumoniae, M. mycoides subsp. capri, and M. mycoides subsp. mycoides (large colony type). Clinical signs. Contagious caprine pleuropneumonia is characterized by severe dyspnea, nasal discharge, cough, and fever ( McMartin et al., 1980 ). Infections with other Mycoplasma species also have similar clinical signs. Septicemia without respiratory involvement may also be a presentation. Epizootiology and transmission. This disease is highly contagious, with high morbidity and mortality. Transmission is by aerosols. Mycoplasma mycoides subsp. mycoides has become a serious cause of morbidity and mortality of goat kids in the United States. Necropsy. Large amounts of pale straw-colored fluid and fibrinous pneumonia and pleurisy are typical. Some lung consolidation may be present. Meningitis, fibrinous pericarditis, and fibrinopurulent arthritis may also be found. Diagnosis is usually made at necropsy by culture of the organism from lungs and other internal organs. Differential dagnosis. In the United States, the principal differential for M. mycoides subsp. mycoides is caprine arthritis encephalitis. Treatment. Tylosin and oxytetracycline are effective. Some infections are slow to resolve. Prevention and control. Vaccines are available in some areas. Infected herds are quarantined. New goats should be quarantined before introduction to the herd. Research complications. The worldwide distribution of the F38 biotype, as well as the aerosol transmission and high morbidity and mortality characteristics of mycoplasmal infectious, make these infections economically important diseases. Considerable attention is presently given to this genus as a source of morbidity and mortality in goats. iv. Mycoplasma conjunctivae (mycoplasmal keratoconjunctivitis) Etiology. Mycoplasma conjunctivae causes infectious conjunctivitis, or pinkeye, in sheep and goats with associated hyperemia, edema, lacrimation, and corneal lesions. Mycoplasma mycoides subsp. mycoides, M. agalactiae, M. arginini, and Acholeplasma oculusi have also been associated with keratoconjunctivitis in these species. Respiratory disease and other infections, such as mastitis, may also be observed. Clinical signs and diagnosis. All ages of animals may be affected. Initially, lacrimation, conjunctival vessel injection, and then keratitis and neovascularization are seen. Sometimes uveitis is evident. Although the presentation is usually unilateral, bilateral involvement is possible. Recurring infections are common. Culturing provides the better diagnostic information, and cultures will be positive even after clinical signs have diminished. Epizootiology and transmission. The infection is passed easily between animals by direct contact. Animals can become reinfected, and carrier animals may be a factor in outbreaks. Necropsy. It is unlikely that animals would die or be euthanized and undergo necropsy for this problem. Conjunctival scrapings would include neutrophils during earlier stages and lymphocytes during later stages. Epithelial cell cytoplasm should be examined for organisms. Differential diagnosis. The primary differential in sheep and goats is Chlamydia, as well as Branhamella, Rickettsia (Colesiota) conjunctivae, and infectious bovine rhinotracheitis in goats only. It is important to consider these differentials if arthritis, pneumonia, or mastitis is present in the group or the individual. Treatment. Animals do recover spontaneously within about 10 weeks. Tetracycline ointments and powders are also used. Third-eyelid flaps may be necessary if corneal ulceration develops. Prevention and control. New animals should be quarantined and, if necessary treated, before introduction to the flock or herd. ff. Rickettsial Diseases i. Eperythrozoonosis (Eperythrozoon, Haemobartonella) Etiology. Eperythrozoonosis is a rare, sporadic, noncontagious, blood-borne disease in ruminants worldwide caused by the rickettsial agent Eperythrozoon. Host-specific species of importance are E. ovis, the causative species in sheep and goats, and E. wenyoni, E. tegnodes, and E. tuomii, the causative agents in cattle. Although the disease is of minor importance, it can cause severe anemia and debilitation in affected animals. Haemobartonella bovis is also rare, and is usually found only in association with other rickettsial diseases. Clinical signs and diagnosis. The disease is more severe in sheep. Following an incubation period of 1–3 weeks, infected animals exhibit episodic hyperthermia, weakness, and anemia. Losses may be greater in younger lambs. Cattle are usually latently infected but may have swollen and tender teats and legs. Fever, anemia, and depression will be present if the cattle are stressed by another systemic disease. Diagnosis is based on clinical evidence of anemia and is confirmed by observing the rickettsiae on the surface of red blood cells in a blood smear. Epizootiology and transmission. The rickettsial organisms are transmitted typically to young sheep by biting insects, ticks, contaminated needles or blood-contaminated surgical instruments. Necropsy findings. Necropsy findings include splenic enlargement and tissue icterus. Pathogenesis. The organism invades and destroys red blood cells. It is believed that intravascular hemolysis and erythrophagocytosis contribute to the macrocytic anemia. As with other red blood cell parasites, splenectomy aggravates the disease. Differential diagnosis. Clontridium novyi type D, babesiosis, and leptospirosis are the primary differentials. Prevention and control. Following strict sanitation practices for surgical procedures and controlling external parasites prevent the disease. Treatment. Treatment is not usually recommended, but Oxytetracycline has been used. Sheep will develop immunity if supported nutritionally during the disease. Research complications. Splenectomized animals are the experimental models used to study these diseases. ii. Q fever, or query fever (Coxiella burnetii) Etiology. Coxiella burnetii is a small, gram-negative, obligate intracellular rickettsial organism that causes query fever and is regarded as a major cause of late abortion in sheep. Clinical signs. Infection of ruminants with C. burnetii is usually asymptomatic. Experimental inoculation in other mammals has resulted in transient hyperthermia, mild respiratory disease, and mastitis. Abortions, stillbirths, and births of weak lambs are also seen. Epizootiology and transmission. Coxiella burnetii is extremely resistant to environmental changes as well as to disinfectants; persistence in the environment for a year or longer is possible. The organism is associated with either a free-living or an arthropod-borne cycle. Coxiella burnetii is found in a variety of tick species, such as ixodid or argasid, where it replicates and is excreted in the feces. Once introduced into a mammal, Coxiella may be maintained without a tick intermediate. The organism is especially concentrated in placental tissues, replicates in trophoblasts, and will be in reproductive fluids. Additionally, the organism is shed in milk, urine, feces, and oronasal secretions. Necropsy findings. No specific lesion will be seen in aborted or stillborn fetuses, but necrotizing placentitis will be a finding in cases of abortion. The placenta will contain white chalky plaques and a red-brown exudate. The disease can be diagnosed by identifying the rickettsial organisms in smears of placental secretions. The organism has been found in the placentas of clinically normal animals. The organism stains red with modified Ziehl-Neelsen and Macchiavello stains and purple with Giemsa stain. Differential diagnosis. Because of the organisms' similarity to Chlamydia, confirmation must be made by culture techniques, immunofluorescent procedures, ELISA, and complement fixation tests. Treatment. Coxiella can be treated with oxytetracyclines. A vaccine is not commercially available. Prevention and control. Any aborting animals should be segregated from other animals, and other pregnant animals should be treated prophylactically with tetracycline. Serologic screening of ruminant sources should be performed routinely. Barrier housing, a review of ventilation exhaust, and defined handling procedures are often required. All placentas and all aborted tissues should be handled and disposed of carefully. Q fever has been reported in many mammalian species, including cats. Research complications. Coxiella burnetii–hee animals are particularly important in studies involving fetuses and placentation. Because of its zoonotic potential, C. burnetii presents a unique problem in the animal research facility environment. A single organism has been shown to cause disease. Some of the greatest concerns are the risk to immunocompromised individuals, pregnant women, and other animals, and the presence of carrier animals or those that may shed the organism in placentas, for example. 2. Viral Diseases a. Adenovirus Infections Etiology. The ruminant adenoviruses are DNA viruses that cause respiratory and reproductive tract diseases. Nine antigenic types of the bovine adenovirus have been identified, with type 3 associated with respiratory disease. Two of the ovine and two of the caprine antigenic types have been identified. Clinical Signs. Signs of infection range from subclinical to severe, including pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, weak calf syndrome, and abortion. Respiratory tract and intestinal tract diseases may be concurrent. Infections caused by this virus are often found associated with other viral and bacterial infections. Epizootiology and transmission. The virus is believed to be widespread, but prevalence and characteristics of infection have not been characterized. Transmission of adenoviruses in other species (e.g., canine) is by aerosols or fecal-oral routes. Necropsy findings. Lesions found after experimental infections include atelectasis, edema, and consolidation of the lungs. b. Bluetongue Virus Infection (Reoviridae) Etiology. The bluetongue virus is an RNA virus in the Orbivirus genus and Reoviridae family. Five serotypes (2, 10, 11, 13, and 17) have been identified in the United States, where it is seen mostly in western states. Bluetongue is an acute arthropod-borne viral disease of ruminants, characterized by stomatitis, depression, coronary band lesions, and congenital abnormalities ( Bulgin, 1986 ). Clinical signs and diagnosis. Sheep are the most likely to show clinical signs. Clinical disease is less common in goats and cattle. Early in the infection, animals will spike a fever and will develop hyperemia and congestion of tissues of the mouth, lips, and ears. The virus name, bluetongue, is associated with the typical cyanotic membranes. The fever may subside, but tissue lesions erode, causing ulcers. Increased salivary discharges and anorexia are often related to ulcers of the dental pad, lips, gums, and tongue, although salivation and lacrimation may precede apparent ulceration. Chorioretinitis and conjunctivitis are also common signs in cattle and sheep. Lameness may be observed associated with coronitis and is evident in the rear legs. Skin lesions such as drying and cracking of the nose, alopecia, and mammary glands are also observed. Secondary bacterial pneumonia may also occur. Animals may also develop severe diarrhea and become recumbent. Sudden deaths due to cardiomyopathy may occur at any time during the disease. Hematologically, animals will be leukopenic. The course of the disease is about 2 weeks, and mortality may reach 80%. If animals are pregnant, the virus crosses the placenta and causes central nervous system lesions. Abortions may occur at any stage of gestation in cattle. Prolonged gestation may result from cerebellar hypoplasia and lack of normal sequence to induce parturition. Cerebellar hypoplasia will also be present in young born of the infected dams, as well as hydrocephalus, cataracts, gingival hyperplasia, or arthrogryposis. Diagnosis is suspected with the characteristic clinical signs and exposure to viral vectors. Virus isolation is the best diagnostic approach if blood is collected during the febrile stage of the disease or brains from aborted fetuses. Fluorescent antibody tests, ELISA, virus neutralization tests, PCR, and agar gel immunodiffusion (AGID) tests are also used to confirm the diagnosis. Epizootiology and transmission. Severe outbreaks have occurred in other countries during this century. Screening for this disease has limited the strains present in the United States. The disease is most common in outdoor-housed animals primarily in the western United States. The virus is primarily transmitted by biting midges, Culicoides. Culicoides variipennis is the most common vector in the United States. A combination of factors associated with viral strain, available and susceptible hosts, environmental conditions (such as damp areas where flies breed), and vector presence are factors in the severity of outbreaks. The disease is rarely transmitted by animal-to-animal contact or by infected animal products. Virus-contaminated semen, transplacental transfer, and carriage on transferred embyros are other possible means of transmission. Necropsy findings. At necropsy, erosive lesions may be observed around the mouth, tongue, palate, esophagus, and pillars of the rumen. Ulceration or hyperemia of the coronary bands may also be seen. Many of the internal organs will contain petechial and ecchymotic hemorrhages of the surfaces, and hemorrhage may be seen at the base of the pulmonary artery. Pathogenesis. The virus multiplies in the hemocoel and salivary glands of the fly and is excreted in transmissible form in the insect's saliva. After entering the host, the virus causes prolonged viremia. The incubation period is 6–14 days. The virus migrates to and attacks the vascular endothelium. The resulting vasculitis accounts for the lesions of the skin, mouth, tongue, esophagus, and rumen and the edema often found in many tissues. Ballooning degeneration of affected tissues, followed by necrosis and ulceration, occurs. The effects on fetuses appear to be due to generalized infections of developing organs. Differential diagnosis. Differentials include other infectious vesicular diseases such as foot-and-mouth disease, contagious ecthyma, bovine viral diarrhea virus-mucosal disease, infectious bovine rhinotracheitis, bovine papular stomatitis, and malignant catarrhal fever. Rinderpest is a differential in countries where it is endemic. Photosensitization should be considered. Foot rot is a differential for the lameness and coronitis. Differentials for the manifestations such as arthrogryposis include border disease virus and genetic predispositions of some breeds such as Charolais cattle and Merino sheep. Prevention and control. Cellular and humoral immunity are necessary for protection from infection. The bluetongue virus is insidious because the genome is capable of reassortment, and some vaccines will not have the antigenic components represented in the local infection. In addition, there is little to no cross protection between strains. Modified live vaccines are available in some parts of the United States but should not be used in pregnant animals. Vaccinating lambs and rams in an outbreak is worthwhile, for example, but vaccinating late-gestation ewes may cause birth defects or abortions. Congenital defects are more common from vaccine use than from naturally occurring infection. Minimizing exposure to the vector in endemic areas will decrease the incidence of the disease. Treatment. Supportive care and nursing care are helpful, including gruels or softer feeds, easily accessed water, and shaded resting places. Nonsteroidal anti-inflammatory drugs are often administered. For the cases of secondary bacterial pneumonia and some cases of bluetongue conjunctivitis, antibiotics may be administered. Research complications. This is a reportable disease because clinical signs resemble foot-and-mouth disease and other exotic vesicular diseases. c. Bovine Lymphosarcoma (Bovine Leukemia Virus Infection, Bovine Leukosis) Etiology. Bovine lymphosarcoma refers to lymphoproliferative diseases in young cattle that are not associated with bovine leukemia virus (BLV) infection, and those in older cattle that are associated with BLV. BLV is a B lymphocyte-associated retrovirus ( Johnson and Kaneene, 1993 a,b,c). Clinical signs. Forms of bovine lymphosarcoma that are not associated with BLV infection are calf, or juvenile; thymic, or adolescent (animals 6 months to 2 years old); and cutaneous (any age). The calf form is rare and characterized by generalized lymphadenopathy. Onset may be sudden, and the disease is usually fatal within a few weeks. Signs include lymphadenopathy, anemia, weight loss, and weakness. Some animals may be paralyzed because of spinal cord compression from subperiosteal infiltration of neoplastic cells. The adolescent form is also rare, the course rapid, and the prognosis poor. The disease is seen most often in beef breeds such as Hereford cattle and is characterized by space-occupying masses in the neck or thorax. These masses are also often present in the brisket. Secondary effects of the masses are loss of condition, dysphagia, rumen tympany, and fatal bloat. The cutaneous presentation has a longer course and may wax and wane. The masses are found at the anus, vulva, escutcheon, shoulder, and flank; they are painful when palpated, raised, and often ulcerated. The animals are anemic, and neoplastic involvement may affect cardiac function. Generalized or limited lymphadenopathy may be apparent. Only the adult, or enzootic, form of bovine lymphosarcoma is associated with BLV infection. Many animals do not develop any malignancies or clinical signs of infection and simply remain permanently infected. Some cows manifest disease only during the periparturient period. Malignant lymphoma is the more common, whereas leukosis, due to B-lymphocyte proliferation, is rare. Clinical signs are loss of condition and a drop in production of dairy cattle, anorexia, diarrhea, ataxia, paresis, and other signs dependent on the location of the neoplastic tissue. Tumors are associated with lymphoid tissues. Common sites also include the abomasum, spinal canal, and uterus. Cardiac tumors develop at the right atrial or left ventricular myocardium, and associated beat and rate abnormalities may be auscultated. The common ocular manifestation of the disease is exophthalmos due to retrobulbar masses. Many internal organs may be involved, and tumors may be palpable per rectum. Secondary infections will be due to immunosuppression and the weakened state of the animal. Sheep have acquired BLV infection naturally and have been used as experimental models; in both situations, this species is susceptible to tumor and leukemia development. Goats seroconvert but do not develop the clinical syndromes. Diagnosis is based on the animal's age, clinical signs, serology, hematology findings according to the form, aspirates or biopsies of masses, and necropsy findings. Kits are available for running AGID, for which the BLV antigens gp-51 and gp-24 are used; antibodies may be detected within weeks after exposure and may also help in predicting disease in clinically normal cattle. ELISA and PCR diagnostic aids will also be helpful. Epizootiology and transmission. This disease is present worldwide. It is estimated that at least 50% of the cattle in the United States are infected with BLV. As few as 1% of these animals develop lymphosarcoma, but the adult form of the disease described here is the most common bovine neoplastic disease in the United States. Larger herds tend to have higher rates. Genetic predisposition may be involved; in addition to the presence of BLV, the type of bovine lymphocyte antigen (BoLA) may be correlated to resistance or susceptibility and to the course of the disease. Transmission is believed to be by inhalation of BLV in secretions; in colostrum; horizontally by contaminated equipment not sanitized between cattle; and by rectum (e.g., mucosal irritation during per-rectum exams or procedures). Natural-service bulls may transmit the infection to cows. Cows infected with BLV may transmit the infection to their calves in utero. Tabanid and other flies also serve as vectors, but these represent a minor means of transmission. Necropsy findings. Neoplastic infiltration of many organs and tissues are found in the calf form and the cutaneous forms. Tumors may be local or widely distributed in the enzootic form. Definitive diagnosis of neoplastic tissue specimens is by histology. Pathogenesis. As with other retroviruses, the BLV integrates viral DNA into host target cell DNA by means of the reverse transcriptase enzyme, creating a provirus. Prevention and control. There is no vaccine for this disease. Development and maintenance of a BLV-free herd, or controlling infection within a herd, requires financial and programmatic commitments: BLV-positive and BLV-negative animals maintained separately; serologic testing (such as at least every 6 months) and separating positive animals; and washing and then disinfecting instruments, needles (or using sterile single-use products), and equipment for ear tagging and dehorning and other such equipment between animals. A fresh rectal exam sleeve and lubricant should be used for each animal examined. Otherwise serologically positive cows may have undetectable antibodies during the periparturient period. Embryo transfer recipients should be negative, and the virus will not be transferred by the embryonic stage. Calves should be fed colostrum from serologically negative cows. Treatment. Treatment regimens of corticosteroids and cancer chemotherapeutic agents provide only short-term improvement. In cases where ova, embryos, or semen need to be collected, supportive care for the affected animals is essential. Research complications. The United States and several countries, some in Europe, have official programs for eradication of enzootic bovine leukosis. d. Bovine Herpes Mammillitis (Bovine Herpesvirus 2 Bovine Ulcerative Mammillitis) Etiology. Bovine herpesvirus 2 causes bovine herpes mammillitis, a widespread disease characterized by teat and udder lesions, as well as oral and skin lesions. Clinical signs and diagnosis. Lesions begin suddenly with teat swelling; the tissue will be edematous and tender when touched. The udder lesions may extend to the perineum. The lesions progress to vesicles, then to ulcers; these may take 10 weeks to heal. Lesions rarely may also develop focally around the mouth and generally on the skin of the udder. Secondary mastitis may occur, because of bacteria associated with the scabs. Diagnosis is by clinical signs and serologically. Epizootiology and transmission. The virus is reported to be widespread. Occurrence is often seasonal, and biting insects may be vectors. Transmission with successful infection requires deep penetration of the skin. Transmission may be by contaminated milkers' hands, contaminated equipment, and other fomites. Differential diagnosis. Differential diagnoses include other diseases that cause lesions on teats such as pseudocowpox, papillomatosis, and vesicular stomatitis. Other vesicular diseases may be considered, but other more severe clinical signs might be associated with those. Prevention and control. Established milking hygiene practices are important control measures: having milkers wash their hands with germicidal solutions or wear gloves, cleaning equipment between animals, and separating affected animals. Treatment. There is no treatment, and affected animals should be separated from the herd and milked last. Lesions can be cleaned and treated with topical antibacterials. e. Bovine Viral Diarrhea Virus Etiology. The bovine viral diarrhea virus (BVDV) is a pestivirus of the Flaviviridae family. The Flaviviridae include hog cholera virus and border disease virus of sheep. The virus contains a single strand of positive-sense RNA. A broad range of disease and immune effects is produced by BVDV only in cattle. In addition, this virus is important in the etiology of bovine pneumonias. Bovine viral diarrhea/mucosal disease (BVD/MD) is one of the most important viral diseases and one of the most complex diseases of cattle. Strains of BVDV are characterized as cytopathic (CP) and noncytopathic (NCP), based on cell-culture growth characteristics. The virus has also been categorized as type 1 and type 2 isolates. Heterologous strains exist that may confound even sound vaccination programs. Clinical signs and diagnosis. Signs of BVDV infections may be subclinical but also include abortions, congenital abnormalities, reduced fertility, persistent infection (PI) with gradual debilitation, and acute and fatal disease. The presence of antibodies, whether from passive transfer or immunizations, does not necessarily guarantee protection from the various forms of the disease. An acute form of the disease, caused by type 2 BVDV, occurs in cattle without sufficient immunity. After an incubation period of 5–7 days, clinical signs include fever, anorexia, oculonasal discharge, oral erosions (including on the hard palate), diarrhea, and decreased milk production. The disease course may be shorter with hemorrhagic syndrome and death within 2 days. Clinical signs of BVDV in calves also include severe enteritis and pneumonia. When susceptible cows are infected in utero from gestational days 50–100, or gestational cows are vaccinated with a modified live vaccine, abortion or stillbirth result. Congenital defects caused by BVDV during gestational days 90–170 include impaired immunity (thymic atrophy), cerebellar hypoplasia, ocular defects, alopecia or hypotrichosis, dysmyelinogenesis, hydranencephaly, hydrocephalus, and intrauterine growth retardation. Typical signs of cerebellar dysfunction will be evident in calves, such as wide-based stance, weakness, opisthotonus, hyperflexion, hypermetria, nystagmus, or strabismus. Some severely affected calves will not be able to stand. Ophthalmic effects include retinal degeneration and microphthalmia. Fetuses can also be infected in utero, normal at birth, immunotolerant to the virus, and persistently infected (PI). The term mucosal disease is commonly associated with this form of the infection. Many PI animals do not survive to maturity, however, and many have weakened immune systems. The PI animals are important because they shed virus and will probably show the clinical signs of mucosal disease (MD) caused by a CP BVDV strain derived from an NCP BVDV strain. These MD clinical signs include fever, anorexia, and profuse diarrhea that may include blood and fibrin casts, and oral and pharyngeal erosions, as well as erosion at the interdigital spaces and on the teats and vulva. Many other associated clinical signs include anemia, bloat, lameness, or corneal opacities and discharges. Secondary effects of hemorrhage and dehydration also contribute to the morbidity and mortality. Animals that do not succumb to the disease will be chronically unthrifty, debilitated, and infection-prone. Diagnosis in affected calves is based on herd health history, clinical signs, and antibodies to BVDV in precolostral serum. Viral culturing from blood may be useful. In older animals, oral lesions, serology, detection of viral antigen, and virus isolation contribute to the diagnosis. Leukopenia, and especially lymphopenia, are seen. Serology must be interpreted with the awareness of the possibility of PI immunotolerant animals. Vaccination against the disease carries its own set of side effects and potential problems, especially when using modified live vaccines, whether against CP or NCP strains. The condition of the animals is also a variable. Epizootiology and transmission. BVDV is present throughout the world. Transmission occurs easily by direct contact between cattle, from feed contaminated with secretions or feces, and by aborted fetuses and placentas. PI females transmit the virus to their fetuses. Semen also is a source of virus. Necropsy findings. In affected calves, histopathologic findings include necrosis of external germinal cells, focal hemorrhages, and folial edema. Later in the disease, large cavities develop in the cerebellum, and atrophy of the cerebellar folia and thin neuropil are evident. Older calves may have areas of intestinal necrosis. In cases where oral erosions occur, erosions will be found extending throughout the gastrointestinal tract to the cecum. The respiratory tract lesions will often be complicated by secondary bacterial pneumonia. When the hemorrhagic syndrome develops, petechiation and mucosal bleeding will be present. Pathogenesis. The CP and NCP strains are thought to be related mutations of the BVDV; the CP short-lived isolates are believed to arise from the NCP strains. The NCP strains are those present in the PI animals, and the strains are maintained in cattle populations. CP and NCP isolates vary in virulence, and classification of these types is based on viral surface proteins. Considerable antigenic variation also exists between strains and types. Other viral infections, such as bovine respiratory syncytial virus and infectious bovine rhinotracheitis, may also be present in the same animals. The pathology caused by BVDV is due to its ability to infect epithelial cells and impair the functioning of immune cell populations through out the bovine system. In type 2 BVDV hemorrhagic syndrome, death results from viral-induced thrombocytopenia. In fetuses, the virus infects developing germinal cells of the cerebellum. The Purkinje's cells in the granular layer are killed, and necrosis and inflammation follow. The immune effects are the result of the virus's interfering with neutrophil and macrophage functions and of lymphocyte blastogenesis. All of these predispose the affected animals to bacterial infections with Pasteurella haemolytica. BVDV damages dividing cells in fetal organ systems, resulting in abortions and congenital effects. Differential diagnosis. Many differentials must be considered for the clinical manifestations of BVDV infections. Differentials for enteritis of calves include viral infections, Cryptosporidia, Escherichia coli, Salmonella, and Coccidia. Salmonella, winter dysentery, Johne's disease, intestinal parasites, malignant catarrhal fever (MCF), and copper deficiency are differentials for the diarrhea seen in the disease in adult animals. Respiratory tract pathogens such as bovine respiratory syncytial virus, Pasteurella, Haemophilus, and Mycoplasma must be considered for the respiratory tract manifestations. Oral lesions are also produced by MCF, vesicular stomatitis, bluetongue, and papular stomatitis. Infectious bovine herpesvirus 1, leptospirosis, brucellosis, trichomoniasis, and mycosis should be considered in cases of abortion. Prevention and control. Combined with sound management in a typical cattle herd, vaccination is the best way to prevent BVDV and should be integrated into the herd health program, timed appropriately preceding breeding, gestation, or stressful events. Vaccine preparations for BVDV are modified live virus (MLV) or killed virus. Each has advantages and disadvantages. The former induces rapid immunity (within 1 week) after a single dose, provides longer duration of immunity against several strains, and induces serum neutralizing antibodies. MLV vaccines are not recommended for use in pregnant cattle, may induce mucosal disease, and may be immunosuppressive at the time of vaccination. The immunosuppression is detrimental if cattle are concurrently exposed to field-strain virus because it will facilitate infection and possible clinical disease. The MLV strains may cross the placenta, resulting in fetal infections. The killed vaccines are safer in pregnant animals but require booster doses after the initial immunization, may need to be given 2–3 times per year, and do not induce cell-mediated immunity. Passive immunity may protect most calves for up to 6–8 months of age. Subsequent vaccination with MLV may provide lifelong immunity, but this is not guaranteed. Annual boosters are recommended to protect against vaccine breaks. The virus persists in the environment for 2 weeks and is susceptible to the disfectants chlorhexidine, hypochlorite, iodophors, and aldehydes. Maintenance of a closed herd to prevent any possibility of the introduction of the virus is difficult. Isolation of new animals, avoidance of the purchase of pregnant cows, scrutiny of records from source farms, use of semen tested bulls, minimization of stress, testing of embryo-recipient cows, and maintainenance of populations of ruminants (smaller or wild species) separately on the premises will minimize viral exposure. Other management strategies may require a program for testing and culling PI cattle. This can be expensive but may be a worthwhile investment to remove the virus shedders from a herd. Treatment. No specific treatment is available. Supportive care and treatment with antibiotics to prevent secondary infection are recommended. Animals that survive the infection should be evaluated a month after recovery to determine their status as PI or virus-free. f. Cache Valley Virus Etiology. Cache Valley virus (CVV), of the arbovirus genus of the Bunyaviridae family, is a cause of congenital defects in lambs. Clinical signs and diagnosis. Teratogenic effects of in utero CVV infection in fetal and newborn lambs include arthrogryposis, microencephaly, hydranencephaly, porencephaly, cerebellar hypoplasia, and micromyelia. Stillbirths and mummified fetuses are seen. Lambs will be born weak and will act abnormally. Diagnosis is by evidence of seroconversion in precolostral blood samples or fetal fluids, as the result of in utero infection. Epizootiology and transmission. The virus is present in the western United States, although it has been isolated in a few Midwestern states. Although considered a disease of sheep, virus has been isolated from cattle and from wild ruminants and antibodies found in white-tailed deer. Transmission is by arthropods during the first trimester of pregnancy. g. Caprine Arthritis Encephalitis Virus Etiology. Caprine arthritis encephalitis virus (CAEV) occurs worldwide, with a high prevalence in the United States. Caprine arthritis encephalitis (CAE) is considered the most important viral disease of goats. The CAEV is in the Lentivirus genus of the Retroviridae family. It causes chronic arthritis in adults and encephalitis in young. CAEV is in the same viral genus as the ovine progressive pneumonia virus (OPPV). Clinical signs and diagnosis. The most common presentation in goats is an insidious, progressive arthritis in animals 6 months of age and older. Animals become stiff, have difficulty getting up, and may be clinically lame in one or both forelimbs. Carpal joints are so swollen and painful that the animal prefers to eat, drink, and walk on its "knees." In dairy goats, milk production decreases, and udders may become firmer. This retrovirus also causes neurological clinical signs in young kids 2–6 months old. Kids may be bright and alert, afebrile, and able to eat normally even when recumbent. Some kids may initially show unilateral weakness in a rear limb, which progresses to hemiplegia or tetraplegia. Mild to severe lower motor neuron deficits may be noted, but spinal reflexes are intact. Clinical signs may also include head tilt, blindness, ataxia, and facial nerve paralysis. Older animals in the group may experience interstitial pneumonia or chronic arthritis. The pneumonia is similar to the pneumonia in sheep caused by OPPV; the course is gradual but progressive, and animals will eventually lose weight and have respiratory distress. Some animals in a herd may not develop any clinical signs. Diagnosis is based on clinical signs, postmortem lesions, and positive serology for viral antibodies to CAEV. An agar gel immunodiffusion (AGID) test identifies antibodies to the virus and is used for diagnosis. Kids acquire an anti-CAEV antibody in colostrum, and this passive immunity may be interpreted as indicative of infection with the virus. The antibody does not prevent viral transmission. Epizootiology and transmission. The virus is prevalent in most industrialized countries. The common means of transmission, from adults to kids, is in the colostrum and milk in spite of the presence of anti-CAEV antibody in the colostrum. Transmission may occur among adult goats by contact. Intrauterine transmission is believed to be rare. Transmission to sheep has occurred only experimentally; there is no documented case of natural transmission. Necropsy findings. Necropsy and histopathology reveal a striking synovial hyperplasia of the joints with infiltrates of lymphocytes, macrophages, and plasma cells. Other histologic lesions include demyelination in the brain and spinal cord, with multifocal invasion of lymphocytes, macrophages, and plasma cells. In severe cases of mastitis, the udder may appear to be composed of lymphoid tissue. Pathogenesis. The virus infects cells of the mononuclear system, resulting in the formation of non-neutralizing antibody to viral core proteins and envelope proteins. Immune complex formation in synovial, mammary gland, and neurological tissue is thought to result in the clinical changes observed. Most commonly, the carpal joint is affected, followed by the stifle, hock, and hip. The infection is lifelong. Differential diagnosis. The differential diagnosis for the neurologic form of CAEV should include copper deficiency, enzootic pneumonia, white muscle disease, listeriosis, and spinal cord disease or injury. The differential diagnosis for CAEV arthritis should include chlamydia and mycoplasma. Prevention and control. Herds can be screened for CAE by testing serologically, using an AGID or an enzyme-linked immunosorbent assay (ELISA) test. The ELISA is purported to be more sensitive, whereas the AGID is more specific. Individual animals show great variation in development of antibody. Because CAE is highly prevalent in the United States, and because seronegative animals can shed organisms in the milk, retesting herds at least annually may be necessary. Recently, an immuno-precipitation test for CAE has been developed that has high sensitivity and specificity. Control measures include management practices such as test and cull, prevention of milk transmission, and isolation of affected animals. Parturition must be monitored, and kids must be removed immediately and fed heat-treated colostrum (56° C for 1 hr). CAEV-negative goats should be separated from CAEV-positive goats. Treatment. There is no treatment for CAEV. h. Infectious Bovine Rhinotracheitis Virus (Infectious Bovine Rhinotracheitis-Infectious Pustular Vulvovaginitis) Etiology. The infectious bovine rhinotracheitis virus (IBRV) is also referred to as bovine herpesvirus 1 (BHV-1) and is an alphaherpesvirus. IBRV causes or contributes to several bovine syndromes, including respiratory and reproductive tract diseases. It is one of the primary pathogens in the bovine respiratory disease complex. Strains include BHV-1.1 (associated with respiratory disease), BHV 1.2 (associated with respiratory and genital diseases), and BHV 1.4 (associated with neurological diseases), which has been reclassified as bovine herpesvirus 5. Clinical signs and diagnosis. Diseases caused by the virus include conjunctivitis, rhinotracheitis, pustular vulvovaginitis, balanoposthitis, abortion, encephalomyelitis, and mastitis. The respiratory form is known as infectious bovine rhinotracheitis, and clinical signs may range from mild to severe, the latter particularly when there are additional respiratory viral infections or secondary bacterial infections. The mortality rate in more mature cattle is low, however, unless there is secondary bacterial pneumonia. Fever, anorexia, restlessness, hyperemia of the muzzle, gray pustules on the muzzle (that later form plaques), nasal discharge (that may progress from serous to mucopurulent), hyperpnea, coughing, salivation, conjunctivitis with excessive epiphora, and decreased production in dairy animals are typical signs. Open-mouth breathing may be seen if the larynx or nasopharygneal areas are blocked by mucopurulent discharges. Neonatal calves may develop respiratory as well as general systemic disease. In these cases, in addition to the symptoms already noted, the soft palate may become necrotic, and gastrointestinal tract ulceration occurs. Young calves are most susceptible to the encephalitic form; signs include dull attitude, head pressing, vocalizations, nystagmus, head tilt, blindness, convulsions, and coma, as well as some signs, such as discharges, seen with respiratory tract presentations. This form is usually fatal within 5 days. Abortion may occur simultaneously with the conjunctival or respiratory tract diseases, when the respiratory infection appears to be mild, or may be delayed by as much as 3 months after the respiratory tract disease signs. Infectious pustular vulvovaginitis is most commonly seen in dairy cows, and clinical signs may be mild and not noticed. Otherwise, signs are fever, depression, anorexia, swelling of the vulvar labia, vulvar discharge, and vestibular mucosa reddened by pustules. The cow will often carry her tail elevated away from these lesions. These soon coalesce, and a fibrous membrane covers the ulcerated area. If uncomplicated, the infection lasts about 4–5 days, and lesions heal in 2 weeks. Younger infected bulls may develop balanoposthitis with edema, swelling, and pain such that the animals will not service cows. Epizootiology and transmission. IBRV is widely distributed throughout the world, and adult animals are the reservoirs of infection. The disease is more common in intensive calf-rearing situations and in grouped or stressed cattle. Transmission is primarily by secretions, such as nasal, during and after clinical signs of disease. Modified live vaccines are capable of causing latent infections. Necropsy findings. Fibrinonecrotic rhinotracheitis is considered pathognomic for IBRV respiratory tract infections. There will be adherent necrotic lesions in the respiratory, ocular, and reproductive mucosa. When there are secondary bacterial infections, such as Pasteurella bronchopneumonia, findings will include congested tracheal mucosa and petechial and ecchymotic hemorrhages in that tissue. Lesions from the encephalitic form include lymphocytic meningoencephalitis and will be found throughout the gray matter (neuronal degeneration, perivascular cuffing) and white matter (myelitis, demyelination). Intranuclear inclusion bodies are not a common finding with this herpesvirus. Pathogenesis. In the encephalitic form, the virus first grows in nasal mucosa and produces plaques. These resolve within 11 days, and the encephalitis develops after the virus spreads centripetally to the brain stem by the trigeminal nerve dendrites. Latent infections are also established in neural tissue. Differential diagnosis. The severe oral erosions seen with BVDV infections are rare with infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV). The conjunctivitis of IBR may initially be mistaken for that of a Moraxella bovis (pinkeye) infection; the IBR will be peripheral, and there will not be corneal ulceration. Bovine viral diarrhea virus and IBRV are the most common viral causes of bovine abortion. Differentials for balanoposthitis include trauma from service. Prevention and control. Vaccination options include inactivated, attenuated, modified live, and genetically altered preparations. Some are in combination with parainfluenza 3 (PI-3) virus. The MLV preparations are administered intranasally; these are advantageous in calves for inducing mucosal immunity even when serologic passive immunity is already present and adequate. Some newer vaccines, with gene deletion, allow for serologic differentiation between antibody responses from infection or immunization. Bulls with the venereal form of the infection will transmit the virus in semen; intranasal vaccine may be used to provide some immunity. Treatment. Uncomplicated mild infections will resolve over a few weeks; palliative treatments, such as cleaning ocular discharges and supplying softened food, are helpful in recovery. Antibiotics are usually administered because of the high likelihood of secondary bacterial pneumonia. The encephalitic animals may need to be treated with anticonvulsants. i. Parainfluenza 3 (PI-3) Etiology. Parainfluenza 3, an RNA virus of the family Paramyxoviridae, causes mild respiratory disease of ruminants when it is the sole pathogen. The viral infection often predisposes the respiratory system to severe disease associated with concurrent viral or bacterial pathogens. Viral strains are reported to vary in virulence. Serotypes seen in the smaller ruminants are distinct from those isolated from cattle. Clinical signs and diagnosis. Infections ranging from asymptomatic to mild signs of upper respiratory tract disease are associated with this virus by itself; infections are almost never fatal. Clinical signs include ocular and nasal discharges, cough, fever, and increased respiratory rate and breath sounds. In pregnant animals, exposure to PI-3 can result in abortions. Clinical signs become apparent or more severe when additional viral pathogens are present, such as bovine viral diarrhea virus, or a secondary bacterial infection, such as Pasteurella haemolytica infection, is involved. Greater morbidity and mortality will be sequelae of the bacterial infections. Viral isolation or direct immunofluorescence antibody (IFA) from nasal swabs can be used for definitive diagnosis. Epizootiology and transmission. The virus is considered ubiquitous in cattle and is a common infection in sheep. Presently it is assumed that the virus is widespread in goats, but firm evidence is lacking. Necropsy findings. For an infection of PI-3 only, findings will be negligible. Some congestion of respiratory mucosa, swelling of respiratory tract-associated lymph nodes, and mild pneumonitis may be noted grossly and histologically. Intranuclear and intracytoplasmic inclusion bodies may be present in the mucosal epithelial cells. Findings will be similar but not as severe as those caused by bovine respiratory syncytial virus. Immunohistochemistry may also be used. Pathogenesis. PI-3 infects the epithelial mucosa of the respiratory tract; however, the disease is often asymptomatic when uncomplicated. Differential diagnosis. Differentials, particularly in cattle, include infections with other respiratory tract viruses of ruminants: IBRV, BVDV, bovine respiratory syncytial virus, and type 3 bovine adenovirus. Prevention and control. Immunization, management, and nutrition are important for this respiratory pathogen, as for others. In cattle, modified live vaccines for intramuscular (IM), subcutaneous (SC), or intranasal (IN) administration are available. The IM and SC routes provide immune protection within 1 week after administration but will not provide protection in the presence of passively acquired antibodies. It is contraindicated for pregnant animals because it will cause abortion. The IN route immunizes in the presence of passively acquired antibodies, provides immunity within 3 days of administration, and stimulates the production of interferon. Other vaccine formulations, about which less information is reported, include inactivated or chemically altered live-virus preparations; both are administered IM, and followup immunizations are needed within 4 weeks. Booster vaccinations are recommended for all preparations within 2–6 months after the initial immunization. All presently marketed vaccine products come in combination with other bovine respiratory viruses as multivaccine products. The humoral immunity protects against PI-3 abortions. There is no approved PI-3 vaccine for sheep and goats. The use of the cattle formulation in these smaller ruminants is not recommended. Sound management of housing, sanitation, nutrition, and preventive medicine programs are all equally important components in prevention and control. Treatment. Uncomplicated disease is not treated. j. Respiratory Syncytial Viruses of Ruminants Etiology. The respiratory syncytial viruses are pneumoviruses of the Paramyxoviridae family and are common causes of severe disease in ruminants, especially calves and yearling cattle. Two serotypes of the bovine respiratory syncytial virus (BRSV) have been described for cattle; these may be similar or identical to the virus seen in sheep and goats. Clinical findings and diagnosis. Infections may be subclinical or develop into severe illness. Clinical signs include fever, hyperpnea, spontaneous or easily induced cough, nasal discharge, and conjunctivitis. Interstitial pneumonia usually develops, and harsh respiratory sounds are evident on auscultation. Development of emphysema indicates a poor prognosis, and death may occur in the severe cases of the viral infection. Secondary bacterial pneumonia, especially with Pasteurella haemolytica, with morbidity and mortality, is also a common sequela. Abortions have been assciated with BRSV outbreaks. Diagnosis is based on virus isolation and serology (acute and convalescent). Nasal swabs for virus isolation should be taken when animals have fever and before onset of respiratory disease. Epizootiology and transmission. These viruses are considered ubiquitous in domestic cattle and are transmitted by aerosols. Necropsy findings. Gross lesions include consolidation of anteroventral lung lobes. Edema and emphysema are present. As the name indicates, syncytia, which may have inclusions, form in areas of the lungs infected with the virus. Necrotizing bronchiolitis, bronchiolitis obliterans, and hyaline membrane formation will be evident microscopically. Pathogenesis. The severe form of the disease, which often follows a mild preliminary infection, is thought to be caused by immune-mediated factors during the process of infection in the lung. Virulence may vary greatly among viral strains. Differential diagnosis. Differentials should include other ruminant respiratory tract viruses. Prevention and control. Vaccination should be part of the standard health program, and all animals should be vaccinated regularly. Vaccinations should be administered within 1–2 months of stressful events, such as weaning, shipping, and introduction to new surroundings. Currently available vaccines include an inactivated preparation and a modified live virus preparation administered intramuscularly or subcutaneously; immunity develops well in yearling animals, and colostral antibodies develop when cows are vaccinated during late gestation. Passive immunity from colostrum provides at least partial protection to calves in herds where disease is prevalent. But this immunity suppresses the mucosal IgA response and serum antibody responses. The basis for successful immune protection is the mucosal memory IgA, but this is difficult to achieve with present vaccine formulations. The virus is easily inactivated in the environment. Preventive measures in preweaning animals should include preconditioning to minimize weaning stress. Treatment. Recovery can be spontaneous; however, antibiotics and supportive therapy are useful to prevent or control secondary bacterial pneumonia. In severe cases, antihistamines and corticosteriods may also be necessary. Use of vaccine during natural infection is not productive and may result in severe disease. k. Ulcerative Dermatosis (Ovine Venereal Disease, Balanoposthitis) Etiology. Ulcerative dermatosis is a contagious disease of sheep only. It is caused by a poxvirus similar to but distinct from the causative agent of contagious ecthyma ( "Current Veterinary Therapy," 1993 ). Clinical signs and diagnosis. Lesions include ulcers and crusts associated with the skin and mucous membranes of the genitalia, face, and feet ( Bulgin, 1986 ). Genital lesions are much more common than the facial or coronal lesions. Discomfort may be associated with the lesions. Paraphimosis occasionally occurs. These lesions are painful; during breeding season, animals will avoid coitus. Morbidity is low to moderate, and mortality negligible if the flock is otherwise healthy. Diagnosis is based on clinical signs. Epizootiology and transmission. Endemic to the western United States, ulcerative dermatosis is transmitted through direct contact with abraded skin of the prepuce, vulva, face, and feet. Necropsy findings. Necropsy would rarely be necessary to diagnose an outbreak in a healthy flock. Findings will be similar to those described for contagious ecthyma. Pathogenesis. Following an incubation period of 2–5 days, the virus replicates in the epidermal cells and leads to necrosis and pustule formation. Pustules rapidly break, forming weeping ulcers. The ulcers scab over and eventually form a fibrotic scar. The disease usually resolves in 2–6 weeks. Rarely, the disease will persist for many months to more than a year. Differential diagnosis. The main differential is contagious ecthyma, which is grossly and histopathologically associated with epithelial hyperplasia. This is also a feature of ulcerative dermatosis. Prevention and control. No vaccine is available. Affected animals, especially males, should not be used for breeding. Treatment. Affected animals should be separated from the rest of the flock. Treatment is supportive, including antiseptic ointments and astringents. Research complications. Breeding and maintenance of the flocks' condition, because of the pain associated with eating, will be compromised during an outbreak. l. Border Disease Etiology. Border disease, also known as hairy shaker disease (or "fuzzies" in the southwestern United States), is a disease of sheep caused by a virus closely related to the bovine viral diarrhea virus (BVDV), a pestivirus of the Togaviridae family. Goats are also affected. The virus causes few pathogenic effects in cattle. Clinical signs and diagnosis. Border disease in ewes causes early embryonic death, abortion of macerated or mummified fetuses, or birth of lambs with developmental abnormalities. Lambs infected in utero that survive until parturition may be born weak and often exhibit a number of congenital defects such as tremor, hirsutism (sometimes darkly pigmented over the shoulders and head), hypothyroidism, central nervous system defects, and joint abnormalities, including arthrogryposis. Later, survivors may be more susceptible to diseases and may develop persistent, sometimes fatal, diarrhea. The virus infection produces similar clinical manifestations in goats, except that the hair changes are not seen. Diagnosis includes the typical signs described above, as well as serological evidence of viral infection. Virus isolation confirms the diagnosis. Epizootiology and transmission. The virus is present worldwide, and reports of disease are sporadic. Disease has occurred when no contact with cattle has occurred. Persistently infected animals, such as lambs, are shedding reservoirs of the virus in urine, feces, and saliva throughout their lives. Necropsy findings. Lesions include placentitis, and characteristic joint and hair-coat changes in the fetus. Histologically, axonal swelling, neuronal vacuolation, dysmyelination, and focal microgliosis are observed in central nervous system structures. Pathogenesis. The virus entering the ewe via the gastrointestinal or respiratory tracts penetrates the mucous membranes and causes maternal and fetal viremia. Infection during the first 45 days of gestation causes embryonic death. In lambs infected between 45 and 80 days, the virus activates follicular development, diminishes the myelination of neurons, and causes dysfunction of the thyroid gland. Infection after 80 days of gestation results in lambs that are born persistently infected. Infected lambs have high perinatal mortality; survivors have diminished signs over time but, as noted, continue to shed the virus. Prevention and control. Border disease can be prevented by vaccinating breeding ewes with killed-BVDV vaccine. Congenitally affected lambs should be maintained separately and disposed of as soon as humanely possible. New animals to the flock should be screened serologically. If cattle are housed nearby, vaccination programs for BVDV should be maintained. Treatment. There is no treatment other than supportive care for affected animals. m. Contagious Ecthyma (Contagious Pustular Dermatitis, Sore Mouth, Orf) Etiology. Contagious ecthyma, also known as contagious pustular dermatitis, sore mouth, or orf, is an acute dermatitis of sheep and goats caused by a parapoxvirus. This disease occurs worldwide and is zoonotic. Naturally occurring disease has also been reported in other species such as musk ox and reindeer. Other parapoxviruses infect the mucous membranes and skin of cattle, causing the diseases bovine pustular dermatitis and pseudocowpox. Clinical signs and diagnosis. The disease is characterized by the presence of papules, vesicles, or pustules and subsequently scabs of the skin of the face, genitals of both sexes, and coronary bands of the feet. Lesions develop most frequently at mucocutaneous junctions and are found most commonly at the commissures of the mouth. Orf is usually found in young animals less than 1 year of age. Younger lambs and kids will have difficulty nursing and become weak. Lesions may also develop on udders of nursing dams, which may resist suckling by offspring to nurse, leading to secondary mastitis. The scabs may appear nodular and raised above the surface of the surrounding skin. Morbidity in a susceptible group of animals may exceed 90%. Mortality is low, but the course of the disease may last up to 6 weeks. Diagnosis is based on characteristic lesions. Biopsies may reveal eosinophilic cytoplasmic inclusions and proliferative lesions under the skin. Electron microscopy will reveal the virus itself. Disease is confirmed by virus isolation. Epizootiology and transmission. All ages of sheep and goats are susceptible. Seasonal occurrences immediately after lambing and after entry into a feedlot are common; stress likely plays a role in susceptibility to this viral disease. Older animals develop immunity that usually prevents reinfection for at least 1 or more years. Resistant animals may be present in some flocks or herds. The virus is very resistant to environmental conditions and may contaminate small-ruminant facilities, pens, feedlots, and the like for many years as the result of scabs that have been shed from infected animals. Transmission occurs through superficial lesions such as punctures from grass awns, scrapes, shearing, and other common injuries. Necropsy findings. Necropsy findings include ballooning degeneration of epidermal and dermal layers, edema, granulomatous inflammation, vesiculation, and cellular hyperplasia. Secondary bacterial infection may also be evident. Pathogenesis. The virus is typical of the Poxviridae, resembling sheep poxvirus (not found in the United States) and vaccinia virus and replicating in the cytoplasm of epithelial cells. Following an incubation period of 2–14 days, papules and vesicles develop around the margins of the lips, nostrils, eyelids, gums, tongue, or teats; skin of the genitalia; or coronary band of the feet. The vesicles form pustules that rupture and finally scab over. Differential diagnosis. Ulcerative dermatosis and bluetongue virus should be considered in both sheep and goats. An important differential in goats is staphylococcal dermatitis. Prevention and control. Individuals handling infected animals should be advised of precautions beforehand, should wear gloves, and should separate work clothing and other personal protective equipment. Clippers, ear tagging devices, and other similar equipment should always be cleaned and disinfected after each use. Colostral antibodies may not be protective. Vaccinating lambs and kids with commercial vaccine best prevents the disease. Dried scabs from previous outbreaks may also be used by rubbing the material into scarified skin on the inner thigh or axilla. Animals newly introduced to infected premises should be vaccinated upon arrival. Precautions must be taken when vaccinating animals, because the vaccine may induce orf in the animal handlers; it is not recommended to vaccinate animals in flocks already free of the disease. Affected dairy goats should be milked last, using disposable towels for cleaning teat ends. Treatment. Affected animals should be isolated and provided supportive care, especially tube feeding for young animals whose mouths are too sore to nurse. Treatment should also address secondary bacterial infections of the orf lesions, including systemic antibiotics for more severe infections. Treatment for myiasis may also be necessary. The viral infection is self-limiting, with recovery in about 4 weeks. Research complications. Carrier animals may be a factor in flock or herd outbreaks. Contagious ecthyma is a zoonotic disease, and human-to-human transmission can also occur. The virus typically enters through abrasions on the hands and results in a large (several centimeters) nodule that is described as being extremely painful and lasting for as many as 6 weeks. Lesions heal without scarring. n. Foot-and-Mouth Disease Etiology. Foot-and-mouth disease (FMD) is caused by the foot-and-mouth disease virus, a Picornavirus in the Aphthovirus genus. The disease is also referred to as aftosa or aphthous fever. Seven immunologically distinct types of the virus have been identified, with 60 subtypes within those 7. Epidemics of the disease have occurred worldwide. North and Central America have been free of the virus since the mid-1950s. This is a reportable disease in the United States; clinical signs are very similar to other vesicular diseases. Cattle (and swine) are primarily affected, but disease can occur in sheep and is usually subclinical in goats. Clinical signs and diagnosis. In addition to vesicle formation around and in the mouth, hooves, and teats, fever, anorexia, weakness, and salivation occur. Vesicles may be as large as 10 cm, rupture after 2 days, and subsequently erode. Secondary bacterial infections often occur at the erosions. Anorexia is likely due to the pain associated with the oral lesions. High morbidity and low mortality, except for the high mortality in young cattle, are typical. Diagnosis must be based on ELISA, virus neutralization, fluorescent antibody tests, and complement fixation. Epizootiology and transmission. Domestic and wild ruminants and several other species, such as swine, rats, bears, and llamas are hosts. Asymptomatic goats can serve as virus reservoirs for more susceptible cohoused species such as cattle. Greater mortality occurs in younger animals. The United States, Great Britain, Canada, Japan, New Zealand, and Australia are FMD-free, whereas the disease is endemic in most of South America, parts of Europe, and throughout Asia and Africa. The virus is very contagious and is spread primarily by the inhalation of aerosols, which can be carried over long distances. Transmission may also occur by fomites, such as shoes, clothing, and equipment. Human hands, soiled bedding, and animal products such as frozen or partially cooked meat and meat products, hides, semen, and pasteurized milk also serve as sources of virus. Necropsy findings. Vesicles, erosions, and ulcers are present in the oral cavity as well as on the rumen pillars and mammary alveolar epithelium. Myocardial and skeletal muscle degeneration (Zenker's) is most common (and accounts for the greater mortality) in younger animals. Histological findings include lack of inclusion bodies. Vesicular lesions include intracellular and extracellular edema, cellular degeneration, and separation of the basal epithelium. Pathogenesis. The incubation period is 2–8 days. The virus replicates in the pharynx and digestive tract in the cells of the stratum spinosum, and viremia and spread of virus to many tissues occur before clinical signs develop. Virus shedding begins about 24 hr before clinical signs are apparent. Vesicles result from the separation of the superficial epithelium from the basal epithelium. Fluid fills the basal epithelium, and erosions develop when the epithelium sloughs. Persistent infection also occurs, and virus can be found for months or years in the pharnyx; the mechanisms for the persistence are not known. Differential diagnosis. Vesicular stomatitis is the principal differential. Other differentials include contagious ecthyma (orf), rinderpest, bluetongue, malignant catarrhal fever, bovine papular stomatitis, bovine herpes mammillitis, and infectious bovine rhinotracheitis virus infection. Prevention and control. Movement of animals and animal products from endemic areas is regulated. Quarantine and slaughter are practiced in outbreaks in endemic areas. Quarantine and vaccination are also used in endemic areas, but vaccines must be type-specific and repeated 2 or 3 times per year to be effective and will provide only partial protection. Autogenous vaccines are best in an outbreak. Passive immunity protects calves for up to 5 months after birth. The virus is inactivated by extremes of pH, sunlight, high temperatures, sodium hydroxide, sodium carbonate, and acetic acid. Treatment. Nursing care and antibiotic therapy to minimize secondary reactions help with recovery. Humoral immunity is considered the more important immune mechanism, with cell-mediated immunity of less importance. Research complications. Rare cases in humans have been reported. Importation into the United States of animal products from endemic areas is prohibited. o. Malignant Catarrhal Fever Etiology. Malignant catarrhal fever (MCF) is a severe disease primarily of cattle. The agents of MCF are viruses of the Gammaherpesvirinae subfamily. Alcelaphine herpesvirus 1 and 2 and ovine herpesvirus 2 are known strains. The alcelaphine strains are seen in Africa. The ovine strain is seen in North America. The alcelaphine and ovine strains differ in incubation times and duration of illness. Disease may occur sporadically or as outbreaks. Clinical signs and diagnosis. Signs range from subclinical to recrudescing latent infections to the lethal disease seen in susceptible species, such as cattle. Sudden death may also occur in cattle. Presentations of the disease may be categorized as alimentary, encephalitis, or skin forms; all three may occur in an animal. Corneal edema starting at the limbus and progressing centripetally is a nearly pathognomonic sign; photophobia, severe keratoconjunctivitis, and ocular involvement may follow. Other signs include prolonged fever, oral mucosal erosions, salivation, lacrimation, purulent nasal discharge, encephalitis, and pronounced lymphadenopathy. As the disease progresses, cattle may shed horns and hooves. In North America, cattle will also have severe diarrhea. The course of the disease may extend to 1 week. Recovery is usually prolonged, and some permanent debilitation may occur. The disease is fatal in severely affected individuals. History of exposure, as well as the clinical signs and lesions, contributes to the diagnosis. Serology, PCR-based assays, viral isolation, and cell-culture assays, such as cytopathic effects on thyroid cell cultures, are also used. Because of the susceptibility of rabbits, inoculation of this species may be used. In less severe outbreaks or individual animal disease, definitive diagnosis may never be made. Epizootiology and transmission. Most ruminant species are susceptible to MCF. Sheep are sources of infection for cattle, which are dead-end hosts. Other ruminants, including goats, may harbor the virus. Both the African and North American strains are transmissible to rabbits; these animals develop a fatal lymphoproliferative disease. The virus is shed from the nasopharynx. Infection of lambs is horizontal from direct contact. Other sources of the virus include water troughs, placental tissues, contaminated fomites, aerosols, birds, and caretakers. Necropsy. Gross findings at necropsy include necrotic and ulcerated nasal and oral mucosa; thickened, edematous, ulcerated, and hemorrhagic areas of the intestinal tract; swollen, friable, and hemorrhagic lymph nodes and other lymphatic tissues; and erosion of affected mucosal surfaces. Lymph nodes should be submitted for histological examination. Histological findings include nonsuppurative vasculitis and encephalitis; large numbers of lymphocytes and lymphoblasts will be present without evidence of virus. Pathogenesis. The incubation period may be up to 3 months. Vascular endothelium and all epithelial surfaces will be affected. The virus is believed to cause proliferation of cytotoxic T lymphocytes with natural killer cell activities, and the resulting lesions are due to an autoimmune type of phenomenon. Differential diagnoses. The differentials for this disease are bovine viral diarrhea/mucosal disease, bovine respiratory disease complex, infectious bovine rhinotracheitis, bluetongue, vesicular stomatitis, and foot-and-mouth disease. Causes of encephalitis, such as bovine spongiform encephalopathy and rabies, should be considered. In Africa, rinderpest is also a differential. Other differentials are arsenic toxicity and chlorinated naphthalene toxicity. Prevention and control. No vaccine is available at this time. In North America, sheep, as well as cattle that have been either exposed or that have survived the disease, are reservoirs for outbreaks in other cattle. If there is concern regarding presence of the virus, animals should be screened serologically; once an animal has been infected, it remains infected indefinitely. Lambs can be free of the infection if removed from the flock at weaning. The virus is very fragile outside of host's cells and will not survive in the environment for more than a few hours. Treatment. Affected and any exposed animals should be isolated from healthy animals. There is no specific treatment for MCF; supportive treatment may improve recovery rates. Corticosteroids may be useful. p. Ovine Progressive Pneumonia (Maedi/Visna) Etiology. An RNA virus in the lentivirus group of the Retroviridae family causes ovine progressive pneumonia (OPP), or maedi/visna. Maedi refers to the progressive pneumonia presentation of the disease; visna refers to the central nervous system disease, which is reported predominantly in Iceland. Visna has been reported in goats but may have been due to caprine arthritis encephalitis infection. Clinical signs and diagnosis. OPP is a viral disease of adult sheep characterized by weakness, unthriftiness, weight loss, and pneumonia ( Pepin et al., 1998 ; de la Concha Bermejillo, 1997 ). Clinically, animals exhibit signs of progressive pulmonary disease after an extremely long incubation period of up to 2 years. Respiratory rate and dyspnea gradually increase as the disease progresses. The animal continues to eat throughout the disease; however, animals progressively lose weight and become weak. Additionally, mastitis is a common clinical feature. Thoracic auscultation reveals consolidation of ventral lung lobes; and hematological findings indicate anemia and leukocytosis. The rare neurological signs include flexion of fetlock and pastern joints, tremors of facial muscles, progressive paresis and paralysis, depression, and prostration. Death occurs in weeks to months. The disease can be serologically diagnosed with agar gel immunodiffusion (AGID) tests, virus isolation, serum neutralization, complement fixation, and enzyme-linked immunosorbent assay (ELISA) tests. Epizootiology and transmission. Sixty-eight percent of sheep in some states have been infected with the virus ( Radostits et al., 1994 ). It is transmitted horizontally via inhalation of aerosolized virus particles and vertically between the infected dam and fetus. In addition, transmission through the milk or colostrum is considered common ( Knowles, 1997 ). Necropsy findings. Lesions are observed in lungs, mammary glands, joints, and the brain. Pulmonary adhesions, ventral lung lobe consolidation, bronchial lymph node enlargement, mastitis, and degenerative arthritis are visualized grossly. Meningeal edema, thickening of the choroid plexus, and foci of leukoencephalomalacia are seen in the central nervous system (CNS). Histologically, interalveolar septal thickening, lymphoid hyperplasia, histiocyte and fibrocyte proliferation, and squamous epithelial changes are seen in the lungs. Meningitis, lymphoid hyperplasia, demyelination, and glial fibrosis are seen in the CNS. Pathogenesis. The virus has a predilection for the lungs, mediastinal lymph nodes, udder, spleen, joints, and rarely the brain. After initial infection, the virus integrates into the DNA of mature monocytes and persists as a provirus. Later in the animal's life, infected monocytes mature as lung (and other tissue) macrophages and establish active infection. The virus induces lymphoproliferative disease, histiocyte and fibrocyte proliferation in the alveolar septa, and squamous metaplasia. Pulmonary alveolar and vascular changes impinge on oxygen and carbon dioxide exchange and lead to serious hypoxia and pulmonary hypertension. Secondary bacterial pneumonia may contribute to the animal's death. Differential diagnosis. Pulmonary adenomatosis is the differential diagnosis. Prevention and control. Isolating or removing infected animals can prevent the disease. Facilities and equipment should also be disinfected. Treatment. Treatment is unsuccessful. q. Poxviruses of Ruminants i. Ovine viral dermatosis. Ovine viral dermatosis is a venereal disease of sheep caused by a parapoxvirus distinct from contagious ecthyma. The disease resolves within 2 weeks in healthy animals, but lesions are painful and resemble those of Corynebacterium renale posthitis/vulvovaginitis. Symptomatic treatment may be necessary in some cases. There is no vaccine. Animals should not be used for breeding while clinical signs are present. ii. Proliferative stomatitis (bovine papular stomatitis) Etiology. A parapoxvirus is the causative agent of bovine papular stomatitis. This virus is considered to be closely related to the parapoxvirus that causes contagious ecthyma and pseudocowpox. It is also a zoonotic disease. The disease is not considered of major consequence, but high morbidity and mortality may be seen in severe outbreaks. In addition, lesions are comparable in appearance to those seen with vesicular stomatitis, bovine viral diarrhea virus, and foot-and-mouth disease. The disease occurs worldwide. Clinical signs and diagnosis. Raised red papules or erosions or shallow ulcers on the muzzle, nose, oral mucosa (including the hard palate), esophagus, and rumen of younger cattle are the most common findings. In some outbreaks, the papules will be associated with ulcerative esophagitis, salivation, diarrhea, and subsequent weight loss. Lesions persist or may come and go over a span of several months. Morbidity among herds may be 100%. Mortalities are rare. Bovine papular stomatitis is associated with "rat tail" in feedlot cattle. Animals continue to eat and usually do not show a fever. No lesion is seen on the feet. The infection may also be asymptomatic. Diagnosis is based on clinical signs, histological findings, and viral isolation. Epizootiology and transmission. Cattle less than 1 year of age are most commonly affected, and disease is rare in older cattle. Transmission is by animal-to-animal contact. Necropsy findings. Raised papules may be found around the muzzle and mouth and involve the mucosa of the esophagus and rumen. Histologically, epithelial cells will show hydropic degeneration and hyperplasia of the lamina propria. Eosinophilic inclusions will be in the cytoplasm of infected epithelial cells. Pathogenesis. Following exposure to the virus, erythematous macules most commonly appear on the nares, followed by the mouth. These become raised papules within a day, regressing after days to weeks; the lesions that remain will be persistent yellow, red, or brown spots. Some infections may recur or persist, with animals showing lesions intermittently or continuously over several months. Differential diagnosis. Pseudocowpox, vesicular stomatitis, foot-and-mouth disease, and bovine viral diarrhea virus infection are the differentials for this disease. The differential for the "rat tail" clinical sign is Sarcocystis infection. Prevention and control. There is no vaccine available for bovine papular stomatitis. Because of the similarity of this virus to the parapoxvirus of contagious ecthyma, it is important to be aware of the persistence in the environment and susceptibility of younger cattle. Vaccination using the local strain, and the skin scarification technique for orf, have been protective. Handlers should wear gloves and protective clothing. Treatment. Cattle usually will not require extensive nursing care, but lesions with secondary bacterial infections should be treated with antibiotics. Research complications. Handlers may develop lesions on their hands at sites of contact with lesions of cattle. iii. Pseudocowpox Etiology. Pseudocowpox is a worldwide cattle disease caused by a parapoxvirus related to the causative agents of contagious ecthyma and bovine papular stomatitis (see Sections III,A,2,m and III,A,2,q,ii). Lesions are confined to the teats. This is also a zoonotic disease. Clinical signs and diagnosis. Minor lesions are usually confined to the teats. These are distinctive because of the ring- or horseshoe-shaped scab that develops after 10 days. Additional lesions sometimes develop on the udder, the medial aspect of the thighs, and the scrotum. The teat lesions may predispose to mastitis. Pathogenesis. The virus is spread by contaminated hands, equipment, and fomites. Differential diagnosis. Differentials include bovine herpes mammillitis and papillomatosis. Prevention and control. Milking hygiene is helpful in control. Treatment. Lesions should be treated symptomatically, and affected animals milked last. Research complications. Like other related poxviruses, this virus causes nodular lesions on humans. r. Pulmonary Adenomatosis (Jaagsiekte) Etiology. Pulmonary adenomatosis is a rare but progressive wasting disease of sheep, with worldwide distribution. Pulmonary adenomatosis is caused by a type D retrovirus antigenically related to the Mason-Pfizer monkey virus. Jaagsiekte was the designation when the disease was described originally in South Africa. Clinical signs and diagnosis. Typical clinical signs include progressive respiratory signs such as dyspnea, rapid respiration, and wasting. The disease is diagnosed by these chronic clinical signs and histology. Epizootiology and transmission. The disease is transmitted by aerosols. Body fluids of viremic animals, such as milk, blood, saliva, tears, semen, and bronchial secretions, will contain the virus or cells carrying the virus. Necropsy. The adenomas and adenocarcinomas will be small firm lesions distributed throughout the lungs. The adenocarcinomas metastasize to regional lymph nodes. Pathogenesis. As with ovine progressive pneumonia (OPP), the incubation period is up to 2 years long. Adenocarcinomatous lesions arising from type II alveolar epithelial cells may be discrete or confluent and involve all lung lobes. Differential diagnosis. This disease occurs coincidentally with or is a differential diagnosis for OPP. Treatment. No treatment is effective. s. Papillomatosis (Warts, Verrucae) Etiology. Cutaneous papillomatosis is a very common disease in cattle and is much less common among sheep and goats. The disease is a viral-induced proliferation of the epithelium of the neck, face, back, and legs. These tumors are caused by a papillomavirus (DNA virus) of the Papovaviridae family, and the viruses are host-specific and often body site-specific. Most are benign, although some forms in cattle and one form in goats can become malignant. In cattle, the site specificity of the papillomavirus strains are particularly well recognized. Designations of the currently recognized bovine papillomavirus (BPV) types are BPV-1 through BPV-5. Clinical signs and diagnosis. The papillomas may last up to 12 months and are seen more frequently in younger animals. Lesions have typical wart appearances and may be single or multiple, small (1 mm) or very large (500 mm). The infections will generally be benign, but pain will be evident when warts develop on occlusal surfaces or within the gastrointestinal tract. In addition, when infections are severe, weight loss may occur. When warts occur on teats, secondary mastitis may develop. In cattle, BPV-1 and BPV-2 cause fibropapillomas on teats and penises or on head, neck, and dewlap, respectively. BPV-3 causes flat warts that occur in all body locations, BPV-4 causes warts in the gastrointestinal tract, and BPV-5 causes small white warts (called rice-grain warts) on teats. Warts caused by BPV-3 and BPV-5 do not regress spontaneously. Prognosis in cattle is poor only when papillomatosis involves more than 20% of the body surface. In sheep, warts are the verrucous type. The disease is of little consequence unless the warts develop in an area that causes discomfort or incapacitation such as between the digits, on the lips, or over the joints. In adult sheep, warts may transform to squamous cell carcinoma. In goats, the disease is rare, and the warts are also of the verrucous type and occasionally may develop into squamous cell carcinoma. Warts on goat udders tend to be persistent. Diagnosis is made by observing the typical proliferative lesions. Epizootiology and transmission. Older animals are less sensitive to papillomatosis than young animals, although immunosu-pressed animals of any age may develop warts as the result of harbored latent infections. The virus is transmitted by direct and indirect (fomite) contact, entering through surface wounds and sites such as tattoos. Pathogenesis. The incubation period ranges from 1 to 6 months. The virus induces epidermal and fibrous tissue proliferation, often described as cauliflower-like skin tumors. The disease is generally self-limiting. Differential diagnosis. In sheep and goats, differentials include contagious ecthyma, ulcerative dermatosis, strawberry foot rot, and sheep and goat pox. Prevention and control. Commercial vaccines (available only for cattle) or autogenous vaccines must be used with a recognition that papovavirus strains are host-specific and that immunity from infection or vaccination is viral-type-specific. Autogenous vaccines are generally considered more effective. Some vaccine preparations are effective at prevention but not treatment of outbreaks. Viricidal products are recommended for disinfection of contaminated environments. Minimizing cutaneous injuries and sanitizing equipment (tattoo devices, dehorners, ear taggers, etc.) in a virucidal solution between uses are also recommended preventive and control measures. Halters, brushes, and other items may also be sources of virus. Treatment. Warts will often spontaneously resolve as immunity develops. In severe cases or with flockwide or herdwide problems, affected animals should be isolated from nonaffected animals, and premises disinfected. Warts can be surgically excised and autogenous vaccines can be made and administered to help prevent disease spread. Cryosurgery with liquid nitrogen or dry ice has also proven to be successful for wart removal. Topical agents such as podophyllin (various formulations) and dimethyl sulfoxide may be applied to individual lesions once daily until regression. t. Pseudorabies (Mad Itch, Aujeszky's Disease) Etiology. Pseudorabies is an acute encephalitic disease caused by a neurotropic alphaherpesvirus, the porcine herpesvirus 1. One serotype is recognized, but strain differences exist. The disease has worldwide distribution. It is a primarily a clinical disease of cattle, with less frequent reports (but no less severe clinical manifestations) in sheep and goats. Clinical signs and diagnosis. A range of clinical signs is seen during the rapid course of this usually fatal disease. At the site of virus inoculation or in other locations, abrasions, swelling, intense pruritus, and alopecia are seen. Pruritus will not be asymmetric. Animals will also become hyperthermic and will vocalize frantically. Other neurological signs range from hoof stamping, kicking at the pruritic area, salivation, tongue chewing, head pressing and circling, to paresthesia or hyperesthesia, ataxia, and conscious proprioceptive deficits. Nystagmus and strabismus are also seen. Animals will be fearful or depressed, and aggression is sometimes seen. Recumbency and coma precede death. Diagnostic evidence includes clinical findings; virus isolation from nasal or pharyngeal secretions or postmortem tissues; and histological findings at necropsy. Serology of affected animals is not productive, because of the rapid course. If swine are housed nearby, or if swine were transported in the same vehicles as affected animals, serological evaluations are worthwhile from those animals. Epizootiology and transmission. Swine are the primary hosts for pseudorabies virus, but they are usually asymptomatic and serve as reservoirs for the virus. The infection can remain latent in the trigeminal ganglion of pigs and recrudesce during stressful conditions. Other animals are dead-end hosts. The unprotected virus will survive only a few weeks in the environment but may remain viable in meat (including carcasses) or saliva and will survive outside the host, in favorable conditions, in the summer for several weeks and the winter for several months. Transmission is by oral, intranasal, intradermal, or subcutaneous introduction of the virus. When the virus is inhaled, the clinical signs of pruritus are less likely to be seen. Transmission can also be by inadvertent exposure (e.g., contaminated syringes) of ruminants to the modified live vaccines developed for use in swine. Spread between infected ruminants is a less likely means of transmission, because of the relatively short period of virus shedding. Transport vehicles used for swine may also be sources of the virus. Raccoons are believed to be vectors of the virus. Horses are resistant to infection. Necropsy findings. There is no pathognomonic gross lesion. Definitive histologic findings include severe, focal, nonsuppurative encephalitis and myelitis. Eosinophilic intranuclear inclusion bodies (Cowdry type A) may be present in some affected neurons. Methods such as immunofluorescence and immunoperoxidase staining can be used to show presence of the porcine herpesvirus 1. Pathogenesis. The incubation period is 90–156 hr and duration of the illness is 8–72 hr. The longest duration is seen in animals with pruritus around the head. Differential diagnoses. Differentials for the neurologic signs of pseudorabies infection include rabies, polioencephalomalacia, salt poisoning, meningitis, lead poisoning, hypomagnesemia, and enterotoxemia. Those for the intense pruritus include psoroptic mange and scrapie in sheep, sarcoptic mange, and pediculosis. Prevention and control. Pseudorabies is a reportable disease in the United States, where a nationwide eradication program exists; states are rated regarding status. Effective disinfectants include sodium hypochlorite (10% solution), formalin, peracetic acid, tamed iodines, and quaternary ammonium compounds. Five minutes of contact time is required, and then surfaces must be rinsed. Other disinfectant methods for viral killing include 6 hr of formaldehyde fumigation, or 360 min of ultraviolet light. Transport vehicles should be cleaned and disinfected between species. Serological screening for pseudorabies of swine housed near ruminants is essential. Treatment. There is no treatment, and most affected animals die. Research complications. Swine housed close to research ruminants should be serologically screened prior to purchase, and all transport vehicles should be cleaned and disinfected between loads of large animals. Humans have been reported to seroconvert. The porcine herpesvirus 1 shares antigens with the infectious bovine rhinotracheitis virus. u. Rabies (Hydrophobia) Etiology. Rabies is a sporadic but fatal, acute viral disease affecting the central nervous system. The rabies virus is a neurotropic RNA virus of the Lyssavirus genus and the Rhabdoviridae family. Sheep, goats, and cattle are susceptible. The zoonotic potential of this virus must be kept in mind at all times when handling moribund animals with neurological signs characteristic of the disease. Rabies is endemic in many areas of the world and within areas of the Unites States. This is a reportable disease in North America. Clinical findings and diagnosis. Animals generally progress through three phases: prodromal, excitatory, and paralytic. Many signs in the different species during these stages are nonspecific, and forms of the disease are also referred to as dumb or furious. During the short prodromal phase, animals are hyperthermic and apprehensive. Animals progress to the excitatory phase, during which they refuse to eat or drink and are active and aggressive. Repeated vocalizations, tenesmus, sexual excitement, and salivation occur during this phase. The final paralytic stage, with recumbency and death, occurs over several hours to days. This paralytic stage is common in cattle, and animals may simply be found dead. The clinical course is usually 1–4 days. Diagnosis is based on clinical signs, with a progressive and fatal course. Confirmation presently is made with the fluorescent antibody technique on brain tissue. Epizootiology and transmission. The rabies virus is transmitted via a bite wound inflicted by a rabid animal. Cats, dogs, raccoons, skunks, foxes, wild canids, and bats are the common disease vectors in North America. Virus is also transmitted in milk and aerosols. Necropsy findings. Few lesions are seen at necropsy. Many secondary lesions from manic behaviors during the course of disease may be evident. Histological findings will include nonsuppurative encephalitis. Negri bodies in the cytoplasm of neurons of the hippocampus and in Purkinje's cells are pathognomonic histologic findings. Pathogenesis. After exposure, the incubation period is variable, from 2 weeks to several months, depending on the distance that the virus has to travel to reach the central nervous system. The rabies virus proliferates locally, gains access to neurons by attaching to acetylcholine receptors, via a viral surface glycoprotein, migrates along sensory nerves to the spinal cord and brain, and then descends via cranial nerves (trigeminal, facial, olfactory, glossopharyngeal) to oral and nasal cavity structures (i.e., salivary glands). The fatal outcome is currently believed to be multifactorial, related to anorexia, respiratory paralysis, and effects on the pituitary. Differential diagnosis. Rabies should be included on the differential list when clinical signs of neurologic disease are evident. Other differentials for ruminants include herpesvirus encephalitis, thromboemobolic meningoencephalitis, nervous ketosis, grass tetany, and nervous cocciodiosis. Prevention and control. Vaccines approved for use cattle and sheep are commercially available and contain inactivated virus; there is not one available in the United States for goats. Ruminants in endemic areas, such as the East Coast of the United States, should be routinely vaccinated. Any animals housed outside that may be exposed to rabid animals should be vaccinated. Vaccination programs generally begin at 3 months of age, with a booster at 1 year of age and then annual or triennial boosters. Awareness of the current rabies case reports for the region and wildlife reservoirs, however, is important. Monitoring for and exclusion of wildlife from large-animal facilities are worthwhile preventive measures. The virus is fragile and unstable outside of a host animal. Research complications. Aerosolized virus is infective. Personal protective equipment, including gloves, face mask, and eye shields, must be worn by individuals handling animals that are manifesting neurological disease signs. v. Transmissible Spongiform Encephalopathies i. Bovine spongiform encephalopathy (mad cow disease). Bovine spongiform encephalopathy, a transmissible spongiform encephalopathy (TSE), is not known to occur in the United States, where since 1989 it has been listed as a reportable disease. The profound impact of this disease on the cattle industry in Great Britain during the past two decades is well known. The disease may be caused by a scrapielike (prion) agent. It is believed that the source of infection for cattle was feedstuff derived from sheep meat and bonemeal that had been inadequately treated during processing. The incubation period of years, the lack of detectable host immune response, the debilitating and progressive neurological illness, and the pathology localized to the central nervous system are characteristics of the disease, and are is comparable to the characteristics of other TSE diseases such as scrapie, which affects sheep and goats. In addition, the infectious agent is extremely resistant to dessication and disinfectants. Confirmation of disease is by histological examination of brain tissue collected at necropsy; the vacuolation that occurs during the disease will be symmetrical and in the gray matter of the brain stem. Molecular biology techniques, such as Western blots and immunohistochemistry, may also be used to identify the presence of the prion protein. Differentials include many infectious or toxic agents that affect the bovine nervous and musculoskeletal systems, such as rabies, listeriosis, and lead poisoning. Metabolic disorders such as ketosis, milk fever, and grass tetany are also differentials. There is no vaccine or treatment. Prevention focuses on import regulations and not feeding ruminant protein to ruminants; recent USDA regulations prohibit feeding any mammalian proteins to ruminants. ii. Scrapie Etiology. Scrapie is a sporadic, slow, neurodegenerative disease caused by a prion. Scrapie is a reportable disease. It is much more common in sheep than in goats. The disease is similar to transmissible mink encephalopathy, kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy (mad cow disease). Prions are nonantigenic, replicating protein agents. Clinical signs and diagnosis. During early clinical stages, animals are excitable and hard to control. Tremors of head and neck muscles, as well as uncoordinated movements and unusual "bunny-hopping" gaits are observed. In advanced stages of the disease, animals experience severe pruritus and will self-mutilate while rubbing on fences, trees, and other objects. Blindness and abortion may also be seen. Morbidity may reach 50% within a flock. Most animals invariably die within 4–6 weeks; some animals may survive 6 months. In goats, the disease is also fatal. Pruritus is generally less severe but may be localized. A wide range of clinical signs have also been noted in goats, including listlessness, stiffness or restlessness, or behavioral changes such as irritability, hunched posture, twitching, and erect tail and ears. As with sheep, the disease gradually progresses to anorexia and debilitation. Diagnosis can be made by clinical signs and histopathological lesions. A newer diagnostic test in live animals is based on sampling from the third eyelid. Tests for genetic resistance or susceptibility require a tube of EDTA blood and are reasonably priced. Epizootiology and transmission. The Suffolk breed of sheep tends to be especially susceptible. Scrapie has also been reported in several other breeds, including Cheviot, Dorset, Hampshire, Corriedale, Shropshire, Merino, and Rambouillet. It is believed that there is hereditary susceptibility in these breeds. Targhees tend to be resistant. Genomic research indicates there are two chromosomsal sites governing this trait; these sites are referred to codons 171 (Q, R, or H genes can be present) and 136 (A or V genes can be present). Of the five genes, R genes appear to confer immunity to clinical scrapie in Suffolks in the United States. Affected Suffolks in the United States that have been tested have been AA QQ. The disease is also enzootic is many other countries. The disease tends to affect newborns and young animals; however, because the incubation period tends to range from 2 to 5 years, adult animals display signs of the disease. Scrapie is transmitted horizontally by direct or indirect contact; nasal secretions or placentas serve as sources of the infectious agent. Vertical transmission is questioned, and transplacental transmission is considered unlikely. Necropsy findings. At necropsy, no gross lesion is observed. Histopathologically, neuronal vacuolization, astrogliosis, and spongiform degeneration are visualized in the brain stem, the spinal cord, and especially the thalamus. Inflammatory lesions are not seen. Pathogenesis. Replication of the prions probably occurs first in lymphoid tissues throughout the host's body and then progresses to neural tissue. Differential diagnosis. In sheep and goats, depending on the speed of onset, differentials for the pruritus include ectoparasites, pseudorabies, and photosensitization. Prevention and control. If the disease diagnosed in a flock, quarantine and slaughter, followed by strict sanitation, are usually required. The U.S. Department of Agriculture has approved the use of 2% sodium hydroxide as the only disinfectant for sanitation of scrapie-infected premises. Prions are highly resistant to physicochemical means of disinfection. Artificial insemination or embryo transfer has been shown to decrease the spread of scrapie ( Linnabary et al., 1991 ). Treatment. No vaccine or treatment is available. Research complications. As noted, this is a reportable disease. Stringent regulations exist in the United States regarding importation of small ruminants from scrapie-infected countries. w. Vesicular Stomatitis Virus Etiology. Vesicular stomatitis (VS) is caused by the vesicular stomatitis virus (VSV), a member of the Rhabdoviridae. Three serotypes are recognized: New Jersey, Indiana, and Isfahan. The New Jersey and Indiana strains cause sporadic disease in cattle in the United States. The disease is rare in sheep. Clinical signs and diagnosis. Adult cattle are most likely to develop VS. Fever and development of vesicles on the oral mucous membranes are the initial clinical signs. Lesions on the teats and interdigital spaces also develop. The vesicles progress quickly to ulcers and erosions. The animal's tongue may be severely involved. Anorexia and salivation are common. Weight loss and decreased milk production are noticeable. Morbidity will be high in an outbreak, but mortality will be low to nonexistent. Diagnostic work should be initiated as soon as possible to distinguish this from foot-and-mouth disease. Diagnosis is based on analysis of fluid, serum, or membranes associated with the vesicles. Virus isolation, enzyme-linked immunosorbent assay (ELISA), competitive ELISA (CELISA), complement fixation, and serum neutralization are used for diagnosis. Epizootiology and transmission. This disease occurs in several other mammalian species, including swine, horses, and wild ruminants. VSV is an enveloped virus and survives well in different environmental conditions, including in soil, extremes of pH, and low temperatures. Outbreaks of VS occur sporadically in the United States, but it is not understood how or in what species the virus survives between these outbreaks. Incidence of disease decreases during colder seasons. Equipment, such as milking machines, contaminated by secretions is a mechanical vector, as are human hands. Transmission may also be from contaminated water and feed. Transmission is also believed to occur by insects (blackflies, sand flies, and Culicoides) that may simply be mechanical vectors. It is believed that carrier animals do not occur in this disease. Necropsy. It is rare for animals to be necropsied as the result of this disease. Typical vesicular lesion histology is seen, with ballooning degeneration and edema. There is no inclusion body formation. Pathogenesis. Lesions often begin within 24 hr after exposure. The virus invades oral epithelium. Injuries or trauma in any area typically affected, such as mouth, teats, or interdigital areas, will increase the likelihood of lesions developing there. Animals will develop a long-term immunity; this immunity can be overwhelmed, however, by a large dose of the virus. Differential diagnosis. Foot-and-mouth disease lesions are identical to VS lesions. Other differentials in cattle include bovine viral diarrhea, malignant catarrhal fever, contagious ecthyma, photosensitization, trauma, and caustic agents. Prevention and control. Quarantine and restrictions on shipping infected animals or animals from the premises housing affected animals are required in an outbreak. Vaccines are available for use in outbreaks and have decreased the severity of lesions. Phenolics, quaternaries, and halogens are effective for inactivating and disinfecting equipment and facilities. Treatment. Affected animals should be segregated from the rest of the herd and provided with separate water and softened feed. These animals should be cared for after unaffected animals. Any feed or water contaminated by these animals should not be used for other animals; contaminated equipment should be disinfected. Topical or systemic antibiotics control secondary bacterial infections. Cases of mastitis secondary to teat lesions must be treated as necessary. Any abrasive materials that could cause further trauma to the animals should be removed. Research complications. Animals developing vesicular lesions must be reported promptly to eliminate the possibility of an outbreak of foot-and-mouth disease. Personal protective equipment, especially gloves, should be worn when handling any animals with vesicular lesions. VSV causes a flulike illness in humans. x. Viral Diarrhea Diseases i. Ovine. Rotavirus, of the family Reoviridae, induces an acute, transient diarrhea in lambs within the first few weeks of life. Four antigenic groups (A-D) have been identified by differences in capsid antigens VP3 and VP7. Primarily group A, but also groups B and C, have been isolated from sheep. The disease is characterized by yellow, semifluid to watery diarrhea occurring 1–4 days after infection. The disease can progress to dehydration, anorexia and weight loss, acidosis, depression, and occasionally death. The virus is ingested with contaminated feed and water and selectively infects and destroys the enterocytes at the tips of the small intestinal villi. The villi are replaced with immature cells that lack sufficient digestive enzymes; osmotic diarrhea results. Virus may remain in the environment for several months. The disease is diagnosed by virus isolation, electron microscopy of feces, fecal fluorescent antibody, fecal ELISA tests (marketed tests generally detect group A rotavirus), and fecal latex agglutination tests. Rotavirus diarrhea is treated by supportive therapy, including maintaining hydration, electrolyte, and acid-base balance. A rotavirus vaccine is available for cattle; because of cross-species immunity, oral administration of high-quality bovine colostrum from vaccinated cows to infected sheep may be helpful ( "Current Veterinary Therapy," 1993 ). Coronavirus, of the family Coronaviridae, produces a more severe, long-lasting disease when compared with rotavirus. Clinical signs are similar to above, although the incubation period tends to be shorter (20–36 hr), and animals exhibit less anorexia than those with rotavirus. Additionally, mild respiratory disease may be noted ( Janke, 1989 ). Like rotavirus, coronavirus also destroys enterocytes of the villus tips. The virus can be visualized with electron microscopy. Treatment is supportive; close consideration of hydration and acid-base status is essential. Bovine vaccines are available. ii. Caprine. Rotavirus, coronavirus, and adenoviruses affect neonatal goats; however, little has been documented on the pathology and significance of these agents in this age group. It appears that bacteria play a more important role in neonatal kid diarrheal diseases then in neonatal calf diarrheas. iii. Bovine. Rotaviruses, coronaviruses, parvoviruses, and bovine viral diarrhea virus (BVDV) are associated with diarrheal disease in calves. Each pathogen multiplies within and destroys the intestinal epithelial cells, resulting in villous atrophy and clinical signs of diarrhea (soft to watery feces), dehydration, and abdominal pain. These viral infections may be complicated by parasitic infections (e.g., Cryptosporidium, Eimeria) or bacterial infections (e.g., Escherichia coli, Salmonella, Campylobacter). Treatment is aimed at correcting dehydration, electrolyte imbalances, and acidosis; cessation of milk replacers and administration of fluid therapy intravenously and by stomach tube may be necessary, depending on the presence of suckle reflex and the condition of the animals. Diagnosis is by immunoassays available for some viruses, viral culture, exclusion or identification of presence of other pathogens (by culture or fecal exams), and microscopic examination of necropsy specimens. Prevention focuses on calves suckling good-quality colostrum; other recommendations for calf care are in Section II,B,5. Combination vaccine products are available for immunizing dams against rotavirus, coronavirus, and enterotoxigenic E. coli. Additional supportive care for calves includes providing calves with sufficient energy and vitamins until milk intake can resume. Rotaviruses of serogroup A are the most common type in neonatal calves; 4- to 14-day old calves are typically affected, but younger and older animals may also be affected. The small intestine is the site of infection. Antirotavirus antibody is present in colostrum, and onset of rotavirus diarrhea coincides with the decline of this local protection. Transmission is likely from other affected calves and asymptomatic adult carriers. The diarrhea is typically a distinctive yellow. Colitis with tenesmus, mucus, and blood may be seen. This virus may be zoonotic. Coronaviruses are commonly associated with disease in calves during the first month of life, and they infect small- and large-intestinal epithelial cells. The virus infection may extend to mild pneumonia. Transmission is by infected calves and also by asymptomatic adult cattle, including dams excreting virus at the time of parturition. Calves that appear to have recovered continue to shed virus for several weeks. Parvovirus infections are usually associated with neonatal calves. BVDV infections also are seen in neonates and also affect many systems and produce other clinical signs and syndromes that are described in Section III,A,2,e. iv. Winter Dysentery. Winter dysentery is an acute, winter-seasonal, epizootic diarrheal disease of adult cattle, although it has been reported in 4-month-old calves. The etiology has not yet been defined, but a viral pathogen is suspected. Coronavirus-like viral particles have been isolated from cattle feces, either the same as or similar to the coronavirus of calf diarrhea. Outbreaks typically last a few weeks, and first-lactation or younger cattle are affected first, with waves of illness moving through a herd. Individual cows are ill for only a few days. The incubation period is estimated at 2–8 days. The outbreaks of disease are often seen in herds throughout the local area. Clinical signs include explosive diarrhea, anorexia, depression, and decreased production. The diarrhea has a distinctive musty, sweet odor and is light brown and bubbly, but some blood streaks or clots may be mixed in with the feces. Animals will become dehydrated quickly but are thirsty. Respiratory symptoms such as nasolacrimal discharges and coughing may develop. Recovery is generally spontaneous. Mortalities are rare. Diagnosis is based on characteristic patterns of clinical signs, and elimination of diarrheas caused by parasites such as coccidia, bacterial organisms such as Salmonella or Mycobacterium paratuberculosis, and viruses such as BVDV. Pathology is present in the colonic mucosa, and necrosis is present in the crypts. 3. Chlamydial Diseases a. Enzootic Abortion of Ewes (Chlamydial Abortion) Etiology. Chlamydia psittaci is a nonmotile, obligate, intracytoplasmic, gram-negative bacterium. Clinical signs. Enzootic abortion in sheep and goats is a contagious disease characterized by hyperthermia and late abortion or by birth of stillborn or weak lambs or kids ( Rodolakis et al., 1998 ). The only presenting clinical sign may be serosanguineous vulvar discharges. Other animals may present with arthritis or pneumonia. Infection of animals prior to about 120 days of gestation results in abortion, stillbirths, or birth of weak lambs. Infection after 120 days results in potentially normal births, but the dams or offspring may be latently infected. Latently infected animals that were infected during their dry period may abort during the next pregnancy. Ewes or does generally only abort once, and thus recovered animals will be immune to future infections. Epizootiology and transmission. Chlamydia possess group and specific antigens associated with the cell surface. The group antigen is common among all Chlamydia; the specific antigen is common to related subgroups. Two subgroups are recognized, one that causes EAE and one that causes polyarthritis and conjunctivitis. The disease is transmitted by direct contact with infectious secretions such as placental, fetal, and uterine fluids or by indirect contact with contaminated feed and water. Necropsy. Placental lesions include intercotyledonary plaques and necrosis and cotyledonary hemorrhages. Histopathological evidence of leukocytic infiltration, edema, and necrosis is found throughout the placentome. Fetal lesions include giant-cell accumulation in mesenteric lymph nodes and lymphohistiocytic proliferations around the blood vessels within the liver. Diagnosis is based on clinical signs and laboratory (serological or histopathological) identification of the organism. Impression smears in placental tissues stained with Giemsa, Gimenez, or modified Ziehl-Neelsen can provide preliminary indications of the causative agent. Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) methods also aid in diagnosis. Differential diagnosis. Q fever will be the major differential for late-term abortion and necrotizing placentitis. Campylobacter and Toxoplasma should also be considered for late-term abortion. Treatment. Animals may respond to treatment with oxytetra cycline. Abortions are prevented through administration of a commercial vaccine, but the vaccine will not eliminate infections. This is a sheep vaccine and should be administered before breeding and annually to at least the young females entering the breeding herd or flock. Research complications. In addition to losses or compromise of research animals, pregnant women should not handle aborted tissues. b. Chlamydial Polyarthritis of Sheep Etiology. Chlamydia psittaci is a nonmotile, obligate intracellular, gram-negative bacterium. Chlamydial polyarthritis is an acute, contagious disease characterized by fever, lameness ( Bulgin, 1986 ), and conjunctivitis (see Section III,A,3,c) in growing and nursing lambs. Clinical signs. Clinically, animals will appear lame on one or all legs and in major joints, including the scapulohumeral, humeroradioulnar, coxofemoral, femorotibial, and tibiotarsal joints. Lambs may be anorexic and febrile. Animals frequently also exhibit concurrent conjunctivitis. The disease usually resolves in approximately 4 weeks. Joint inflammation usually resolves without causing chronic articular changes. Epizootiology and transmission. The disease is transmitted to susceptible animals by direct contact as well as by contaminated feed and water. The organism penetrates the gastrointestinal tract and migrates to joints and synovial membranes as well as to the conjunctiva. The organism causes acute inflammation and associated fibrinopurulent exudates. Necropsy findings. Lesions are found in joints, tendon sheaths, conjunctiva, and lungs. Pathological sites will be edematous and hyperemic, with fibrinous exudates but without articular changes. Lesions will be infiltrated with mononuclear cells. Lung lesions include atelectasis and alveolar inspissation. Diagnosis is based on clinical signs. Synovial taps and subsequent smears may allow the identification of chlamydial inclusion bodies. Treatment. Animals respond to treatment with parenteral oxytetracycline. c. Chlamydial Conjunctivitis (Infectious Keratoconjunctivitis, Pinkeye) Etiology. Chlamydia psittaci, a nonmotile, obligate intracellular, gram-negative bacterium, is the most common cause of infectious keratoconjunctivitis in sheep. Chlamydia and Mycoplasma are considered to be the most common causes of this disease in goats. Chlamydial conjunctivitis is not a disease of cattle. Clinical signs. Infectious keratoconjunctivitis is an acute, contagious disease characterized in earlier stages by conjunctival hyperemia, epiphora, and edema and in later stages by, corneal edema, ulceration, and opacity. Perforation may result from the ulceration. Animals will be photophobic. In less severe cases, corneal healing associated with fibrosis and neovascularization occurs in 3–4 days. Lymphoid tissues associated with the conjunctiva and nictitating membrane may enlarge and prolapse the eyelids. Morbidity may reach 80–90%. Bilateral and symmetrical infections characterize most outbreaks. Relapses may occur. Other concurrent systemic infections may be seen, such as polyarthritis or abortion in sheep and polyarthritis, mastitis, and uterine infections in goats. Epizootiology and transmission. Direct contact, and mechanical vectors such as flies easily spread the organism. Necropsy. If the chlamydial or mycoplasmal agents are suspected, diagnostic laboratories should be contacted for recommendations regarding sampling. Conjunctival smears are also useful. Pathogenesis. The pathogen penetrates the conjunctival epithelium and replicates in the cytoplasm by forming initial and elementary bodies. The infection moves from cell to cell and causes an acute inflammation and resultant purulent exudate. The chlamydial organism may penetrate the bloodstream and migrate to the opposite eye or joints, leading to arthritis. Diagnosis is suggested by the clinical signs. Cytoplasmic inclusions observed on conjunctival scrapings and immunofluorescent techniques help confirm the diagnosis. Differential diagnosis. Nonchlamydial keratoconjunctivitis also occurs in sheep and goats. The primary agents involved include Mycoplasma conjunctiva, M. agalactiae in goats, and Branhamella (Neisseria) ovis. A less common differential for sheep and cattle is Listeria monocytogenes. Other differentials include eye worms, trauma, and foreign bodies such as windblown materials (pollen, dust) and poor-quality hay; these latter irritants and stress may predispose the animals' eyes to the infectious agents. Prevention and control. Source of mechanical irritation should be minimized whenever possible. Quarantine of new animals and treatment, if necessary, before introduction into the flock or herd are important measures. Shade should be provided for all animals. Treatment. The infections can be self-limiting in 2–3 weeks without treatment. Treatment consists of topical application of tetracycline ophthalmic ointments. Systemic or oral oxytetracycline treatments have been used with the topical treatment. Atropine may be added to the treatment regimen when uveitis is present. Shade should be provided. 4. Parasitic Diseases a. Protozoa i. Anaplasmosis Etiology. Anaplasmosis is an infectious, hemolytic, noncontagious, transmissible disease of cattle caused by the protozoan Anaplasma marginale. Anaplasma is a member of the Anaplas-matacae family within the order Rickettsiales. In sheep and goats, the disease is caused by A. ovis and is an uncommon cause of hemolytic disease. Anaplasmosis has not been reported in goats in the United States. Some controversy exists regarding the classification. Most recently it is classified as a protozoal disease because of similarities to babesiosis. It has also been classified as a rickettsial pathogen. This summary addresses the disease in cattle with limited reference to A. ovis infections, but there are many similarities to the disease in cattle. Clinical signs and diagnosis. Acute anemia is the predominant sign in anaplasmosis, and fever coincides with parasitemia. Weakness, pallor, lethargy, dehydration, and anorexia are the result of the anemia. Four disease stages—incubation, developmental, convalescent, and carrier—are recognized. The incubation stage may be long, 3–8 weeks, and is characterized by a rise in body temperature as the infection moves to the next stage. Most clinical signs occur during the 4- to 9-day developmental stage, with hemolytic anemia being common. Death is most likely to occur at this stage or at the beginning of the convalescent stage. Death may also occur from anoxia, because of the animal's inability to handle any exertion or stress, especially if treatment is initiated when severe anemia exists. Reticulocytosis characterizes the convalescent stage, which may continue for many weeks. Morbidity is high, and mortality is low. The carrier stage is defined as the time in the convalescent stage when the animal host becomes a reservoir of the disease, and Anaplasma organisms and any parasitemia are not discernible. Common serologic tests are the complement fixation test and the rapid card test. These become positive after the incubation phase and do not distinguish between the later three stages of disease. Definitive diagnosis is made by clinical and necropsy findings. Staining of thin blood smears with Wright's or Giemsa stain allows detection of basophilic, spherical A. marginale bodies near the red blood cell peripheries. Evidence will most likely be found before a hemolytic episode. A negative finding should not eliminate the pathogen from consideration. Epizootiology and transmission. The disease is common in cattle in the southern and western United States. Anaplasma organisms are spread biologically or mechanically. Mechanical transmission occurs when infected red blood cells are passed from one host to another on the mouthparts of seasonal biting flies. Sometimes mosquitoes or instruments such as dehorners or hypodermic needles may facilitate transfer of infected red cells from one animal to another. Biological transmission occurs when the tick stage of the organism is passed by Dermacentor andersoni and D. occidentalis ticks. The carrier stage covers the time when discernible Anaplasma organisms can be found on host blood smears. Recovered animals serve as immune carriers and disease reservoirs. Necropsy. Pale tissues and watery, thin blood are typical findings. Splenomegaly, hepatomegaly, and gallbladder distension are common findings. Pathogenesis. The parasites infect the host's red blood cells, and acute hemolysis occurs during the parasites' developmental stage. The four stages of the parasite's life cycle are described above because these are closely linked to the clinical stages. Differential diagnosis. The clinical disease closely resembles the protozoal disease babesiosis. Prevention and control. Offspring of immune carriers resist infection up to 6 months of age because of passive immunity. Vector control and attention to hygiene are essential, such as between-animal rinsing in disinfectant of mechanical vectors such as dehorners. There is no entirely effective means, however, to prevent and control the disease. Vaccination (killed whole organism) programs are not entirely effective, and vaccine should not be administered to pregnant cows. Neonatal isoerythrolysis may occur because of the antierythrocyte antibodies stimulated by one vaccine product. Vaccinated animals can still become infected and become carriers. The cattle vaccine has shown no efficacy in smaller ruminants, and there is no A. ovis vaccine. Identifying carriers serologically and treating with tetracycline during and/or after vector seasons may be an option. Removing carriers to a separate herd is also an approach. Interstate movement of infected animals is regulated. Treatment. Oxytetracycline, administered once, helps reduce the severity of the infection during the developmental stage. Other tetracycline treatment programs have been described to help control carriers. ii. Babesiosis (red water, Texas cattle fever, cattle tick fever) Etiology. Babesia bovis and Ba. bigemina are protozoa that cause subclinical infections or disease in cattle. These are intraerythrocytic parasites. Babesia bovis is regarded as the more virulent of the two organisms. This disease is not seen in the smaller ruminants in the United States. Clinical signs and diagnosis. The more common presentation is liver and kidney failure due to hemolysis with icterus, hemoglobinuria, and fever. Hemoglobinuria indicates a poor prognosis. Acute encephalitis is a less common presentation and begins acutely with fever, ataxia, depression, deficits in conscious proprioception, mania, convulsions, and coma. The encephalitic form generally also has a poor prognosis. Sudden death may occur. Thin blood smears stained with Giemsa will show Babesia trophozoites at some stages of the disease, but lack of these cannot be interpreted as a negative. The trophozoites occur in a variety of shapes, such as piriform, round, or rod. Complement fixation, immunofluorescent antibody, and enzyme immunoassay are the most favored of the available serologic tests. Epizootiology and transmission. Babesiosis is present on several continents, including the Americas. In addition to domestic cattle, some wild ruminants, such as white-tailed deer and American buffalo, are also susceptible. Bos indicus breeds have resistance to the disease and the tick vectors. Innate resistance factors have been found in all calves. If infected, these animals will not show many signs of disease during the first year of life and will become carriers. Stress can cause disease development. Necropsy findings. Signs of acute hemolytic crisis are the most common findings, including hepatomegaly, splenomegaly, dark and distended gallbladder, pale tissues, thin blood, scattered hemorrhages, and petechiation. Animals dying after a longer course of disease will be emaciated and icteric, with thin blood, pale kidneys, and enlarged liver. Pathogenesis. The protozoon is transmitted by the cattle fever ticks Boophilus annulatus, B. microplus, and B. decoloratus; these one-host ticks acquire the protozoon from infected animals. It is passed transovarially, and both nymph and adult ticks may transmit to other cattle. Only B. ovis is transmitted by the larval stage. Clinical signs develop about 2 weeks after tick infestations or mechanical transmission but may develop sooner with the mechanical transmission. Hemolysis is due to intracellular reproduction of the parasites and occurs intra- and extravascularly. In addition to the release of merozoites, proteolytic enzymes are also released, and these contribute to the clinical metabolic acidosis and anoxia. The development of the encephalitis form is believed to be the result of direct invasion of the central nervous system, disseminated intravascular coagulation, capillary thrombosis by the parasites and infarction, and/or tissue anoxia. Differential diagnosis. In addition to anaplasmosis, other differentials for the hemolytic form of the disease are leptospirosis, chronic copper toxicity, and bacillary hemoglobinuria. Several differentials in the United States for the encephalitic presentation include rabies, nervous system coccidiosis, polioencephalomalacia, lead poisoning, infectious bovine rhinotracheitis, salt poisoning, and chlorinated hydrocarbon toxicity. Prevention and control. Control or eradication of ticks and cleaning of equipment to prevent mechanical transmission, as noted in Section III,A,3,a,i, are important preventive measures. Some vaccination approaches have been effective, but a commercial product is not available. Treatment. Supportive care is indicated, including blood transfusions, fluids, and antibiotics. Medications such as diminazene diaceturate, phenamidine diisethionate, imidocarb diprionate, or amicarbalide diisethionate are most commonly used. Treatment outcomes will be either elimination of the parasite or development of a chronic carrier state immune to further disease. Research complications. This is a reportable disease in the United States. iii. Coccidiosis Etiology. Coccidiosis is an important acute and chronic protozoal disease of ruminants. In young ruminants, it is characterized primarily by hemorrhagic diarrhea. Adult ruminants may carry and shed the protozoa, but they rarely display clinical signs. Intensive rearing and housing conditions and stress increase the severity of the disease in all age groups. Coccidia are protozoal organisms of the phylum Apicomplexa, members of which are obligatory intracellular parasites. There are at least 11 reported species of coccidia in sheep, of which several are considered pathogenic: Eimeria ashata, E. crandallis, and E. ovinoidalis ( Schillhorn van Veen, 1986 ). At least 9 species of Eimeria have been recognized in the goat ( Foreyt, 1990 ). Eimeria ninakohlyakimovae, E. arloingi, and E. christenseni are regarded as the most pathogenic. Eimeria bovis and E. zuernii (highly pathogenic), and E. auburnensis and E. alabamensis (moderately pathogenic), are among the 13 species known to infect cattle. Eimeria zuernii is more commonly seen in older cattle and is the agent of "winter coccidiosis." Clinical signs and diagnosis. Hemorrhagic diarrhea develops 10 days to 3 weeks after infection. Fecal staining of the tail and perineum will be present. Animals will frequently display tenesmus; rectal prolapses may also develop. Anorexia, weight loss, dehydration, anemia, fever (infrequently), depression, and weakness may also be seen in all ruminants. The diarrhea is watery and malodorous and will contain variable amounts of blood and fibrinous, necrotic tissues. The intestinal hemorrhage may subsequently lead to anemia and hypoproteinemia. Depending on the predilection of the coccidial species for small and/or large intestines, malabsorption of nutrients or water may occur, and electrolyte imbalances may be severe. Concurrent disease with other enteropathogens may also be part of the clinical picture. In sheep, secondary bacterial infection with organisms such as Fusobacterium necrophorum may ensue. Young goats may die peracutely or suffer severe anemia from blood loss into the bowel. Older goats may lose the pelleted form of feces. Cattle may have explosive diarrhea and develop anal paralysis. The disease is usually diagnosed by history and clinical signs. Numerous oocysts will frequently be observed in fresh fecal flotation (salt or sugar solution) samples as the diarrhea begins. Laboratory results are usually reported as number of oocysts per gram of feces. Coccidia seen on routine fecal evaluations reflect shedding, possibly of nonpathogenic species, without necessarily being indicative of impending or resolving mild disease. Epizootiology and transmission. As noted, coccidiosis is a common disease in young ruminants. In goats, young animals aged 3 weeks to 5 months are primarily affected, but isolated outbreaks in adults may occur after stressful conditions such as transportation or diet changes. Coccidia are host-specific and also host cell-specific. The disease is transmitted via ingestion of sporulated oocysts. Coccidial oocysts remain viable for long periods of time when in moist, shady conditions. Necropsy. Necropsies provide information on specific locations and severity of lesions that correlate with the species involved. Ileitis, typhlitis, and colitis with associated necrosis and hemorrhage will be observed. Mucosal scrapings will frequently yield oocysts. Various coccidial stages associated with schizogony or gametogony may be observed in histopathological sections of the intestines. Fibrin and cellular infiltrates will be found in the lamina propria. Pathogenesis. This parasite has a complex life cycle in which sexual and asexual reproduction occurs in gastrointestinal enterocytes ( Speer, 1996 ). The severity of the disease is correlated primarily with the number of ingested oocysts. Specifics of life cycles vary with the species, and those characteristics contribute to the pathogenicity. In most cases, the disease is well established by the time clinical signs are seen. Oocysts must undergo sporulation over a 3- to 10-day period in the environment. After ingestion of the sporulated oocysts, sporozoites are released and penetrate the intestinal mucosa and form schizonts. Schizonts initially undergo replication by fission to form merozoites and eventually undergo sexual reproduction, forming new oocysts. The organisms cause edema and hyperemia; penetration into the lamina propria may lead to necrosis of capillaries and hemorrhage. Differential diagnosis. Differential diagnoses include the many enteropathogens associated with acute diarrhea in young ruminants: cryptosporidia, colibacilli, salmonella, enterotoxins, Yersinia, viruses, and other intestinal parasites such as helminths. In cattle, for example, bovine viral diarrhea virus and helminthiasis caused by Ostergia must be considered. Management factors, such as dietary-induced diarrheas, are also differentials. In older animals, differentials in addition to stress are malnutrition, grain engorgement, and other intestinal parasitisms. Prevention and control. Good management practices will help prevent the disease. Oocysts are resistant to disinfectants but are susceptible to dry or freezing conditions. Proper sanitation of animal housing and minimizing overcrowding are essential. Coccidiostats added to the feed and water are helpful in preventing the disease in areas of high exposure. Treatment. Affected animals should be isolated. On an individual basis, treatment should also include provision of a dry, warm environment, fluids, electrolytes (orally or intravenously), antibiotics (to prevent bacterial invasion and septicemia), and administration of coccidiostats. Coccidiostats are preferred to coccidiocidals because the former allow immunity to develop. Although many coccidial infections tend to be self-limiting, sulfonamides and amprolium may be used to aid in the treatment of disease. Other anticoccidial drugs include decoquinate, lasalocid, and monensin; labels should be checked for specific approval in a species or specific indications. Animals treated with amprolium should be monitored for development of secondary polioencephalomalacia. Pen mates of affected animals should be considered exposed and should be treated to control early stages of infection. Mechanisms of immunity have not been well defined but appear to be correlated with the particular coccidial species and their characteristics (for example, the extent of intracellular penetration). Immunity may result when low numbers are ingested and there is only mild disease. Immunity also may develop after more severe infections. iv. Cryptosporidiosis Etiology. Cryptosporidium organisms are a very common cause of diarrhea in young ruminants. Four Cryptosporidium species have been described in vertebrates: C. baileyi and C. meleagridis in birds and C. parvum and C. muris in mammals. Cryptosporidium parvum is the species affecting sheep ( Rings and Rings, 1996 ). Debate continues regarding whether there are definite host-specific variants. Clinical signs and diagnosis. Cryptosporidiosis is characterized by protracted, watery diarrhea and debilitation. The diarrhea may last only 6–10 days or may be persistent and fatal. The diarrhea is watery and yellow, and blood, mucus, bile, and undigested milk may also be present. Infected animals will display tenesmus, anorexia and weight loss, dehydration, and depression. In relapsing cases, animals become cachectic. Overall, morbidity will be high, and mortality variable. Mucosal scrapings or fixed stained tissue sections may be useful in diagnosis. The disease is also diagnosed by detecting the oocysts in iodine-stained feces or in tissues stained with periodic acid-Schiff stain or methenamine silver. Cryptosporidium also stains red on acid-fast stains such as Kinyoun or Ziehl-Neelsen. Fecal flotations should be performed without sugar solutions or with sugar solutions at specific gravity of 1.27 (Foryet, 1990). Fecal immunofluorescent antibody (IFA) techniques have also been described. Epizootiology and transmission. Younger ruminants are commonly affected: lambs, kids (especially kids between the ages of 5 and 10 days old), and calves less than 30 days old. Like other coccidians, Cryptosporidium is transmitted via the fecal-oral route. In addition to local contamination, water supplies have also been sources of the infecting oocysts. The oocysts are extremely resistant to desiccation in the environment and may survive in the soil and manure for many months. Necropsy findings. The lesions caused by Cryptosporidium are nonspecific. Animals will be emaciated. Moderate enteritis and hyperplasia of the crypt epithelial cells with villous atrophy as well as villous fusion, primarily in the lower small intestines, will be present. Cecal and colonic mucosae may sometimes be involved. Gastrointestinal smears may be made at necropsy and stained as described above. Pathogenesis. Although Cryptosporidium infections are clinically similar to Eimeria infections ( Moore, 1989 ), Cryptosporidium, in contrast to Eimeria, invades just under the surface but does not invade the cytoplasm of enterocytes. There is no intermediate host. The oocysts are half the size of Eimeria oocysts and are shed sporulated; they are, therefore, immediately infective. Within 2–7 days of exposure, diarrhea and oocyst shedding occur. The diarrhea is the result of malabsorption and, in younger animals, intraluminal milk fermentation. Autoinfection within the lumen of the intestines may also occur and result in persistent infections. In addition, several other pathogens may be involved, such as concurrent coronavirus and rotavirus infections in calves. Environmental stressors such as cold weather increase mortality. Intensive housing arrangements increase morbidity and mortality. Differential diagnosis. Other causes of diarrhea in younger ruminants include rotavirus, coronavirus, and other enteric viral infections; enterotoxigenic Escherichia coli; Clostridium; other coccidial pathogens; and dietary causes (inappropriate use of milk replacers). In addition, these other agents may also be causing illness in the affected animals and may complicate the diagnosis and the treatment picture. Eimeria is more likely to cause diarrhea in calves and lambs at 3–4 weeks of age. Giardia organisms may be seen in fecal preparations from young ruminants but are not considered to play a significant role in enteric disease. Prevention and control. Precautions should be taken when handling infected animals. Affected animals must be removed and isolated as soon as possible. Animal housing areas should be disinfected with undiluted commercial bleach or 5% ammonia. Formalin (10%) fumigation has proven successful (Foryet, 1990). After being cleaned, areas should be allowed to dry thoroughly and should remain unpopulated for a period of time. Because enteric disease often is multifactorial, other pathogens should also be considered, and management and husbandry should be examined. Treatment. No known drug treatment is available. The disease is generally self-limiting, so symptomatic, supportive therapy aimed at rehydrating, correcting electrolyte and acid-base balance, and providing energy is often effective. Supplementation with vitamin A may be helpful. Age resistance begins to develop when the animals are about 1 month old. Research complications. Cryptosporidiosis is a zoonotic disease. It is easily spread from calves to humans, for example, even as the result of simply handling clothing soiled by calf diarrhea. Adult immunocompetent humans are reported to experience watery diarrhea, cramping, flatulence, and headache. The disease can be life-threatening in immunocompromised individuals. v. Giardiasis Etiology. Giardia lamblia (also called G. intestinalis and G. duodenalis) is a flagellate protozoon. Giardiasis is a worldwide protozoal-induced diarrheal disease of mammals and some birds ( Kirkpatrick, 1989 ), but it not considered to be a significant pathogen in ruminants. Clinical signs and diagnosis. Diarrhea may be continuous or intermittent, is pasty to watery, is yellow, and may contain blood. Animals exhibit fever, dehydration, and depression. Chronic cases may result in a "poor doer" syndrome with weight loss and unthriftiness. Giardia can be diagnosed by identifying the motile piriform trophozoites in fresh fecal mounts. Oval cysts can be floated with zinc sulfate solution (33%). Standard solutions tend to be too hyperosmotic and to distort the cysts. Newer enzyme-linked immunosorbent assay (ELISA) and IFA tests are sensitive and specific. Epizootiology and transmission. Giardia infection may occur at any age, but young animals are predisposed. Chronic oocyst shedding is common. Transmission of the cyst stage is fecal-oral. Wild animals may serve as reservoirs. Necropsy findings. Gross lesions may not be evident. Villous atrophy and cuboidal enterocytes may be evident histologically. Pathogenesis. Following ingestion, each Giardia cyst releases four trophozoites, which attach to the enterocytes of the duodenum and proximal jejunum and subsequently divide by binary fission or encyst. The organism causes little intestinal pathology, and the cause of diarrhea is unknown but is thought to be related to disruption of digestive enzyme function, leading to malabsorption. Disturbances in intestinal motility may also occur ( Rings and Rings, 1996 ). Prevention and control. Intensive housing and warm environments should be minimized. Cysts can survive in the environment for long periods of time but are susceptible to desiccation. Effective disinfectants include quaternary ammonium compounds, bleach-water solution (1:16 or 1:32), steam, or boiling water. After cleaning, areas should be left empty and allowed to dry completely. Treatment. Giardia has been successfully treated with oral metronidazole. Benzimidazole anthelmintics are also effective, but these are not approved for use in animals for this purpose. Research complications. Giardia is zoonotic. Precautions should be taken when handling infected animals. vi. Neosporosis Etiology. Neosporosis is a common, worldwide cause of bovine abortion caused by the protozoal species Neospora caninum. Abortions have also been reported in sheep and goats. Neonatal disease is seen in lambs, kids, and calves. Until 1988, these infections were misdiagnosed as caused by Toxoplasma gondii. Some similarities exist between the life cycles and pathogeneses of both organisms. Clinical signs and diagnosis. Abortion is the only clinical sign seen in adult cattle and occurs sporadically, endemically, or as abortion storms. Bovine abortions occur between the third and seventh month of gestation; fetal age at abortion correlates with the parity of the dam as well as with pattern of abortion in the herd. Although cows that abort tend to be culled after the first or second abortion, repeated N. caninum- caused abortions will occur progressively later in gestation (up to about 6 months) and within a shorter time frame in the same cow ( Thurmond and Hietala, 1997 ). Although infections in adults are asymptomatic other than the abortions, decreased milk production has been noted in congenitally infected cows. Many Neospo ra-infected calves will be born asymptomatic. Weakness will be evident in some infected calves, but this resolves. Rare clinical signs include exophthalmos or asymmetric eyes, weight loss, ataxia, hyperflexion or hyperextension of all limbs, decreased patellar reflexes, and loss of conscious proprioception. Some fetal deaths will occur, and resorption, mummification, autolysis, or stillbirth will follow. Immunohistochemistry and histopathology of fetal tissue are the most efficient and reliable means of establishing a postmortem diagnosis. Serology (IFA and ELISA) is useful, including precolostral levels in weak neonates, but this indicates only exposure. Titers of dams will not be elevated at the time of abortion; fetal serology is influenced by the stage of gestation and course of infection. Earlier and rapid infections are less likely to yield antibodies against Neospora. None of the currently available tests is predictive of disease. Epizootiology and transmission. The parasite is now acknowledged to be widespread in dairy and cattle herds. The life cycle of N. caninum is complex, and many aspects remain to be clarified. The definitive host is the dog ( McAllister et al., 1998 ). Placental or aborted tissues are the most likely sources of infection for the definitive host and play a minor role in transmission to the intermediate hosts. The many intermediate hosts include ruminants, deer, and horses. Transplacental transmission is the major mode of transmission in dairy cattle and is the means by which a herd's infection is perpetuated. A less significant mode of transmission is by ingestion of oocysts, which sporulate in the environment or in the intermediate host's body. Reactivation in a chronically infected animal's body is the result of rupture of tissue cysts in neural tissue. Seropositive immunity does not protect a cow from future abortions. Many seropositive cows and calves will never abort or show clinical signs, respectively. Some immunological cross-reactivity may exist among Neospora, Cryptosporidia, and Coccidium. Necropsy findings. Aborted fetuses will usually be autolysed. In those from which tissue can be recovered, tissue cysts are most commonly found in the brain. Spinal cord is also useful. Histological lesions include mild to moderate gliosis, nonsuppurative encephalitis, and perivascular infiltration by mixed mononuclear cells. Pathogenesis. As with Toxoplasma, cell death is the result of intracellular multiplication of Neospora tachyzoites. Neospora undergoes sexual replication in the dog's intestinal tract, and oocysts are shed in the feces. The intermediate hosts develop nonclinical systemic infections, with tachyzoites in several organs, and parasites then localize and become encysted in particular tissues, especially the brain. Infections of this type are latent and lifelong. Except when immunocompromised, most cattle do not usually develop clinical signs and do not have fetal loss. Fetuses become infected, leading to fetal death, mid-gestation abortions, or live calves with latent infections or congenital brain disease. It usually takes 2–4 weeks for a fetus to die and to be expelled. Many aspects of the role of the maternal immune response and pregnancy-associated immunodeficiency in the patterns of Neospora abortions remain to be elucidated. Differential diagnosis. Even when there is a herd history of confirmed Neospora abortions, leptospirosis, bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), salmonellosis, and campylobacteriosis should be considered. BVDV in particular should be considered for abortion storms. Differentials for weak calves are BVDV, perinatal hypoxia following dystocia (immediate postpartum time), bluetongue virus, Toxoplasma, exposure to teratogens, or congenital defects. Prevention and control. The primary preventive measure is preventing contact with contaminated feces. Oocysts will not survive dry environments or extremes of temperature. Dog populations should be controlled, and dogs and other canids should not have access to placentas or aborted fetuses. Dogs should also be restricted from feed bunks and other feed storage areas. Preventive culling is not economically practical for most producers. A vaccine recently became available. If embryo transfer is practiced, recipients should be screened serologically before use. Treatment. There is no known treatment or immunoprophylaxis. vii. Sarcocystosis Etiology. Sarcocystosis is the disease caused by the cyst-forming sporozoon Sarcocystis. Sarcocystis capricanus, S. ovi-canus, and S. tenella are the species that infect sheep and goats. Sarcocystis cruzi, S. hirsuta, and S. hominis are the species that infect cattle. Definitive hosts are carnivores, and all ruminant species are intermediate hosts. Clinical signs and diagnosis. Clinical signs of sarcocystosis infection are seen in cattle during the stage when the parasite encysts in soft tissues. Often the infections are asymptomatic. Fever, anemia, ataxia, symmetric lameness, tremors, tail-switch hair loss, excessive salivation, diarrhea, and weight loss are clinical signs. Abortions in cattle occur during the second trimester and in smaller ruminants 28 days after ingestion of the sporulated oocysts. Definitive diagnosis is based on finding merozoites and meronts in neural tissue lesions. Clinical hematology results include decreased hematocrit, decreased serum protein, and prolonged prothrombin times. Sarcocystis-specific IgG will increase dramatically by 5–6 weeks after infection. There is no cross-reaction between Sarcocystis and Toxoplasma. Epizootiology and transmission. Infection rates among cattle in the United States are estimated to be very high. Transmission is by ingestion of feed and water contaminated by feces of the definitive hosts. Dogs are the definitive hosts for the species that infect the smaller ruminants. Cats, dogs, and primates (including humans when S. hominis is involved) are the definitive hosts for the species that infect cattle. Necropsy. Aborted fetuses may be autolysed. Lesions in neural tissues, including meningoencephalomyelitis, focal malacia, perivascular cuffing, neuronal degeneration, and gliosis, are most marked in the cerebellum and midbrain. Lesions may be found in other tissues, such as lymphadenopathy, and hemorrhages may be found in muscles and on serous surfaces. Cysts in cardiac and skeletal muscles are common incidental findings during necropsies. Pathogenesis. Ingestion of muscle flesh from an infected ruminant results in Sarcocystis cysts' being broken down in the carnivore's digestive system, release of bradyzoites, infection of intestinal mucosal cells by the bradyzoites, differentiation into sexual stages, fusion of the male and female gametes to form oocysts, and shedding as sporocysts by the definitive hosts. The sporocysts are eaten by the ruminant and penetrate the bowel walls; several stages of development occur in endothelial cells of arteries. Merozoites are the form that enters soft tissues, such as muscle, and subsequently encysts. Prevention and control. Feed supplies of ruminants must be protected from fecal contamination by domestic and wild carnivores. These animals should be controlled and must also not have access to carcasses. In larger production situations, monensin may be fed as a prophylactic measure. Treatment. Monensin fed during incubation is prophylactic, but the efficacy in clinically affected cattle is not known. viii. Toxoplasmosis Etiology. Toxoplasmosis is caused by the obligate intracellular protozoon Toxoplasma gondii, a coccidial parasite of the family Eimeridae. Cats are the only definitive hosts, and several warm-blooded animals, including ruminants, have been shown to be intermediate hosts. The disease is a major cause of abortion in sheep and goats and less common in cattle. Clinical signs and diagnosis. Clinical signs depend on the organ or tissue parasitized. Toxoplasmosis is typically associated with placentitis, abortion, stillbirths, or birth of weak young ( Underwood and Rook, 1992 ; Buxton, 1998 ). It has also been shown to cause pneumonia and nonsuppurative encephalitis. The enteritis at the early stage of infection may be fatal in some hosts. Hydrocephalus does not occur in animals as it does in human fetal Toxoplasma infections. Rare clinical presentations in ruminants include retinitis and chorioretinitis; these are usually asymptomatic. Infection of the ewe during the first trimester usually leads to fetal resorption, during the second trimester leads to abortion, and during the third trimester leads to birth of weak to normal lambs with subsequent high perinatal mortality. Congenitally infected lambs may display encephalitic signs of circling, incoordination, muscular paresis, and prostration. In sheep, weak young will develop normally if they survive the first week after birth. Infected adult sheep show no systemic illness. Infected adult goats, however, may die. Diagnosis may be difficult, and biological, serological, and histological methods are helpful. Serological tests are the most readily available. Complement fixation and the Sabin-Feldman antibody test may assist in diagnosis. Antibodies found in fetuses are indicative of congenital infection and are typically detectable 35 days after infection; fetal thoracic fluid is especially useful in demonstrating serological evidence of exposure. Biological methods, such as tissue culture or inoculation of mice with maternal body fluids, or with postmortem or necropsy tissues, are more time-consuming and expensive. Epizootiology and transmission. This protozoon is considered ubiquitous. Fifty percent (50%) of adult western sheep and 20% of feedlot lambs have positive hemagglutination titers (1:64 or higher) ( Jensen and Swift, 1982 ). Transmission among the definitive host is by ingestion of tissue cysts. Necropsy findings. At necropsy, placental cotyledons contain multiple small white areas that are sites of necrosis, edema, and calcification. Fetal brains may show nonspecific lesions such as coagulative necrosis, nonsuppurative encephalomyelitis, pneumonia, myocarditis, and hepatitis. Histologically, granulomas with Toxoplasma organisms may be seen in the retina, myocardium, liver, kidney, brain, and other tissues. Impression smears of these tissues, stained appropriately (e.g., with Giemsa), provide a rapid means of diagnosis. Identification of the organism in tissue sections (especially of the heart and the brain) also confirms the findings. Toxoplasma gondii is crescent-shaped, with a clearly visible nuclei, and will be found within macrophages. Pathogenesis. The protozoon has three infectious stages: the tachyzoite, the bradyzoite, and the sporozoite within the oocyst. The definitive hosts, felids, become infected by ingesting cyst stages in mammalian tissues, by ingesting oocysts in feces, and by transplacental transfer. Ingested zoites invade epithelial cells and eventually undergo sexual reproduction, resulting in new oocysts, which the cats will shed in the feces. Cats rarely show clinical signs of infection. One cat can shed millions of oocysts in 1 gm of feces, but the asymptomatic shedding takes place for only a few weeks in its life. Oocysts sporulate in cat feces after 1 day. Ruminants are intermediate hosts of toxoplasmosis and become infected by ingesting sporulated oocyst-contaminated water or feed. As in the definitive host, the ingested sporozoite invades epithelial cells within the intestine but also further invades the bloodstream and is transported throughout the host. The organism migrates to tissues such as the brain, liver, muscles, and placenta. Placental infection develops about 14 days after ingestion of the oocysts. The damage caused by an infection is due to multiplication within cells. Toxoplasma does not produce any toxin. Differential diagnosis. Differentials for abortion include Campylobacter, Chlamydia, and Q fever. Prevention and control. Feline populations on source farms should be controlled. Eliminating contamination of feed and water with cat feces is the best preventive measure. Sporulated oocysts can survive in soil and other places for long periods of time and are resistant to desiccation and freezing. Vaccines for abortion prevention in sheep are available in New Zealand and Europe. Treatment. Toxoplasmosis treatment is ineffective, although feeding monensin during pregnancy may be helpful ( Underwood and Rook, 1992 ). (Monensin is not approved for this use in the Unites States.) Weak lambs that survive the first week after birth will mature normally and will not deliver Toxoplasma- infected young. Research complications. Because toxoplasmosis is zoonotic, precautions must be taken when handling tissues from any abortions or neurological cases. Infections in immunocompromised humans have been fatal. ix. Trichomoniasis Etiology. Trichomoniasis is an insidious venereal disease of cattle caused by Tritrichomonas (also referred to as Trichomonas) fetus, a large, pear-shaped, flagellated protozoon. The organism is an obligate parasite of the reproductive tract, and it requires a microaerophilic environment to establish chronic infections. In the United States, it is now primarily a disease seen in western beef herds. There are many similarities between trichomoniasis and campylobacteriosis; both diseases cause herd infertility problems. Clinical signs and diagnosis. Clinical signs include infertility manifested by high nonpregnancy rates as well as periodic pyometras and abortions during the first half of gestation. Often the problem is not recognized until herd pregnancy checks indicate many "open," delayed-estrus, late-bred cows, or cows with postcoital pyometras. The abortion rate varies from 5% to 30%, and placentas will be expelled or retained. Tritrichomonas fetus also causes mild salpingitis but this does not result in permanent damage. Other than these manifestations, infection with T. fetus causes no systemic signs. Diagnosis is based on patterns of infertility and pyometras. For example, pyometras in postcoital heifers or cows are suggestive of this pathogen. Diagnostic methods include identifying or culturing the trichomonads from preputial smegma, cervicovaginal mucus, uterine exudates, placental fluids, or abomasal contents of aborted fetuses. Other nonpathogenic protozoa from fecal contamination may be present in the sample. The trichomonad has three anterior flagellae, one posterior flagella, and an undulating membrane; it travels in fluids with a characteristic jerky movement. Culturing must be done on specific media, such as Diamond's or modified Pastridge. Epizootiology and transmission. All transmission is by venereal exposure from breeding bulls or cows or, in some cases, contaminated breeding equipment. Necropsy findings. Nonspecific lesions, such as pyogranulomatous bronchopneumonia of fetuses and placentitis, may be seen in aborted material; some cases will have no gross lesions. Histologically, trichomonads may be visible in the fetal lung lesions and the placenta; those tissues are also the most useful for culturing. Pathogenesis. Tritrichomonas fetus colonizes the female reproductive tract, and subsequent clinical manifestations may be related to the size of the initial infecting dose. Tritrichomonas fetus does not interfere with conception. Embryonic death occurs within the first 2 months of infection. Affected cows will clear the infection over a span of months and maintain immunity for about 6 months. Infections in younger bulls are transient; apparently organisms are cleared by the bulls' immune systems and are dependent on exposure to infected females. Older bulls become chronic carriers, probably because of the ability of T. fetus to colonize deeper epithelial crypts of the prepuce and penis. Differential diagnosis. Campylobacteriosis is the other primary differential for reduced reproductive efficiency of a herd. Other venereal diseases should be considered when infertility problems are noted in a herd: brucellosis, mycoplasmosis, ureaplasmosis, and infectious pustular vulvovaginitis. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. A bacterin vaccine is available. Heifers, cows, and breeding bulls are vaccinated subcutaneously twice at 2 to 4 week intervals, with the booster dose administered 4 weeks before breeding season starts. Similar timing is recommended for administration of the annual booster; a long, anamnestic response does not occur. Bulls used for artificial insemination (AI) are screened routinely for T. fetus (and Campylobacter). AI reduces but does not eliminate the disease. The use of younger, vaccinated bulls is recommended in all circumstances. New animals should be tested before introduction to the herd. Control measures also include culling affected cows or else removing them from the breeding herd for 3 months to rest and clear the infection. Culling chronically infected bulls is strongly recommended. Treatment. Imidazole compounds have been effective, but the use of these is not permitted in food animals in the United States. Therapeutic immunizations are worthwhile when a positive diagnosis has been made. These will not curtail fetal losses but will shorten the convalescence of the affected cows and improve immunity of breeding bulls. Research complications. Trichomoniasis should be considered whenever natural service is used and fertility problems are encountered. b. Nematodes Nematodes are important ruminant pathogens that cause acute, chronic, subclinical, and clinical disease in adults and adolescents. The major helminths may cause gastroenteritis associated with intestinal hemorrhage and malnutrition. Nematodiasis is associated with grazing exposure to infective larvae; animals procured for research may have had exposure to these helminths. Mixed infections of these parasites are common. Generally, older animals develop resistance to some of the species; thus, animals between about 2 months and 2 years of age are most susceptible to infection. Because of the parasites' effects on the animals' physiology, infection in these younger animals is a major contributor to a cycle of poor nutrition and digestion, compromised immune responses, and impaired growth and development. Diagnosis is primarily based on fecal flotation techniques; however, because many of these nematodes have similar-appearing ova, hatching the ova and identifying the larvae are often required (Baermann technique). A number of anthelmintics can be used to interrupt nematode life cycles. See Zajac and Moore (1993) and Pugh et al. (1998 ) for comprehensive reviews of treatment and control of nematodiasis. i. Haemonchus contortus, H. placei (barber's pole worm, large stomach worm). Haemonchus contortus is the most important internal parasite of sheep and goats, and the brief description here focuses on the disease in the smaller ruminants. Haemonchus contortus and H. placei infections do occur in younger cattle and are similar to the disease in sheep. Haemonchus is extremely pathogenic, and the adults feed by sucking blood from the mucosa of the abomasum. Severe anemia may lead to death. Weight loss, decreased milk production, poor wool growth, and intermandibular and cervical edema due to hypoproteinemia ("bottle jaw") are also common clinical signs. Diarrhea is not seen in all cases but may sometimes be severe or chronic. The life cycle is direct. Under optimal conditions, a complete life cycle, from ingestion of larvae to eggs passed in the feces, occurs in 3 weeks. Embryonated eggs may develop into infective larvae within a week. Hypobiotic (arrested) larvae may exist for several months in animal tissues, serving as a reservoir for future pasture contamination. Periparturient increases in egg shedding by ewes contribute to large numbers of eggs spread on spring pastures ("spring rise"). Resistance to common anthelmintics has developed; currently ivermectin or benzimidazole products are used, with a minimum of 2 dosings given 2–3 weeks apart. Levamisole is also used. In severe cases, animals may benefit from blood transfusions and iron supplementation. Because animals may easily acquire infective larvae from ingestion of contaminated feed and from contaminated pastures, general facility sanitation and pasture management and rotation are important preventive and control measures. Haemonchus contortus is susceptible to destruction by freezing temperatures and dry conditions. ii. Ostertagia (Teladorsagia) circumcincta (medium stomach worm). Ostertagia circumcincta is also highly pathogenic for sheep and goats and, like Haemonchus, attaches to the abomasal mucosa and ingests blood. The life cycle is comparable to that of Haemonchus, including the phenomenon of hypobiosis. Larvae are especially resistant to cool temperatures, however, and will overwinter on pastures. Larvae-induced hyperplasia of abomasal epithelial glands results in a change of gastric pH from about 2.0 to near 7.0, leading to decreased digestive enzyme activity and malnutrition. Clinical syndromes are categorized as type 1 or type 2. The former type is associated with infections acquired in fall or spring and is seen in younger animals. The latter type is associated with emergence of the arrested larvae during spring or fall. Clinical signs include anemia, weight loss, decreased milk production, and unthriftiness. Diarrhea is usually seen in type 1 only; the symptoms of type 2 are comparable to those of Haemonchus infections. Anthelmintic drug therapy is comparable to that for Haemonchus, and drug resistance is also a problem with Ostertagia. iii. Ostertagia ostertagi (cattle stomach worm). Ostertagia ostertagi is the most pathogenic and most costly of the cattle nematodes. Ostertagia leptospicularis and O. bisonis also cause disease. The life cycle is direct, and egg shedding by the cattle may occur within 3–4 weeks of ingestion of infective larvae. Hypobiosis is also a characteristic of O. ostertagi. In the initial steps of infection, the normal processes of the abomasum are profoundly disrupted and cells are destroyed as the larvae develop within and emerge from the glands. Moroccan leather appearance is the term to describe the result of cellular hyperplasia and loss of cell differentiation. Cycles of infection and morbidity depend on geographic location, climate, and production cycles. Type 1 cattle ostertagiasis is associated with ingestion of large numbers of infective larvae, occurs in animals less than 2 years old, and causes diarrhea and anorexia. Type 2 ostertagiasis occurs in cattle 2–4 years old and older adults, is the result of the emergence and development of hypobiotic larvae, and in addition to signs seen with type 1, hypoproteinemia with development of submandibular edema, fever, and anemia is a clinical sign. Treatment options include ivermectin, fenbendazole, and levamisole; all are effective against the arrested larvae. Ostertagia is susceptible to desiccation but is resistant to freezing. iv. Trichostrongylus vitrinus, T. axei, T. colubriformis (hair worms). Trichostrongylus species favor cooler conditions, and some larvae may overwinter. Although the different species may affect different segments of the gastrointestinal tract, the nematode attaches to the mucosa and affects secretion and/or absorption. Trichostrongylus vitrinus and T. colubriformis infect the small intestine of sheep and goats. Trichostrongylus axei infects the abomasum of cattle, sheep, and goats and causes increases in abomasal pH similar to those seen with Ostertagia. Mucosal hyperplasia is not seen. The prepatent period is about 3 weeks. Affected animals display unthriftiness, anorexia, decreased milk production, weight loss, diarrhea, and dehydration. These worms show intermediate resistance to freezing temperatures and dry conditions. v. Nematodirus spathiger, N. battus (thread-necked worms). Nematodirus has lower pathogenicity compared with other gastrointestinal nematodes. The larvae cause small-intestinal necrosis and inflammation. The larvae are especially resistant to desiccation and freezing. Clinical signs include depression, weight loss, anorexia, and diarrhea. vi. Cooperia (small intestinal worms). Cooperia primarily affects younger animals less than 1 year of age. Cooperia curticei infects the small intestine of sheep and goats; C. punctata and C. oncophora infect the small intestines of cattle, sheep, and goats. Cooperia pectinata infects the stomach of cattle. Large numbers lead to clinical infection, and the prepatent period is about 3 weeks. Cooperia and Osteragia infections, like infections of some other nematode species, may act synergistically. Because these nematodes suck blood, clinical signs include anemia, gastrointestinal hemorrhage, and malnutrition. Animals exhibit weight loss, diarrhea, and depression. Cooperia species are intermediate to resistant to the effects of cold temperatures. vii. Strongyloides papillosus. Strongyloides papillosus is a small-intestinal parasite of sheep and cattle. Strongyloides has a different life cycle from that of many nematodes. The eggs, expelled in the feces, are larvated, and when they hatch, they form both free-living males and females or parasitic females only. The parasitic females may enter the gastrointestinal tract through oral ingestion, such as in milk during nursing, or through direct penetration of the skin. Penetrating larvae enter the bloodstream and are transported to the lungs, where they penetrate the alveoli, are coughed up, and then swallowed to ultimately enter the gastrointestinal tract. Adult females may reproduce in the small intestines by parthenogenesis. Clinical signs associated with Strongyloides include weight loss, diarrhea, unthriftiness, and dermatitis in cases where large numbers migrate through the skin. The current broad-spectrum anthelmintics are effective against Strongyloides. viii. Bunostomum trigonocephalum (hookworm). Bunostomum trigonocephalum is a hookworm that occasionally infects sheep in locales in the southwestern United States. Like Strongyloides, Bunostomum infection may involve oral ingestion or direct penetration of the skin (followed by tracheal migration and swallowing). The larvae mature in the small intestines and suck blood. Larvae are susceptible to desiccation and freezing. Heavy infection with Bunostomum may result in anemia, diarrhea, intestinal hemorrhage, edema, and weight loss. ix. Oesophagostomum columbianum, O. venulosum (nodule worms). Oesophagostomum spp. primarily infect the large intestine and occasionally the distal small intestine, causing nodule worm disease, or simply gut. Oesophagostomum columbianum and O. venulosum infect sheep and cattle. These nematodes may affect sheep from 3 months to 2 years of age, and the prepatent period is about 6 weeks. Larvae are highly sensitive to freezing and desiccation and rarely overwinter. Larvae penetrate the large-intestinal mucosa but occasionally move into the deeper areas of the intestinal wall near the serosa. The resultant inflammatory reaction may lead to the formation of a caseous nodule that may mineralize over time. Intestinal lesions may accelerate peristalsis, leading to diarrhea, or may inhibit peristalsis (later stages), resulting in constipation. Clinical signs include weakness, unthriftiness, alternating episodes of diarrhea and constipation, and severe weight loss. Nodular lesions are typical at necropsy. x. Chabertia ovis (large-mouth bowel worm). Chabertia ovis is a minor colon parasite of sheep, goats, and cattle and is seen primarily in sheep. Signs of infection are not usually seen in cattle. Prepatent periods are up to 50 days. Heavy infection, which may result from as few as 100 worms located at the proximal end of the colon, may lead to hemorrhagic mucoid diarrhea, weight loss, weakness, colitis, and mild anemia. xi. Trichuris (whipworms). Trichuris spp. are mildly pathogenic nematodes and are usually attached to the cecal mucosa. Trichuris has a rather long prepatent period, extending from 1 to 3 months. The oval eggs are double-operculated and survive well in pasture environmental extremes. The adult worms also have a characterisitic morphology, with one thicker end appearing as a whip handle. The nematodes cause a minor cecitis and will feed on blood. Clinical infection is rare and results in diarrhea with mucus and blood. Treatment and prevention methods are similar to those for other nematodes. xii. Dictyocaulus (lungworms). Dictyocaulus spp., or lungworms, are nematodes that cause varying clinical signs in ruminants. In sheep, Dictyocaulus filaria, Protostrongylus rufescens, and Muellerius capillaris cause disease; Dictyocaulus is the most pathogenic. Goats are infected by the same species as sheep, but infections are uncommon. Dictyocaulus viviparus is the only lungworm found in cattle, causing "fog fever." Infections with these parasites in the United States tend to be associated with cooler, moister climates. Lungworms induce a severe parasitic bronchitis (known as husk, or verminous pneumonia) in sheep between approximately 2 and 18 months of age. Sheep infected with any of the lungworm species may display coughing, dyspnea, nasal discharge, weight loss, unthriftiness, and occasionally fever. Coughing and dyspnea are symptoms in goats. Diagnosis is suggested by persistent coughing and nasal discharge and is confirmed by identifying larvae in the feces or adults in pathological samples. The Baermann technique, involving prompt examination of room-temperature feces, is usually used; zinc sulfate flotation is also used. Dictyocaulus has a direct life cycle. The adult worms reside in the large bronchi. Dictyocaulus produces embryonated eggs that are coughed up and swallowed; the eggs then hatch in the intestines, and larvae are expelled in the feces. The expelled larvae are infectious in about 7–10 days and, after ingestion, penetrate the intestinal mucosa and move through the lymphatics and blood into the lungs, where they develop into adults in about 5 weeks. Dictyocaulus filaria causes an especially severe bronchitis in sheep. Protostrongylus inhabits smaller bronchioles. Muellerius is of minor pathogenicity. Protostrongylus and Muellerius require the snail or slug as an intermediate host. Infection occurs through ingestion of infected snails; infections are less likely than those caused by the direct ingestion of Dictyocaulus larvae. Immunity wanes over a year. Viral and bacterial respiratory tract infections may be associated with the parasitic infection. Dictyocaulus viviparus causes the obvious signs in cattle. More severe illness is seen after infections with Cooperia and Ostertagia, because of a synergism between the nematodes even if the cattle are not currently infected with those parasites. Hypobiosis (arrested development of immature worms in lung tissue) is associated with Dictyocaulus infections; cattle will be silent carriers, showing no clinical signs and serving as a means for the infection to survive over winter or a dry season. Pastures can be heavily contaminated during the next grazing season. Necropsy lesions include bronchiolitis and bronchitis, atelectasis, and hyperplasia of peribronchiolar lymphoid tissue. Nematodes frequently reside in the bronchi of the diaphragmatic lung lobes and are frequently enmeshed with frothy exudate. Prevention and control of the disease involve appropriate pasture management. Elimination of intermediate hosts is important in sheep and goat pastures. In a laboratory setting, animals may be procured that are already harboring the disease. Infected animals can be treated with anthelmintics such as ivermectin or levamisole. Muellerius tends to be resistant to levamisole. There is no anthelmintic currently approved for goats, but fenbendazole, administered 2 weeks apart, has been effective for all three nematodes. Treating D. viviparus depends on the type and stage of life of the cattle; label directions must be followed. There is no vaccine for D. viviparus in the United States. Even if infections are not severe and do resolve with treatment, permanent lesions may be inflicted on the lung tissue. c. Cestodes (Tapeworms) i. Moniezia expansa and Thysanosoma actinoides infections. Tapeworms are rarely of clinical or economic importance. In younger animals, heavy infections result in potbellies, constipation or mild diarrhea, poor growth, rough coat, and anemia. Moniezia expansa, and less commonly Moniezia benedini, inhabit the small intestines of grazing ruminants. Moniezia expansa has the widest distribution of the tapeworm species in North America. Soil mites (Galumna spp. and Oribatula spp.) contribute to the life cycle as intermediate hosts, a period that lasts up to 16 weeks. Cysticercoids released from the mites are grazed, pass into the small intestines, and mature. No clinical or pathological sign is usually observed with Moniezia infection; diagnosis is made by observing the characteristic triangular-shaped eggs in fecal flotation examinations. Infection is treated with cestocides. Thysanosoma actinoides, or the fringed tapeworm, is a cestode that resides in the duodenum, bile duct, and pancreatic duct of sheep and cattle raised primarily west of the Mississippi River in the United States. Thysanosoma is of the family Anoplocephalidae. The life cycle is indirect, and the intermediate host is the psocid louse. Larval forms, or cysticercoids, are ingested by grazing animals, and the prepatent period is several months. Typically, no clinical signs are observed with Thysanosoma infection; nonetheless, liver damage, resulting in liver condemnation at slaughter, occurs. Necropsy lesions include bile and/or ductal hyperplasia and fibrosis. Thysanosoma is diagnosed premortem by identifying the gravid segments in the feces. ii. Abdominal or visceral cysticercosis. Abdominal or visceral cysticercosis is an occasional finding at slaughter. The so-called bladder worms typically affect the liver or peritoneal cavity and are the larval form of Taenia hydatigena, the common tapeworm of the dog family. Taenia hydatigena resides in the small intestines of canids, and its gravid segments, oncospheres, contaminate feed and water sources. After ingestion, the larvae penetrate the intestinal mucosa, are transported via the bloodstream to the liver, and cause migration tracts throughout the liver parenchyma. The larvae may leave the liver and migrate into the peritoneal cavity, where they attach and develop over the next 1–9 months into small fluid-filled bladders. The life cycle is completed only after these bladders are ingested by a carnivore, thus completing the maturation of the adult tapeworms. Although larval migration may cause nonspecific signs such as anorexia, hyperthermia, and weight loss, affected animals are usually asymptomatic. At necropsy, the bladder worms will be observed attached to the peritoneal or organ surfaces. Migration tracts may result in fibrosis and inflammation. Diagnosis is usually made at necropsy. Because of the migration through the liver, Fasciola hepatica is a differential diagnosis. Minimizing exposure to canine feces-contaminated feeds and water effectively interrupts the life cycle. Research animals may have been exposed prior to purchase. iii. Echinococcosis (hydatidosis, hydatid cyst disease). Echinococcosis, like cysticercosis, is an occasional finding at slaughter or necropsy. The hydatid cyst is the larval intermediate of the adult tapeworm Echinococcus granulosus, which resides in the small intestines of dogs and wild canids. Embryonated ova are expelled in the feces of the primary host and are ingested by herbivores, swine, and potentially humans. The eggs hatch in the gastrointestinal tract, and the oncospheres penetrate the mucosal lining, enter the bloodstream, and are transported to various organs such as the liver and lungs. The cystic structure develops and potentially ruptures, forming new cystic structures. Clinically, echinococcosis presents minimal clinical signs; unthriftiness or pneumonic lesions may be associated with infected organs. Cysts are typically observed at necropsy. Prevention should be aimed at decreasing fecal contamination of feed and water by canids. Additionally, tapeworm-infected dogs can be treated with standard tapeworm therapies. Treatment of infected ruminants is uncommon. iv. Gid. Coenuris cerebralis, the larval form of the canid tapeworm Taenia (Multiceps) multiceps, is the causative agent of the rare condition called gid. The disease occurs in ruminants as well as many other mammalian species. The larval parasite, ingested from fecal-contaminated food and water, invades the brain and spinal cord and develops as a bladder worm that causes pressure necrosis of the nervous tissues. The resultant signs of hyperesthesia, meningitis, paresis, paralysis, ataxia, and convulsions are observed. Diagnosis is usually made at necropsy. Eliminating transfer from the canid hosts prevents the disease. d. Trematodes i. Fascioliasis (liver fluke disease). Liver flukes are an important cause of acute and chronic disease in grazing sheep and cattle. There are three common species of flukes in ruminants of the continental United States: Fasciola hepatica, Fascioloides magna, and Dicrocoelium dendriticum. Fasciola hepática infections are primarily seen in Gulf Coast and western states. Fascioloides magna infections are typically seen in Gulf, Great Lake, and northwestern states, where ruminants share pasture with deer, elk, and moose. Dicrocoelium dendriticum infections occur only in New York State. Liver fluke eggs are passed in the bile and feces and hatch in 2–3 weeks to form the free-swimming miracidia. It is important to note that each fluke egg represents the source of eventually thousands of cercariae or metacercariae. The miracidia penetrate the body of an intermediate host (usually freshwater snails) and develop through sporocyst and redia stages, finally forming cercariae. (Dicrocoelium is unique because it utilizes a land snail that expels slime balls, each containing several hundred cercariae. These are eaten by a second intermediate host, the ant Formica fusca.) The cercariae leave the intermediate host, swim to grassy vegetation, lose their tail, and become a cystlike metacercaria. The metacercariae may remain in a dormant stage on the grass for 6 months or longer until ingested by a ruminant. The ingested metacercariae penetrate the small-intestinal wall and migrate through the abdominal cavity to the liver. There they locate in a bile duct, mature, and remain for up to 4 years. Acute liver fluke disease is related to the damage caused by the migration of immature flukes. Migratory flukes may lead to liver inflammation, hemorrhage, necrosis, and fibrosis. Fascioloides magna infections in sheep and goats can be fatal as the result of just one fluke tunneling through hepatic tissue. In cattle, infections are often asymptomatic because of the host's encapsulation of the parasite. Liver fluke damage may predispose to invasion by anaerobic Clostridium species such as C. novyi that could lead to fatal black disease or bacillary hemoglobinuria. Chronic disease may result from fluke-induced physical damage to the bile ducts and cholangiohepatitis. Blood loss into the bile may lead to anemia and hypoproteinemia. Liver damage also is evidenced by increases in liver enzymes such as γ-glutamyl transpeptidase (GGT). Persistent eosinophilia is also seen with liver fluke disease. Other clinical signs of liver fluke disease include anorexia, weight loss, unthriftiness, edema, and ascites. At necropsy, livers will be pale and friable and may have distinct migration tunnels along the serosal surfaces. Bile ducts will be enlarged, and areas of fibrosis will be evident. Diagnosis can be made from clinical signs and postmortem analyses. Blood chemistries suggestive of liver disease and eosinophilia support the diagnosis. Liver fluke control involves removal of the intermediate hosts. In a laboratory setting, liver fluke infection is unlikely. Nonetheless, incoming animals from pasture environments may be infected. Liver flukes can be treated by using the anthelmintic albendazole. ii. Rumen fluke infections (paramphistomosis). Paramphistomosis is an uncommon disease found in sheep and cattle in southern states. Paramphistomum microbothrioides and P. cervi inhabit the duodenum and rumen of affected sheep. Eggs are passed in the feces and hatch in approximately 1 month, and the miracidia penetrate the intermediate snail hosts. Cercariae develop in the snail over the next month, emerge, and encyst on grasses as metacercariae. When eaten, the metacercariae develop into adult flukes and attach to the mucosal lining. The life cycle is complete in approximately 100 days. The flukes cause localized injury to the mucosa and, by interfering with digestive processes, cause diarrhea and protein loss. Clinically, animals may experience anorexia, dehydration, weight loss, and diarrhea with or without blood. Mortality may reach 25%. Diagnosis is based on clinical findings as well as the identification of flukes or eggs in the feces. Animals can be treated with fluki-cides. Eliminating the intermediate host prevents the disease. e. Mites (Mange) Mites cause a chronic dermatitis. The principal symptom of these infections is intense pruritus. In addition, papules, crusts, alopecia, and secondary dermatitis are seen. Anemia, disruption of reproductive cycles, and increased susceptibility to other diseases may also occur. Mites are rare in ruminants in the United States, but infections of Sarcoptes and Psorergates mange must be reported to animal health officials. Ruminants in poorly managed facilities are generally the most susceptible to infection, and infections are more frequent during winter months. Diagnosis is based on signs, examination of skin scrapings, and response to therapy. No effective treatment for demodectic mange in large animals has been found. The differential for mite infestations is pediculosis. Several genera of mites may affect sheep. These have been eradicated from flocks in the United States or are very rare and include Psoroptes ovis (common scabies), Sarcoptes scabiei (head scabies, barn itch), Psorergates ovis (sheep itch mite), Chorioptes ovis (foot scabies, tail mange), and Demodex ovis (follicular mange). Goats can also be infected by sarcoptic, chorioptic, and psoroptic mange. The scabies mite Sarcoptes rupicaprae invades epidermal tissue and causes focal pruritic areas around the head and neck. The chorioptic mite, either Chorioptes bovis or C. caprae, does not invade epidermal tissue but rather feeds on dead skin tissue. The chorioptic mite prefers distal limbs, the udder, and the scrotum and can be a significant cause of pruritus. The psoroptic mite Psoroptes cuniculi commonly occurs in the ear canal and causes head shaking and scratching. Repeated treatments of lime sulfur, amitraz, or ivermectin may be effective ( Smith and Sherman, 1994 ). Goats are also susceptible to demodectic mange caused by Demodex caprae. Adult mites invade hair follicles and sebaceous glands. Pustules may develop with secondary bacterial infection. Psoroptes bovis continues to be present in cattle in the United States, although it has been eradicated from sheep. Chorioptes bovis typically infects lower hindlimbs, perineum, tail, and scrotum but can become generalized. The sarcoptic mange mite S. scabei can survive off the host, so fomite transmission is a factor. The mange usually begins around the head but then spreads. This parasite can be transmitted to humans. Demodex bovis infects cattle; nodules on the face and neck are typical. Demodex bovis infections may resolve without treatment. Lindane, coumaphos, malathion, and lime sulfur are used to treat Psoroptes and Psorergates. Ivermectin is effective against Sarcoptes and is approved for use in cattle. f. Lice (Pediculosis) Lice that infect ruminants are of the orders Mallophaga, biting or chewing lice, and Anoplura, sucking lice. These are wingless insects. Members of the Mallophaga are colored yellow to red; members of the Anoplura are blue gray. Lice produce a seasonal (winter-to-spring), chronic dermatitis. In sheep, biting lice include Damalinia (Bovicola) ovis (sheep body louse). Sucking lice that infect sheep include Linognathus ovillus (blue body louse) and L. pedalis (sheep foot louse). In goats, biting lice infection are caused by D. caprae (goat biting louse), D. limbatus (Angora goat biting louse), and D. crassipes. Sucking louse infections in goats are caused by L. stenopis and L. africanus. Damalinia bovis is the cattle biting louse. Sucking lice include L. vituli, Solenopotes capillatus, Haematopinus eurysternus, and H. quadripertusus. Pruritus is the most common sign and often results in alopecia and excoriation. The host's rubbing and grooming may not correlate with the extent of infestation. Hairballs can result from overgrooming in cattle. In severe cases, the organisms can lead to anemia, weight loss, and damaged wool in sheep and damaged pelts in other ruminants. Young animals with severe infestations of sucking lice may become anemic or even die. Pregnant animals with heavy infestations may abort. In sheep infected with the foot louse, lameness may result. Lice are generally species-specific. Those infecting ruminants are usually smaller than 5 mm. Goats may serve as a source of infection for sheep by harboring Damalinia ovis. Transmission is primarily by direct contact between animals. Transmission can also occur by attachment to flies or by fomites. Some animals are identified as carriers and seem to be particularly susceptible to infestations. Biting or chewing lice inhabit the host's face, lower legs, and flanks and feed on epidermal debris and sebaceous secretions. Sucking lice inhabit the host's neck, back, and body region and feed on blood. Lice eggs or nits are attached to hairs near the skin. Three nymphal stages, or instars, occur between egg and adult, and the growth cycle takes about 1 month for all species. Lice cannot survive for more than a few days off the host. All ruminant mite infestations are differentials for the clinical signs seen with pediculosis. Animals that are carriers should be culled, because these individuals may perpetuate the infection in the group. Lice are effectively treated with a variety of insecticides, including coumaphos, dichlorvos, crotoxyphos, avermectin, and pyrethroids. Label directions should be read and adhered to, including withdrawal times. Products should not be used on female dairy animals. Treatments must be repeated at least twice at intervals appropriate for nit hatches (about every 16 days) because nits will not be killed. Fall treatments are useful in managing the infections. Systemic treatments in cattle are contraindicated when there may be concurrent larvae of cattle grubs (Hypoderma lineatum and H. bovis). Back rubbers with insecticides, capitalizing on self-treatment, are useful for cattle. Sustained-release insecticide-containing ear tags are approved for use in cattle. g. Ticks Etiology. Ruminants are susceptible to many species of Ixodidae (hard-shell ticks) and Argasidae (softshell ticks). Many diseases, including anaplasmosis, babesiosis, and Q fever are transmitted by ticks. Clinical signs and diagnosis. Tick infestations are associated with decreased productivity, loss of blood and blood proteins, transmission of diseases, debilitation, and even death. Feeding sites on the host vary with the tick species. Ticks are associated with an acute paralytic syndrome called tick paralysis. This disease is characterized by ascending paralysis and may lead to death if the tick is not removed before the paralysis reaches the respiratory muscles. Diagnosis is based on identification of the species. Epizootiology and transmission. Ticks are not as host-specific as lice. Ticks are classified as one-host, two-host, or three-host; this refers to whether they drop off the host between larval and nymphal stages to molt. Pathogenesis of tick infestations. Patterns of feeding on the host differ between Argasidae and Ixodidae. The former feed repeatedly, whereas the latter feed once during each life stage. Pathogenesis of tick paralysis. Following a tick-feeding period of 4–6 days, the tick salivary toxin travels hematogenously to the myoneural junctions and spinal cord and inhibits nerve transmission. Removal of the ticks reverses the syndrome unless paralysis has migrated anteriorly to the respiratory centers of the medulla. In these cases, death due to respiratory failure occurs. Treatment. Ticks can be treated using systemic or topical insecticides. h. Other Parasites i. Nasal bots (nasal myiasis, head grubs). Nasal myiasis causes a chronic rhinitis and sinusitis. The disease is caused by the larval forms of the botfly Oestrus ovis. The botfly deposits eggs around the nostrils of sheep. The ova hatch, and the larvae migrate throughout the nasal cavity and sinuses, feeding on mucus and debris. In 2–10 months, the larvae complete their growing phase, migrate back to the nasal cavity, and are sneezed out. The mature larvae penetrate the soil and pupate for 1–1.5 months and emerge as botflies. Clinically, early in the disease course, animals display unique behaviors such as stamping, snorting, sneezing, and rubbing their noses against each other or objects. Hypersensitivity to the larvae occurs ( Dorchies et al., 1998 ). Later, mucopurulent nasal discharges associated with the larval-induced inflammation of mucosal linings will be observed. At necropsy, larvae will be observed in the nasal cavity or sinuses. Mild inflammatory reactions, mucosal thickening, and exudates will accompany the larvae. The disease is diagnosed by observing the behaviors or identifying organisms at necropsy. Up to 80% of a flock will potentially be infected; treatment should be employed on the rest of the flock. Ivermectins and other insecticides will eliminate the larvae; but treatment should be done in the early fall, when larvae are small. Fly repellents may be helpful at preventing additional infections. ii. Screwworm flies. Cochliomyia hominivorax (Callitroga americana) is the the screwworm that causes occasional disease in the southwestern United States along the Mexico border. Eradication programs have been pursued, and the disease is reportable. Large greenish flies lay large numbers of white eggs as shinglelike layers at the edges of open wounds (including docking and castration sites), soiled skin, or abrasions. Eggs hatch within 24 hr. Larvae are obligate parasites of living tissue, and the cycle is perpetuated because the increasingly large wound continues to be attractive to the next generation of flies. Larvae eventually drop off, pupate best in hot climates, and hatch in 3 weeks. Large cavities in parasitized tissue are formed, and lesions are characterized by malodor, large volumes of brown exudate, and necrosis. Single animals or entire herds may be affected. Treatment is intensive, with dressings and larvicidal applications. If there is no intervention, the host succumbs to secondary infections and fluid loss. Effective current control regimens include subcutaneous injection of ivermectin and programs that release sterile male flies. iii. Sheep keds ("sheep ticks"). In sheep and goats, sheep keds produce a chronic irritation and dermatitis with associated pruritus. The disease is caused by Melophagus ovinus, which is a flat, brown, blood-sucking, wingless fly; the term sheep tick is incorrectly used. The adult fly lives entirely on the skin of sheep. Females mate and produce 10–15 larvae following a gestation of about 10–12 days. The larvae attach to the wool or hair and then pupate for about 3 weeks. The adult female feeds on blood and lives for 4–5 months; the life cycle is completed in about 5–6 weeks. Infection is highest in fall and winter. Pruritus develops around the neck, sides, abdomen, and rump. In severe cases, anemia may occur. Keds can transmit bluetongue virus. Keds are diagnosed by gross or microscopic identification. Ivermectin or other insecticides are useful treatment agents. 5. Fungal Disease: Dermatophytes (Ringworm) Etiology. Dermatophytosis, or infection of the keratinized layers of skin, is caused mostly by species of the genera Trichophyton and Microsporum. The primary causes in sheep are T. mentagrophytes and T. verrucosum. In goats, the agents are T. mentagrophytes, M. canis, M. gypseum, T. verrucosum, T. schoenleinii, and Epidermophyton floccosum. In cattle, T. verrucosum is the primary causative agent. Dermatophytosis is a common fungal infection of the epidermis of cattle and is less common in sheep and goats. Clinical signs and diagnosis. Multiple, gray, crusty, circumscribed, hyperkeratotic lesions are characteristic of infection. Lesions will vary in size. In all ruminants, lesions will be around the head, neck, and ears. In goats and cattle, lesions will extend down the neck, and in cattle, lesions develop particularly around the eyes and on the thorax. Cattle lesions are unique in the marked crustiness, which progressively appears wartlike. Hair shafts become brittle and break off. Intense pruritus is often associated with the alopecic lesions. The disease can be diagnosed by microscopic identification of hyphae and conidia on the hairs following skin scraping and 20% potassium hydroxide digestion. Dermatophyte test media (DTM) cultures are the most reliable means to diagnose the fungus. Broken hairs from the periphery of the lesion are the best sources of the fungus. Epizootiology and transmission. Younger animals are more susceptible, and factors such as crowding, indoor housing, warm and humid conditions, and poor nutrition are also important. Transmission is by direct contact or by contact with contaminated fomites, such as equipment, fencing, or feed bunks. Pathogenesis. Incubation can be as long as 6 weeks. The organisms invade and multiply in hair shafts. Treatment. Spontaneous recovery occurs in all species in 1–4 months. Although cell-mediated immunity is considered important, other immune mechanisms are not well understood. Immunity may not be of long duration. Recovery is enhanced by correcting nutritional deficiencies and improving housing and ventilation problems. A number of topical treatments, such as 2–5% lime-sulfur solution, 3% captan, iodophors, thiabendazole, and 0.5% sodium hypochlorite, can be used. In severe cases, systemic therapy with griseofulvin may be successful. Prevention and control. The animals' environment and overall physical condition should be reassessed with particular attention to ventilation, crowding, sanitation, and nutrition. Pens should be thoroughly cleaned and disinfected. Research complications. Ringworm is a zoonotic disease. 1. Bacterial, Mycoplasmal, and Rickettsial Diseases a. Actinobacillosis ("Wooden Tongue") Etiology. Actinobacillus lignieresii is an aerobic, nonmotile, non-spore-forming, gram-negative rod that is widespread in soil and manure and is found as normal flora of the respiratory, gastrointestinal, and reproductive tracts of ruminants. In sheep and cattle, A. lignieresii causes sporadic, noncontagious, and potentially chronic disease characterized by diffuse abscess and granuloma formation in tissues of the head and occasionally other body organs. This disease, called wooden tongue, has not been documented in goats. Clinical signs. Skin lesions are common. Tongue lesions are more common in cattle than in sheep. Lip lesions are more common in sheep. Soft-tissue or lymph node swelling accompanied by draining tracts is observed in the head and neck regions, as well as other areas. Animals may have difficulty prehending food; may be anorexic, weak, unthrifty and depressed; and may salivate excessively. Diagnosis is made based on clinical signs and is confirmed by culture. Epizootiology and transmission. The organism penetrates wounds of the skin, mouth, nose, gastrointestinal tract, testicles, and mammary gland. Rough feed material and foreign bodies may play a role in causing abrasions. Actino bacillus lignieresii then enters into deeper tissues, where it causes chronic inflammation and abscess formation. Lymphatic spread may occur, leading to abscessation of lymph nodes or infection of other organs. Necropsy findings. Purulent discharges of white-green exudate drain from the tracts that often extend from the area of colonization to the skin surface. Exudates will also contain characteristic small white-gray (sulfurlike) granules. The pus is usually nonodorous. Differential diagnosis. Contagious ecthyma and caseous lymphadenitis are the primary differentials. Diseases or injuries causing oral pain and discomfort, such as dental infections, foreign bodies, and trauma, should be considered. Treatment. Animals should be fed softer feeds. Antibiotics such as sulfonamides, tetracyclines, and ampicillin are effective, although high doses and long durations of therapy are required. Penicillin is not effective. Weekly systemic administration of sodium iodide for several weeks is not as effective as antibiotic therapy. Surgical excision and drainage are not recommended. Prevention and control. Because the organism enters through tissue wounds, especially those associated with oral trauma, feedstuffs should be closely monitored for coarse material and foreign bodies. b. Arcanobacterium Infection (Formerly actinomycosis, or "Lumpy Jaw ") Etiology. Arcanobacterium (formerly known as Actinomyces or Corynebacterium) pyogenes and A. bovis are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Arcanobacterium bovis is a normal part of the ruminant oral microflora and is the organism associated with "lumpy jaw" in cattle; this syndrome is rarely seen in sheep and goats. This organism has also been associated with pharyngitis and mastitis in cattle. Clinical signs and diagnosis. Arcanobacterium bovis causes mandibular lesions primarily. The mass will be firm, non-painful, and immovable. Draining tracts may develop over time. If teeth roots become involved, painful eating and weight loss are evident. Radiographic studies are helpful for determining fistulas. Diagnosis is based on clinical signs, and culture is required to confirm Arcanobacterium. The prognosis is poor for lumpy jaw. Epizootiology and transmission. These organisms are normal flora of the gastrointestinal tracts of ruminants and gain entrance into the tissues through abrasions and penetrating wounds. Necropsy. Draining lesions with sulfurlike granules (as with actinobacillosis) are frequently observed. Pathogenesis. Arcanobacterium pyogenes is known to produce an exotoxin, which may be involved in the pathogenesis. Differential diagnosis. Actinobacillus lignieresii and caseous lymphadenitis are important differentials for draining tracts. A major differential for omphalophlebitis is an umbilical hernia, which will typically not be painful or infected. There are many differentials for septic joints and polyarthritis: Chlamydia spp., Mycoplasma spp., streptococci, coliforms, Erysipelothrix rhusiopathiae, Fusobacterium necrophorum, and Salmonella spp. Tumors, trauma to the affected area, such as the mandible, and dental disease or oral foreign body should also be considered. Prevention and control. Arcanobacterium bovis lesions can be prevented or minimized by feeds without coarse or sharp materials. Treatment. Penicillin or derivatives such as ampicillin or amoxicillin are treatments of choice. Sodium iodides (intravenous) and potassium iodides (orally) have been utilized also. Extended antibiotic therapy may be necessary. Surgical excision is an option. In addition to medications noted above, isoniazid is somewhat effective for A. bovis infections in nonpregnant cattle. Research complications. The possibility of long-term infection and long therapy are factors that will diminish the value of affected research animals. c. Actinomycosis Omphalophlebitis, omphaloarteritis, omphalitis, and navel ill are terms referring to infection of the umbilicus in young animals. Arcanobacterium pyogenes is the most common organism causing omphalophlebitis, an acute localized inflammation and infection of the external umbilicus. Most cases occur within the first 3 months of age, and animals are presented with a painful enlargement of the umbilicus. Animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, hematuria, and so on. Severe sequelae may include septicemia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, and endocarditis. Research complications. Young stock affected by omphalophlebitis may be inappropriate subjects because of growth setbacks and physiologic stresses from the infection. Affected adult animals will not thrive and, even with therapy, may not be appropriate research subjects. d. Anthrax Etiology. Bacillus anthracis is a nonmotile, capsulated, spore-forming, aerobic, gram-positive bacillus that is found in alkaline soil, contaminated feeds (such as bonemeal), and water. Common names for the disease anthrax include woolsorters' disease, splenic fever, charbon, and milzbrand. Clinical signs and diagnosis. Anthrax is a sporadic but very serious infectious disease of cattle, sheep, and goats characterized by septicemia, hyperthermia, anorexia, depression, listlessness, depression, and tremors. Subacute and chronic cases may occur also and are characterized by swelling around the shoulders, ventral neck, and thorax. The incubation period is 1 day to 2 weeks. Bloody secretions such as hematuria and bloody diarrhea often occur. Abortion and blood-tinged milk may also be noted. The disease is usually fatal, especially in sheep and goats, after 1–3 days. Death is the result of shock, renal failure, and anoxia. Diagnosis is based on the clinical signs of peracute deaths and hemorrhage. Stained blood smears may show short, single to chained bacilli. Blood may be collected from a superficial vein and submitted for culture. Epizootiology and transmission. Cattle and sheep tend to be affected more commonly than goats, because of grazing habits. Older animals are more vulnerable than younger, and bulls are more vulnerable than cows. Although the disease occurs worldwide, and even in cold climates, most cases in the United States occur in the central and western states, and outbreaks usually occur as the result of spore release after abrupt climatic changes such as heavy rainfall after droughts or during warmer, dryer months. Spores survive very well in the environment. The anthrax organisms (primarily spores) are generally ingested, sporulate, and replicate in the local tissues. Abrasive forages may play a role in infection. Transmission via insect bites or through skin abrasions rarely occurs. Necropsy. Necropsies should not be done around animal pens or pastures, and definitive diagnoses may be made without opening the animals. Incomplete rigor mortis, rapid putrefaction, and dark, uncoagulated blood exuding from all body orifices are common findings. Blood collected carefully and promptly from peripheral veins of freshly dead animals can be used diagnostically. Splenomegaly, cyanosis, epicardial and subcutaneous hemorrhages, and lymphadenopathy are characterisitic of the disease. Pathogenesis. The rapidly multiplying organisms enter the lymphatics and bloodstream and result in a severe septicemia and neurotoxicosis. Encapsulation protects the organisms from phagocytosis. Liberated toxins cause local edema. Differential diagnosis. Although anthrax should always be considered when an animal healthy the previous day dies acutely, other causes of acute death in ruminants should be considered, e.g., bloat, poisoning, enterotoxemia, malignant edema, blackleg, and black disease. Prevention and control. Outbreaks must be reported to state officials. Anthrax is of particular concern as a bioterrorism agent. Any vaccination programs should also be reviewed with regulatory personnel. Herds in endemic areas and along waterways are usually vaccinated routinely with the Sterne-strain spore vaccine (virulent, nonencapsulated, live). Careful hygiene and quarantine practices are crucial during outbreaks. Dead animals and contaminated materials should be incinerated or buried deeply. Biting insects should be controlled. The disease is zoonotic and a serious public health risk. Treatment. Treatment of animals in early stages with penicillin and anthrax antitoxin (hyperimmune serum, if available) may be helpful. Amoxicillin, erythromycin, oxytetracycline, gentamicin, and fluoroquinolones are also good therapeutic agents. During epidemics, animals should be vaccinated with the Sterne vaccine. Research complications. Natural and experimental anthrax infections are a risk to research personnel; the pathogen may be present in many body fluids and can penetrate intact skin. The organism sporulates when exposed to air, and spores may be inhaled during postmortem examinations. e. Brucellosis Etiology. Brucella is a nonmotile, non-spore-forming, nonencapsulated, gram-negative coccobacillus. Brucella abortus is one of several Brucella species that infects domestic animals but cross-species infections occur rarely. Brucella abortus or B. melitensis may cause brucellosis in sheep, cattle, and goats. Brucella melitensis (biovar 1, 2, or 3) is the primary cause of sheep disease ( Garin-Bastuji et al., 1998 ). Brucella ovis is more commonly associated with ovine epididymitis or orchitis than abortion. In the United States, clusters of brucellosis are still found in western areas contiguous to Yellowstone National Park. Bang's disease is the common name given to the disease in ruminants. Clinical signs and diagnosis. Brucella melitensis in the adult ewe is generally asymptomatic and self-limiting within about 3 months. However, because the organism may enter and cause necrosis of the chorionic villi and fetal organs, abortion or stillbirths may occur. Abortion usually occurs in the third trimester, after which the ewe will appear to recover. It has been reported that up to 20% of infected ewes may abort more than once. Rams will also be infected and may develop orchitis or pneumonia. The disease caused by B. ovis is manifested by clinical or subclinical infection of the epididymis, leading to epididymal enlargement and testicular atrophy. Brucella ovis causes decreased fertility. Brucella melitensis is the more common cause of brucellosis in goats. Brucella abortus has been shown to infect goats in natural and experimental infections, and B. ovis has also been shown to infect goats experimentally. Does infected with B. melitensis will also abort during the third trimester. Infections with B. abortus in cattle produce few clinical signs. There may be a brief septicemia during which organisms are phagocytosed by neutrophils and fixed macrophages in lymph nodes. In cows, the organism localizes in supramammary lymph nodes and udders and in the endometrium and placenta of pregnant cows. Infection may cause abortions after the fifth month, with resulting retained placentas. Permanent infection of the udder is common and results in shedding of organisms in milk. In bulls, the organism may cause unilateral orchitis and epidydimitis and involvement of the secondary sex organs. Organisms may be in the semen. In infected herds, lameness may also be a clinical sign. Diagnosis of brucellosis can be made by bacterial isolation of the Brucella organism from necropsy samples (especially the fetal stomach contents), as well as by supportive serological evidence. Many serological tests are available, such as the tube and plate agglutination tests, the card or rose bengal test, the rivanol precipitation test, complement fixation, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and others. Test selection is often dependent on state requirements in the United States. Epizootiology and transmission. The primary route of transmission of B . abortus is ingestion of the organism from infected tissues and fluids (milk, vaginal and uterine discharges) during and for a few weeks after abortion or parturition; contaminated semen is considered to be a minor source of infection. Exposure to the organism may occur via the gastrointestinal tract (contaminated feed or water), the respiratory tract (droplet infection), or the reproductive tract (contaminated semen) and through other mucous membranes such as the conjunctiva. Brucella ovis is transmitted in the semen, as well as orally or nasally through contaminated feed and bedding. Necropsy findings. A sheep fetus aborted due to Brucella will exhibit generalized edema. The liver and spleen will be swollen, and serosal surfaces will be covered with petecchial hemorrhages. Peritoneal and pleural cavities often contain serofibrinous exudates. The placenta will be leathery. Pathogenesis. Ruminants are considered especially susceptible to Brucella infection, because of higher levels of erythritol (a sugar alcohol), which is a growth stimulant for the organism. Brucella utilizes erythritol preferentially over glucose as an energy source. Placentas and male genitalia also contain high levels of erythritol. Brucella organisms also evade lysis when phagocytosed by macrophages and neutrophils and survive intracellularly in phagosomes. Abortion is the result of placentitis, typically during the third trimester of gestation. Brucella ovis enters the host through the mucous membranes, then passes into the lymphatics, causes hyperplasia of reticuloendothelial cells, and is spread to various organs via the blood. The organism localizes in the epididymides, the seminal vesicles, the bulbourethral glands, and the ampullae. Orchitis may be a sequelae of the disease. Epididymitis can be diagnosed by identifying gross lesions by palpation of the epididymides, by serological evidence of antibodies to B. ovis, and by semen cultures. Differential diagnosis. Differential diagnoses include all other abortion-causing diseases. Many other agents, such as Actinobacillus spp., Arcanobacterium (Actinomyces) pyogenes, Eschericia coli, Pseudomonas spp., Proteus mirabilis, Chlamydia, Mycoplasma, and others may be associated with ovine epididymitis and orchitis. A clinically and pathologically similar agent, Actinobacillus seminis, has been isolated from virgin rams. This organism has morphological and staining characteristics similar to those of B. ovis and complicates the diagnosis ( Genetzky, 1995 ). Prevention and control. The Rev 1 vaccine has been recommended for vaccination of ewe lambs in endemic areas, but this vaccine is not used in the United States. Separating young rams from potentially infected older males, sanitizing facilities, and vaccinating them with B. ovis bacterin can prevent the disease. Over the past 20 years, aggressive federal and state regulatory and cattle herd health programs in the United States have provided control and prevention mechanisms for this pathogen through a combination of serological monitoring of herds, slaughter of diseased animals, herd management, vaccination programs, and monitoring of transported animals. Most states are considered brucellosis-free in the cattle populations; thus, procurement of ruminants that have been exposed to this infectious agent will be unlikely. Cattle vaccination programs can be very successful when conducted on a herd basis to reduce likelihood of exposure. Strain 19 and the recently validated attentuated strain RB51 are live vaccines and can be used in healthy heifer calves 4–12 months old. Vaccination for older animals may be done under certain circumstances. Vaccination of bull calves is not recommended, because of low likelihood of spread through semen and possibility of vaccination-induced orchitis. The strain 19 vaccine induces long-term cell-mediated immunity, protects a herd from abortions, and protects the majority of a herd from reactors during a screening and culling program. The vaccine will not, however, protect the animals from becoming infected with B. abortus. Strain 19 vaccine induces an antibody response in cattle. The RB51 vaccine does not result in antibody titers and therefore is advantageous because infection with Brucella can be determined serologically. The RB51 vaccine has been designated as the official calfhood bovine brucellosis vaccine in the United States by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS) ( Stevens et al., 1997 ). Brucella vaccine should be administered to unstressed, healthy cattle, with attention to particular side effects of the vaccination material and to prevention of compounding stresses associated with weaning, regrouping, other management changes, and shipping. The RB51 is regarded as less pathogenic and abortigenic in cattle. Treatment. Definite confirmation of Brucella infection is important from the standpoint of public and herd health. Culling is considered the treatment of choice in cattle herds. Rams infected with B. ovis should be isolated and treated with tetracyclines. Research complications. Brucellosis represents a research complication as a cause of abortions and of infections in male ruminants. Impairment of the infected host's immune system, especially alteration of phagocytic cells where the bacteria stay in membrane-bound vesicles, should be considered. The potential complications of needle sticks by large-animal veterinarians with the strain 19 vaccine and the public health risks (undulant fever) are well known. Less is known presently regarding the RB51 vaccine effects in humans. Epidemiologic and diagnostic methodologies are being developed to track and monitor these cases. There is also a risk of human infection from handling infected materials during a dystocia or postmortem. Worldwide, B. melitensis is the leading cause of human brucellosis. f. Campylobacteriosis (Vibriosis) i. Campylobacter fetus subsp. intestinalis; C. jejuni infection (ovine vibriosis) Etiology. Campylobacter (Vibrio) fetus subsp. intestinalis, a pleomorphic curved to coccoid, motile, non-spore-forming, gram-negative bacterium, causes campylobacteriosis, the most important cause of ovine abortion in the United States. There are few reports of campylobacteriosis in goats in the United States. Vibriosis is derived from the name formerly given to the genus; the term is still frequently used. Clinical signs and diagnosis. Ovine vibriosis is a contagious disease that causes abortion, stillbirths, and weak lambs. The organism inhabits the intestines and gallbladder in subclinical carriers. Abortion generally occurs in the last trimester, and abortion storms may occur as more susceptible animals, such as maiden ewes, become exposed to the infectious tissues. It is reported that 20–25% of the flock may become infected and up to 5% of the ewes will die ( Jensen and Swift, 1982 ). Some lambs may be born alive but will be weak, and dams will not be able to produce milk. Diagnosis is achieved by microscopic identification or isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharges. Tentative identification of the organism can be made by observing curved ("gull-wing") rods in Giemsa-stained or Ziehl–Neelsen–stained smears from fetal stomach contents, placentomes, or maternal uterine fluids. Epizootiology and transmission. Campylobacteriosis occurs worldwide. Campylobacter spp., such as C. jejuni, normally inhabit ovine gastrointestinal tracts. Transmission of the disease occurs through the gastrointestinal tract, followed by shedding, especially associated with aborted tissues and fluids. In abortion storms, considerable contamination of the environment will occur due to placenta, fetuses, and uterine fluids. Ewes may have active Campylobacter organisms in uterine discharges for several months after abortion. The bacteria will also be shed in feces, and feed and water contamination serve as another source. There is no venereal transmission in the ovine. Necropsy. Aborted fetuses will be edematous, with accumulation of serosanguinous fluids within the subcutis and muscle tissue fascia. The liver may contain 2–3 cm pale foci. Placental tissues will be thickened and edematous and will contain serous fluids similar to those of the fetus. The placental cotyledons may appear gray. Pathogenesis. The organism enters the bloodstream and causes a short-term bacteremia (1–2 weeks) prior to the localizing of the bacteria in the chorionic epithelial cells and finally passing into the fetus. Differential diagnosis. Toxoplasma, Chlamydia, and Listeria should be considered in late gestation ovine abortions. Prevention and control. A bacterin is available to prevent the disease. Carrier states have been cleared by treating with a combination of antibiotics, including penicillin and oral Chlortetracycline. Aborting ewes should be isolated immediately from the rest of the flock. After an outbreak, ewes will develop immunity lasting 2–3 years. Treatment. Infected animals should be isolated and provided with supportive therapy. Prompt decontamination of the area and disposal of the aborted tissues and discharges are important. Research complications. Losses from abortion may be considerable. Campylobacter ssp. are zoonotic agents, and C. fetus subsp. intestinalis may be the cause of "shepherd's scours." ii. Campylobacter fetus subsp. venerealis infection (bovine vibriosis) Etiology. Campylobacter fetus subsp. venerealis is the main cause of bovine campylobacteriosis abortions. It does not cause disease in other ruminant species. Clinical signs and diagnosis. Preliminary signs of a problem in the herd will be a high percentage of cows returning to estrus after breeding and temporary infertility. This will be particularly apparent in virgin heifers that may return to estrus by 40 days after breeding. Long interestrous intervals also serve an indication of a problem. Spontaneous abortions will occur in some cases, typically during the fourth to eighth months of gestation. Severe endometritis may lead to salpingitis and permanent infertility. Demonstration or isolation of the organism, a curved rod with corkscrew motility, is the basis for diagnosis. The vaginal mucous agglutination test is used to survey herds for campylobacteriosis. Serology will not be worthwhile, because the infection does not trigger a sufficient antibody response. Culture from breeding animals may be difficult because Campylobacter will be overgrown by faster-growing species also present in the specimens. Epizootiology and transmission. The bacteria is an obligate, ubiquitous organism of the genital tract. Transmission is from infected bulls to heifers. Older cows develop effective immunity. Necropsy findings. Necrotizing placentitis, dehydration, and fibrinous serositis will be found grossly. In addition, bronchopneumonia and hepatitis will be seen histologically. Pathogenesis. Campylobacter organisms grow readily in the genital tract, and infection is established within days of exposure. The resulting endometritis prevents conception or causes embyronic death. Differential diagnosis. The primary differential diagnosis for campylobacteriosis is trichomoniasis. Other venereal diseases should be considered when infertility problems are noted in a herd. These include brucellosis, mycoplasmosis, ureaplasmosis, infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV), and bovine virus diarrhea (BVD). Leptospirosis should also be considered. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. Killed bacterin vaccines are available, either as oil adjuvant or as aluminum hydroxide adsorbed. The former is preferred because of duration of immunity but causes granulomas. That vaccine also has specific recommendations regarding administration several months before the breeding season. The latter product is administered closer to the breeding season, and the duration of immunity is not as prolonged. In both cases, boosters should be given after the initial immunization and as part of the regular prebreeding regimen. Only one bacterin product is approved for use in bulls. Many combination vaccine products contain only the aluminum hydroxide adsorbed product. Artificial insemination (AI) is particularly useful at controlling the disease, but bulls used for AI must be part of a screening program for this and other venereal diseases such as trichomoniasis. Treatment. Cows will usually recover from the infection, and treatment with antibiotics such as penicillin, administered as an intrauterine infusion, improve the chances of returning to breeding condition. g. Caprine Staphylococcal Dermatitis Etiology. The most common caprine bacterial skin infection is caused by Staphylococcus intermedius or S. aureus and is known as staphylococcal dermatitis ( Smith and Sherman, 1994 ). The Staphylococcus organisms are cocci and are categorized as primary pathogens or ubiquitous skin commensals of humans and animals. Staphylococcus aureus and S. intermedius are classified as primary pathogens and produce coagulase, a virulence factor. Clinical signs and diagnosis. Small pustular lesions, caused by bacterial infection and inflammation of the hair follicle, occur around the teats and perineum. Occasionally, the infection may involve the flanks, underbelly, axilla, inner thigh, and neck. Staphylococcal dermatitis may occur secondary to other skin lesions. Diagnosis is based on lesions. Culture will distinguish S. aureus. Pathogenesis. Simple boredom may cause rubbing, followed by staphylococcal infection of damaged epidermis. Differential diagnosis. The presence of scabs makes contagious ecthyma a differential diagnosis, along with fungal skin infections and nutritional causes of skin disease. Treatment. Severe infections should be treated with antibiotics based on culture and sensitivity. Severe lesions and lesions localized to the underbelly, thighs, and udder benefit by periodic cleaning with an iodophor shampoo and spraying with an antibiotic and an astringent ( Smith and Sherman, 1994 ). h. Clostridial Diseases i. Clostridium perfringens type C infection (enterotoxemia and struck) Etiology. Clostridium perfringens is an anaerobic, gram-positive, nonmotile, spore-forming bacterium that lives in the soil, in contaminated feed, and in gastrointestinal tracts of ruminants. The bacteria is categorized by toxin production. Toxins include alpha (hemolytic), beta (necrotizing), delta (cytotoxic and hemoltyic), epsilon, and iota. Types of C. perfingens are A, B, C, D, and E. This is a common and economically significant disease of sheep, goats, and cattle. Clinical signs and diagnosis. The beta toxin associated with overgrowth of this bacterium results in a fatal hemorrhagic enterocolitis within the first 72 hr of a young ruminant's life. Many animals may be found dead, with no clinical presentation. Affected animals are acutely anemic, dehydrated, anorexic, restless, and depressed and may display tremors or convulsions as well as abdominal pain. Feces may range from loose gray-brown to dark red and malodorous. Morbidity and mortality may be nearly 100%. A similar noncontagious but acutely fatal form of enterotoxemia in adult sheep, called struck, occurs in yearlings and adults. Struck is rare in the United States. The disease is also caused by the beta toxin of C. perfringens type C and is often associated with rapid dietary changes or shearing stresses in sheep. Although affected animals are usually found dead, clinical signs include uneasiness, depression, and convulsions. Mortality is usually less than 15%. Diagnosis is usually based on necropsy findings, although confirmation can be made by culture of the organism. Identification of the beta toxin in intestinal contents may be difficult because of instability of the toxin. Epizootiology and transmission. Clostridial organisms are ubiquitous in the environment as well as in the gastrointestinal tract and contaminated feeds. Confinement and poor sanitation predisposes to infection with C. perfringens. Transmission is by ingestion of contaminated material. Necropsy findings. Necropsy findings include a milk-filled abomasum, and hemorrhage in the distal small intestine and throughout the large intestine. Petechial hemorrhages of the serosal surfaces of many organs, especially the thymus, heart, and gastrointestinal tract, will be visible. Hydropericardium, hydroperitoneum, and hemorrhagic mesenteric lymph nodes will also be present. Pulmonary and brain edema may also be seen. Histologically, the gram-positive C. perfringens organisms may be visible in excess numbers along the mucosal surface of the swollen, congested, necrotic intestines. In cases of struck, necropsy findings include congestion and erosions of the mucosa of the gastrointestinal tract, serosal hemorrhages, and serous peritoneal and pericardial fluids. In late stages of the disease and especially if prompt necropsy is not performed, the organism will infiltrate the muscle fascial layers and produce serohemorrhagic and gaseous infiltration of perimysial and epimysial spaces. Pathogenesis. Hemorrhagic enterotoxemia is an acute, sporadic disease caused by the beta toxin of Clostridium perfringens type C. Neonates ingest the organism, which then proliferates and attaches to the gastrointestinal microvilli and elaborates primarily the beta toxins. The trypsin inhibitors present in colostrum prevent inactivation of the beta toxin. The toxins injure intestinal epithelial cells and then enter the blood, leading to acute toxemia. The intestinal injury may result in diarrhea, with small amounts of hemorrhage. Associated electrolyte and water loss result in dehydration, acidosis, and shock. Differential diagnosis. Differential diagnoses include other clostridial diseases such as blackleg and black disease, as well as coccidiosis, salmonellosis, anthrax, and acute poisoning. Prevention and control. A commercial toxoid is available and should be administered to the pregnant animals prior to parturition. An alternative includes administration of an antitoxin to the newborn lambs. The disease may become endemic once it is on the premises. Treatment. Treatment is difficult and usually unsuccessful. Antitoxin may be useful in milder cases, and the antitoxin and toxoid can also be administered during an outbreak. Research complications. This disease can be costly in losses of neonates and younger animals. ii. Clostridium perfringens type D infection (pulpy kidney disease) Etiology. Clostridium perfringens type D releases epsilon toxin that is proteolytically activated by trypsin. This disease caused by C. perfringens tends to be associated with sheep and is of less importance in goats and cattle. Clinical signs. The peracute condition in younger animals is characterized by sudden deaths, which are occasionally preceded by neurological signs such as incoordination, opisthotonus, and convulsions. Because the disease progresses so rapidly to death (within 1–2 hr), clinical signs are rarely observed. Hypersalivation, rapid respirations, hyperthermia, convulsions, and opisthotonus have been noted. In acute cases, hyperglycemia and glucosuria are considered almost pathognomonic. Clinical signs in chronic cases in older animals, such as adult goats, include soft stools, weight loss, anorexia, depression, and severe diarrhea, sometimes with mucus and blood. Mature affected sheep may be blind and anorectic and may head-press. Necropsy findings. Necropsy findings are similar to those seen with C. perfringens type C. Additionally, extremely necrotic, soft kidneys ("pulpy kidneys") are usually observed immediately following death. (This phenomenon is in contrast to what is normally associated with later stages of postmortem autolysis.) Focal encephalomalacia, and petechial hemorrhages on serosal surfaces of the brain, diaphragm, gastrointestinal tract, and heart are common findings. Diagnosis can be made from the typical clinical signs and necropsy findings as well as the observation of glucose in the urine at necropsy. Pathogenesis. The epsilon toxin causes neuronal death and shock, probably through vascular damage. The noncontagious, peracute form of enterotoxemia occurs in suckling, fast-growing animals, either nursing from their dams or on high-protein, high-energy concentrates. The largest, fastest-growing animals generally are predisposed to this condition; for example, lambs, fat ewe lambs, and usually singleton lambs tend to be most susceptible. The hyperglycemia and glucosuria seen in acute cases are due to epsilon toxin effects on liver glycogen metabolism. Differential diagnosis. Tetanus, enterotoxigenic E. coli, botulism, polioencephalomalacia, grain overload, and listeriosis are differentials. Prevention and control. Vaccination prevents the disease. Maternal antibodies last approximately 5 weeks postpartum; thus young animals should be vaccinated at about this time. Feeding regimens to young, fast-growing animals and feeding of concentrates to adults should be evaluated carefully. Treatment. Treatment consists of support (fluids, warmth), antitoxin administration, oral antibiotics, and diet adjustment. iii. Clostridium tetani infection (tetanus, lockjaw) Etiology. Clostridium tetani is a strictly anaerobic, motile, spore-forming, gram-positive rod that persists in soils and manure and within the gastrointestinal tract. At least 10 serotypes of C. tetani exist. Clinical signs. Infection by C. tetani is characterized by a sporadic, acute, and fatal neuropathy. After an incubation period of 4 days to 3 weeks, the animal exhibits bloat; muscular spasticity; prolapse of the third eyelid; rigidity and extension of the limbs, leading to a stiff gate; an inability to chew; and hyperthermia. Erect or drooped ears, retracted lips, drooling, hypersensitivity to external stimuli, and a "sawhorse" stance are frequent signs. The animal may convulse. Death occurs within 3–10 days, and mortality is nearly 100%, primarily from respiratory failure. Diagnosis is based on clinical signs. Muscle-related serum enzymes such as aspartate aminotransferase (AST), creatinine kinase (CK), and lactate dehydrogenase (LDH) might be elevated. ( Jensen and Swift, 1982 ). Serum cortisol may also be elevated, and stress hyperglycemia may be evident. Permanent lameness may result in survivors. Epizootiology and transmission. Clostridium tetani is a soil contaminant and is often found as part of the gut microflora of herbivores. The organisms sporulate and persist in the environment. All species of livestock are susceptible, but sheep and goats are more susceptible than cattle. Individual cases may occur, or herd outbreaks may follow castration, tail docking, ear tagging, or dehorning. Mouth wounds may also be sites of entry. Pathogenesis. Tetanus, or lockjaw, is caused by the toxins of C. tetani. All serovars produce the same exotoxin, which is a multiunit protein composed of tetanospasmin, which is neurotoxic, and tetanolysin, which is hemolytic. A nonspasmogenic toxin is also produced. Contamination of wounds results in anaerobic proliferation of the bacterium and liberation of the tetanospasmin, which diffuses through motor neurons in a retrograde direction to the spinal cord. The toxin inhibits the release of glycine and γ-aminobutyric acid from Renshaw cells; this results in hypertonia and muscular spasms. Proliferation of C. tetani in the gut of affected animals may also serve as a source and may produce clinical signs. The uterus is the most common site of infection in postparturient dairy cattle with retained placentas. Differential diagnoses. Early in the course of the infection, differential diagnoses include bloat, rabies, hypomagnesemic tetany, polioencephalomalacia, white muscle disease, enterotoxemia in lambs, and lead poisoning. Polyarthritis of cattle is a differential for the gait changes in that species. Necropsy findings. Findings are nonspecific except for the inflammatory reaction associated with the wound. Because of the low number of organisms necessary to cause neurotoxicosis, isolation of C. tetani from the wound may be difficult. Treatment. Treatment consists of cleaning the infected wound; administering tetanus antitoxin (e.g., at least 500 IU in an adult sheep or goat); vaccinating with tetanus toxoid; administering of antibiotics (penicillin, both parenterally [potassium penicillin intravenously and procaine penicillin intramuscularly] and flushed into the cleaned wound), a sedative or tranquilizer (e.g., acepromazine or chlorpromazine) and a muscle relaxant; and keeping the animal in a dark, quiet environment. Supportive fluids and glucose must be administered until the animal is capable of feeding. If the animal survives, revaccination should be done 14 days after the previous dose. Prevention and control. Like other ubiquitous clostridial diseases, tetanus is impossible to eradicate. The disease can be controlled and prevented by following good sanitation measures, aseptic surgical procedures, and vaccination programs. Tetanus toxoid vaccine is available and very effective for stimulating long-term immunity. Tetanus antitoxin can be administered (200 IU in lambs) as a preventive or in the face of disease as an adjunct to therapy. Both the toxoid and the antitoxin can be administered to an animal at the same time, but they should not be mixed in the syringe, and each should be administered at different sites, with a second toxoid dose administered 4 weeks later. Animals should be vaccinated 2 or 3 times during the first year of life. Does and ewes should receive booster vaccinations within 2 months of parturition to ensure colostral antibodies. Research complications. Unprotected, younger ruminants may be affected following routine flock or herd management procedures. Contaminated or inadequately managed open wounds or lesions in older animals may provide anaerobic incubation sites. iv. Clostridium novyi infection (bighead; black disease; bacillary hemoglobinuria, or red water) and C. chauvoei infection (blackleg) Etiology. Clostridium novyi, an anaerobic, motile, spore-forming, gram-positive bacteria, is the agent of bighead and black disease. Clostridium novyi type D (C. hemolyticum) is the cause of bacillary hemoglobinuria, or "red water." Clostridium chauvoei is the causative agent of blackleg. Clinical signs. Bighead is a disease of rams characterized by edema of the head and neck. The edema may migrate to ventral regions such as the throat. Additional clinical signs include swelling of the eyelids and nostrils. Most animals will die within 48–72 hours. Black disease, or infectious necrotic hepatitis, is a peracute, fatal disease associated with C. novyi. It is more common in cattle and sheep but may be seen in goats. The clinical course is 1–2 days in cattle and slightly shorter in sheep. Otherwise healthy-appearing adult animals are often affected. Clinical signs are rarely seen, because of the peracute nature of the disease. Occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency are observed prior to death. Bacillary hemoglobinuria is an acute disease seen primarily in cattle and characterized by fever and anorexia, in addition to the hemoglobinemia and hemoglobinuria indicated by the name. Animals that survive a few days will develop icterus. Mortality may be high. Blackleg, a disease similar to bighead, causes necrosis and emphysema of muscle masses, serohemorrhagic fluid accumulation around the infected area, and edema ( Jackson et al., 1995 ). Blackleg is more common in cattle than in sheep. The incubation period is 2–5 days and is followed by hyperthermia, muscular stiffness and pain, anorexia, and gangrenous myositis. The clinical course is short, 24–48 hr, and untreated animals invariably die. Blackleg in cattle can be associated with subcutaneous edema or crepitation; these do not usually occur in sheep. Most lesions are associated with muscles of the face, neck, perineum, thigh, and back. Epizootiology and transmission. Bighead is caused by the toxins of C. novyi, which enters through wounds often associated with horn injuries during fighting. The C. novyi type B organisms produce alpha and beta toxins, and the alpha toxins are mostly responsible for toxemia, tissue necrosis, and subsequent death. Clostridium novyi type D is endemic in the western United States. It is hypothesized that the C. chauvoei organisms enter through the gastrointestinal tract. Black disease and bacillary hemoglobinuria are associated with concurrent liver disease, often associated with Fasciola infections (liver flukes); it is sometimes seen as a sequela to liver biopsies. The diseases are more common in summer months, and fecal contamination of pastures, flooding, and infected carcasses are sources of the organism. Birds and wild animals may be vectors of the pathogen. Ingested spores are believed to develop in hepatic tissue damaged and anoxic from the fluke migrations. Necropsy. Diagnosis of black disease is usually based on postmortem lesions. Subcutaneous vessels will be engorged with blood, resulting in dried skin with a dark appearance. Carcasses putrefy quickly. In addition, hepatomegaly and endocardial hemorrhages are common, and hepatic damage from flukes may be so severe that diagnosis is difficult. Blood coagulates slowly in affected animals. Pathogenesis. The propagation of the clostridial organisms is self-promoted by the damage caused by the toxins and the increased local anaerobic environment created. Clostridium novyi proliferates in the soft tissues of the head and neck, and the resultant clostridial toxin causes increased capillary permeability and the liberation of serous fluids into the tissues. Mixed infections with related clostridial organisms may lead to increasing hemorrhage and necrosis in the affected tissues. Diagnosis is based on clinical signs. In black disease and bacillary hemoglobinuria disease, the ingested clostridial spores are absorbed, enter the liver, and cause hepatic necrosis. Associated toxemia causes subcutaneous vascular dilatation; increased pericardial, pleural, and peritoneal fluid; and endocardial hemorrhages. The toxins produced by C. novyi, identified as beta, eta, and theta, and each having enzymatic or lytic properties or both, also contribute to the hemolytic disease. Clostridium chauvoei spores proliferate in traumatized muscle areas damaged by transportation, rough handling, or injury. Differential diagnosis. Differential diagnoses include other clostridial diseases as well as photosensitization. Hemolytic diseases such as babesiosis, leptospirosis, and hemobartonellosis should be included as differentials. Treatment. For C. chauvoei infection (blackleg), early treatment with penicillin or tetracycline may be helpful. Treatment for black disease is not rewarding even if the animal is found before death. Carcasses from bacillary hemoglobinuria losses should be burned, buried deeply, or removed from the premises. Prevention and control. Vaccinating animals with multivalent clostridial vaccines can prevent these diseases. Subcutaneous administration of vaccine material is recommended over intramuscular. Vaccinations may be useful in an outbreak. Careful handling of ruminants during shipping and transfers will contribute to fewer muscular injuries. For bighead, mature rams penned together should be monitored for lesions, especially during breeding season. Control of fascioliasis is very important in prevention and control of black disease and in the optimal timing of vaccinations. v. Clostridium septicum infection (malignant edema) Etiology. Clostridium septicum is the species usually associated with malignant edema, but mixed infections involving other clostridial species such as C. chauvoei, C. novyi, C. sordellii, and C. perfringens may occur. Clostridium spp. are motile (C. chauvoei, C. septicum) or nonmotile, anaerobic, spore-forming, gram-positive rods. Clinicial signs. Malignant edema, or gas gangrene, is an acute and often fatal bacterial disease caused by Clostridium spp. The incubation period is approximately 2–4 days. The affected area will be warm and will contain gaseous accumulations that can be palpated as crepitation of the subcutaneous tissue around the infected area. Regional lymphadenopathy and fever may occur. The animal becomes anorexic, severely depressed, and possibly hyperthermic. Edema and crepitation may be noted around the wound; death occurs within 12 hr to 2 days. Epizootiology and transmission. The organisms are ubiquitous in the environment and may survive in the soil for years. The disease is especially prevalent in animals that have had recent wounds such as those that have undergone castration, docking, ear notching, shearing, or dystocia. Necropsy findings. The tissue necrosis and hemorrhagic serous fluid accumulations resemble those of other clostridial diseases. Pathogenesis. In most cases, the clostridial organisms cause a spreading infection through the fascial planes around the area of the injury; vegetative organisms then produce potent exotoxins, which result in necrosis (alpha toxin) and/or hemolysis (beta toxin). Furthermore, the toxins enter the bloodstream and central nervous system, resulting in systemic collapse and high mortality. Necropsy. Spreading, crepitant lesions around wounds are suggestive of malignant edema. Affected tissues are inflamed and necrotic. Gas and serosanguineous fluids with foul odors infiltrate the tissue planes. Large rod-shaped bacteria may be observed on histopathology; confirmation is made through culture and identification. Intramuscular inoculation of guinea pigs causes a necrotizing myositis and death. Organisms can be cultured from guinea pig tissues. Treatment. Infected animals can be treated with large doses of penicillin and fenestration of the wound is recommended. Prevention and control. Proper preparation of surgical sites, correct sanitation of instruments and the housing environment, and attention to postoperative wounds will help prevent this disease. Multivalent clostridial vaccines are available. Research complications. Morbidity or loss of animals from lack of or unsuccessful vaccination and from contaminated surgical sites or wounds may be consequences of this disease. i. Colibacillosis Etiology. Escherichia coli is a motile, aerobic, gram-negative, non-spore-forming coccobacillus commonly found in the environment and gastrointestinal tracts of ruminants. Escherichia coli organisms have three areas of surface antigenic complexes (O, somatic; K, envelope or pili; and H, flagellar), which are used to "group" or classify the serotypes. Colibacillosis is the common term for infections in younger animals caused by this bacteria. Clinical signs. Presentation of E. coli infections vary with the animal's age and the type of E. coli involved. Enterotoxigenic E. coli infection causes gastroenteritis and/or septicemia in lambs and calves. Colibacillosis generally develops within the first 72 hr of life when newborn animals are exposed to the organism. The enteric infection causes a semifluid, yellow to gray diarrhea. Occasionally blood streaking of the feces may be observed. The animal may demonstrate abdominal pain, evidenced by arching of the back and extension of the tail, classically described as "tucked up." Hyperthermia is rare. Severe acidosis, depression, and recumbancy ensue, and mortality may be as high as 75%. The septicemic form generally occurs between 2 and 6 weeks of age. Animals display an elevated body temperature and show signs suggestive of nervous system involvement such as incoordination, head pressing, circling, and the appearance of blindness. Opisthotonos, depression, and death follow. Occasionally, swollen, painful joints may be observed with septicemic colibacillosis. Blood cultures may be helpful in identifying the septicemic form. In ruminants, E. coli is is a less common cause of cystitis and pyelonephritis. The cystitis is characterized by dysuria and pollakiuria; gross hematuria and pyuria may be present. The infection may or may not be restricted to the bladder; in the later presentation, and in cases of pyelonephritis, a cow will be acutely depressed, have a fever and ruminal stasis, and be anorexic. In chronic cases, animals will be polyuric and undergo weight loss. Escherichia coli may also cause in utero disease in cattle, resulting in abortion or weakened offspring. Epizootiology and transmission. Escherichia coli is one of the most common gram-negative pathogens isolated from ruminant neonates. Zeman et al. (1989 ) classify E. coli infections into four groups: enterotoxigenic, enterohemorrhagic, enteropathogenic, and enteroinvasive. Enterotoxigenic E. coli (ETEC) attach to the enterocytes via pili, produce enterotoxins, and are the primary cause of colibacillosis in animals and humans. Fimbrial (pili) antigens associated with ovine disease include K99 and F41. Enterohemorrhagic E. coli (EHEC) attach and efface the microvillus, produce verotoxins, and occasionally cause disease in humans and animals. Enteropathogenic E. coli (EPEC) colonize and efface the microvillus but do not produce verotoxins. EPEC are associated with disease in humans and rabbits and cause a secretory diarrhea. Enteroinvasive E. coli (EIEC) invade the enterocytes of humans and cause a shigella-like disease. Overcrowding and poor sanitation contribute significantly to the development of this disease in young animals. The organism will be endemic in a contaminated environment and present on dams' udders. The bacteria rapidly proliferate in the neonates' small intestines. The bacteria and associated toxins cause a secretory diarrhea, resulting in the loss of water and electrolytes. If the bacteria infiltrate the intestinal barrier and enter the blood, septicemia results. Diagnosis of the enteric form can be made by observation of clinical signs, including diarrhea and staining of the tail and wool. Necropsy findings. Swollen, yellow to gray, fluid-filled small and large intestines, swollen and hemorrhagic mesenteric lymph nodes, and generalized tissue dehydration are common. Septicemic lambs may have serofibrinous fluid in the peritoneal, thoracic, and pericardial cavities; enlarged joints containing fibrinopurulent exudates; and congested and inflamed meninges. Isolation and serotyping of E. coli confirm the diagnosis. ELISA and latex agglutination tests are available diagnostic tools. Differential diagnosis. Differential diagnoses include the enterotoxemias caused by C. perfringens type A, B, or C; Campylobacter jejuni; Coccidia, rotavirus, coronavirus, Salmonella, and Cryptosporidia. Other contributing causes of abomasal tympany in young ruminants, such as dietary changes, copper deficiency, excessive intervals between feedings of milk replacer, or feeding large volumes should be considered. Prevention and control. The best preventive measures are maintenance of proper housing conditions, limiting overcrowding, and frequently sanitizing lambing areas. Attention to colostrum feeding techniques and colostral quality are important means of preventing disease. Treatment must include intravenous fluid hydration and reestablishment of acid-base and electrolyte abnormalities. Treatment. Antibiotics such as trimethoprim-sulfadiazine, enrofloxacin, cephalothin, amikacin, and apramycin may be helpful; oral antibiotics are not recommended. Vaccines are available for prevention of colibacillosis in cattle. j. Corynebacterium pseudotuberculosis Infection (Caseous Lymphadenitis) Etiology. Corynebacterium pseudotuberculosis (previously C. ovis) are nonmotile, non-spore-forming, aerobic, short and curved, gram-positive coccobacilli. Caseous lymphadenitis (CLA) is such a common, chronic contagious disease of sheep and goats that any presentation of abscessing and draining lymph nodes should be presumed to be this disease until proven otherwise. The disease has been reported occasionally in cattle. Clinical signs and diagnosis. Abscessation of superficial lymph nodes, such as the superficial cervical, retropharyngeal, subiliacs (prefemoral), mammary, superficial inguinals, and popliteal nodes, and of deep nodes, such as mediastinal and mesenteric lymph nodes, is typical. Radiographs may be helpful in identifying affected central nodes. Peripheral lymph nodes may erode and drain caseous, "cheesy," yellow-green-tan secretions. The incubation period may be weeks to months. Over time, an infected animal may become exercise-intolerant, anorexic, and debilitated. Fever, increased respiratory rates, and pneumonia may also be common signs. Exotoxin-induced hemolytic crises may occur occasionally. Morbidity up to 15% is common, and morbid animals will often eventually succumb to the disease. Diagnosis is based on clinical lesions; ELISA serological testing is also available. Smears of the exudate or lymph nodes aspirates can be Gram-stained. Lymph node aspirates may also be sent for culturing. Epizootiology and transmission. The organism can survive for 6 months or more in the environment and enters via skin wounds, shearing, fighting, castration, and docking. Ingestion and aerosolization (leading to pulmonary abscesses) have been reported as alternative routes of entry. Necropsy findings. Disseminated superficial abscesses as well as lesions of the mediastinal and mesenteric lymph nodes will be identified. Cut surfaces of the affected lymph nodes may appear lamellated. Lungs, liver, spleen, and kidneys may also be affected. Cranioventral lung consolidation with hemorrhage, fibrin, and edema are seen histologically. Pathogenesis. Corynebacterium pseudotuberculosis produces an exotoxin (phospholipase D) that damages endothelial and blood cell membranes. This process enhances the organisms' ability to withstand phagocytosis. The infection spreads through the lymphatics to local lymph nodes. The necrotic lymph nodes seed local capillaries and hematogenously and lymphatically spread the organisms to other areas, especially the lungs. Differential diagnosis. Differentials include pathogens causing lymphadenopathy and abscessation. Treatment. Antibiotic therapy is not usually helpful. Abscesses can be surgically lanced and flushed with iodine-containing and/or hydrogen peroxide solutions. Abscessing lymph nodes can be removed entirely from valuable animals. During warmer months, an insect repellent should be applied to and around healing lesions. All materials used to treat animals should be disposed of properly. Because of the contagious nature of the disease, animals with draining and lanced lesions should be isolated from CLA-negative animals at least until healed. Commercial vaccines are available ( Piontkowski and Shivvers, 1998 ). Prevention and control. Minimizing contamination of the environment, using proper sanitation methods for facilities and instruments, segregating affected animals, and taking precautions to prevent injuries are all important. Research complications. This pathogen is a risk for animals undergoing routine management procedures or invasive research procedures, because of its persistence in the environment, its long clinical incubation period, and its poor response to antibiotics. k. Corynebacterium renale, C. cystitidis, and C. pilosum Infections (Pyelonephritis; Posthitis and Ulcerative Vulvovaginitis) Etiology. Corynebacterium renale, C. cystitidis, and C. pilosum are sometimes referred to as the C. renale group. These are piliated and nonmotile gram-positive rods and are distinguished biochemically. Corynebacterium renale causes pyelonephritis in cattle, and C. pilosum and C. cystitidis cause posthitis, also known as pizzle rot or sheath rot, in sheep and goats. In many references, all these clinical presentations are attributed to C. renale. Clinical signs and diagnosis. Acute pyelonephritis is characterized by fever, anorexia, polyuria, hematuria, pyuria, and arched back posture. Untreated infections usually become chronic, with weight loss, anorexia, and loss of production in dairy animals. Relapses are common, and some infections are severe and fatal. Diagnosis of pyelonephritis is based on urinalysis (proteinuria and hematuria) and rectal or vaginal palpation (assessing ureteral enlargement). Urine culturing may not be productive. In chronic cases, E. coli and other gram-negatives may be present. Posthitis and vulvovaginitis are characteriazed by ulcers, crusting, swelling and pain. The area may have a distinct malodor. Necrosis and scarring may be sequelae of more severe infections. Fly-strike may also be a complication. Diagnosis is based on clinical signs and on investigation of feeding regimens. Epizootiology and transmission. Ascending urinary tract infections with cystitis, ureteritis, and pyelonephritis are widespread problems, but incidence is relatively low. The vaginitis and posthitis contribute to the venereal transmission, but indirect transmission is possible because the organisms are stable in the environment and present on the wool or scabs shed from affected animals. Posthitis occurs in intact and castrated sheep and goats. Necropsy findings. Pyelonephritis, multifocal kidney abscessation, dilated and thickened ureters, cystitis, and purulent exudate in many sections of the urinary tract are common finding at gross necropsy. Pathogenesis. Corynebacterium renale is a normal inhabitant of bovine genitourinary tracts. The pilus mediates colonization. Conditions such as trauma, urinary tract obstruction, and anatomic anomalies may predispose to infection. In addition, more basic pH urine levels may block some immune defenses. Infections ascend through the urinary tract. The bacteria are urease-positive when tested in vitro, and the ammonia produced in vivo during an infection damages mucosal linings, with subsequent inflammation. Corynebacterium cystitidis and C. pilosum are normally found around the prepuce of sheep and goats. High-protein diets, resulting in higher urea excretion and more basic urine, are contributing factors. Posthitis and vulvovaginitis may develop within a week of change to the more concentrated or richer diet, such as pasture or the addition of high-protein forage. The ammonia produced irritates the preputial and vulvar skin, increasing the vulnerability to infection. Differential diagnosis. Urolithiasis is a primary consideration for these diseases. Contagious ecthyma should be considered for the crusting that is seen with posthitis and vulvovaginitis, although the lesions of contagious ecthyma are more likely to develop around the mouth. Ovine viral ulcerative dermatosis is also a differential for the lesions of posthitis and vulvovaginitis. Prevention and treatment. Because high-protein feed is often associated with posthitis and vulvovaginitis, feeding practices must be reconsidered. Clipping long wool and hair also is helpful. Treatment. Long-term (3 weeks) penicillin treatment is effective for pyelonephritis. Reduction of dietary protein, clipping and cleaning skin lesions, treating for or preventing fly-strike, and topical antibacterial treatments are effective for posthitis and vulvovaginitis; systemic therapy may be necessary for severe cases. Surgical debridement or correction of scarring may also be indicated in severe cases. l. Erysipelas Etiology. Erysipelothrix rhusiopathiae is a nonmotile, non-spore-forming, gram-positive rod that resides in alkaline soils. Clinical signs. Erysipelothrix causes sporadic but chronic polyarthritis in lambs less than 3 months of age. In older goats, erysipelas has been associated with joint infections. Epizootiology and transmission. The disease may follow wound inoculation associated with castration, docking, or improper disinfection of the umbilicus. Following wound contamination and a 1- to 5-day incubation period, the lamb exhibits a fever and stiffness and lameness in one or more limbs. Joints, especially the stifle, hock, elbow, and carpus, are tender but not greatly enlarged. Necropsy findings. Thickened articular capsules, mild increases in normal-appearing joint fluid and erosions of the articular cartilage are usually found. The joint capsule is infiltrated with mononuclear cells, but bacteria are difficult to find. Diagnosis is based on clinical signs of polyarthritis, and confirmation is made by culturing the organism from the joints. Differential diagnosis. Differential diagnoses include polyarthritis caused by chlamydia or other bacteria and stiffness caused by white muscle disease. Other bacteria causing septic joints include Areanobacterium pyogenes and Fusobacterium necrophorum. Caprine arthritis encephalitis (CAE) should also be considered. Prevention and control. Proper sanitation and prevention of wound contamination are important in preventing the infection in lambs. Screening of goat herds for CAE is recommended. Treatment. Erysipelas is sensitive to penicillin antibiotic therapy. m. Dermatophilosis (Cutaneous Streptothricosis, Lumpy Wool, Strawberry Foot Rot) Etiology. Dermatophilus congolensis is an aerobic, gram-positive, filamentous bacterium with branching hyphae. Dermatophilosis is a chronic bacterial skin disease characterized by crustiness and exudates accumulating at the base of the hair or wool fibers ( Scanlan et al., 1984 ). Clinical signs. Animals will be painful but will not be pruritic. Two forms of the disease exist in sheep: mycotic dermatitis (also known as lumpy wool) and strawberry foot rot. Mycotic dermatitis is characterized by crusts and wool matting, with exudates over the back and sides of adult animals and about the face of lambs. Strawberry foot rot is rare in the United States but is characterized by crusts and inflammation between the carpi and/or tarsi and the coronary bands. Animals will be lame. In goats and cattle, similar clinical signs of crusty, suppurative dermatitis are seen; the disease is often referred to as cutaneous streptothricosis in these species. Lesions in younger goats are seen along the tips of the ears and under the tail. Diagnosis is based on clinical signs as well as the typical microscopic appearance on stained skin scrapings, cultures, and serology. Epizootiology and transmission. The disease occurs worldwide, and the Dermatophilus organism is believed to be a saprophyte. Transmission occurs by direct or indirect contact and is aggravated by prolonged wet wool or hair associated with inclement weather. Biting insects may aid in transmission. Necropsy findings. Lymphadenopathy as well as liver and splenic changes may be observed. Histopathologically, superficial epidermal layers are necrotic and crusted with serum, white blood cells, and wool or hair. Dermal layers are hyperemic and edematous and may be infiltrated with mononuclear cells. Pathogenesis. Lesions typically begin around the muzzle and hooves and the dorsal midline. Prevention and control. Potash alum and aluminum sulfate have been used as wool dusts in sheep to prevent dermatophilosis. Minimizing moist conditions is helpful in controlling and preventing the disease. In addition, controlling external parasites or other factors that cause skin lesions is important. Lesions will resolve during dry periods. Treatment. Animals can be treated with antibiotics such as penicillin and oxytetracycline. Treating the animals with povidone-iodine shampoos or chlorhexidine solutions is also useful in clearing the disease. n. Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum Infection (Virulent Foot Rot; Contagious Foot Rot of Sheep and Goats; Foot Scald) Etiology. Two bacteria, Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum, work synergistically in causing contagious foot rot in sheep and goats. Other organisms may be involved as secondary invaders. Both Dichelobacter and Fusobacterium are nonmotile, non-spore-forming, anaerobic, gram-negative bacilli. Foot rot is a contagious, acute or chronic dermatitis involving the hoof and underlying tissues ( Bulgin, 1986 ). It is the leading cause of lameness in sheep. At least 20 serotypes of Dichelobacter are known. Arcanobacterium pyogenes may also contribute to the pathogenicity or to foot abscesses in goats. Foot scald, an interdigital dermatitis, is caused primarily by D. nodosus alone. Clinical signs. Varying degrees of lameness are observed in all ages of animals within 2–3 weeks of exposure to the organisms. Severely infected animals will show generalized signs of weight loss, decreased productivity, and anorexia associated with an inability to move. The interdigital skin and hooves will be moist, with a distinct necrotic odor. Morbidity may reach 70% in susceptible animals. Diagnosis is based on clinical signs. Smears and cultures confirm the definitive agents. Clinical signs of the milder disease, foot scald, include mild lameness, redness and swelling, and little to no odor. Epizootiology and transmission. Fusobacterium necrophorum is ubiquitous in soil and manure, in the gastrointestinal tract, and on the skin and hooves of domestic animals. In contrast, Dichelobacter contaminates the soil and manure but rarely remains in the environment for more than about 2 weeks. Some animals may be chronic carriers. Overcrowded, warm, and moist environments are key elements in transmission. Outbreaks are likely in the spring season. Shipping trailers and contaminated pens or yards should be considered also as likely sources of the bacteria. Pathogenesis. Both organisms are transmitted to the susceptible animal by direct or indirect contact. The organisms enter the hoof through injuries or through sites where Strongyloides papillosus larvae have penetrated. Fusobacterium necrophorum initiates the colonization and is followed by D. nodosus. The latter attaches and releases proteases; these cause necrosis of the epidermal layers and separation of the hoof from the underlying dermis. The pathogenicity of the serotypes of D. nodosus is correlated with the production of these proteases and numbers of pili. Additionally, F. necrophorum causes a severe, damaging inflammatory reaction. Differential diagnosis. Foot abscesses, tetanus, selenium/vitamin E deficiencies, copper deficiency, strawberry foot rot, bluetongue virus infection (manifested with myopathy and coronitis), and trauma are among the many differentials that must be considered. Treatment. Affected animals are best treated by manually trimming the necrotic debris from the hooves, followed by application of local antibiotics and foot wraps. Systemic antibiotics such as penicillin, oxytetracycline, and erythromycin may be used. Goats have improved dramatically when given a single dose of penicillin (40,000 U/kg) ( Smith and Sherman, 1994 ). Footbaths containing 10% zinc sulfate, 20% copper sulfate, or 10% formalin (not legal in all states) can be used for treatment as well as for prevention of the disease. Affected animals should be separated from the flock. Vaccination has been shown to be effective as part of the treatment regimen. Some breeds of sheep and some breeds and lines of goats are resistant to infection. Individual sheep may recover without treatment or are resistant to infection. Prevention and control. Prevention and control programs involve scrutiny of herd and flock management; quarantine of incoming animals; vaccination; segregation of affected animals; careful and regular hoof trimming; discarding trimmings from known or suspected infected hooves; maintaining animals in good body condition; avoiding muddy pens and holding areas; and culling individuals with chronic and nonresponsive infections. Dichelobacter nodosus bacterins are commercially available; cross protection between serotypes varies. Biannual vaccinination in wet areas may be essential. Some breeds may develop vaccination site lumps. Footbaths of 10% zinc sulfate, 10% formalin (where allowed by state regulations), or 10% copper sulfate are also considered very effective preventive measures. Goats are less sensitive than sheep to the copper in the footbaths. Research complications. Treating and controlling foot rot is costly in terms of time, initial handling and treatments and their follow-up, housing space, and medications. o. Fusobacterium necrophorum and Bacteroides melaninogenicus Infection (Foot Rot of Cattle, Interdigital Necrobacillosis of Cattle) Etiology. Interdigital necrobacillosis of cattle is caused by the synergistic infection of traumatized interdigital tissues by Fusobacterium necrophorum and Bacteroides melaninogenicus. Like F. necrophorum, B. melaninogenicus is a nonmotile, anaerobic, gram-negative bacterium. Dichelobacter nodosus, the agent of interdigital dermatitis, may be present in some cases. This is a common cause of lameness in cattle. Clinical signs. Clinical signs include mild to moderate lameness of sudden onset. Hindlimbs are more commonly affected, and cattle will often flex the pastern and bear weight only on the toe. The interdigital space will be swollen, as will be the coronet and bulb areas. Characteristic malodors will be noted, but there will be little purulent discharge. In more severe cases, animals will have elevated body temperature and loss of appetite. The lesions progress to fissures with necrosis until healing occurs. The diagnosis is by the odor and appearance. Anaerobic culturing confirms the organisms involved. Epizootiology and transmission. Cases may be sporadic, or epizootics may occur. Bos taurus dairy breeds and animals with wide interdigital spaces are more commonly affected. The factors here are comparable to those present in foot rot of smaller ruminants. Necropsy findings. Findings at necropsy include dermatitis and necrosis of the skin and subcutaneous tissues. Although necropsy would rarely be performed, secondary osteomyelitis may be noted in severe cases by sectioning limbs. Pathogenesis. The bacteria enter through the skin of the interdigital area after trauma to the interdigital skin, from hardened mud, or from softening of the skin due to, for example, constant wet conditions in pens. Colonization leads to cellulitis. In addition, F. necrophorum releases a leukocidal exotoxin that reduces phagocytosis and causes the necrosis, whereas the tissues and tendons are damaged by the proteases and collagenases produced by B. melaninogenicus. Zinc deficiency may play a role in the pathogenesis in some situations. Differential diagnoses. The most common differentials for sudden lameness include hairy heel warts and subsolar abcesses. Bluetongue virus should also be considered. Grain engorgement and secondary infection from cracks caused by selenium toxicosis should also be considered. The exotic foot-and-mouth disease virus would be considered in areas where that pathogen is found. Prevention and control. As with foot rot in smaller ruminants, management of the area and herd are important. Paddocks and pens should be kept dry, well drained, and free of material that will damage feet. Footbaths and chlortetracycline in the feed have been shown to control incidence. Affected animals should be segregated during treatment. Chronically affected or severely lame animals should be culled. New cattle should be quarantined and evaluated. Treatment. Successful treatment regimens that result in healing within a week include cleaning the feet and trimming necrotic tissue; parenteral antimicrobials, such as oxytetracycline or procaine penicillin, or sulfonomethazine in the drinking water or tetracyclines in feed; and footbaths (such as 10% zinc sulfate, 2.5% formalin, or 5% copper sulfate) twice a day. In severe cases, more aggressive therapy such as bandaging the feet or wiring the digits together may be needed. Animals can recover without treatment but will be lame for several weeks. Acquired immunity is reported to be poor. Research complications. Research complications are comparable to those noted for foot rot in smaller ruminants. p. Fusobacterium necrophorum infection (Foot Abscesses) Fusobacterium necrophorum is also associated with foot abscesses, the infection of the deeper structures of the foot, in sheep and goats. Only one claw of the affected hoof may be involved. The animals will be three-legged lame, and the affected hoof will be hot. Pockets of purulent material may be in the heel or toe. q. Heel Warts (Bovine Digital Dermatitis, Interdigital Papillomatosis, Papillomatous Digital Dermatitis, Foot Warts, Heel Warts, Hairy Foot Warts, Mortellaro's Disease) Etiology. Bacteria such as Fusobacterium spp., Bacteroides spp., and Dichelobacter nodosus have been isolated from bovine heel lesions. Spirochete-like organisms have also been shown in the lesions of cows with papillomatous digital dermatitis (PDD), in the United States and Europe; these have culturing requirements similar to those of Treponema species. Clinical signs. All lesions occur on the haired, digital skin. One or all feet may be affected. Most lesions occur on the plantar surface of the hindfoot (near the heel bulbs and/or extending from the interdigital space), but the palmar and dorsal aspect of the interdigital spaces may also be involved. Progression of lesions, typically over 2–3 weeks, includes erect hairs, loss of hair, and thickening skin. Moist plaques begin as red and remain red or turn gray or black. Exudate or blood may be present on the plaque. Plaques enlarge and "hairs" protrude from the roughened surface. Lesioned areas are painful when touched. The lesions may or may not be malodorous. Epizootiology and transmission. Facility conditions and herd management are considered contributing factors. The following have been examined as contributing factors: nutrition, particularly zinc deficiency; poorly drained, low-oxygen, organic material underfoot; poor ventilation; rough flooring; damp and dirty bedding areas; and overcrowding. These interdigital lesions occur commonly in young stock and in dairy facilities throughout the world. The disease is seen only in cattle. Pathogenesis. The organisms noted above, combined with poor facility and herd management, are critical in the pathogenesis. Differential diagnosis. Differentials for lameness will include sole abscesses, laminitis, and trauma. Prevention and control. Each facility and management condition noted above should be addressed in conjunction with appropriate antibiotic and/or antiseptic treatment regimens. All equipment used for hoof trimming must be cleaned and disinfected after every use. Trucks and trailers should also be sanitized between groups of animals. Treatment. Antibiotic and antiseptic regimens have been used successfully for this problem. Antibiotics include parenteral cephalosporins and pencillins, as well as topical tetracyclines with bandaging. Antiseptic or antibiotic solutions in footbaths include tetracyclines, zinc sulfate, lincomycin, spectinomycin, copper sulfate, and formalin. The footbaths must be well maintained, minimizing contamination by feces and other materials. Tandem arrangements, such as the cleaning footbaths and then the medicated footbaths, and preventing dilution from precipitation are useful. Other treatments such as surgical debridement, cryotherapy, and caustic topical solutions have been successful. Research complications. Infectious, contagious PPD is one of the major causes of lameness among heifers and dairy cattle and is a costly problem to treat. The outbreaks are generally worse in younger animals in chronically infected herds. The immune response is not well understood, and it may be temporary in older animals. r. Haemophilus somnus infection (Thromboembolic Meningoencephalitis) Etiology. Haemophilus somnus is a pleomorphic, nonencapsulated, gram-negative bacterium. Diseases caused by this organism include thromboembolic meningoencephalitis (TEME), septicemia, arthritis, and reproductive failures due to genital tract infections in males and females. Haemophilus somnus is a also major contributor to the bovine respiratory disease complex. Haemophilus spp. have been associated with respiratory disease in sheep and goats. Clinical signs. The neurologic presentation may be preceded by 1–2 weeks of dry, harsh coughing. Neurologic signs include depression, ataxia, falling, conscious proprioceptive deficits; signs such as head tilt from otitis interna or otitis media, opisthotonus, and convulsions may be seen as the brain stem is affected. High fever, extreme morbidity, and death within 36 hr may occur. Respiratory tract infections are usually part of the complex with infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza 3, Mycoplasma, and Pasteurella, and the synergism among these contributes to the signs of bovine respiratory disease complex (BRDC). In acute neurologic as well as chronic pneumonic infections, polyarthritis may develop. Abortion, vulvitis, vaginitis, endometritis, placentitis, and failure to conceive are manifestations of reproductive tract disease. In all cases, asymptomatic infections may also occur. Diagnosis based on culture findings is difficult because H. somnus is part of the normal nasopharyngeal flora. Paired serum samples are recommended; single titers in some animals seem to be high because of passive immunity, previous vaccination, or previous exposure. In cases of abortion, other causes should be eliminated from consideration. Epizootiology and transmission. Because the organism is considered part of the normal flora of cattle and can be isolated from numerous tissues, the distinction between the normal flora and the status of chronic carrier is not clear. Outbreaks are associated with younger cattle in feedlots in western United States, but stresses of travel and coinfection with other respiratory pathogens are involved in some cases. Adult cattle have also been affected. Vaccination for viral respiratory pathogens may increase susceptibility. Transmission is by respiratory and genital tract secretions. The organism does not persist in the environment. Necropsy findings. Pathognomonic central nervous system lesions include multifocal red-brown foci of necrosis and inflammation on and within the brain and the meninges. Many thrombi with bacterial colonies will be seen in these affected areas. Ocular lesions may also be seen, including conjunctivitis, retinal hemorrhages, and edema. Usually animals with neurological disease will not have respiratory tract lesions. The respiratory tract lesions include bronchopneumonia and suppurative pleuritis. When combined with Pasteurella infection, the pathology becomes more severe. Aborted fetuses will not show lesions, but necrotizing placentitis will be evident histologically. Pure cultures of H. somnus may be possible from these tissues. Pathogenesis. Inhalation of contaminated respiratory secretions from carrier animals is the primary means of transmission. The anatomical location of bacterial residence within the carriers has not been identified. After gaining access by way of the respiratory tract, the bacteria proliferate, and a bacteremia develops. The bacteria are phagocytosed by neutrophils but are not killed. The thrombosis formation is due to the adherence by the nonphagocytosed organisms to vascular endothelial cells, degeneration and desquamation of these cells, and exposure of subendothelial collagen, with subsequent initiation of the intrinsic coagulation pathway. Antigen-antibody complex formation, resulting in vasculitis, is also correlated with high levels of agglutinating antibodies. Differential diagnosis. Differentials in all ruminants include other pathogens associated with neurological disease and respiratory disease such as Pasteurella hemolytica, P. multocida, and P. aeruginosa. In smaller ruminants, Corynebacterium pseudotuberculosis should be considered. Prevention and control. Stressed animals or those exposed to known carriers can be treated prophylactically with tetracycline administered parenterally or orally (in the feed or water). The late-stage polyarthritis is resistant to antibiotic therapy, because of failure of the antibiotic to reach the site of infection. Planning vaccination programs carefully will decrease chances of outbreaks. For example, avoiding vaccinating animals for infectious bovine rhinotrachetitis and bovine viral diarrhea during times of stress to the cattle is worthwhile. Killed whole-cell bacterins are commercially available; these have been shown to be effective in controlling the respiratory disease presentation. Control of other clinical aspects of the H. somnus disease by these bacterins has not been well described. Treatment. Rapid treatment at the first signs of neurologic disease is important in an outbreak. Haemophilus somnus is susceptible to several antibiotics, such as Oxytetracycline and penicillin, and these are often used in sequence until the cattle are recovered. s. Leptospirosis Etiology. Seven different species of the spirochete genus Leptospira are now recognized, and pathogenic serovars exist within each species; previously pathogenic leptospires were all classified as members of the species L. interrogans. The serovars pomona, icterohaemorrhagiae, grippotyphosa, interrogans, and hardjo are recognized pathogens. Leptospira hardjo and L. pomona are the serovars most commonly diagnosed in cattle, with L. hardjo causing endemic infection. Leptospira hardjo is also the major sheep serovar. Goats are susceptible to several serovars. Clinical signs. Leptospirosis is a contagious but uncommon disease in sheep and goats. The disease may cause abortion, anemia, hemoglobinuria, and icterus and is often associated with a concurrent fever. After a 4- to 10-day incubation period, the organism enters the bloodstream and causes bacteremia, fever, and red-cell hemolysis. Leptospiremia may last up to 7 days. Immune stimulation is apparently rapid, and antibodies are detectable at the end of the first week of infection; cross-serovar protection does not occur. During active bacteremia, hemolysis may result in hemoglobin levels of 50% below normal. Hyperthermia, hemoglobinuria, icterus, and anemia may be observed during this phase, and ewes in late gestation may abort. Abortion usually occurs only once. Mortality rates of above 50% have been reported in infected ewes and lambs ( Jensen and Swift, 1982 ). Subclinical infection is more common in nonpregnant and nonlactating animals. Sheep infected with leptospirosis may display a hemolytic crisis associated with IgM acting as a cold-reacting hemagglutinin. Acute and chronic infections in cattle are more common than infections in sheep and goats. Acute forms in cattle display signs similar to those in sheep. Acute infection in calves may progress to meningitis and death. Lactating cows will have severe drops in production. Chronic cases may lead to abortion, with retained placenta, and weakened calves or animals that carry the infection. Infertility may also be a sequela. Epizootiology and transmission. Leptospires are a large genus, and leptospirosis is a complicated disease to prevent, treat, and control. The organism survives well in the environment, especially in moist, warm, stagnant water. Cattle, swine, and other domestic and wild animals are potential carriers of serovars common to particular regions. Wild animals often serve as maintenance hosts, but domestic livestock may be reservoirs also. Organisms are shed in urine, in uterine discharges, and through milk. Animals become carriers when they are infected with a host-adapted serovar; sporadic clinical disease is more commonly associated with exposure to a non-host-adapted serovar ( Heath and Johnson, 1994 ). Infection may occur via oral ingestion of contaminated feed and water, via placental fluids, or through the mucous membranes of the susceptible animal. Placental or venereal transmission may occur. As the organisms are cleared from the bloodstream, they chronically infect the renal convoluted tubules and the reproductive tract (and occasionally the cerebrospinal fluid or vitreous humor). Chronically infected animals may shed the organism in the urine for 60 days or longer. Necropsy. Diagnosis is confirmed by identification of leptospires in fetal tissues. The leptospires are visible in silver- or fluorescent antibody-stained sections of liver or kidney. Leptospires may also be seen under dark-field or phase-contrast microscopy of fetal stomach contents. Fetal and maternal serology, and diagnostic tests such as the microscopic agglutination test, are useful; interpretation is complicated because of cross reaction of antibodies to many serovars. Differential diagnosis. More than one serovar may cause infection in one animal, and each serovar should be considered as a separate pathogen. Because of the associated anemia, differential diagnoses should include copper toxicity and parasites, in addition to other abortifacient diseases. Prevention and control. Polyvalent vaccines, tailored to common serovars regionally, are available and effective for preventing leptospirosis in cattle. Immunity is serovar specific. Because serological titers tend to diminish rapidly (40–50 days in sheep [ Jensen and Swift, 1982 ]), frequent vaccination may be necessary. Other prevention measures such as species-specific housing, control of wild rodents, and proper sanitation should be instituted. Treatment. Antibiotic treatment is aimed at treating ill animals and trying to clear the carrier state. Treatment methods for acute leptospirosis include oxytetracycline for 3–6 days. Addition of oxytetracycline or chlortetracycline to the feed for 1 week may be helpful. These antibiotics are considered best for removal of the carrier state of some serovars. Vaccination and antibiotic therapy can be combined in an outbreak. Research complications. Leptospirosis is zoonotic and may be associated with flulike symptoms, meningitis, or hepatorenal failure in humans. t. Listeria (Circling Disease, Silage Disease) Etiology. Listeria monocytogenes is a pleomorphic, motile, non-spore-forming, β-hemolytic, gram-positive bacillus that inhabits the soil for long periods of time and has been often found in fermented feedstuffs such as spoiled silage. Of the 16 known serovars, several produce clinical signs in ruminants. Listeria ivanovii (associated with abortions in sheep) is serovar 5. Clinical signs. Listeriosis is an acute, sporadic, noncontagious disease associated with neurological signs or abortions in sheep and other ruminants. The overall case rate is low. The disease may present as an isolated case or with multiple animals affected. Three forms of disease are described: encephalitis, placentitis with abortion, and septicemia with hepatitis and pneumonia. The encephalitic form is most common in sheep; septicemic forms may occur in neonatal lambs ( Scarratt, 1987 ). Clinically, the encephalitic form begins with depression, anorexia, and mild hyperthermia after an incubation period of 2–3 weeks. As the disease progresses, animals exhibit nasal discharges and conjunctivitis and begin to walk in circles, as if disoriented. Facial paralytic lesions, including drooping of an ear or eyelid, dilation of a nostril, or strabismus occur unilaterally on the affected side as the result of dysfunction of some or all the cranial nerves V-XII. The neck will by flexed away from the affected side. Facial muscle twitching, protrusion of the tongue, dysphagia, hypersalivation, and nasal discharges may be noted. The hypersalivation may lead to metabolic acidosis in advanced cases in cattle. Anorexia, prostration, coma, and death follow. The placental form usually results in last-trimester abortions in ewes and does, which typically survive this form of the disease. The affected females may be asymptomatic or may show severe clinical signs such as fever and depression, with subsequent retained placenta or endometritis. Abortion usually occurs within 2 weeks of Listeria infection. In cattle, abortion occurs during the last 2 months of gestation and has been induced experimentally 6–8 days after exposure. Cows present with the range of clinical signs seen in smaller-ruminant dams. There is no long-term effect on the fertility of affected dams. Epizootiology and transmission. The organism is transmitted by oral ingestion of contaminated feeds and water or possibly by inhalation. By the oral route, the organism enters through breaks in the oral cavity and ascends to the brain stem by way of nerves. When severe outbreaks occur, feedstuffs should be assessed for spoilage. Listeria organisms can be shed by asymptomatic carriers, especially at the end of pregnancy and at lambing. Diagnosis and necropsy findings. Diagnosis is usually made from clinical signs. Culture confirms the diagnosis (cold enrichment at 20° C is preferable but not essential for isolation). Impression smears will show the pleomorphic gram-positive characterisitics of the pathogen. Tissue fluorescent antibody techniques may also be utilized. Gross lesions are not observed with the encephalitic form. Microscopic lesions include thrombosis, neutrophilic or mononuclear foci in areas of inflammation, and neuritis. The pons, medulla, and anterior spinal cord are primarily affected in the encephalitic form. Microabscesses of the midbrain are characteristic of Listeria encephalitis in sheep. Aborted fetuses that are intact may show fibrinous polyserositis, with excessive serous fluids; small, necrotic foci of the liver; and small abomasal erosions. Necrotic lesions of the fetal spleen and lungs may also be seen. In goats, Listeria-induced neurological lesions occur only in the brain stem. Placentitis, focal bronchopneumonia, hepatitis, splenitis, and nephritis may be seen with other forms. Pathogenesis. With the encephalitic form, the organism penetrates mucosal abrasions and enters the trigeminal or hypoglossal nerves. The Listeria organisms then migrate along the nerves and associated lymphatics to the brain stem (medulla and pons). In the septicemic form, the organism penetrates tissues of the gastrointestinal tract and enters the bloodstream, to be distributed to the liver, spleen, lungs, kidneys, and placenta. After infection, organisms are shed in all body secretions (infected milk is an important risk factor for zoonosis). A toxin produced by Listeria monocytogenes is correlated with pathogenicity, but the mechanism of the pathogenesis of this molecule has not been elucidated. Differential diagnoses. Rabies, bacterial meningitis, brain abscess, lead toxicity, and otitis media must be considered as differentials. In sheep, the differentials include organisms that cause abortion, and neurological signs, such as enterotoxemia due to Clostridium perfringens type D. In goats, the major differentials include caprine arthritis encephalitis viral infection and chlamydial and mycoplasmal infections. In both species, scrapie is a differential. In cattle, aberrant parasite migration or Hemophilus somnus infection must also be considered. Prevention and control. Affected dams should be segregated and treated. Other animals in the group may be treated with oxytetracycline as needed. Aborted tissues should be removed immediately. Proper storage of fermented feeds minimizes this source of contamination. When silage spoils, the pH increases, producing a suitable growth environment for the organism. Commercial vaccines are not available in the United States. Treatment. Affected animals can be treated aggressively with penicillin, ampicillin, oxytetracycline, or erythromycin. Exceptionally high levels of penicillin are required for treating affected cattle. Severely affected animals should receive appropriate fluid support and other nursing care. Treatment is less successful, and mortality is especially high in sheep. Recovered animals tend to resist reinfection. Research complications. In addition to the loss of fetal animals, stress to the dams, and risks to other animals, any aborted tissue by a ruminant should be regarded as a potential zoonotic risk. Listeria can cause mild to severe flulike symptoms in humans and may be a particular risk for pregnant women and for older or immune-compromised individuals. Listeriosis in humans is a reportable disease. u. Lyme Disease (Borrelia burgdorferi Infection, Borreliosis) Etiology. Lyme disease is caused by the spirochete Borrelia burgdorferi. Clinical signs and diagnosis. Reports in ruminants indicate seroconversion to B. burgdorferi, but there are few definitive correlations to the arthritis that is present. Diagnosis requires culturing from the affected joints and diagnostic elimination of other causes of lameness and arthritis. Epizootiology and transmission. The organism is present throughout much of the Northern Hemisphere and has been reported in many mammals and also in birds. Ticks of the Ixodes ricinus complex are the major vectors of the spirochete and must be attached for 24 hr for successful transmission. Pathogenesis. The Ixodes ticks have three life stages: larval, nymphal, and adult. Feeding occurs once during each stage, and wild animals are the source of blood meals. The larval stages feed from rodents, such as the white-footed deer mouse, Peromyscus leucopus, from which they acquire the spirochete. The nymphal stage is that which usually infects other animals. The adult ticks are usually found on deer. Differential diagnosis. Seroconversion to B. burgdorferi does not necessarily confirm the cause of arthritis. Other causes of arthritis and lameness in ruminants include trauma, caprine arthritis encephalitis virus, Mycoplasma spp., Chlamydia psittaci, Erysipelothrix spp., Arcanobacterium pyogenes, Brucella spp., and rickets. Prevention and control. Control of the tick vector is the most important factor in preventing the possibility of exposure or disease. Treatment. Antibiotic therapy, with tetracycline, penicillin, amoxicillin, and cephalosporins, is used for diagnosed or suspected Lyme arthritis. Research complications. Lyme disease is zoonotic, and the Ixodes ticks transmit the disease to humans. v. Mastitis i. Ovine mastitis Mastitis in ewes may be acute, subclinical, or chronic. Acute mastitis often results in anorexia, fever, abnormal milk, and swelling of the mammary gland. Pasteurella haemolytica is the most common cause of acute mastitis. Additional isolates may include, in order of prevalence, Staphylococcus aureus, Actinomyces (Corynebacterium) spp., and Histophilus ovis. Escherichia coli and Pseudomonas aeruginosa have also been found to cause acute mastitis. As many as six serotypes of Pasteurella haemolytica have been isolated from the mammary glands of mastitic ewes. Furthermore, intramammary inoculation of these organisms isolated from ovine and bovine pulmonary lesions has resulted in clinical mastitis in ewes ( Watkins and Jones, 1992 ). Subclinical mastitis is detected only indirectly, by counting somatic cells. The most common isolate from ewes with subclinical mastitis is coagulase-negative staphylococci. Other isolates include Actinomyces bovis, Streptococcus uberis, S. dysgalactiae, Micrococcus spp., Bacillus spp., and fecal streptococci. Most of these organisms are commonly found in the environment. Diffuse chronic mastitis, or hardbag, results from interstitial accumulations of lymphocytes in the udder. Both glands are usually affected, but no inflammation is present. Serological evidence suggests that diffuse chronic mastitis is caused by the retrovirus that causes ovine progressive pneumonia (OPP or maedi/visna virus). Other bacterial agents or Mycoplasma have not usually been isolated from udders with this type of mastitis. Acute mastitis occurs in approximately 5% of lactating ewes annually, and it usually occurs either soon after lambing or when lambs are 3–4 months old ( Lasgard and Vaabenoe, 1993 ). Subclinical mastitis occurs in 4–50% of lactating ewes ( Kirk and Glenn, 1996 ). Subclinical mastitis is more common in ewes from high-milk-producing breeds. Skin or teat lesions and dermatitis increase the prevalence of disease. Acute mastitis can be diagnosed in ewes with associated systemic signs of disease by physical examination of the udder and inspection of the milk. Subclinical mastitis is often suggested by somatic cell counts elevated above 1 × 10 6 cells/ml. When high somatic cell counts are identified, subclinical mastitis can be diagnosed by milk culture. The California mastitis test may also be helpful as an indicator of mastitis. Manual palpation of a hard, indurated udder as well as serological testing for the maedi/visna virus is helpful in confirming the diagnosis of diffuse chronic mastitis. Treatment for acute bacterial mastitis should include aggressive application of broad-spectrum antibiotics (intramammary and systemic) and supportive therapy such as fluids and anti-inflammatory drugs. It is may be helpful to milk out the infected udder frequently; oxytocin injections preceding milking will improve gland evacuation. Because somatic cell counting is often not routinely performed, treatment of subclinical mastitis is seldom done. There is currently no treatment available for diffuse chronic mastitis. ii. Caprine mastitis Lactating goats are subject to inflammation of mammary gland, or mastitis. The primary causative organisms are Staphylococcus epidermidis and other coagulase-negative Staphylococcus spp. Clinical signs of mastitis include abnormal coloration or composition of milk, mammary gland redness, heat and pain, enlargement of the mammary gland, discoloration of the mammary gland, and systemic signs of septicemia. Large abscesses may be present in the affected gland. Staphylococcus aureus is also associated with caprine mastitis, and toxemia may be part of the clinical picture. This organism produces a necrotizing alpha toxin that can result in gangrenous mastitis. Caprine mastitis may be clinical or subclinical, and the first indication of mastitis may be weak, depressed, or thin kids. Diagnosis is based on careful culture of mastitic milk. Treatment includes frequent stripping, intramammary antibiotics, and nonsteroidal anti-inflammatory drugs. Oxytocin (5–10 U) may help milk letdown for frequent strippings. Bovine mastitis products can be used in the goat; however, care should be taken not to insert the mastitis tube tip fully, because damage to the protective keratin layer lining the teat canal may occur. In severe acute systemic cases, steroids, fluids, and systemic antibiotics may be necessary. Other less common causes of mastitis in goats include Streptococcus spp. (S. agalactiae, S. dysgalactiae, S. uberis, and zooepidemicus). Gram-negative causes of caprine mastitis include Escherichia coli, Klebsiella pneumoniae, Pasteurella spp., Pseudomonas, and Proteus mirabilis. Corynebacterium pseudotuberculosis can cause mammary gland abscessation, whereas Mycoplasma mycoides may cause agalactia and systemic disease. "Hard udder" can be caused by caprine arthritis encephalitis virus (CAEV). Brucellosis and listeriosis can cause a subclinical interstitial mastitis ( Smith and Sherman, 1994 ). iii. Bovine mastitis Mastitis is the disease of greatest economic importance for the dairy cattle industry. The majority of the impact will be on the production and overall health of the cows, but low-incidence herds also diminish the risk of calves' ingesting or being exposed to pathogens. The most common bovine mastitis pathogens include Staphylococcus aureus and Streptococcus agalactiae, S. dysgalactiae, and S. uberis; coliform agents such as Escherichia coli, Enterobacter aerogenes, Serratia marcescens, and Klebsiella pneumoniae; mycoplasmal species such as Mycoplasma bovis, M. bovigenitalium, M. californicum, M. canadensis, and M. alkalescens; and Salmonella spp. such as S. typhimurium, S. newport, S. enteritidis, S. dublin, and S. muenster. Many of these agents such as Staphylococcus spp., Salmonella spp., and the coliforms can cause both acute and chronic mastitis, as well as severe systemic disease, including fever and anorexia. These must be regarded as herd and environmental pathogens in terms of treatment and prevention. The pathogenesis of staphylococcal infections is comparable to that in goats. Staphylococcus agalactiae can be cleared from udders because it does not invade other tissues, is an obligate resident of the glands, and is susceptible to penicillin. In contrast, S. uberis and S. dysgalactiae are environmental organisms and can be highly resistant to pencillin. Mycoplasma bovis is the more common of the mycoplasmal pathogens and can cause severe infections. Transmission of the mycoplasmas is not well defined but may be related to their presence in other organ systems. Treatments for mycoplasmal mastitis are not successful; culling is recommended. There are many interrelated factors associated with prevention and control of mastitis in a herd, including herd health and dry cow management, order of animals milked, milking procedures, milking equipment, condition of the teats, and the condition of the environment. Management of the overall herd includes aspects such as vaccination programs, nutrition, isolation of incoming animals, and quarantine and treatment of or culling diseased individuals. Culturing or testing newly freshened cows and monitoring the bulk milk tank serve as indicators of subclinical mastitis. Herd management will diminish teat lesions. Bacterin vaccines are available for preventing and controlling coliform mastitis and S. aureus mastitis. At the time of dry-off, all cows must be treated by intramammary route. Some infections can be successfully cleared during this time. Younger, disease-free animals should be milked first; any animals with diagnosed problems should be milked after the rest of the herd and/or segregated during treatment. Milkers' hands easily serve as a means of pathogen transmission, and wearing rubber gloves is recommended. Teat and udder cleaning practices include washing and drying with single-service paper or cloth towels or pre-and postmilking dipping. Milking equipment must be maintained to provide proper vacuum levels and pumping rates, and liners should be the appropriate size. Facilities that provide clean and dry areas for the animals to rest, feed, and move will diminish teat injuries and reduce exposures to mastitis pathogens. In that regard, inorganic bedding such as clean sand harbors few pathogens in contrast to shavings and sawdust. w. Moraxella bovis Infection (Infectious Bovine Keratoconjunctivitis, Pinkeye) Etiology. Moraxella bovis, a gram-negative coccobacillus, is the most common cause of infectious bovine keratoconjunctivitis (IBK) in cattle. This organism is not a cause of keratoconjunctivitis in sheep and goats. The disease includes conjunctivitis and ulcerative keratitis. The pathogenic M. bovis strain is piliated, and at least seven serotypes exist. Clinical signs. Lacrimation, photophobia, and blepharospasm are seen initially. Conjunctival injection and chemosis develop within a day of exposure, and then keratitis with corneal edema and ulcers. Anterior uveitis may be a sequela within a few days, and thicker mucopurulent ocular discharge may be seen. Corneal vascularization begins by 10 days after onset. Reepithelialization of the corneal ulcers occurs by 2–3 weeks after onset. Diagnosis is usually based on clinical signs, but culturing is helpful and fluorescein staining is useful for demonstrating corneal ulceration. Epizootiology and transmission. The disease is more severe in younger cattle. The clinical signs of IBK tend to be more severe in cattle that are also infected with infectious bovine rhinotracheitis (IBR) virus or those that have been vaccinated recently with modified live IBR vaccine. The bacteria are shed in nasal secretions and cattle with no clinical symptoms may be carriers. Transmission is by fomites, flies, aerosols, and direct contact. Incidence in winter months is very low. Nonhemolytic strains are associated with the winter epidemics, and hemolytic strains are associated with summer epidemics. Necropsy findings. Necropsy is not typically performed on these cases. Corneal edema, ulceration, hypopyon, and uveitis would be noted, depending on the stage of infection. Pathogenesis. The pili of M. bovis bind to receptors of corneal epithelium. The virulent strains of the bacteria then release the enzymes that damage the corneal epithelial cells. Other factors contributing to infection include ultraviolet light and trauma from dust and plant materials. Differential diagnoses. Infectious bovine rhinotrachetitis virus causes conjunctivitis, but the central corneal ulceration that is characteristic of IBK is not seen with M. bovis infections. Mycoplasma, Listeria, Branhamella (Neisseria), and adenovirus may be cultured from affected bovine eyes but none has been shown to produce the corneal lesions when inoculated into susceptible animals. Prevention and control. Cattle should not be immunized intranasally with modified live infectious bovine rhinotracheitis vaccine during IBK outbreaks; this will likely exacerbate the infection. New animals should be quarantined and treated prophylactically before introduction to herds. The available vaccines, containing. M. bovis pili or killed M. bovis, help decrease incidence and severity of disease; these preparations are not completely effective, because the M. bovis strain may not be homologous to that used for the vaccine preparation. Other preventive measures include 10% permethrin-impregnated bilateral ear tags, pour-on avermectins, or dust bags or face rubbers containing insecticide (such as 5% coumaphos) to control flies throughout the season and premises; mowing of high pasture grass to minimize ocular trauma; provision of shade; control of dust and sources of other mechanical trauma; and segregation of animals by age. Treatment. Cattle can recover without treatment, but younger animals should be treated as soon as the infection is detected. Antibiotic treatments include topical, subconjunctival administration and intramuscular dosing. Several standard topical antibiotics have been shown to be effective, including oxytetracycline, gentamicin, and triple antibiotic combinations. These should be administered twice per day. Subconjunctival injections of antibiotics, such as penicillin G, provide higher corneal levels of drug; these are typically administered only once or twice in severe cases. Intramuscular doses of long-acting oxytetracycline, given on alternate days, are effective in larger herds, and 2 doses 72 hr apart eliminate carriers. Third-eyelid flaps, temporary tarsorrhaphy, or eye patches may be useful in certain cases. Research complications. This pathogen does present a complication due to the carrier status of some animals, the likelihood of herd outbreaks, the severity of disease in younger animals, and the morbidity, possible progression to uveitis, and time and treatment costs associated with infections. The overall condition of the cattle will be affected for several weeks, and permanent visual impairment or loss, as well as ocular disfigurement, may occur. x. Mycobacterial Diseases Mycobacterium bovis Infection (Tuberculosis) Etiology. Mycobacteria are aerobic, nonmotile, non-spore-forming, acid-fast pleomorphic bacteria. Most cases of tuberculosis in sheep are related to Mycobacterium bovis or M. avium. Cases in goats have been attributed to M. bovis, M. avium, or M. tuberculosis. Mycobacterium bovis, or the bovine tubercle bacillus, is the cause in cattle but has been isolated from many domestic and wild mammals. Other agents of mammalian tuberculosis include M. microti and M. africanum. Clinical signs. Tuberculosis is a sporadic, chronic, contagious disease of ruminants and is zoonotic. The infection is often asymptomatic later in the illness, and it may be diagnosed only at necropsy. The respiratory system (M. bovis) or the digestive system (M. avium) is the primary site of infection; other tissues such as mammary tissue and reproductive tract may be infrequently involved. Locations of the characteristic tubercles will determine whether clinical signs are seen. Respiratory signs may include dyspnea, coughing, and pneumonia. Digestive tract signs include diarrhea, bloat, or constipation; diarrhea is most common. Lymphadenopathy occurs in advanced cases. Fever and generalized disease may be seen after calving. Infected goats lose weight and develop a persistent cough. Epizootiology and transmission. Although M. bovis can be killed by sunlight, it otherwise survives a long time in the environment and in cattle feces. Animals acquire the infection from the environment or from other animals via aerosols, from contaminated feed and water, and from secretions such as milk, semen, genital discharges, urine, and feces. Clinically normal animals may serve as carriers. The bacilli stimulate an initial neutrophilic tissue response. Neutrophils become necrotic and are phagocytosed by macrophages, forming giant epithelioid cells called Langhans' giant cells. An outer lymphocytic zone is formed, and fibrotic encapsulation creates the classical caseous nodules. Vascular erosion and hematogenous migration of the organisms may lead to lesions throughout the body. Necropsy findings. Yellow primary tubercles (granulomas) with central areas of caseous necrosis and calcification are present in the lungs. Caseous nodules are also associated with gastrointestinal organs and mesenteric lymph nodes. Prevention and control. Significant progress has been made in eradication programs in the United States during the past several decades, but during the 1990s, infected animals continued to be found in domestic cattle herds and particularly in captive deer herds in hunting preserves. The intradermal tuberculin test, using purified protein derivative (PPD), is usually used as a diagnostic indicator in live animals. This test should be performed annually on bovine and caprine dairy herds (and bison herds); the official tests are the caudal fold, comparative cervical, and single cervical tests. Notification to state officials is required following identification of intradermal-positive animals. Great care must be exercised in any handling of tissue or necropsies of reactors, and state animal health officials should be consulted regarding disposal of materials and cleaning of premises following depopulation of positive animals. Treatment. No treatment is recommended, and treatment is usually not allowed, because of the zoonotic potential, chronicity of the disease, and the treatment costs. Slaughter is preferred, to prevent potential transmission to humans. Research complications. The pathogen is zoonotic. Paratuberculosis, or Johne's disease (Mycobacterium paratuberculosis) Etiology. Mycobacterium paratuberculosis, the causative agent of Johne's disease, is a fastidious, non-spore-forming, acid-fast, gram-positive rod. The organism is actually a subspecies of M. avium, but M. paratuberculosis does not produce the siderophore mycobactin (an iron-binding molecule) of M. avium. Clinical signs and diagnosis. Johne's disease is a chronic, contagious, granulomatous disease of adult ruminants and is characterized by unthriftiness, weight loss, and intermittent diarrhea. In sheep and goats, chronic wasting is usually seen, occasionally with pasty feces or diarrhea. In cattle, chronic diarrhea and rapid weight loss are the most common clinical signs of the disease. Usually older adult animals are infected, but over time in an infected herd, younger animals will become infected when sufficient doses of organisms are ingested. Although clinical signs are nonspecific, Johne's disease should be considered if the affected diarrheic animals have a good appetite and are on a good anthelmintic program. The disease is diagnosed based on clinical signs and laboratory analyses, although none of the tests is more than 50% sensitive. In addition, the sensitivity of the serological tests differs between species. The standard is the fecal culture that takes 8–12 weeks. The enzyme-linked immunosorbent assay (ELISA) is now considered the most reliable serological test, but false negatives do occur. Other serological tests such as agar gel immunodiffusion (AGID) and complement fixation are useful. Herd screening may be done using the AGID or ELISA serological tests. Identification of the organism on culture, or the presence of acid-fast organisms on mucosal or mesenteric lymph node smears or from rectal biopsies, helps confirm the diagnosis. Some animals serologically negative for Johne's disease, however, have been found to be positive on fecal culture. Commercial AGID tests approved for use in cattle may be useful in diagnosing Johne's disease in sheep ( Dubash et al., 1996 ). Serological tests cross-react with other species of Mycobacterium, especially M. avium. Epizootiology and transmission. The organism is prevalent in the environment and is transmitted to young animals by direct or indirect contact. Although vertical transmission has been reported, the organism more commonly enters the gastrointestinal tract and penetrates the mucosa of the distal small intestine, primarily the ileum. Chronic carriers may intermittently shed the organisms. Pathogenesis. Mycobacterium paratuberculosis is an obligate parasite that grows only in macrophages of infected animals. Nursing infected dams are a primary source of infection of neonates. If the organism is not cleared, it proliferates slowly in the tissue, leading to inflammatory reactions that progress through neutrophilic to mononuclear stages. The organism may penetrate the lymphatics and proliferate in mesenteric lymph nodes. After an incubation period of a year or more, some of the carriers will progress to clinical disease manifested by fibrotic and hyperplastic changes in the ileum, leading to the classic thickening in the region. Gut changes result in intermittent diarrhea, with subsequent dehydration, electrolyte imbalances, and malnutrition, although this clinical sign is more common in cattle than in sheep or goats. Necropsy and diagnosis. The ileum from infected cattle is grossly thickened; this is not seen in sheep and goats. Ileal and ileocecal lymph nodes provide the best samples for histology and acid-fast staining. Differential diagnosis. Diseases causing chronic wasting and poor coat and body condition of all ruminants should be considered. These include chronic salmonellosis, peritonitis, severe parasitism, winter dysentery, and pyelonephritis. Deer can be infected, and the lesions can be confused with those of tuberculosis. Treatment. Treatment is not worthwhile. Prevention and control. Prevention is the most effective method to manage this pathogen. Efforts should be focused on eliminating the disease through test and slaughter. Neonates should not be reared by infected dams. Some states have Johne's disease eradication programs. Facilities and pastures where animals testing positive for Johne' disease were maintained should be thoroughly cleaned and kept vacant for a year after culling. Other considerations. Mycobacterium paratuberculosis is being investigated as a factor in the development of Crohn's disease in humans. y. Navel Ill (Omphalitis, Omphalophlebitis, Omphaloarteritis, Joint Ill) Etiology. The most common organism causing infection of the umbilicus is Arcanobacterium (formerly Actinomyces, Corynebacterium) pyogenes; other bacteria may be present. Arcanobacterium spp. are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Other environmental contaminants are also associated with this disease, such as Escherichia coli, Enterococcus spp., Proteus, Streptococcus spp., and Staplylococcus spp. Clinical signs and diagnosis. Navel ill is an acute localized inflammation and infection of the external umbilicus. Animals present with fever and painful enlargement of the umbilicus. Animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, and hematuria. Other common severe sequelae include septicemia, pneumonia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, uveitis, endocarditis, and diarrhea. Epizootiology and transmission. Many cases occur in neonates, and most cases occur within the first 3 months of age. Cleanliness of the birthing and housing environment and successful transfer of passive immunity are important factors in the occurrence of the disease. Dystocia resulting in weak neonates can be a factor predisposing to the development of the disease. Navel ill is diagnosed by typical clinical signs. The presence of microabscesses and palpation of the umbilical area for firm intra-abdominal structures extending from the umbilicus are abnormal. Assessment of colostral immunoglobulin transfer may contribute to determination of the prognosis. Navel ill should always be considered for young ruminants with fever of unknown origin during the first week of life and for slightly older lambs, kids, or calves that are not thriving. Arthrocentesis of affected joints and culture of the fluid for identification of the pathogen are also diagnostic options and essential for effective antimicrobial selection. Differential diagnosis. The major differential is an umbilical hernia, which will typically not be painful or infected and can often be reduced. Mycoplasmal arthritis is a differential in kids. In the past, Erysipelothrix rhusopathiae was a common navel ill pathogen in sheep. Treatment. Omphalitis can be treated with a 10 to 14 day course of broad-spectrum antibiotics such as ampicillin, amoxicillin, penicillin, ceftiofur, florfenicol, and erythromycin. If an isolated abscess is palpable, it should be surgically opened and repeatedly flushed with iodine solutions. Surgical reduction of the infected umbilicus is indicated if intra-abdominal structures are involved. The prognosis for recovery is good if systemic involvement has not occurred. Prevention and control. The disease is best prevented and controlled by providing clean birthing environments, ensuring adequate colostral immunity, thoroughly dipping the umbilicus of newborns in tincture of iodine or strong iodine solution (Lugol's), monitoring for dystocias, and maintaining young growing animals in noncontaminated environments. Research complications. The disease can be costly to treat, and the toll taken on young animals due to the consequences of systemic infection may detract from their research value. z. Pasteurellosis (Shipping Fever, Hemorrhagic Septicemia, Enzootic Pneumonia) Etiology. Pasteurella hemolytica and P. multocida are aerobic, nonmotile, non-spore-forming, bipolar, gram-negative rods. Biotype A serotypes are associated with pneumonia and septicemia in all ruminants ( Ellis, 1984 ). Serotype 1 of P. hemolytica is considered a major cause of pulmonary lesions of bovine bronchopneumonia and fibrinous bronchopneumonia. Clinical signs. Pasteurellosis is an acute bacterial disease characterized by bronchopneumonia, septicemia, and sudden death. The organism invades the mucosa of the gastrointestinal tract or respiratory tract and causes localized areas of necrosis, hemorrhage, and thrombosis. The lungs and liver are frequent areas of formation of microabscesses. Acute rhinitis or pharyngitis often precedes the respiratory form. The organism also may invade the bloodstream, causing disseminated septicemia. Clinically, the lambs may exhibit nasal discharge of mucopurulent to hemorrhagic exudate, hyperthermia, coughing, dyspnea, anorexia, and depression. With the respiratory form, auscultation of the thorax suggests dullness and consolidation of anteroventral lobes; this will be confirmed by radiographs. The disease is diagnosed by clinical signs, blood cultures from septicemic animals, blood smears showing bipolar organisms, and history of predisposing stressors. In cultures, P. hemolytica is distinguished from P. multocida by hemolysis on blood agar; only P. multocida produces indole. Epizootiology and transmission. The organism is ubiquitous in the environment and in the respiratory tracts of these animals. Younger ruminants, between 2 and 12 months of age, are especially prone to infection during times of stress, such as weaning, transportation, dietary changes, weather changes, and overcrowding. The pneumonic form appears as a complex associated with concurrent infections such as parainfluenza 3, adenovirus type 6, respiratory syncytial virus, mycoplasmas, chlamydia, Pasteurella multocida and Bordetella parapertussis ( Martin, 1996 ; Brogden et al., 1998 ). The organism is transmitted between animals by direct and indirect contact, through inhalation or ingestion. Necropsy findings. Necropsy lesions include areas of necrosis and hemorrhage in the small intestines and multifocal 1 mm lesions distributed on the surfaces of the lungs and liver. With the pneumonic form, serofibrinous exudates fill the alveoli; ventral lung lobes are consolidated and are congested and purple-gray in color. Fibrinous pleuritis, pericarditis, and hematogenously induced arthritis also may be evident. Pathogenesis. A leukotoxin is considered to be a key factor in the pathogenesis of the P. hemolytica infection. Macrophages and neutrophils are lysed by the toxin as they arrive at the lung, and the enzymes released by the neutrophils cause additional damage to the tissue. Treatment. Treatment may include the use of antibiotics such as penicillin, ampicillin, tylosin, sulfonamides, or oxytetracycline. Newer antibiotics, such as ceftiofur, tilmicosin, spectinomycin, and florfenicol, are very effective and approved for use in cattle. In outbreaks, cultures from fresh necropsies are helpful for determining sensitivities useful for the remaining group. Prevention and control. The incidence of disease can be decreased by minimizing the degree of stress; by improving management, such as nutrition and control of parasitism; and, in cattle and sheep, by vaccinating for viral respiratory infections such as parainfluenza. Early Pasteurella hemolytica bacterin vaccines for use in cattle are not considered effective, but newer products based on immunizing against the leukotoxin and some bacterial capsule surface antigens are effective. Pasteurella multocida bacterins and live streptomycin-dependent mutant vaccines are available. In young animals, passive immunity is protective. Preventive measures also include maintaining good ventilation in enclosures and barns. New animals to the flock or herds should be quarantined for at least 2 weeks before introduction. aa. Salmonellosis Etiology. Salmonella typhimurium is a motile, aerobic to facultatively anaerobic, non-spore-forming, gram-negative bacillus and is the organism associated with enteric disease and some abortions in ruminants. It is a common inhabitant of the gastrointestinal tract of ruminants. Current nomenclature categorizes S. typhimurium as a serovar within the species S. enteritidis (the other two species are S. typhi and S. choleraesuis). Salmonella typhimurium, S. dublin, and S. newport are the common species seen in bovine cases. Salmonella typhimurium, S. dublin, S. anatum, and S. montevideo are seen in ovine and caprine cases, although a host-adapted species has not been identified in the goat. Ovine abortions due to various Salmonella species are not reported in the United States but are enzootic in other countries. Salmonella serotypes have been associated with aborted fetuses in all ruminant species. Clinical signs and diagnosis. Salmonellosis causes acute gastroenteritis, dysentery, and septicemia ( Anderson and Blanchard, 1989 ). Clinically, the animals become anorexic and hyperthermic. Diarrhea or dysentery develops; feces may contain mucus and/or blood and have a putrid odor. Animals become severely depressed and weak, losing a high percentage of their body weight. Animals may die in 1–5 days because of dehydration associated with dysenteric fluid loss, septicemia, shock, and acidosis. Morbidity may be 25%, and mortality may be high. Septicemia may result in subsequent meningitis, polyarthritis, and pneumonia. Chronically infected animals may have intermittent diarrhea. In goats, salmonellosis may be recognized as diarrhea and septicemia in neonates, as enteritis in preweaned kids and mature goats, and, rarely, as abortion. Adult cases may be sporadic, with intermittent bouts of diarrhea, subacute or even chronic. Morbidity and mortality will be highest in neonates, and some may simply be found dead. The older animals generally tend to fare better during the disease. Abdominal distension with profuse yellow feces is common. Kids become severely depressed, anorexic, febrile (with temperatures as high as 106°–107°F), dehydrated, acidotic, recumbent, and comatose. Salmonella abortions may occur throughout gestation. There may not be any other clinical signs, or abortion may be seen with diarrhea, fever, and vulvar discharges. Hemorrhage, placental necrosis, and edema will be present. Metritis and placental retention may occur. Some mortality of dams may occur. Diagnosis is based on clinical signs and can be confirmed by culturing fresh feces or at necropsy. Because of intermittent shedding of organisms, culture may be difficult; repeated cultures are recommended. Leukopenia and a degenerative shift to the left are not uncommon hematological findings. Epizootiology and transmission. Stresses associated with recent shipping, overcrowding, and inclement weather may predispose the animal to enteric infection. Birds and rodents may be natural reservoirs of Salmonella in external housing environments. Transmission is fecal-oral. After ingestion, the organisms may proliferate throughout the gastrointestinal tract and may penetrate the mucosa of the intestines, invade the Peyer's patches and lymphatics, and migrate to the spleen, liver, and other organs. Animals that survive may become chronic carriers and shedders of the organisms, and this has been demonstrated experimentally ( Arora, 1983 ). Fecal-oral transmission is also associated with Salmonella abortion; veneral transmission has not been reported. Necropsy findings and diagnosis. Animals will have noticeable perineal staining. Intestines (particularly the ileum, cecum, and colon) may contain mucoid feces with or without hemorrhages. Petechial hemorrhages and areas of necrosis may be noticed on the surface of the liver, heart, and mesenteric lymph nodes. The wall of the intestines, gallbladder, and mesenteric lymph nodes will be edematous, and a pseudodiphtheritic membrane lining the distal small intestines and colon may be observed. This membrane is not normally seen in the goat ( Smith and Sherman, 1994 ). Splenomegaly may be present. Aborted fetuses will often be autolysed. Placentitis, placental necrosis, and hemorrhage are commonly seen. Serologic evidence of recent infection can be demonstrated in the dam. Salmonella can be isolated from the aborted tissues. Pathogenesis. After ingestion, the organism proliferates in the intestine. Damage to the intestines and the resulting diarrhea are due to the bacterial production of cytoxin and endotoxin. Although the Salmonella organisms will be taken up by phagocytic cells involved in the inflammatory response, they survive and multiply further. Septicemia is a common sequela, with the bacteria localizing throughout the body. In latently infected animals, it is often shed from the gallbladder and mesenteric lymph nodes. Younger animals may be susceptible because of immature immunity and intestinal flora and higher intestinal pH. Carriers may develop clinical disease when stressed. Differential diagnoses. In young animals, differentials include other enteropathogens: Escherichia coli, rotavirus and coronavirus, clostridia, cryptosporidia, and other coccidial forms. These pathogens may also be present in the affected animals. Differentials in adults include bovine viral diarrheas and winter dysentery in cattle and parasitemia and enterotoxemia in all ruminants. Prevention and control. Affected animals should be isolated during herd outbreaks. Samples for culture should include herd-mates, water and feed sources, recently arrived livestock (other species), and area wildlife, including birds and rodents. Repeated cultures, culling of animals, intensive cleaning, and disinfection of facilities are all important during outbreaks. The bacteria survive for about a week in moist cow manure. Vaccination using the commercially available killed bacterin or autologous bacterins may be useful in outbreaks involving pregnant cattle, although the J-5 bacterin is now considered better. Treatment. Nursing care includes rehydration and correction of acid-base abnormalities. Antibiotic therapy may be useful in cases with septicemia, but it is controversial because it may induce carrier animals. Gentamicin, trimethoprim-sulfadiazine, ampicillin, enrofloxacin, and amikacin antibiotics may be successful. Research complications. Salmonellosis is zoonotic, and some serotypes of the organism have caused fatalities even in immunocompetent humans. Attempts should be made to identify and cull carrier animals. bb. Spirochete-Associated Abortion in Cattle (Epizootic Foothill Abortion) Etiology. Spirochete-like organisms are associated with this disease; it is now recognized that the agent is not a chlamydial organism. The disease has been reported only in the foothills bordering the central valley of California. Clinical signs. Cows that become infected with the causative agent before 6 months of gestation abort or give birth to weak calves without any clinical sign of infection. Cows infected after 6 months of gestation give birth to normal calves. Affected cows rarely abort in subsequent pregnancies. Epizootiology and transmission. The tick vector is Ornithodorus coriaceus. Necropsy. Fetuses show several pathological changes, including enlargement of the cervical lymph nodes, spleen, and liver. The calf's thymus will be small, and histologically there will be losses of thymic cortical lymphocytes. Histologic changes in lymph nodes and spleen include vasculitis, necrosis, and histiocytosis. Treatment. Chlortetracycline treatment has been effective in controlling this disease. cc. Tularemia Etiology. Tularemia is caused by Pasteurella (Francisella) tularensis a nonmotile, non-spore-forming, aerobic, gram-negative, rod-shaped bacterium. Type A is more virulent than type B. Clinical Signs. Although tularemia is a disease of livestock, pets, and wild animals, sheep are most commonly affected. The disease is characterized by hyperthermia, muscular stiffness, and lymphadenopathy. Infected animals move stiffly, are depressed, and are hyperthermic. Anemia and diarrhea may develop, and infected lymph nodes enlarge and may ulcerate. Mortality may reach 40%. Animals that recover will have immunity of long duration. Epizootiology and transmission. The disease is most commonly transmitted by ticks or biting flies. The wood tick, Dermacentor andersoni, is an important vector in transmitting the disease in the western United States, and, as natural hosts, wild rodents and rabbits tend to be reservoirs of the pathogen. Pathogenesis. The organisms, entering the tick bite wound, move via lymphatics to lymph nodes and subsequently to the bloodstream, where they cause septicemia. The organisms can also be transmitted orally through contaminated water. Necropsy findings. Ticks may also be present on the carcasses. Suppurative, necrotic lymph nodes are typical. Lungs will be congested and edematous. Diagnosis is confirmed by prompt culturing of the organism from lymph nodes, spleen, or liver where granulomatous lesions form; P. tularensis does not survive for long periods in carcasses. Serological findings may also be helpful. Treatment. Infected animals can be treated with oxytetracycline, aminoglycosides, or cephalosporins. Differential diagnosis. When tick infestations are heavy, P. tularensis should be suspected. Pasteurella haemolytica (sheep), Haemophilus somnus (cattle), and Mycoplasma mycoides (goats), and anthrax (all ruminant species) should be considered as differentials. Control and prevention. Eliminating the tick vectors can prevent tularemia. Animals should be provided with fresh water frequently. The organism can survive in freezing conditions and in water and mud for long periods of time. Caretakers, veterinarians, and researchers should take special precautions before handling the tissues of infected sheep, because this is a method of zoonotic spread. Research complications. The disease is zoonotic, and transmission to people may result from tick bites or from handling contaminated tissues. Although not a major disease of concern in sheep, researchers using potentially infected animals from western range states of the United States should be aware of it. The organism is antigenically related to Brucella spp. dd. Yersinia Etiology. Yersiniosis is caused by infections with Yersinia enterocolitica, a gram-negative, aerobic, and facultative anaerobe of the family Enterobacteriaceae. There are 50 serotypes reported for Y. enterocolitica. Yersinia pseudotuberculosis infections have also been seen in ruminants. Enteric infections predominate in the diseases caused by these bacteria. Clinical signs and diagnosis. Clinical disease may be seen rarely in many groups of ruminants. Goats of 1–6 months old suffer from the enteric form of the disease, which is characterized by sudden death or the acute onset of watery diarrhea lasting 1 or more days. Spontaneous abortions and weak neonates are also clinical manifestations of infection. Lactating does may have mastitis that becomes chronically hemorrhagic. Bacteremia results in internal abscesses, abortion, and acute deaths. Yersinia pseudotuberculosis has been associated with laboratory goat epizootics ( Obwolo, 1976 ). Diarrhea in pastured sheep, stressed by other factors, has also been reported. Diagnosis is based on culture and serology. Epizootiology and transmission. The bacteria are carried by wild birds and rodents, and transmission is by ingestion of contaminated feed and water. Necropsy findings. Edema of mesenteric lymph nodes is the most common postmortem finding. Liver abscesses, micro-absecesses in the intestines, and granuloma formation have also been reported. Placentas are white, with opaque white foci found on cotyledons. Histologically, suppurative placentitis and suppurative pneumonia are found in the fetal tissue. Pathogenesis. After ingestion, the bacteria cause an enteric infection, and bacteremia follows. Differential diagnoses. Other causes of abortions, including abortion storms, acute deaths, enteritis, neonatal deaths, and white foci on cotyledons, should be considered. In young animals, differentials include coccidiosis and nematode parasitism. Corynebacterium pseudotuberculosis and tuberculosis are differentials for the internal abscesses. Prevention and control. Control measure are not well defined, because the epidemiology of the disease is poorly understood ( Smith and Sherman, 1994 ). Tissues from affected goats must be handled and disposed of properly. Areas housing affected goats must be thoroughly sanitized. Treatment. In case of an abortion storm, treatment of goats with tetracycline has been useful. Other broad-spectrum antibiotics may also be useful. Research complications. Yersinia is zoonotic. The risk of severe enteric disease is considered particularly great for immunocompromised persons. ee. Mycoplasmal Diseases i. Mycoplasma bovigenitalium and M. bovis infections Etiology. Mycoplasma bovigenitalium and M. bovis are associated sporadically with bovine infertility and abortions. This pathogen has also been reported associated with similar clinical signs in sheep and goats. Clinical signs and diagnosis. Infertility is more commonly caused by M. bovigenitalium infections, and granular vulvovaginitis and endometritis will be present. Granular vulvovaginitis is characterized by raised papules on the mucous membranes and mucopurulent exudate. Abortions and mastitis are associated with M. bovis infections. Calves that are born may be weak. It is rare to have a definitive diagnosis of an abortion due to Mycoplasma. After consideration of other causes of abortion and evaluation of tissues for placentitis or fetal inflammation, diagnosis is confirmed by isolation of Mycoplasma from the genital tract or aborted tissues. Epidemiology and transmission. Mycoplasmal species are considered ubiquitous, are carried in the genital tracts of males and females, and are transmitted during natural breeding or through contaminated insemination materials. Aerosols also serve as a means of transmission. In addition, transmission occurs by passage through the birth canal, by direct contact, and by contamination from urine of infected animals. Pathophysiology. Experimental infections of M. bovis have resulted in placentitis and fetal pneumonia. Differential diagnoses. Acholeplasma, Ureaplasma, and Haemophilus somnus are differentials for granular vulvovaginitis. Treatment. Fluoroquinolone antibiotics may be useful for treating Mycoplasma-induced reproductive diseases. ii. Mycoplasma ovipneumoniae (ovine mycoplasmal pneumonia) Etiology. Mycoplasma ovipneumoniae causes acute or chronic pneumonia in lambs. Clinical signs. Mycoplasmas induce serious diseases in sheep, causing pneumonia, conjunctivitis, and genitourinary disease. The disease may be coincidental with pasteurellosis. Respiratory distress, coughing, and nasal discharge are observed in infected animals. Bronchoalveolar lavage followed by culture is the best method for diagnosis (mycoplasmas are fastidious organisms requiring special handling techniques). Mycoplasmas are isolated from the genitourinary tract of sheep. Vulvovaginitis and reproductive problems are associated conditions. Treatment. Tylosin, quinolones, oxytetracycline, and gentamicin are good choices for therapy. Prevention. No vaccine is available. iii. Mycoplasma mycoides biotype F38 (contagious caprine pleuropneumonia, caprine pneumonia, pleuritis, and pleuropneumonia) Etiology. Mycoplasma mycoides biotype F38 is the agent of contagious caprine pleuropneumonia and is found worldwide. In the United States, caprine pneumonia is also caused by M. ovipneumoniae, M. mycoides subsp. capri, and M. mycoides subsp. mycoides (large colony type). Clinical signs. Contagious caprine pleuropneumonia is characterized by severe dyspnea, nasal discharge, cough, and fever ( McMartin et al., 1980 ). Infections with other Mycoplasma species also have similar clinical signs. Septicemia without respiratory involvement may also be a presentation. Epizootiology and transmission. This disease is highly contagious, with high morbidity and mortality. Transmission is by aerosols. Mycoplasma mycoides subsp. mycoides has become a serious cause of morbidity and mortality of goat kids in the United States. Necropsy. Large amounts of pale straw-colored fluid and fibrinous pneumonia and pleurisy are typical. Some lung consolidation may be present. Meningitis, fibrinous pericarditis, and fibrinopurulent arthritis may also be found. Diagnosis is usually made at necropsy by culture of the organism from lungs and other internal organs. Differential dagnosis. In the United States, the principal differential for M. mycoides subsp. mycoides is caprine arthritis encephalitis. Treatment. Tylosin and oxytetracycline are effective. Some infections are slow to resolve. Prevention and control. Vaccines are available in some areas. Infected herds are quarantined. New goats should be quarantined before introduction to the herd. Research complications. The worldwide distribution of the F38 biotype, as well as the aerosol transmission and high morbidity and mortality characteristics of mycoplasmal infectious, make these infections economically important diseases. Considerable attention is presently given to this genus as a source of morbidity and mortality in goats. iv. Mycoplasma conjunctivae (mycoplasmal keratoconjunctivitis) Etiology. Mycoplasma conjunctivae causes infectious conjunctivitis, or pinkeye, in sheep and goats with associated hyperemia, edema, lacrimation, and corneal lesions. Mycoplasma mycoides subsp. mycoides, M. agalactiae, M. arginini, and Acholeplasma oculusi have also been associated with keratoconjunctivitis in these species. Respiratory disease and other infections, such as mastitis, may also be observed. Clinical signs and diagnosis. All ages of animals may be affected. Initially, lacrimation, conjunctival vessel injection, and then keratitis and neovascularization are seen. Sometimes uveitis is evident. Although the presentation is usually unilateral, bilateral involvement is possible. Recurring infections are common. Culturing provides the better diagnostic information, and cultures will be positive even after clinical signs have diminished. Epizootiology and transmission. The infection is passed easily between animals by direct contact. Animals can become reinfected, and carrier animals may be a factor in outbreaks. Necropsy. It is unlikely that animals would die or be euthanized and undergo necropsy for this problem. Conjunctival scrapings would include neutrophils during earlier stages and lymphocytes during later stages. Epithelial cell cytoplasm should be examined for organisms. Differential diagnosis. The primary differential in sheep and goats is Chlamydia, as well as Branhamella, Rickettsia (Colesiota) conjunctivae, and infectious bovine rhinotracheitis in goats only. It is important to consider these differentials if arthritis, pneumonia, or mastitis is present in the group or the individual. Treatment. Animals do recover spontaneously within about 10 weeks. Tetracycline ointments and powders are also used. Third-eyelid flaps may be necessary if corneal ulceration develops. Prevention and control. New animals should be quarantined and, if necessary treated, before introduction to the flock or herd. ff. Rickettsial Diseases i. Eperythrozoonosis (Eperythrozoon, Haemobartonella) Etiology. Eperythrozoonosis is a rare, sporadic, noncontagious, blood-borne disease in ruminants worldwide caused by the rickettsial agent Eperythrozoon. Host-specific species of importance are E. ovis, the causative species in sheep and goats, and E. wenyoni, E. tegnodes, and E. tuomii, the causative agents in cattle. Although the disease is of minor importance, it can cause severe anemia and debilitation in affected animals. Haemobartonella bovis is also rare, and is usually found only in association with other rickettsial diseases. Clinical signs and diagnosis. The disease is more severe in sheep. Following an incubation period of 1–3 weeks, infected animals exhibit episodic hyperthermia, weakness, and anemia. Losses may be greater in younger lambs. Cattle are usually latently infected but may have swollen and tender teats and legs. Fever, anemia, and depression will be present if the cattle are stressed by another systemic disease. Diagnosis is based on clinical evidence of anemia and is confirmed by observing the rickettsiae on the surface of red blood cells in a blood smear. Epizootiology and transmission. The rickettsial organisms are transmitted typically to young sheep by biting insects, ticks, contaminated needles or blood-contaminated surgical instruments. Necropsy findings. Necropsy findings include splenic enlargement and tissue icterus. Pathogenesis. The organism invades and destroys red blood cells. It is believed that intravascular hemolysis and erythrophagocytosis contribute to the macrocytic anemia. As with other red blood cell parasites, splenectomy aggravates the disease. Differential diagnosis. Clontridium novyi type D, babesiosis, and leptospirosis are the primary differentials. Prevention and control. Following strict sanitation practices for surgical procedures and controlling external parasites prevent the disease. Treatment. Treatment is not usually recommended, but Oxytetracycline has been used. Sheep will develop immunity if supported nutritionally during the disease. Research complications. Splenectomized animals are the experimental models used to study these diseases. ii. Q fever, or query fever (Coxiella burnetii) Etiology. Coxiella burnetii is a small, gram-negative, obligate intracellular rickettsial organism that causes query fever and is regarded as a major cause of late abortion in sheep. Clinical signs. Infection of ruminants with C. burnetii is usually asymptomatic. Experimental inoculation in other mammals has resulted in transient hyperthermia, mild respiratory disease, and mastitis. Abortions, stillbirths, and births of weak lambs are also seen. Epizootiology and transmission. Coxiella burnetii is extremely resistant to environmental changes as well as to disinfectants; persistence in the environment for a year or longer is possible. The organism is associated with either a free-living or an arthropod-borne cycle. Coxiella burnetii is found in a variety of tick species, such as ixodid or argasid, where it replicates and is excreted in the feces. Once introduced into a mammal, Coxiella may be maintained without a tick intermediate. The organism is especially concentrated in placental tissues, replicates in trophoblasts, and will be in reproductive fluids. Additionally, the organism is shed in milk, urine, feces, and oronasal secretions. Necropsy findings. No specific lesion will be seen in aborted or stillborn fetuses, but necrotizing placentitis will be a finding in cases of abortion. The placenta will contain white chalky plaques and a red-brown exudate. The disease can be diagnosed by identifying the rickettsial organisms in smears of placental secretions. The organism has been found in the placentas of clinically normal animals. The organism stains red with modified Ziehl-Neelsen and Macchiavello stains and purple with Giemsa stain. Differential diagnosis. Because of the organisms' similarity to Chlamydia, confirmation must be made by culture techniques, immunofluorescent procedures, ELISA, and complement fixation tests. Treatment. Coxiella can be treated with oxytetracyclines. A vaccine is not commercially available. Prevention and control. Any aborting animals should be segregated from other animals, and other pregnant animals should be treated prophylactically with tetracycline. Serologic screening of ruminant sources should be performed routinely. Barrier housing, a review of ventilation exhaust, and defined handling procedures are often required. All placentas and all aborted tissues should be handled and disposed of carefully. Q fever has been reported in many mammalian species, including cats. Research complications. Coxiella burnetii–hee animals are particularly important in studies involving fetuses and placentation. Because of its zoonotic potential, C. burnetii presents a unique problem in the animal research facility environment. A single organism has been shown to cause disease. Some of the greatest concerns are the risk to immunocompromised individuals, pregnant women, and other animals, and the presence of carrier animals or those that may shed the organism in placentas, for example. a. Actinobacillosis ("Wooden Tongue") Etiology. Actinobacillus lignieresii is an aerobic, nonmotile, non-spore-forming, gram-negative rod that is widespread in soil and manure and is found as normal flora of the respiratory, gastrointestinal, and reproductive tracts of ruminants. In sheep and cattle, A. lignieresii causes sporadic, noncontagious, and potentially chronic disease characterized by diffuse abscess and granuloma formation in tissues of the head and occasionally other body organs. This disease, called wooden tongue, has not been documented in goats. Clinical signs. Skin lesions are common. Tongue lesions are more common in cattle than in sheep. Lip lesions are more common in sheep. Soft-tissue or lymph node swelling accompanied by draining tracts is observed in the head and neck regions, as well as other areas. Animals may have difficulty prehending food; may be anorexic, weak, unthrifty and depressed; and may salivate excessively. Diagnosis is made based on clinical signs and is confirmed by culture. Epizootiology and transmission. The organism penetrates wounds of the skin, mouth, nose, gastrointestinal tract, testicles, and mammary gland. Rough feed material and foreign bodies may play a role in causing abrasions. Actino bacillus lignieresii then enters into deeper tissues, where it causes chronic inflammation and abscess formation. Lymphatic spread may occur, leading to abscessation of lymph nodes or infection of other organs. Necropsy findings. Purulent discharges of white-green exudate drain from the tracts that often extend from the area of colonization to the skin surface. Exudates will also contain characteristic small white-gray (sulfurlike) granules. The pus is usually nonodorous. Differential diagnosis. Contagious ecthyma and caseous lymphadenitis are the primary differentials. Diseases or injuries causing oral pain and discomfort, such as dental infections, foreign bodies, and trauma, should be considered. Treatment. Animals should be fed softer feeds. Antibiotics such as sulfonamides, tetracyclines, and ampicillin are effective, although high doses and long durations of therapy are required. Penicillin is not effective. Weekly systemic administration of sodium iodide for several weeks is not as effective as antibiotic therapy. Surgical excision and drainage are not recommended. Prevention and control. Because the organism enters through tissue wounds, especially those associated with oral trauma, feedstuffs should be closely monitored for coarse material and foreign bodies. Etiology. Actinobacillus lignieresii is an aerobic, nonmotile, non-spore-forming, gram-negative rod that is widespread in soil and manure and is found as normal flora of the respiratory, gastrointestinal, and reproductive tracts of ruminants. In sheep and cattle, A. lignieresii causes sporadic, noncontagious, and potentially chronic disease characterized by diffuse abscess and granuloma formation in tissues of the head and occasionally other body organs. This disease, called wooden tongue, has not been documented in goats. Clinical signs. Skin lesions are common. Tongue lesions are more common in cattle than in sheep. Lip lesions are more common in sheep. Soft-tissue or lymph node swelling accompanied by draining tracts is observed in the head and neck regions, as well as other areas. Animals may have difficulty prehending food; may be anorexic, weak, unthrifty and depressed; and may salivate excessively. Diagnosis is made based on clinical signs and is confirmed by culture. Epizootiology and transmission. The organism penetrates wounds of the skin, mouth, nose, gastrointestinal tract, testicles, and mammary gland. Rough feed material and foreign bodies may play a role in causing abrasions. Actino bacillus lignieresii then enters into deeper tissues, where it causes chronic inflammation and abscess formation. Lymphatic spread may occur, leading to abscessation of lymph nodes or infection of other organs. Necropsy findings. Purulent discharges of white-green exudate drain from the tracts that often extend from the area of colonization to the skin surface. Exudates will also contain characteristic small white-gray (sulfurlike) granules. The pus is usually nonodorous. Differential diagnosis. Contagious ecthyma and caseous lymphadenitis are the primary differentials. Diseases or injuries causing oral pain and discomfort, such as dental infections, foreign bodies, and trauma, should be considered. Treatment. Animals should be fed softer feeds. Antibiotics such as sulfonamides, tetracyclines, and ampicillin are effective, although high doses and long durations of therapy are required. Penicillin is not effective. Weekly systemic administration of sodium iodide for several weeks is not as effective as antibiotic therapy. Surgical excision and drainage are not recommended. Prevention and control. Because the organism enters through tissue wounds, especially those associated with oral trauma, feedstuffs should be closely monitored for coarse material and foreign bodies. Etiology. Actinobacillus lignieresii is an aerobic, nonmotile, non-spore-forming, gram-negative rod that is widespread in soil and manure and is found as normal flora of the respiratory, gastrointestinal, and reproductive tracts of ruminants. In sheep and cattle, A. lignieresii causes sporadic, noncontagious, and potentially chronic disease characterized by diffuse abscess and granuloma formation in tissues of the head and occasionally other body organs. This disease, called wooden tongue, has not been documented in goats. Clinical signs. Skin lesions are common. Tongue lesions are more common in cattle than in sheep. Lip lesions are more common in sheep. Soft-tissue or lymph node swelling accompanied by draining tracts is observed in the head and neck regions, as well as other areas. Animals may have difficulty prehending food; may be anorexic, weak, unthrifty and depressed; and may salivate excessively. Diagnosis is made based on clinical signs and is confirmed by culture. Epizootiology and transmission. The organism penetrates wounds of the skin, mouth, nose, gastrointestinal tract, testicles, and mammary gland. Rough feed material and foreign bodies may play a role in causing abrasions. Actino bacillus lignieresii then enters into deeper tissues, where it causes chronic inflammation and abscess formation. Lymphatic spread may occur, leading to abscessation of lymph nodes or infection of other organs. Necropsy findings. Purulent discharges of white-green exudate drain from the tracts that often extend from the area of colonization to the skin surface. Exudates will also contain characteristic small white-gray (sulfurlike) granules. The pus is usually nonodorous. Differential diagnosis. Contagious ecthyma and caseous lymphadenitis are the primary differentials. Diseases or injuries causing oral pain and discomfort, such as dental infections, foreign bodies, and trauma, should be considered. Treatment. Animals should be fed softer feeds. Antibiotics such as sulfonamides, tetracyclines, and ampicillin are effective, although high doses and long durations of therapy are required. Penicillin is not effective. Weekly systemic administration of sodium iodide for several weeks is not as effective as antibiotic therapy. Surgical excision and drainage are not recommended. Prevention and control. Because the organism enters through tissue wounds, especially those associated with oral trauma, feedstuffs should be closely monitored for coarse material and foreign bodies. b. Arcanobacterium Infection (Formerly actinomycosis, or "Lumpy Jaw ") Etiology. Arcanobacterium (formerly known as Actinomyces or Corynebacterium) pyogenes and A. bovis are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Arcanobacterium bovis is a normal part of the ruminant oral microflora and is the organism associated with "lumpy jaw" in cattle; this syndrome is rarely seen in sheep and goats. This organism has also been associated with pharyngitis and mastitis in cattle. Clinical signs and diagnosis. Arcanobacterium bovis causes mandibular lesions primarily. The mass will be firm, non-painful, and immovable. Draining tracts may develop over time. If teeth roots become involved, painful eating and weight loss are evident. Radiographic studies are helpful for determining fistulas. Diagnosis is based on clinical signs, and culture is required to confirm Arcanobacterium. The prognosis is poor for lumpy jaw. Epizootiology and transmission. These organisms are normal flora of the gastrointestinal tracts of ruminants and gain entrance into the tissues through abrasions and penetrating wounds. Necropsy. Draining lesions with sulfurlike granules (as with actinobacillosis) are frequently observed. Pathogenesis. Arcanobacterium pyogenes is known to produce an exotoxin, which may be involved in the pathogenesis. Differential diagnosis. Actinobacillus lignieresii and caseous lymphadenitis are important differentials for draining tracts. A major differential for omphalophlebitis is an umbilical hernia, which will typically not be painful or infected. There are many differentials for septic joints and polyarthritis: Chlamydia spp., Mycoplasma spp., streptococci, coliforms, Erysipelothrix rhusiopathiae, Fusobacterium necrophorum, and Salmonella spp. Tumors, trauma to the affected area, such as the mandible, and dental disease or oral foreign body should also be considered. Prevention and control. Arcanobacterium bovis lesions can be prevented or minimized by feeds without coarse or sharp materials. Treatment. Penicillin or derivatives such as ampicillin or amoxicillin are treatments of choice. Sodium iodides (intravenous) and potassium iodides (orally) have been utilized also. Extended antibiotic therapy may be necessary. Surgical excision is an option. In addition to medications noted above, isoniazid is somewhat effective for A. bovis infections in nonpregnant cattle. Research complications. The possibility of long-term infection and long therapy are factors that will diminish the value of affected research animals. Etiology. Arcanobacterium (formerly known as Actinomyces or Corynebacterium) pyogenes and A. bovis are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Arcanobacterium bovis is a normal part of the ruminant oral microflora and is the organism associated with "lumpy jaw" in cattle; this syndrome is rarely seen in sheep and goats. This organism has also been associated with pharyngitis and mastitis in cattle. Clinical signs and diagnosis. Arcanobacterium bovis causes mandibular lesions primarily. The mass will be firm, non-painful, and immovable. Draining tracts may develop over time. If teeth roots become involved, painful eating and weight loss are evident. Radiographic studies are helpful for determining fistulas. Diagnosis is based on clinical signs, and culture is required to confirm Arcanobacterium. The prognosis is poor for lumpy jaw. Epizootiology and transmission. These organisms are normal flora of the gastrointestinal tracts of ruminants and gain entrance into the tissues through abrasions and penetrating wounds. Necropsy. Draining lesions with sulfurlike granules (as with actinobacillosis) are frequently observed. Pathogenesis. Arcanobacterium pyogenes is known to produce an exotoxin, which may be involved in the pathogenesis. Differential diagnosis. Actinobacillus lignieresii and caseous lymphadenitis are important differentials for draining tracts. A major differential for omphalophlebitis is an umbilical hernia, which will typically not be painful or infected. There are many differentials for septic joints and polyarthritis: Chlamydia spp., Mycoplasma spp., streptococci, coliforms, Erysipelothrix rhusiopathiae, Fusobacterium necrophorum, and Salmonella spp. Tumors, trauma to the affected area, such as the mandible, and dental disease or oral foreign body should also be considered. Prevention and control. Arcanobacterium bovis lesions can be prevented or minimized by feeds without coarse or sharp materials. Treatment. Penicillin or derivatives such as ampicillin or amoxicillin are treatments of choice. Sodium iodides (intravenous) and potassium iodides (orally) have been utilized also. Extended antibiotic therapy may be necessary. Surgical excision is an option. In addition to medications noted above, isoniazid is somewhat effective for A. bovis infections in nonpregnant cattle. Research complications. The possibility of long-term infection and long therapy are factors that will diminish the value of affected research animals. Etiology. Arcanobacterium (formerly known as Actinomyces or Corynebacterium) pyogenes and A. bovis are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Arcanobacterium bovis is a normal part of the ruminant oral microflora and is the organism associated with "lumpy jaw" in cattle; this syndrome is rarely seen in sheep and goats. This organism has also been associated with pharyngitis and mastitis in cattle. Clinical signs and diagnosis. Arcanobacterium bovis causes mandibular lesions primarily. The mass will be firm, non-painful, and immovable. Draining tracts may develop over time. If teeth roots become involved, painful eating and weight loss are evident. Radiographic studies are helpful for determining fistulas. Diagnosis is based on clinical signs, and culture is required to confirm Arcanobacterium. The prognosis is poor for lumpy jaw. Epizootiology and transmission. These organisms are normal flora of the gastrointestinal tracts of ruminants and gain entrance into the tissues through abrasions and penetrating wounds. Necropsy. Draining lesions with sulfurlike granules (as with actinobacillosis) are frequently observed. Pathogenesis. Arcanobacterium pyogenes is known to produce an exotoxin, which may be involved in the pathogenesis. Differential diagnosis. Actinobacillus lignieresii and caseous lymphadenitis are important differentials for draining tracts. A major differential for omphalophlebitis is an umbilical hernia, which will typically not be painful or infected. There are many differentials for septic joints and polyarthritis: Chlamydia spp., Mycoplasma spp., streptococci, coliforms, Erysipelothrix rhusiopathiae, Fusobacterium necrophorum, and Salmonella spp. Tumors, trauma to the affected area, such as the mandible, and dental disease or oral foreign body should also be considered. Prevention and control. Arcanobacterium bovis lesions can be prevented or minimized by feeds without coarse or sharp materials. Treatment. Penicillin or derivatives such as ampicillin or amoxicillin are treatments of choice. Sodium iodides (intravenous) and potassium iodides (orally) have been utilized also. Extended antibiotic therapy may be necessary. Surgical excision is an option. In addition to medications noted above, isoniazid is somewhat effective for A. bovis infections in nonpregnant cattle. Research complications. The possibility of long-term infection and long therapy are factors that will diminish the value of affected research animals. c. Actinomycosis Omphalophlebitis, omphaloarteritis, omphalitis, and navel ill are terms referring to infection of the umbilicus in young animals. Arcanobacterium pyogenes is the most common organism causing omphalophlebitis, an acute localized inflammation and infection of the external umbilicus. Most cases occur within the first 3 months of age, and animals are presented with a painful enlargement of the umbilicus. Animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, hematuria, and so on. Severe sequelae may include septicemia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, and endocarditis. Research complications. Young stock affected by omphalophlebitis may be inappropriate subjects because of growth setbacks and physiologic stresses from the infection. Affected adult animals will not thrive and, even with therapy, may not be appropriate research subjects. Research complications. Young stock affected by omphalophlebitis may be inappropriate subjects because of growth setbacks and physiologic stresses from the infection. Affected adult animals will not thrive and, even with therapy, may not be appropriate research subjects. Research complications. Young stock affected by omphalophlebitis may be inappropriate subjects because of growth setbacks and physiologic stresses from the infection. Affected adult animals will not thrive and, even with therapy, may not be appropriate research subjects. d. Anthrax Etiology. Bacillus anthracis is a nonmotile, capsulated, spore-forming, aerobic, gram-positive bacillus that is found in alkaline soil, contaminated feeds (such as bonemeal), and water. Common names for the disease anthrax include woolsorters' disease, splenic fever, charbon, and milzbrand. Clinical signs and diagnosis. Anthrax is a sporadic but very serious infectious disease of cattle, sheep, and goats characterized by septicemia, hyperthermia, anorexia, depression, listlessness, depression, and tremors. Subacute and chronic cases may occur also and are characterized by swelling around the shoulders, ventral neck, and thorax. The incubation period is 1 day to 2 weeks. Bloody secretions such as hematuria and bloody diarrhea often occur. Abortion and blood-tinged milk may also be noted. The disease is usually fatal, especially in sheep and goats, after 1–3 days. Death is the result of shock, renal failure, and anoxia. Diagnosis is based on the clinical signs of peracute deaths and hemorrhage. Stained blood smears may show short, single to chained bacilli. Blood may be collected from a superficial vein and submitted for culture. Epizootiology and transmission. Cattle and sheep tend to be affected more commonly than goats, because of grazing habits. Older animals are more vulnerable than younger, and bulls are more vulnerable than cows. Although the disease occurs worldwide, and even in cold climates, most cases in the United States occur in the central and western states, and outbreaks usually occur as the result of spore release after abrupt climatic changes such as heavy rainfall after droughts or during warmer, dryer months. Spores survive very well in the environment. The anthrax organisms (primarily spores) are generally ingested, sporulate, and replicate in the local tissues. Abrasive forages may play a role in infection. Transmission via insect bites or through skin abrasions rarely occurs. Necropsy. Necropsies should not be done around animal pens or pastures, and definitive diagnoses may be made without opening the animals. Incomplete rigor mortis, rapid putrefaction, and dark, uncoagulated blood exuding from all body orifices are common findings. Blood collected carefully and promptly from peripheral veins of freshly dead animals can be used diagnostically. Splenomegaly, cyanosis, epicardial and subcutaneous hemorrhages, and lymphadenopathy are characterisitic of the disease. Pathogenesis. The rapidly multiplying organisms enter the lymphatics and bloodstream and result in a severe septicemia and neurotoxicosis. Encapsulation protects the organisms from phagocytosis. Liberated toxins cause local edema. Differential diagnosis. Although anthrax should always be considered when an animal healthy the previous day dies acutely, other causes of acute death in ruminants should be considered, e.g., bloat, poisoning, enterotoxemia, malignant edema, blackleg, and black disease. Prevention and control. Outbreaks must be reported to state officials. Anthrax is of particular concern as a bioterrorism agent. Any vaccination programs should also be reviewed with regulatory personnel. Herds in endemic areas and along waterways are usually vaccinated routinely with the Sterne-strain spore vaccine (virulent, nonencapsulated, live). Careful hygiene and quarantine practices are crucial during outbreaks. Dead animals and contaminated materials should be incinerated or buried deeply. Biting insects should be controlled. The disease is zoonotic and a serious public health risk. Treatment. Treatment of animals in early stages with penicillin and anthrax antitoxin (hyperimmune serum, if available) may be helpful. Amoxicillin, erythromycin, oxytetracycline, gentamicin, and fluoroquinolones are also good therapeutic agents. During epidemics, animals should be vaccinated with the Sterne vaccine. Research complications. Natural and experimental anthrax infections are a risk to research personnel; the pathogen may be present in many body fluids and can penetrate intact skin. The organism sporulates when exposed to air, and spores may be inhaled during postmortem examinations. Etiology. Bacillus anthracis is a nonmotile, capsulated, spore-forming, aerobic, gram-positive bacillus that is found in alkaline soil, contaminated feeds (such as bonemeal), and water. Common names for the disease anthrax include woolsorters' disease, splenic fever, charbon, and milzbrand. Clinical signs and diagnosis. Anthrax is a sporadic but very serious infectious disease of cattle, sheep, and goats characterized by septicemia, hyperthermia, anorexia, depression, listlessness, depression, and tremors. Subacute and chronic cases may occur also and are characterized by swelling around the shoulders, ventral neck, and thorax. The incubation period is 1 day to 2 weeks. Bloody secretions such as hematuria and bloody diarrhea often occur. Abortion and blood-tinged milk may also be noted. The disease is usually fatal, especially in sheep and goats, after 1–3 days. Death is the result of shock, renal failure, and anoxia. Diagnosis is based on the clinical signs of peracute deaths and hemorrhage. Stained blood smears may show short, single to chained bacilli. Blood may be collected from a superficial vein and submitted for culture. Epizootiology and transmission. Cattle and sheep tend to be affected more commonly than goats, because of grazing habits. Older animals are more vulnerable than younger, and bulls are more vulnerable than cows. Although the disease occurs worldwide, and even in cold climates, most cases in the United States occur in the central and western states, and outbreaks usually occur as the result of spore release after abrupt climatic changes such as heavy rainfall after droughts or during warmer, dryer months. Spores survive very well in the environment. The anthrax organisms (primarily spores) are generally ingested, sporulate, and replicate in the local tissues. Abrasive forages may play a role in infection. Transmission via insect bites or through skin abrasions rarely occurs. Necropsy. Necropsies should not be done around animal pens or pastures, and definitive diagnoses may be made without opening the animals. Incomplete rigor mortis, rapid putrefaction, and dark, uncoagulated blood exuding from all body orifices are common findings. Blood collected carefully and promptly from peripheral veins of freshly dead animals can be used diagnostically. Splenomegaly, cyanosis, epicardial and subcutaneous hemorrhages, and lymphadenopathy are characterisitic of the disease. Pathogenesis. The rapidly multiplying organisms enter the lymphatics and bloodstream and result in a severe septicemia and neurotoxicosis. Encapsulation protects the organisms from phagocytosis. Liberated toxins cause local edema. Differential diagnosis. Although anthrax should always be considered when an animal healthy the previous day dies acutely, other causes of acute death in ruminants should be considered, e.g., bloat, poisoning, enterotoxemia, malignant edema, blackleg, and black disease. Prevention and control. Outbreaks must be reported to state officials. Anthrax is of particular concern as a bioterrorism agent. Any vaccination programs should also be reviewed with regulatory personnel. Herds in endemic areas and along waterways are usually vaccinated routinely with the Sterne-strain spore vaccine (virulent, nonencapsulated, live). Careful hygiene and quarantine practices are crucial during outbreaks. Dead animals and contaminated materials should be incinerated or buried deeply. Biting insects should be controlled. The disease is zoonotic and a serious public health risk. Treatment. Treatment of animals in early stages with penicillin and anthrax antitoxin (hyperimmune serum, if available) may be helpful. Amoxicillin, erythromycin, oxytetracycline, gentamicin, and fluoroquinolones are also good therapeutic agents. During epidemics, animals should be vaccinated with the Sterne vaccine. Research complications. Natural and experimental anthrax infections are a risk to research personnel; the pathogen may be present in many body fluids and can penetrate intact skin. The organism sporulates when exposed to air, and spores may be inhaled during postmortem examinations. Etiology. Bacillus anthracis is a nonmotile, capsulated, spore-forming, aerobic, gram-positive bacillus that is found in alkaline soil, contaminated feeds (such as bonemeal), and water. Common names for the disease anthrax include woolsorters' disease, splenic fever, charbon, and milzbrand. Clinical signs and diagnosis. Anthrax is a sporadic but very serious infectious disease of cattle, sheep, and goats characterized by septicemia, hyperthermia, anorexia, depression, listlessness, depression, and tremors. Subacute and chronic cases may occur also and are characterized by swelling around the shoulders, ventral neck, and thorax. The incubation period is 1 day to 2 weeks. Bloody secretions such as hematuria and bloody diarrhea often occur. Abortion and blood-tinged milk may also be noted. The disease is usually fatal, especially in sheep and goats, after 1–3 days. Death is the result of shock, renal failure, and anoxia. Diagnosis is based on the clinical signs of peracute deaths and hemorrhage. Stained blood smears may show short, single to chained bacilli. Blood may be collected from a superficial vein and submitted for culture. Epizootiology and transmission. Cattle and sheep tend to be affected more commonly than goats, because of grazing habits. Older animals are more vulnerable than younger, and bulls are more vulnerable than cows. Although the disease occurs worldwide, and even in cold climates, most cases in the United States occur in the central and western states, and outbreaks usually occur as the result of spore release after abrupt climatic changes such as heavy rainfall after droughts or during warmer, dryer months. Spores survive very well in the environment. The anthrax organisms (primarily spores) are generally ingested, sporulate, and replicate in the local tissues. Abrasive forages may play a role in infection. Transmission via insect bites or through skin abrasions rarely occurs. Necropsy. Necropsies should not be done around animal pens or pastures, and definitive diagnoses may be made without opening the animals. Incomplete rigor mortis, rapid putrefaction, and dark, uncoagulated blood exuding from all body orifices are common findings. Blood collected carefully and promptly from peripheral veins of freshly dead animals can be used diagnostically. Splenomegaly, cyanosis, epicardial and subcutaneous hemorrhages, and lymphadenopathy are characterisitic of the disease. Pathogenesis. The rapidly multiplying organisms enter the lymphatics and bloodstream and result in a severe septicemia and neurotoxicosis. Encapsulation protects the organisms from phagocytosis. Liberated toxins cause local edema. Differential diagnosis. Although anthrax should always be considered when an animal healthy the previous day dies acutely, other causes of acute death in ruminants should be considered, e.g., bloat, poisoning, enterotoxemia, malignant edema, blackleg, and black disease. Prevention and control. Outbreaks must be reported to state officials. Anthrax is of particular concern as a bioterrorism agent. Any vaccination programs should also be reviewed with regulatory personnel. Herds in endemic areas and along waterways are usually vaccinated routinely with the Sterne-strain spore vaccine (virulent, nonencapsulated, live). Careful hygiene and quarantine practices are crucial during outbreaks. Dead animals and contaminated materials should be incinerated or buried deeply. Biting insects should be controlled. The disease is zoonotic and a serious public health risk. Treatment. Treatment of animals in early stages with penicillin and anthrax antitoxin (hyperimmune serum, if available) may be helpful. Amoxicillin, erythromycin, oxytetracycline, gentamicin, and fluoroquinolones are also good therapeutic agents. During epidemics, animals should be vaccinated with the Sterne vaccine. Research complications. Natural and experimental anthrax infections are a risk to research personnel; the pathogen may be present in many body fluids and can penetrate intact skin. The organism sporulates when exposed to air, and spores may be inhaled during postmortem examinations. e. Brucellosis Etiology. Brucella is a nonmotile, non-spore-forming, nonencapsulated, gram-negative coccobacillus. Brucella abortus is one of several Brucella species that infects domestic animals but cross-species infections occur rarely. Brucella abortus or B. melitensis may cause brucellosis in sheep, cattle, and goats. Brucella melitensis (biovar 1, 2, or 3) is the primary cause of sheep disease ( Garin-Bastuji et al., 1998 ). Brucella ovis is more commonly associated with ovine epididymitis or orchitis than abortion. In the United States, clusters of brucellosis are still found in western areas contiguous to Yellowstone National Park. Bang's disease is the common name given to the disease in ruminants. Clinical signs and diagnosis. Brucella melitensis in the adult ewe is generally asymptomatic and self-limiting within about 3 months. However, because the organism may enter and cause necrosis of the chorionic villi and fetal organs, abortion or stillbirths may occur. Abortion usually occurs in the third trimester, after which the ewe will appear to recover. It has been reported that up to 20% of infected ewes may abort more than once. Rams will also be infected and may develop orchitis or pneumonia. The disease caused by B. ovis is manifested by clinical or subclinical infection of the epididymis, leading to epididymal enlargement and testicular atrophy. Brucella ovis causes decreased fertility. Brucella melitensis is the more common cause of brucellosis in goats. Brucella abortus has been shown to infect goats in natural and experimental infections, and B. ovis has also been shown to infect goats experimentally. Does infected with B. melitensis will also abort during the third trimester. Infections with B. abortus in cattle produce few clinical signs. There may be a brief septicemia during which organisms are phagocytosed by neutrophils and fixed macrophages in lymph nodes. In cows, the organism localizes in supramammary lymph nodes and udders and in the endometrium and placenta of pregnant cows. Infection may cause abortions after the fifth month, with resulting retained placentas. Permanent infection of the udder is common and results in shedding of organisms in milk. In bulls, the organism may cause unilateral orchitis and epidydimitis and involvement of the secondary sex organs. Organisms may be in the semen. In infected herds, lameness may also be a clinical sign. Diagnosis of brucellosis can be made by bacterial isolation of the Brucella organism from necropsy samples (especially the fetal stomach contents), as well as by supportive serological evidence. Many serological tests are available, such as the tube and plate agglutination tests, the card or rose bengal test, the rivanol precipitation test, complement fixation, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and others. Test selection is often dependent on state requirements in the United States. Epizootiology and transmission. The primary route of transmission of B . abortus is ingestion of the organism from infected tissues and fluids (milk, vaginal and uterine discharges) during and for a few weeks after abortion or parturition; contaminated semen is considered to be a minor source of infection. Exposure to the organism may occur via the gastrointestinal tract (contaminated feed or water), the respiratory tract (droplet infection), or the reproductive tract (contaminated semen) and through other mucous membranes such as the conjunctiva. Brucella ovis is transmitted in the semen, as well as orally or nasally through contaminated feed and bedding. Necropsy findings. A sheep fetus aborted due to Brucella will exhibit generalized edema. The liver and spleen will be swollen, and serosal surfaces will be covered with petecchial hemorrhages. Peritoneal and pleural cavities often contain serofibrinous exudates. The placenta will be leathery. Pathogenesis. Ruminants are considered especially susceptible to Brucella infection, because of higher levels of erythritol (a sugar alcohol), which is a growth stimulant for the organism. Brucella utilizes erythritol preferentially over glucose as an energy source. Placentas and male genitalia also contain high levels of erythritol. Brucella organisms also evade lysis when phagocytosed by macrophages and neutrophils and survive intracellularly in phagosomes. Abortion is the result of placentitis, typically during the third trimester of gestation. Brucella ovis enters the host through the mucous membranes, then passes into the lymphatics, causes hyperplasia of reticuloendothelial cells, and is spread to various organs via the blood. The organism localizes in the epididymides, the seminal vesicles, the bulbourethral glands, and the ampullae. Orchitis may be a sequelae of the disease. Epididymitis can be diagnosed by identifying gross lesions by palpation of the epididymides, by serological evidence of antibodies to B. ovis, and by semen cultures. Differential diagnosis. Differential diagnoses include all other abortion-causing diseases. Many other agents, such as Actinobacillus spp., Arcanobacterium (Actinomyces) pyogenes, Eschericia coli, Pseudomonas spp., Proteus mirabilis, Chlamydia, Mycoplasma, and others may be associated with ovine epididymitis and orchitis. A clinically and pathologically similar agent, Actinobacillus seminis, has been isolated from virgin rams. This organism has morphological and staining characteristics similar to those of B. ovis and complicates the diagnosis ( Genetzky, 1995 ). Prevention and control. The Rev 1 vaccine has been recommended for vaccination of ewe lambs in endemic areas, but this vaccine is not used in the United States. Separating young rams from potentially infected older males, sanitizing facilities, and vaccinating them with B. ovis bacterin can prevent the disease. Over the past 20 years, aggressive federal and state regulatory and cattle herd health programs in the United States have provided control and prevention mechanisms for this pathogen through a combination of serological monitoring of herds, slaughter of diseased animals, herd management, vaccination programs, and monitoring of transported animals. Most states are considered brucellosis-free in the cattle populations; thus, procurement of ruminants that have been exposed to this infectious agent will be unlikely. Cattle vaccination programs can be very successful when conducted on a herd basis to reduce likelihood of exposure. Strain 19 and the recently validated attentuated strain RB51 are live vaccines and can be used in healthy heifer calves 4–12 months old. Vaccination for older animals may be done under certain circumstances. Vaccination of bull calves is not recommended, because of low likelihood of spread through semen and possibility of vaccination-induced orchitis. The strain 19 vaccine induces long-term cell-mediated immunity, protects a herd from abortions, and protects the majority of a herd from reactors during a screening and culling program. The vaccine will not, however, protect the animals from becoming infected with B. abortus. Strain 19 vaccine induces an antibody response in cattle. The RB51 vaccine does not result in antibody titers and therefore is advantageous because infection with Brucella can be determined serologically. The RB51 vaccine has been designated as the official calfhood bovine brucellosis vaccine in the United States by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS) ( Stevens et al., 1997 ). Brucella vaccine should be administered to unstressed, healthy cattle, with attention to particular side effects of the vaccination material and to prevention of compounding stresses associated with weaning, regrouping, other management changes, and shipping. The RB51 is regarded as less pathogenic and abortigenic in cattle. Treatment. Definite confirmation of Brucella infection is important from the standpoint of public and herd health. Culling is considered the treatment of choice in cattle herds. Rams infected with B. ovis should be isolated and treated with tetracyclines. Research complications. Brucellosis represents a research complication as a cause of abortions and of infections in male ruminants. Impairment of the infected host's immune system, especially alteration of phagocytic cells where the bacteria stay in membrane-bound vesicles, should be considered. The potential complications of needle sticks by large-animal veterinarians with the strain 19 vaccine and the public health risks (undulant fever) are well known. Less is known presently regarding the RB51 vaccine effects in humans. Epidemiologic and diagnostic methodologies are being developed to track and monitor these cases. There is also a risk of human infection from handling infected materials during a dystocia or postmortem. Worldwide, B. melitensis is the leading cause of human brucellosis. Etiology. Brucella is a nonmotile, non-spore-forming, nonencapsulated, gram-negative coccobacillus. Brucella abortus is one of several Brucella species that infects domestic animals but cross-species infections occur rarely. Brucella abortus or B. melitensis may cause brucellosis in sheep, cattle, and goats. Brucella melitensis (biovar 1, 2, or 3) is the primary cause of sheep disease ( Garin-Bastuji et al., 1998 ). Brucella ovis is more commonly associated with ovine epididymitis or orchitis than abortion. In the United States, clusters of brucellosis are still found in western areas contiguous to Yellowstone National Park. Bang's disease is the common name given to the disease in ruminants. Clinical signs and diagnosis. Brucella melitensis in the adult ewe is generally asymptomatic and self-limiting within about 3 months. However, because the organism may enter and cause necrosis of the chorionic villi and fetal organs, abortion or stillbirths may occur. Abortion usually occurs in the third trimester, after which the ewe will appear to recover. It has been reported that up to 20% of infected ewes may abort more than once. Rams will also be infected and may develop orchitis or pneumonia. The disease caused by B. ovis is manifested by clinical or subclinical infection of the epididymis, leading to epididymal enlargement and testicular atrophy. Brucella ovis causes decreased fertility. Brucella melitensis is the more common cause of brucellosis in goats. Brucella abortus has been shown to infect goats in natural and experimental infections, and B. ovis has also been shown to infect goats experimentally. Does infected with B. melitensis will also abort during the third trimester. Infections with B. abortus in cattle produce few clinical signs. There may be a brief septicemia during which organisms are phagocytosed by neutrophils and fixed macrophages in lymph nodes. In cows, the organism localizes in supramammary lymph nodes and udders and in the endometrium and placenta of pregnant cows. Infection may cause abortions after the fifth month, with resulting retained placentas. Permanent infection of the udder is common and results in shedding of organisms in milk. In bulls, the organism may cause unilateral orchitis and epidydimitis and involvement of the secondary sex organs. Organisms may be in the semen. In infected herds, lameness may also be a clinical sign. Diagnosis of brucellosis can be made by bacterial isolation of the Brucella organism from necropsy samples (especially the fetal stomach contents), as well as by supportive serological evidence. Many serological tests are available, such as the tube and plate agglutination tests, the card or rose bengal test, the rivanol precipitation test, complement fixation, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and others. Test selection is often dependent on state requirements in the United States. Epizootiology and transmission. The primary route of transmission of B . abortus is ingestion of the organism from infected tissues and fluids (milk, vaginal and uterine discharges) during and for a few weeks after abortion or parturition; contaminated semen is considered to be a minor source of infection. Exposure to the organism may occur via the gastrointestinal tract (contaminated feed or water), the respiratory tract (droplet infection), or the reproductive tract (contaminated semen) and through other mucous membranes such as the conjunctiva. Brucella ovis is transmitted in the semen, as well as orally or nasally through contaminated feed and bedding. Necropsy findings. A sheep fetus aborted due to Brucella will exhibit generalized edema. The liver and spleen will be swollen, and serosal surfaces will be covered with petecchial hemorrhages. Peritoneal and pleural cavities often contain serofibrinous exudates. The placenta will be leathery. Pathogenesis. Ruminants are considered especially susceptible to Brucella infection, because of higher levels of erythritol (a sugar alcohol), which is a growth stimulant for the organism. Brucella utilizes erythritol preferentially over glucose as an energy source. Placentas and male genitalia also contain high levels of erythritol. Brucella organisms also evade lysis when phagocytosed by macrophages and neutrophils and survive intracellularly in phagosomes. Abortion is the result of placentitis, typically during the third trimester of gestation. Brucella ovis enters the host through the mucous membranes, then passes into the lymphatics, causes hyperplasia of reticuloendothelial cells, and is spread to various organs via the blood. The organism localizes in the epididymides, the seminal vesicles, the bulbourethral glands, and the ampullae. Orchitis may be a sequelae of the disease. Epididymitis can be diagnosed by identifying gross lesions by palpation of the epididymides, by serological evidence of antibodies to B. ovis, and by semen cultures. Differential diagnosis. Differential diagnoses include all other abortion-causing diseases. Many other agents, such as Actinobacillus spp., Arcanobacterium (Actinomyces) pyogenes, Eschericia coli, Pseudomonas spp., Proteus mirabilis, Chlamydia, Mycoplasma, and others may be associated with ovine epididymitis and orchitis. A clinically and pathologically similar agent, Actinobacillus seminis, has been isolated from virgin rams. This organism has morphological and staining characteristics similar to those of B. ovis and complicates the diagnosis ( Genetzky, 1995 ). Prevention and control. The Rev 1 vaccine has been recommended for vaccination of ewe lambs in endemic areas, but this vaccine is not used in the United States. Separating young rams from potentially infected older males, sanitizing facilities, and vaccinating them with B. ovis bacterin can prevent the disease. Over the past 20 years, aggressive federal and state regulatory and cattle herd health programs in the United States have provided control and prevention mechanisms for this pathogen through a combination of serological monitoring of herds, slaughter of diseased animals, herd management, vaccination programs, and monitoring of transported animals. Most states are considered brucellosis-free in the cattle populations; thus, procurement of ruminants that have been exposed to this infectious agent will be unlikely. Cattle vaccination programs can be very successful when conducted on a herd basis to reduce likelihood of exposure. Strain 19 and the recently validated attentuated strain RB51 are live vaccines and can be used in healthy heifer calves 4–12 months old. Vaccination for older animals may be done under certain circumstances. Vaccination of bull calves is not recommended, because of low likelihood of spread through semen and possibility of vaccination-induced orchitis. The strain 19 vaccine induces long-term cell-mediated immunity, protects a herd from abortions, and protects the majority of a herd from reactors during a screening and culling program. The vaccine will not, however, protect the animals from becoming infected with B. abortus. Strain 19 vaccine induces an antibody response in cattle. The RB51 vaccine does not result in antibody titers and therefore is advantageous because infection with Brucella can be determined serologically. The RB51 vaccine has been designated as the official calfhood bovine brucellosis vaccine in the United States by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS) ( Stevens et al., 1997 ). Brucella vaccine should be administered to unstressed, healthy cattle, with attention to particular side effects of the vaccination material and to prevention of compounding stresses associated with weaning, regrouping, other management changes, and shipping. The RB51 is regarded as less pathogenic and abortigenic in cattle. Treatment. Definite confirmation of Brucella infection is important from the standpoint of public and herd health. Culling is considered the treatment of choice in cattle herds. Rams infected with B. ovis should be isolated and treated with tetracyclines. Research complications. Brucellosis represents a research complication as a cause of abortions and of infections in male ruminants. Impairment of the infected host's immune system, especially alteration of phagocytic cells where the bacteria stay in membrane-bound vesicles, should be considered. The potential complications of needle sticks by large-animal veterinarians with the strain 19 vaccine and the public health risks (undulant fever) are well known. Less is known presently regarding the RB51 vaccine effects in humans. Epidemiologic and diagnostic methodologies are being developed to track and monitor these cases. There is also a risk of human infection from handling infected materials during a dystocia or postmortem. Worldwide, B. melitensis is the leading cause of human brucellosis. Etiology. Brucella is a nonmotile, non-spore-forming, nonencapsulated, gram-negative coccobacillus. Brucella abortus is one of several Brucella species that infects domestic animals but cross-species infections occur rarely. Brucella abortus or B. melitensis may cause brucellosis in sheep, cattle, and goats. Brucella melitensis (biovar 1, 2, or 3) is the primary cause of sheep disease ( Garin-Bastuji et al., 1998 ). Brucella ovis is more commonly associated with ovine epididymitis or orchitis than abortion. In the United States, clusters of brucellosis are still found in western areas contiguous to Yellowstone National Park. Bang's disease is the common name given to the disease in ruminants. Clinical signs and diagnosis. Brucella melitensis in the adult ewe is generally asymptomatic and self-limiting within about 3 months. However, because the organism may enter and cause necrosis of the chorionic villi and fetal organs, abortion or stillbirths may occur. Abortion usually occurs in the third trimester, after which the ewe will appear to recover. It has been reported that up to 20% of infected ewes may abort more than once. Rams will also be infected and may develop orchitis or pneumonia. The disease caused by B. ovis is manifested by clinical or subclinical infection of the epididymis, leading to epididymal enlargement and testicular atrophy. Brucella ovis causes decreased fertility. Brucella melitensis is the more common cause of brucellosis in goats. Brucella abortus has been shown to infect goats in natural and experimental infections, and B. ovis has also been shown to infect goats experimentally. Does infected with B. melitensis will also abort during the third trimester. Infections with B. abortus in cattle produce few clinical signs. There may be a brief septicemia during which organisms are phagocytosed by neutrophils and fixed macrophages in lymph nodes. In cows, the organism localizes in supramammary lymph nodes and udders and in the endometrium and placenta of pregnant cows. Infection may cause abortions after the fifth month, with resulting retained placentas. Permanent infection of the udder is common and results in shedding of organisms in milk. In bulls, the organism may cause unilateral orchitis and epidydimitis and involvement of the secondary sex organs. Organisms may be in the semen. In infected herds, lameness may also be a clinical sign. Diagnosis of brucellosis can be made by bacterial isolation of the Brucella organism from necropsy samples (especially the fetal stomach contents), as well as by supportive serological evidence. Many serological tests are available, such as the tube and plate agglutination tests, the card or rose bengal test, the rivanol precipitation test, complement fixation, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and others. Test selection is often dependent on state requirements in the United States. Epizootiology and transmission. The primary route of transmission of B . abortus is ingestion of the organism from infected tissues and fluids (milk, vaginal and uterine discharges) during and for a few weeks after abortion or parturition; contaminated semen is considered to be a minor source of infection. Exposure to the organism may occur via the gastrointestinal tract (contaminated feed or water), the respiratory tract (droplet infection), or the reproductive tract (contaminated semen) and through other mucous membranes such as the conjunctiva. Brucella ovis is transmitted in the semen, as well as orally or nasally through contaminated feed and bedding. Necropsy findings. A sheep fetus aborted due to Brucella will exhibit generalized edema. The liver and spleen will be swollen, and serosal surfaces will be covered with petecchial hemorrhages. Peritoneal and pleural cavities often contain serofibrinous exudates. The placenta will be leathery. Pathogenesis. Ruminants are considered especially susceptible to Brucella infection, because of higher levels of erythritol (a sugar alcohol), which is a growth stimulant for the organism. Brucella utilizes erythritol preferentially over glucose as an energy source. Placentas and male genitalia also contain high levels of erythritol. Brucella organisms also evade lysis when phagocytosed by macrophages and neutrophils and survive intracellularly in phagosomes. Abortion is the result of placentitis, typically during the third trimester of gestation. Brucella ovis enters the host through the mucous membranes, then passes into the lymphatics, causes hyperplasia of reticuloendothelial cells, and is spread to various organs via the blood. The organism localizes in the epididymides, the seminal vesicles, the bulbourethral glands, and the ampullae. Orchitis may be a sequelae of the disease. Epididymitis can be diagnosed by identifying gross lesions by palpation of the epididymides, by serological evidence of antibodies to B. ovis, and by semen cultures. Differential diagnosis. Differential diagnoses include all other abortion-causing diseases. Many other agents, such as Actinobacillus spp., Arcanobacterium (Actinomyces) pyogenes, Eschericia coli, Pseudomonas spp., Proteus mirabilis, Chlamydia, Mycoplasma, and others may be associated with ovine epididymitis and orchitis. A clinically and pathologically similar agent, Actinobacillus seminis, has been isolated from virgin rams. This organism has morphological and staining characteristics similar to those of B. ovis and complicates the diagnosis ( Genetzky, 1995 ). Prevention and control. The Rev 1 vaccine has been recommended for vaccination of ewe lambs in endemic areas, but this vaccine is not used in the United States. Separating young rams from potentially infected older males, sanitizing facilities, and vaccinating them with B. ovis bacterin can prevent the disease. Over the past 20 years, aggressive federal and state regulatory and cattle herd health programs in the United States have provided control and prevention mechanisms for this pathogen through a combination of serological monitoring of herds, slaughter of diseased animals, herd management, vaccination programs, and monitoring of transported animals. Most states are considered brucellosis-free in the cattle populations; thus, procurement of ruminants that have been exposed to this infectious agent will be unlikely. Cattle vaccination programs can be very successful when conducted on a herd basis to reduce likelihood of exposure. Strain 19 and the recently validated attentuated strain RB51 are live vaccines and can be used in healthy heifer calves 4–12 months old. Vaccination for older animals may be done under certain circumstances. Vaccination of bull calves is not recommended, because of low likelihood of spread through semen and possibility of vaccination-induced orchitis. The strain 19 vaccine induces long-term cell-mediated immunity, protects a herd from abortions, and protects the majority of a herd from reactors during a screening and culling program. The vaccine will not, however, protect the animals from becoming infected with B. abortus. Strain 19 vaccine induces an antibody response in cattle. The RB51 vaccine does not result in antibody titers and therefore is advantageous because infection with Brucella can be determined serologically. The RB51 vaccine has been designated as the official calfhood bovine brucellosis vaccine in the United States by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS) ( Stevens et al., 1997 ). Brucella vaccine should be administered to unstressed, healthy cattle, with attention to particular side effects of the vaccination material and to prevention of compounding stresses associated with weaning, regrouping, other management changes, and shipping. The RB51 is regarded as less pathogenic and abortigenic in cattle. Treatment. Definite confirmation of Brucella infection is important from the standpoint of public and herd health. Culling is considered the treatment of choice in cattle herds. Rams infected with B. ovis should be isolated and treated with tetracyclines. Research complications. Brucellosis represents a research complication as a cause of abortions and of infections in male ruminants. Impairment of the infected host's immune system, especially alteration of phagocytic cells where the bacteria stay in membrane-bound vesicles, should be considered. The potential complications of needle sticks by large-animal veterinarians with the strain 19 vaccine and the public health risks (undulant fever) are well known. Less is known presently regarding the RB51 vaccine effects in humans. Epidemiologic and diagnostic methodologies are being developed to track and monitor these cases. There is also a risk of human infection from handling infected materials during a dystocia or postmortem. Worldwide, B. melitensis is the leading cause of human brucellosis. f. Campylobacteriosis (Vibriosis) i. Campylobacter fetus subsp. intestinalis; C. jejuni infection (ovine vibriosis) Etiology. Campylobacter (Vibrio) fetus subsp. intestinalis, a pleomorphic curved to coccoid, motile, non-spore-forming, gram-negative bacterium, causes campylobacteriosis, the most important cause of ovine abortion in the United States. There are few reports of campylobacteriosis in goats in the United States. Vibriosis is derived from the name formerly given to the genus; the term is still frequently used. Clinical signs and diagnosis. Ovine vibriosis is a contagious disease that causes abortion, stillbirths, and weak lambs. The organism inhabits the intestines and gallbladder in subclinical carriers. Abortion generally occurs in the last trimester, and abortion storms may occur as more susceptible animals, such as maiden ewes, become exposed to the infectious tissues. It is reported that 20–25% of the flock may become infected and up to 5% of the ewes will die ( Jensen and Swift, 1982 ). Some lambs may be born alive but will be weak, and dams will not be able to produce milk. Diagnosis is achieved by microscopic identification or isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharges. Tentative identification of the organism can be made by observing curved ("gull-wing") rods in Giemsa-stained or Ziehl–Neelsen–stained smears from fetal stomach contents, placentomes, or maternal uterine fluids. Epizootiology and transmission. Campylobacteriosis occurs worldwide. Campylobacter spp., such as C. jejuni, normally inhabit ovine gastrointestinal tracts. Transmission of the disease occurs through the gastrointestinal tract, followed by shedding, especially associated with aborted tissues and fluids. In abortion storms, considerable contamination of the environment will occur due to placenta, fetuses, and uterine fluids. Ewes may have active Campylobacter organisms in uterine discharges for several months after abortion. The bacteria will also be shed in feces, and feed and water contamination serve as another source. There is no venereal transmission in the ovine. Necropsy. Aborted fetuses will be edematous, with accumulation of serosanguinous fluids within the subcutis and muscle tissue fascia. The liver may contain 2–3 cm pale foci. Placental tissues will be thickened and edematous and will contain serous fluids similar to those of the fetus. The placental cotyledons may appear gray. Pathogenesis. The organism enters the bloodstream and causes a short-term bacteremia (1–2 weeks) prior to the localizing of the bacteria in the chorionic epithelial cells and finally passing into the fetus. Differential diagnosis. Toxoplasma, Chlamydia, and Listeria should be considered in late gestation ovine abortions. Prevention and control. A bacterin is available to prevent the disease. Carrier states have been cleared by treating with a combination of antibiotics, including penicillin and oral Chlortetracycline. Aborting ewes should be isolated immediately from the rest of the flock. After an outbreak, ewes will develop immunity lasting 2–3 years. Treatment. Infected animals should be isolated and provided with supportive therapy. Prompt decontamination of the area and disposal of the aborted tissues and discharges are important. Research complications. Losses from abortion may be considerable. Campylobacter ssp. are zoonotic agents, and C. fetus subsp. intestinalis may be the cause of "shepherd's scours." ii. Campylobacter fetus subsp. venerealis infection (bovine vibriosis) Etiology. Campylobacter fetus subsp. venerealis is the main cause of bovine campylobacteriosis abortions. It does not cause disease in other ruminant species. Clinical signs and diagnosis. Preliminary signs of a problem in the herd will be a high percentage of cows returning to estrus after breeding and temporary infertility. This will be particularly apparent in virgin heifers that may return to estrus by 40 days after breeding. Long interestrous intervals also serve an indication of a problem. Spontaneous abortions will occur in some cases, typically during the fourth to eighth months of gestation. Severe endometritis may lead to salpingitis and permanent infertility. Demonstration or isolation of the organism, a curved rod with corkscrew motility, is the basis for diagnosis. The vaginal mucous agglutination test is used to survey herds for campylobacteriosis. Serology will not be worthwhile, because the infection does not trigger a sufficient antibody response. Culture from breeding animals may be difficult because Campylobacter will be overgrown by faster-growing species also present in the specimens. Epizootiology and transmission. The bacteria is an obligate, ubiquitous organism of the genital tract. Transmission is from infected bulls to heifers. Older cows develop effective immunity. Necropsy findings. Necrotizing placentitis, dehydration, and fibrinous serositis will be found grossly. In addition, bronchopneumonia and hepatitis will be seen histologically. Pathogenesis. Campylobacter organisms grow readily in the genital tract, and infection is established within days of exposure. The resulting endometritis prevents conception or causes embyronic death. Differential diagnosis. The primary differential diagnosis for campylobacteriosis is trichomoniasis. Other venereal diseases should be considered when infertility problems are noted in a herd. These include brucellosis, mycoplasmosis, ureaplasmosis, infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV), and bovine virus diarrhea (BVD). Leptospirosis should also be considered. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. Killed bacterin vaccines are available, either as oil adjuvant or as aluminum hydroxide adsorbed. The former is preferred because of duration of immunity but causes granulomas. That vaccine also has specific recommendations regarding administration several months before the breeding season. The latter product is administered closer to the breeding season, and the duration of immunity is not as prolonged. In both cases, boosters should be given after the initial immunization and as part of the regular prebreeding regimen. Only one bacterin product is approved for use in bulls. Many combination vaccine products contain only the aluminum hydroxide adsorbed product. Artificial insemination (AI) is particularly useful at controlling the disease, but bulls used for AI must be part of a screening program for this and other venereal diseases such as trichomoniasis. Treatment. Cows will usually recover from the infection, and treatment with antibiotics such as penicillin, administered as an intrauterine infusion, improve the chances of returning to breeding condition. i. Campylobacter fetus subsp. intestinalis; C. jejuni infection (ovine vibriosis) Etiology. Campylobacter (Vibrio) fetus subsp. intestinalis, a pleomorphic curved to coccoid, motile, non-spore-forming, gram-negative bacterium, causes campylobacteriosis, the most important cause of ovine abortion in the United States. There are few reports of campylobacteriosis in goats in the United States. Vibriosis is derived from the name formerly given to the genus; the term is still frequently used. Clinical signs and diagnosis. Ovine vibriosis is a contagious disease that causes abortion, stillbirths, and weak lambs. The organism inhabits the intestines and gallbladder in subclinical carriers. Abortion generally occurs in the last trimester, and abortion storms may occur as more susceptible animals, such as maiden ewes, become exposed to the infectious tissues. It is reported that 20–25% of the flock may become infected and up to 5% of the ewes will die ( Jensen and Swift, 1982 ). Some lambs may be born alive but will be weak, and dams will not be able to produce milk. Diagnosis is achieved by microscopic identification or isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharges. Tentative identification of the organism can be made by observing curved ("gull-wing") rods in Giemsa-stained or Ziehl–Neelsen–stained smears from fetal stomach contents, placentomes, or maternal uterine fluids. Epizootiology and transmission. Campylobacteriosis occurs worldwide. Campylobacter spp., such as C. jejuni, normally inhabit ovine gastrointestinal tracts. Transmission of the disease occurs through the gastrointestinal tract, followed by shedding, especially associated with aborted tissues and fluids. In abortion storms, considerable contamination of the environment will occur due to placenta, fetuses, and uterine fluids. Ewes may have active Campylobacter organisms in uterine discharges for several months after abortion. The bacteria will also be shed in feces, and feed and water contamination serve as another source. There is no venereal transmission in the ovine. Necropsy. Aborted fetuses will be edematous, with accumulation of serosanguinous fluids within the subcutis and muscle tissue fascia. The liver may contain 2–3 cm pale foci. Placental tissues will be thickened and edematous and will contain serous fluids similar to those of the fetus. The placental cotyledons may appear gray. Pathogenesis. The organism enters the bloodstream and causes a short-term bacteremia (1–2 weeks) prior to the localizing of the bacteria in the chorionic epithelial cells and finally passing into the fetus. Differential diagnosis. Toxoplasma, Chlamydia, and Listeria should be considered in late gestation ovine abortions. Prevention and control. A bacterin is available to prevent the disease. Carrier states have been cleared by treating with a combination of antibiotics, including penicillin and oral Chlortetracycline. Aborting ewes should be isolated immediately from the rest of the flock. After an outbreak, ewes will develop immunity lasting 2–3 years. Treatment. Infected animals should be isolated and provided with supportive therapy. Prompt decontamination of the area and disposal of the aborted tissues and discharges are important. Research complications. Losses from abortion may be considerable. Campylobacter ssp. are zoonotic agents, and C. fetus subsp. intestinalis may be the cause of "shepherd's scours." Etiology. Campylobacter (Vibrio) fetus subsp. intestinalis, a pleomorphic curved to coccoid, motile, non-spore-forming, gram-negative bacterium, causes campylobacteriosis, the most important cause of ovine abortion in the United States. There are few reports of campylobacteriosis in goats in the United States. Vibriosis is derived from the name formerly given to the genus; the term is still frequently used. Clinical signs and diagnosis. Ovine vibriosis is a contagious disease that causes abortion, stillbirths, and weak lambs. The organism inhabits the intestines and gallbladder in subclinical carriers. Abortion generally occurs in the last trimester, and abortion storms may occur as more susceptible animals, such as maiden ewes, become exposed to the infectious tissues. It is reported that 20–25% of the flock may become infected and up to 5% of the ewes will die ( Jensen and Swift, 1982 ). Some lambs may be born alive but will be weak, and dams will not be able to produce milk. Diagnosis is achieved by microscopic identification or isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharges. Tentative identification of the organism can be made by observing curved ("gull-wing") rods in Giemsa-stained or Ziehl–Neelsen–stained smears from fetal stomach contents, placentomes, or maternal uterine fluids. Epizootiology and transmission. Campylobacteriosis occurs worldwide. Campylobacter spp., such as C. jejuni, normally inhabit ovine gastrointestinal tracts. Transmission of the disease occurs through the gastrointestinal tract, followed by shedding, especially associated with aborted tissues and fluids. In abortion storms, considerable contamination of the environment will occur due to placenta, fetuses, and uterine fluids. Ewes may have active Campylobacter organisms in uterine discharges for several months after abortion. The bacteria will also be shed in feces, and feed and water contamination serve as another source. There is no venereal transmission in the ovine. Necropsy. Aborted fetuses will be edematous, with accumulation of serosanguinous fluids within the subcutis and muscle tissue fascia. The liver may contain 2–3 cm pale foci. Placental tissues will be thickened and edematous and will contain serous fluids similar to those of the fetus. The placental cotyledons may appear gray. Pathogenesis. The organism enters the bloodstream and causes a short-term bacteremia (1–2 weeks) prior to the localizing of the bacteria in the chorionic epithelial cells and finally passing into the fetus. Differential diagnosis. Toxoplasma, Chlamydia, and Listeria should be considered in late gestation ovine abortions. Prevention and control. A bacterin is available to prevent the disease. Carrier states have been cleared by treating with a combination of antibiotics, including penicillin and oral Chlortetracycline. Aborting ewes should be isolated immediately from the rest of the flock. After an outbreak, ewes will develop immunity lasting 2–3 years. Treatment. Infected animals should be isolated and provided with supportive therapy. Prompt decontamination of the area and disposal of the aborted tissues and discharges are important. Research complications. Losses from abortion may be considerable. Campylobacter ssp. are zoonotic agents, and C. fetus subsp. intestinalis may be the cause of "shepherd's scours." ii. Campylobacter fetus subsp. venerealis infection (bovine vibriosis) Etiology. Campylobacter fetus subsp. venerealis is the main cause of bovine campylobacteriosis abortions. It does not cause disease in other ruminant species. Clinical signs and diagnosis. Preliminary signs of a problem in the herd will be a high percentage of cows returning to estrus after breeding and temporary infertility. This will be particularly apparent in virgin heifers that may return to estrus by 40 days after breeding. Long interestrous intervals also serve an indication of a problem. Spontaneous abortions will occur in some cases, typically during the fourth to eighth months of gestation. Severe endometritis may lead to salpingitis and permanent infertility. Demonstration or isolation of the organism, a curved rod with corkscrew motility, is the basis for diagnosis. The vaginal mucous agglutination test is used to survey herds for campylobacteriosis. Serology will not be worthwhile, because the infection does not trigger a sufficient antibody response. Culture from breeding animals may be difficult because Campylobacter will be overgrown by faster-growing species also present in the specimens. Epizootiology and transmission. The bacteria is an obligate, ubiquitous organism of the genital tract. Transmission is from infected bulls to heifers. Older cows develop effective immunity. Necropsy findings. Necrotizing placentitis, dehydration, and fibrinous serositis will be found grossly. In addition, bronchopneumonia and hepatitis will be seen histologically. Pathogenesis. Campylobacter organisms grow readily in the genital tract, and infection is established within days of exposure. The resulting endometritis prevents conception or causes embyronic death. Differential diagnosis. The primary differential diagnosis for campylobacteriosis is trichomoniasis. Other venereal diseases should be considered when infertility problems are noted in a herd. These include brucellosis, mycoplasmosis, ureaplasmosis, infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV), and bovine virus diarrhea (BVD). Leptospirosis should also be considered. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. Killed bacterin vaccines are available, either as oil adjuvant or as aluminum hydroxide adsorbed. The former is preferred because of duration of immunity but causes granulomas. That vaccine also has specific recommendations regarding administration several months before the breeding season. The latter product is administered closer to the breeding season, and the duration of immunity is not as prolonged. In both cases, boosters should be given after the initial immunization and as part of the regular prebreeding regimen. Only one bacterin product is approved for use in bulls. Many combination vaccine products contain only the aluminum hydroxide adsorbed product. Artificial insemination (AI) is particularly useful at controlling the disease, but bulls used for AI must be part of a screening program for this and other venereal diseases such as trichomoniasis. Treatment. Cows will usually recover from the infection, and treatment with antibiotics such as penicillin, administered as an intrauterine infusion, improve the chances of returning to breeding condition. Etiology. Campylobacter fetus subsp. venerealis is the main cause of bovine campylobacteriosis abortions. It does not cause disease in other ruminant species. Clinical signs and diagnosis. Preliminary signs of a problem in the herd will be a high percentage of cows returning to estrus after breeding and temporary infertility. This will be particularly apparent in virgin heifers that may return to estrus by 40 days after breeding. Long interestrous intervals also serve an indication of a problem. Spontaneous abortions will occur in some cases, typically during the fourth to eighth months of gestation. Severe endometritis may lead to salpingitis and permanent infertility. Demonstration or isolation of the organism, a curved rod with corkscrew motility, is the basis for diagnosis. The vaginal mucous agglutination test is used to survey herds for campylobacteriosis. Serology will not be worthwhile, because the infection does not trigger a sufficient antibody response. Culture from breeding animals may be difficult because Campylobacter will be overgrown by faster-growing species also present in the specimens. Epizootiology and transmission. The bacteria is an obligate, ubiquitous organism of the genital tract. Transmission is from infected bulls to heifers. Older cows develop effective immunity. Necropsy findings. Necrotizing placentitis, dehydration, and fibrinous serositis will be found grossly. In addition, bronchopneumonia and hepatitis will be seen histologically. Pathogenesis. Campylobacter organisms grow readily in the genital tract, and infection is established within days of exposure. The resulting endometritis prevents conception or causes embyronic death. Differential diagnosis. The primary differential diagnosis for campylobacteriosis is trichomoniasis. Other venereal diseases should be considered when infertility problems are noted in a herd. These include brucellosis, mycoplasmosis, ureaplasmosis, infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV), and bovine virus diarrhea (BVD). Leptospirosis should also be considered. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. Killed bacterin vaccines are available, either as oil adjuvant or as aluminum hydroxide adsorbed. The former is preferred because of duration of immunity but causes granulomas. That vaccine also has specific recommendations regarding administration several months before the breeding season. The latter product is administered closer to the breeding season, and the duration of immunity is not as prolonged. In both cases, boosters should be given after the initial immunization and as part of the regular prebreeding regimen. Only one bacterin product is approved for use in bulls. Many combination vaccine products contain only the aluminum hydroxide adsorbed product. Artificial insemination (AI) is particularly useful at controlling the disease, but bulls used for AI must be part of a screening program for this and other venereal diseases such as trichomoniasis. Treatment. Cows will usually recover from the infection, and treatment with antibiotics such as penicillin, administered as an intrauterine infusion, improve the chances of returning to breeding condition. g. Caprine Staphylococcal Dermatitis Etiology. The most common caprine bacterial skin infection is caused by Staphylococcus intermedius or S. aureus and is known as staphylococcal dermatitis ( Smith and Sherman, 1994 ). The Staphylococcus organisms are cocci and are categorized as primary pathogens or ubiquitous skin commensals of humans and animals. Staphylococcus aureus and S. intermedius are classified as primary pathogens and produce coagulase, a virulence factor. Clinical signs and diagnosis. Small pustular lesions, caused by bacterial infection and inflammation of the hair follicle, occur around the teats and perineum. Occasionally, the infection may involve the flanks, underbelly, axilla, inner thigh, and neck. Staphylococcal dermatitis may occur secondary to other skin lesions. Diagnosis is based on lesions. Culture will distinguish S. aureus. Pathogenesis. Simple boredom may cause rubbing, followed by staphylococcal infection of damaged epidermis. Differential diagnosis. The presence of scabs makes contagious ecthyma a differential diagnosis, along with fungal skin infections and nutritional causes of skin disease. Treatment. Severe infections should be treated with antibiotics based on culture and sensitivity. Severe lesions and lesions localized to the underbelly, thighs, and udder benefit by periodic cleaning with an iodophor shampoo and spraying with an antibiotic and an astringent ( Smith and Sherman, 1994 ). Etiology. The most common caprine bacterial skin infection is caused by Staphylococcus intermedius or S. aureus and is known as staphylococcal dermatitis ( Smith and Sherman, 1994 ). The Staphylococcus organisms are cocci and are categorized as primary pathogens or ubiquitous skin commensals of humans and animals. Staphylococcus aureus and S. intermedius are classified as primary pathogens and produce coagulase, a virulence factor. Clinical signs and diagnosis. Small pustular lesions, caused by bacterial infection and inflammation of the hair follicle, occur around the teats and perineum. Occasionally, the infection may involve the flanks, underbelly, axilla, inner thigh, and neck. Staphylococcal dermatitis may occur secondary to other skin lesions. Diagnosis is based on lesions. Culture will distinguish S. aureus. Pathogenesis. Simple boredom may cause rubbing, followed by staphylococcal infection of damaged epidermis. Differential diagnosis. The presence of scabs makes contagious ecthyma a differential diagnosis, along with fungal skin infections and nutritional causes of skin disease. Treatment. Severe infections should be treated with antibiotics based on culture and sensitivity. Severe lesions and lesions localized to the underbelly, thighs, and udder benefit by periodic cleaning with an iodophor shampoo and spraying with an antibiotic and an astringent ( Smith and Sherman, 1994 ). Etiology. The most common caprine bacterial skin infection is caused by Staphylococcus intermedius or S. aureus and is known as staphylococcal dermatitis ( Smith and Sherman, 1994 ). The Staphylococcus organisms are cocci and are categorized as primary pathogens or ubiquitous skin commensals of humans and animals. Staphylococcus aureus and S. intermedius are classified as primary pathogens and produce coagulase, a virulence factor. Clinical signs and diagnosis. Small pustular lesions, caused by bacterial infection and inflammation of the hair follicle, occur around the teats and perineum. Occasionally, the infection may involve the flanks, underbelly, axilla, inner thigh, and neck. Staphylococcal dermatitis may occur secondary to other skin lesions. Diagnosis is based on lesions. Culture will distinguish S. aureus. Pathogenesis. Simple boredom may cause rubbing, followed by staphylococcal infection of damaged epidermis. Differential diagnosis. The presence of scabs makes contagious ecthyma a differential diagnosis, along with fungal skin infections and nutritional causes of skin disease. Treatment. Severe infections should be treated with antibiotics based on culture and sensitivity. Severe lesions and lesions localized to the underbelly, thighs, and udder benefit by periodic cleaning with an iodophor shampoo and spraying with an antibiotic and an astringent ( Smith and Sherman, 1994 ). h. Clostridial Diseases i. Clostridium perfringens type C infection (enterotoxemia and struck) Etiology. Clostridium perfringens is an anaerobic, gram-positive, nonmotile, spore-forming bacterium that lives in the soil, in contaminated feed, and in gastrointestinal tracts of ruminants. The bacteria is categorized by toxin production. Toxins include alpha (hemolytic), beta (necrotizing), delta (cytotoxic and hemoltyic), epsilon, and iota. Types of C. perfingens are A, B, C, D, and E. This is a common and economically significant disease of sheep, goats, and cattle. Clinical signs and diagnosis. The beta toxin associated with overgrowth of this bacterium results in a fatal hemorrhagic enterocolitis within the first 72 hr of a young ruminant's life. Many animals may be found dead, with no clinical presentation. Affected animals are acutely anemic, dehydrated, anorexic, restless, and depressed and may display tremors or convulsions as well as abdominal pain. Feces may range from loose gray-brown to dark red and malodorous. Morbidity and mortality may be nearly 100%. A similar noncontagious but acutely fatal form of enterotoxemia in adult sheep, called struck, occurs in yearlings and adults. Struck is rare in the United States. The disease is also caused by the beta toxin of C. perfringens type C and is often associated with rapid dietary changes or shearing stresses in sheep. Although affected animals are usually found dead, clinical signs include uneasiness, depression, and convulsions. Mortality is usually less than 15%. Diagnosis is usually based on necropsy findings, although confirmation can be made by culture of the organism. Identification of the beta toxin in intestinal contents may be difficult because of instability of the toxin. Epizootiology and transmission. Clostridial organisms are ubiquitous in the environment as well as in the gastrointestinal tract and contaminated feeds. Confinement and poor sanitation predisposes to infection with C. perfringens. Transmission is by ingestion of contaminated material. Necropsy findings. Necropsy findings include a milk-filled abomasum, and hemorrhage in the distal small intestine and throughout the large intestine. Petechial hemorrhages of the serosal surfaces of many organs, especially the thymus, heart, and gastrointestinal tract, will be visible. Hydropericardium, hydroperitoneum, and hemorrhagic mesenteric lymph nodes will also be present. Pulmonary and brain edema may also be seen. Histologically, the gram-positive C. perfringens organisms may be visible in excess numbers along the mucosal surface of the swollen, congested, necrotic intestines. In cases of struck, necropsy findings include congestion and erosions of the mucosa of the gastrointestinal tract, serosal hemorrhages, and serous peritoneal and pericardial fluids. In late stages of the disease and especially if prompt necropsy is not performed, the organism will infiltrate the muscle fascial layers and produce serohemorrhagic and gaseous infiltration of perimysial and epimysial spaces. Pathogenesis. Hemorrhagic enterotoxemia is an acute, sporadic disease caused by the beta toxin of Clostridium perfringens type C. Neonates ingest the organism, which then proliferates and attaches to the gastrointestinal microvilli and elaborates primarily the beta toxins. The trypsin inhibitors present in colostrum prevent inactivation of the beta toxin. The toxins injure intestinal epithelial cells and then enter the blood, leading to acute toxemia. The intestinal injury may result in diarrhea, with small amounts of hemorrhage. Associated electrolyte and water loss result in dehydration, acidosis, and shock. Differential diagnosis. Differential diagnoses include other clostridial diseases such as blackleg and black disease, as well as coccidiosis, salmonellosis, anthrax, and acute poisoning. Prevention and control. A commercial toxoid is available and should be administered to the pregnant animals prior to parturition. An alternative includes administration of an antitoxin to the newborn lambs. The disease may become endemic once it is on the premises. Treatment. Treatment is difficult and usually unsuccessful. Antitoxin may be useful in milder cases, and the antitoxin and toxoid can also be administered during an outbreak. Research complications. This disease can be costly in losses of neonates and younger animals. ii. Clostridium perfringens type D infection (pulpy kidney disease) Etiology. Clostridium perfringens type D releases epsilon toxin that is proteolytically activated by trypsin. This disease caused by C. perfringens tends to be associated with sheep and is of less importance in goats and cattle. Clinical signs. The peracute condition in younger animals is characterized by sudden deaths, which are occasionally preceded by neurological signs such as incoordination, opisthotonus, and convulsions. Because the disease progresses so rapidly to death (within 1–2 hr), clinical signs are rarely observed. Hypersalivation, rapid respirations, hyperthermia, convulsions, and opisthotonus have been noted. In acute cases, hyperglycemia and glucosuria are considered almost pathognomonic. Clinical signs in chronic cases in older animals, such as adult goats, include soft stools, weight loss, anorexia, depression, and severe diarrhea, sometimes with mucus and blood. Mature affected sheep may be blind and anorectic and may head-press. Necropsy findings. Necropsy findings are similar to those seen with C. perfringens type C. Additionally, extremely necrotic, soft kidneys ("pulpy kidneys") are usually observed immediately following death. (This phenomenon is in contrast to what is normally associated with later stages of postmortem autolysis.) Focal encephalomalacia, and petechial hemorrhages on serosal surfaces of the brain, diaphragm, gastrointestinal tract, and heart are common findings. Diagnosis can be made from the typical clinical signs and necropsy findings as well as the observation of glucose in the urine at necropsy. Pathogenesis. The epsilon toxin causes neuronal death and shock, probably through vascular damage. The noncontagious, peracute form of enterotoxemia occurs in suckling, fast-growing animals, either nursing from their dams or on high-protein, high-energy concentrates. The largest, fastest-growing animals generally are predisposed to this condition; for example, lambs, fat ewe lambs, and usually singleton lambs tend to be most susceptible. The hyperglycemia and glucosuria seen in acute cases are due to epsilon toxin effects on liver glycogen metabolism. Differential diagnosis. Tetanus, enterotoxigenic E. coli, botulism, polioencephalomalacia, grain overload, and listeriosis are differentials. Prevention and control. Vaccination prevents the disease. Maternal antibodies last approximately 5 weeks postpartum; thus young animals should be vaccinated at about this time. Feeding regimens to young, fast-growing animals and feeding of concentrates to adults should be evaluated carefully. Treatment. Treatment consists of support (fluids, warmth), antitoxin administration, oral antibiotics, and diet adjustment. iii. Clostridium tetani infection (tetanus, lockjaw) Etiology. Clostridium tetani is a strictly anaerobic, motile, spore-forming, gram-positive rod that persists in soils and manure and within the gastrointestinal tract. At least 10 serotypes of C. tetani exist. Clinical signs. Infection by C. tetani is characterized by a sporadic, acute, and fatal neuropathy. After an incubation period of 4 days to 3 weeks, the animal exhibits bloat; muscular spasticity; prolapse of the third eyelid; rigidity and extension of the limbs, leading to a stiff gate; an inability to chew; and hyperthermia. Erect or drooped ears, retracted lips, drooling, hypersensitivity to external stimuli, and a "sawhorse" stance are frequent signs. The animal may convulse. Death occurs within 3–10 days, and mortality is nearly 100%, primarily from respiratory failure. Diagnosis is based on clinical signs. Muscle-related serum enzymes such as aspartate aminotransferase (AST), creatinine kinase (CK), and lactate dehydrogenase (LDH) might be elevated. ( Jensen and Swift, 1982 ). Serum cortisol may also be elevated, and stress hyperglycemia may be evident. Permanent lameness may result in survivors. Epizootiology and transmission. Clostridium tetani is a soil contaminant and is often found as part of the gut microflora of herbivores. The organisms sporulate and persist in the environment. All species of livestock are susceptible, but sheep and goats are more susceptible than cattle. Individual cases may occur, or herd outbreaks may follow castration, tail docking, ear tagging, or dehorning. Mouth wounds may also be sites of entry. Pathogenesis. Tetanus, or lockjaw, is caused by the toxins of C. tetani. All serovars produce the same exotoxin, which is a multiunit protein composed of tetanospasmin, which is neurotoxic, and tetanolysin, which is hemolytic. A nonspasmogenic toxin is also produced. Contamination of wounds results in anaerobic proliferation of the bacterium and liberation of the tetanospasmin, which diffuses through motor neurons in a retrograde direction to the spinal cord. The toxin inhibits the release of glycine and γ-aminobutyric acid from Renshaw cells; this results in hypertonia and muscular spasms. Proliferation of C. tetani in the gut of affected animals may also serve as a source and may produce clinical signs. The uterus is the most common site of infection in postparturient dairy cattle with retained placentas. Differential diagnoses. Early in the course of the infection, differential diagnoses include bloat, rabies, hypomagnesemic tetany, polioencephalomalacia, white muscle disease, enterotoxemia in lambs, and lead poisoning. Polyarthritis of cattle is a differential for the gait changes in that species. Necropsy findings. Findings are nonspecific except for the inflammatory reaction associated with the wound. Because of the low number of organisms necessary to cause neurotoxicosis, isolation of C. tetani from the wound may be difficult. Treatment. Treatment consists of cleaning the infected wound; administering tetanus antitoxin (e.g., at least 500 IU in an adult sheep or goat); vaccinating with tetanus toxoid; administering of antibiotics (penicillin, both parenterally [potassium penicillin intravenously and procaine penicillin intramuscularly] and flushed into the cleaned wound), a sedative or tranquilizer (e.g., acepromazine or chlorpromazine) and a muscle relaxant; and keeping the animal in a dark, quiet environment. Supportive fluids and glucose must be administered until the animal is capable of feeding. If the animal survives, revaccination should be done 14 days after the previous dose. Prevention and control. Like other ubiquitous clostridial diseases, tetanus is impossible to eradicate. The disease can be controlled and prevented by following good sanitation measures, aseptic surgical procedures, and vaccination programs. Tetanus toxoid vaccine is available and very effective for stimulating long-term immunity. Tetanus antitoxin can be administered (200 IU in lambs) as a preventive or in the face of disease as an adjunct to therapy. Both the toxoid and the antitoxin can be administered to an animal at the same time, but they should not be mixed in the syringe, and each should be administered at different sites, with a second toxoid dose administered 4 weeks later. Animals should be vaccinated 2 or 3 times during the first year of life. Does and ewes should receive booster vaccinations within 2 months of parturition to ensure colostral antibodies. Research complications. Unprotected, younger ruminants may be affected following routine flock or herd management procedures. Contaminated or inadequately managed open wounds or lesions in older animals may provide anaerobic incubation sites. iv. Clostridium novyi infection (bighead; black disease; bacillary hemoglobinuria, or red water) and C. chauvoei infection (blackleg) Etiology. Clostridium novyi, an anaerobic, motile, spore-forming, gram-positive bacteria, is the agent of bighead and black disease. Clostridium novyi type D (C. hemolyticum) is the cause of bacillary hemoglobinuria, or "red water." Clostridium chauvoei is the causative agent of blackleg. Clinical signs. Bighead is a disease of rams characterized by edema of the head and neck. The edema may migrate to ventral regions such as the throat. Additional clinical signs include swelling of the eyelids and nostrils. Most animals will die within 48–72 hours. Black disease, or infectious necrotic hepatitis, is a peracute, fatal disease associated with C. novyi. It is more common in cattle and sheep but may be seen in goats. The clinical course is 1–2 days in cattle and slightly shorter in sheep. Otherwise healthy-appearing adult animals are often affected. Clinical signs are rarely seen, because of the peracute nature of the disease. Occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency are observed prior to death. Bacillary hemoglobinuria is an acute disease seen primarily in cattle and characterized by fever and anorexia, in addition to the hemoglobinemia and hemoglobinuria indicated by the name. Animals that survive a few days will develop icterus. Mortality may be high. Blackleg, a disease similar to bighead, causes necrosis and emphysema of muscle masses, serohemorrhagic fluid accumulation around the infected area, and edema ( Jackson et al., 1995 ). Blackleg is more common in cattle than in sheep. The incubation period is 2–5 days and is followed by hyperthermia, muscular stiffness and pain, anorexia, and gangrenous myositis. The clinical course is short, 24–48 hr, and untreated animals invariably die. Blackleg in cattle can be associated with subcutaneous edema or crepitation; these do not usually occur in sheep. Most lesions are associated with muscles of the face, neck, perineum, thigh, and back. Epizootiology and transmission. Bighead is caused by the toxins of C. novyi, which enters through wounds often associated with horn injuries during fighting. The C. novyi type B organisms produce alpha and beta toxins, and the alpha toxins are mostly responsible for toxemia, tissue necrosis, and subsequent death. Clostridium novyi type D is endemic in the western United States. It is hypothesized that the C. chauvoei organisms enter through the gastrointestinal tract. Black disease and bacillary hemoglobinuria are associated with concurrent liver disease, often associated with Fasciola infections (liver flukes); it is sometimes seen as a sequela to liver biopsies. The diseases are more common in summer months, and fecal contamination of pastures, flooding, and infected carcasses are sources of the organism. Birds and wild animals may be vectors of the pathogen. Ingested spores are believed to develop in hepatic tissue damaged and anoxic from the fluke migrations. Necropsy. Diagnosis of black disease is usually based on postmortem lesions. Subcutaneous vessels will be engorged with blood, resulting in dried skin with a dark appearance. Carcasses putrefy quickly. In addition, hepatomegaly and endocardial hemorrhages are common, and hepatic damage from flukes may be so severe that diagnosis is difficult. Blood coagulates slowly in affected animals. Pathogenesis. The propagation of the clostridial organisms is self-promoted by the damage caused by the toxins and the increased local anaerobic environment created. Clostridium novyi proliferates in the soft tissues of the head and neck, and the resultant clostridial toxin causes increased capillary permeability and the liberation of serous fluids into the tissues. Mixed infections with related clostridial organisms may lead to increasing hemorrhage and necrosis in the affected tissues. Diagnosis is based on clinical signs. In black disease and bacillary hemoglobinuria disease, the ingested clostridial spores are absorbed, enter the liver, and cause hepatic necrosis. Associated toxemia causes subcutaneous vascular dilatation; increased pericardial, pleural, and peritoneal fluid; and endocardial hemorrhages. The toxins produced by C. novyi, identified as beta, eta, and theta, and each having enzymatic or lytic properties or both, also contribute to the hemolytic disease. Clostridium chauvoei spores proliferate in traumatized muscle areas damaged by transportation, rough handling, or injury. Differential diagnosis. Differential diagnoses include other clostridial diseases as well as photosensitization. Hemolytic diseases such as babesiosis, leptospirosis, and hemobartonellosis should be included as differentials. Treatment. For C. chauvoei infection (blackleg), early treatment with penicillin or tetracycline may be helpful. Treatment for black disease is not rewarding even if the animal is found before death. Carcasses from bacillary hemoglobinuria losses should be burned, buried deeply, or removed from the premises. Prevention and control. Vaccinating animals with multivalent clostridial vaccines can prevent these diseases. Subcutaneous administration of vaccine material is recommended over intramuscular. Vaccinations may be useful in an outbreak. Careful handling of ruminants during shipping and transfers will contribute to fewer muscular injuries. For bighead, mature rams penned together should be monitored for lesions, especially during breeding season. Control of fascioliasis is very important in prevention and control of black disease and in the optimal timing of vaccinations. v. Clostridium septicum infection (malignant edema) Etiology. Clostridium septicum is the species usually associated with malignant edema, but mixed infections involving other clostridial species such as C. chauvoei, C. novyi, C. sordellii, and C. perfringens may occur. Clostridium spp. are motile (C. chauvoei, C. septicum) or nonmotile, anaerobic, spore-forming, gram-positive rods. Clinicial signs. Malignant edema, or gas gangrene, is an acute and often fatal bacterial disease caused by Clostridium spp. The incubation period is approximately 2–4 days. The affected area will be warm and will contain gaseous accumulations that can be palpated as crepitation of the subcutaneous tissue around the infected area. Regional lymphadenopathy and fever may occur. The animal becomes anorexic, severely depressed, and possibly hyperthermic. Edema and crepitation may be noted around the wound; death occurs within 12 hr to 2 days. Epizootiology and transmission. The organisms are ubiquitous in the environment and may survive in the soil for years. The disease is especially prevalent in animals that have had recent wounds such as those that have undergone castration, docking, ear notching, shearing, or dystocia. Necropsy findings. The tissue necrosis and hemorrhagic serous fluid accumulations resemble those of other clostridial diseases. Pathogenesis. In most cases, the clostridial organisms cause a spreading infection through the fascial planes around the area of the injury; vegetative organisms then produce potent exotoxins, which result in necrosis (alpha toxin) and/or hemolysis (beta toxin). Furthermore, the toxins enter the bloodstream and central nervous system, resulting in systemic collapse and high mortality. Necropsy. Spreading, crepitant lesions around wounds are suggestive of malignant edema. Affected tissues are inflamed and necrotic. Gas and serosanguineous fluids with foul odors infiltrate the tissue planes. Large rod-shaped bacteria may be observed on histopathology; confirmation is made through culture and identification. Intramuscular inoculation of guinea pigs causes a necrotizing myositis and death. Organisms can be cultured from guinea pig tissues. Treatment. Infected animals can be treated with large doses of penicillin and fenestration of the wound is recommended. Prevention and control. Proper preparation of surgical sites, correct sanitation of instruments and the housing environment, and attention to postoperative wounds will help prevent this disease. Multivalent clostridial vaccines are available. Research complications. Morbidity or loss of animals from lack of or unsuccessful vaccination and from contaminated surgical sites or wounds may be consequences of this disease. i. Colibacillosis Etiology. Escherichia coli is a motile, aerobic, gram-negative, non-spore-forming coccobacillus commonly found in the environment and gastrointestinal tracts of ruminants. Escherichia coli organisms have three areas of surface antigenic complexes (O, somatic; K, envelope or pili; and H, flagellar), which are used to "group" or classify the serotypes. Colibacillosis is the common term for infections in younger animals caused by this bacteria. Clinical signs. Presentation of E. coli infections vary with the animal's age and the type of E. coli involved. Enterotoxigenic E. coli infection causes gastroenteritis and/or septicemia in lambs and calves. Colibacillosis generally develops within the first 72 hr of life when newborn animals are exposed to the organism. The enteric infection causes a semifluid, yellow to gray diarrhea. Occasionally blood streaking of the feces may be observed. The animal may demonstrate abdominal pain, evidenced by arching of the back and extension of the tail, classically described as "tucked up." Hyperthermia is rare. Severe acidosis, depression, and recumbancy ensue, and mortality may be as high as 75%. The septicemic form generally occurs between 2 and 6 weeks of age. Animals display an elevated body temperature and show signs suggestive of nervous system involvement such as incoordination, head pressing, circling, and the appearance of blindness. Opisthotonos, depression, and death follow. Occasionally, swollen, painful joints may be observed with septicemic colibacillosis. Blood cultures may be helpful in identifying the septicemic form. In ruminants, E. coli is is a less common cause of cystitis and pyelonephritis. The cystitis is characterized by dysuria and pollakiuria; gross hematuria and pyuria may be present. The infection may or may not be restricted to the bladder; in the later presentation, and in cases of pyelonephritis, a cow will be acutely depressed, have a fever and ruminal stasis, and be anorexic. In chronic cases, animals will be polyuric and undergo weight loss. Escherichia coli may also cause in utero disease in cattle, resulting in abortion or weakened offspring. Epizootiology and transmission. Escherichia coli is one of the most common gram-negative pathogens isolated from ruminant neonates. Zeman et al. (1989 ) classify E. coli infections into four groups: enterotoxigenic, enterohemorrhagic, enteropathogenic, and enteroinvasive. Enterotoxigenic E. coli (ETEC) attach to the enterocytes via pili, produce enterotoxins, and are the primary cause of colibacillosis in animals and humans. Fimbrial (pili) antigens associated with ovine disease include K99 and F41. Enterohemorrhagic E. coli (EHEC) attach and efface the microvillus, produce verotoxins, and occasionally cause disease in humans and animals. Enteropathogenic E. coli (EPEC) colonize and efface the microvillus but do not produce verotoxins. EPEC are associated with disease in humans and rabbits and cause a secretory diarrhea. Enteroinvasive E. coli (EIEC) invade the enterocytes of humans and cause a shigella-like disease. Overcrowding and poor sanitation contribute significantly to the development of this disease in young animals. The organism will be endemic in a contaminated environment and present on dams' udders. The bacteria rapidly proliferate in the neonates' small intestines. The bacteria and associated toxins cause a secretory diarrhea, resulting in the loss of water and electrolytes. If the bacteria infiltrate the intestinal barrier and enter the blood, septicemia results. Diagnosis of the enteric form can be made by observation of clinical signs, including diarrhea and staining of the tail and wool. Necropsy findings. Swollen, yellow to gray, fluid-filled small and large intestines, swollen and hemorrhagic mesenteric lymph nodes, and generalized tissue dehydration are common. Septicemic lambs may have serofibrinous fluid in the peritoneal, thoracic, and pericardial cavities; enlarged joints containing fibrinopurulent exudates; and congested and inflamed meninges. Isolation and serotyping of E. coli confirm the diagnosis. ELISA and latex agglutination tests are available diagnostic tools. Differential diagnosis. Differential diagnoses include the enterotoxemias caused by C. perfringens type A, B, or C; Campylobacter jejuni; Coccidia, rotavirus, coronavirus, Salmonella, and Cryptosporidia. Other contributing causes of abomasal tympany in young ruminants, such as dietary changes, copper deficiency, excessive intervals between feedings of milk replacer, or feeding large volumes should be considered. Prevention and control. The best preventive measures are maintenance of proper housing conditions, limiting overcrowding, and frequently sanitizing lambing areas. Attention to colostrum feeding techniques and colostral quality are important means of preventing disease. Treatment must include intravenous fluid hydration and reestablishment of acid-base and electrolyte abnormalities. Treatment. Antibiotics such as trimethoprim-sulfadiazine, enrofloxacin, cephalothin, amikacin, and apramycin may be helpful; oral antibiotics are not recommended. Vaccines are available for prevention of colibacillosis in cattle. i. Clostridium perfringens type C infection (enterotoxemia and struck) Etiology. Clostridium perfringens is an anaerobic, gram-positive, nonmotile, spore-forming bacterium that lives in the soil, in contaminated feed, and in gastrointestinal tracts of ruminants. The bacteria is categorized by toxin production. Toxins include alpha (hemolytic), beta (necrotizing), delta (cytotoxic and hemoltyic), epsilon, and iota. Types of C. perfingens are A, B, C, D, and E. This is a common and economically significant disease of sheep, goats, and cattle. Clinical signs and diagnosis. The beta toxin associated with overgrowth of this bacterium results in a fatal hemorrhagic enterocolitis within the first 72 hr of a young ruminant's life. Many animals may be found dead, with no clinical presentation. Affected animals are acutely anemic, dehydrated, anorexic, restless, and depressed and may display tremors or convulsions as well as abdominal pain. Feces may range from loose gray-brown to dark red and malodorous. Morbidity and mortality may be nearly 100%. A similar noncontagious but acutely fatal form of enterotoxemia in adult sheep, called struck, occurs in yearlings and adults. Struck is rare in the United States. The disease is also caused by the beta toxin of C. perfringens type C and is often associated with rapid dietary changes or shearing stresses in sheep. Although affected animals are usually found dead, clinical signs include uneasiness, depression, and convulsions. Mortality is usually less than 15%. Diagnosis is usually based on necropsy findings, although confirmation can be made by culture of the organism. Identification of the beta toxin in intestinal contents may be difficult because of instability of the toxin. Epizootiology and transmission. Clostridial organisms are ubiquitous in the environment as well as in the gastrointestinal tract and contaminated feeds. Confinement and poor sanitation predisposes to infection with C. perfringens. Transmission is by ingestion of contaminated material. Necropsy findings. Necropsy findings include a milk-filled abomasum, and hemorrhage in the distal small intestine and throughout the large intestine. Petechial hemorrhages of the serosal surfaces of many organs, especially the thymus, heart, and gastrointestinal tract, will be visible. Hydropericardium, hydroperitoneum, and hemorrhagic mesenteric lymph nodes will also be present. Pulmonary and brain edema may also be seen. Histologically, the gram-positive C. perfringens organisms may be visible in excess numbers along the mucosal surface of the swollen, congested, necrotic intestines. In cases of struck, necropsy findings include congestion and erosions of the mucosa of the gastrointestinal tract, serosal hemorrhages, and serous peritoneal and pericardial fluids. In late stages of the disease and especially if prompt necropsy is not performed, the organism will infiltrate the muscle fascial layers and produce serohemorrhagic and gaseous infiltration of perimysial and epimysial spaces. Pathogenesis. Hemorrhagic enterotoxemia is an acute, sporadic disease caused by the beta toxin of Clostridium perfringens type C. Neonates ingest the organism, which then proliferates and attaches to the gastrointestinal microvilli and elaborates primarily the beta toxins. The trypsin inhibitors present in colostrum prevent inactivation of the beta toxin. The toxins injure intestinal epithelial cells and then enter the blood, leading to acute toxemia. The intestinal injury may result in diarrhea, with small amounts of hemorrhage. Associated electrolyte and water loss result in dehydration, acidosis, and shock. Differential diagnosis. Differential diagnoses include other clostridial diseases such as blackleg and black disease, as well as coccidiosis, salmonellosis, anthrax, and acute poisoning. Prevention and control. A commercial toxoid is available and should be administered to the pregnant animals prior to parturition. An alternative includes administration of an antitoxin to the newborn lambs. The disease may become endemic once it is on the premises. Treatment. Treatment is difficult and usually unsuccessful. Antitoxin may be useful in milder cases, and the antitoxin and toxoid can also be administered during an outbreak. Research complications. This disease can be costly in losses of neonates and younger animals. Etiology. Clostridium perfringens is an anaerobic, gram-positive, nonmotile, spore-forming bacterium that lives in the soil, in contaminated feed, and in gastrointestinal tracts of ruminants. The bacteria is categorized by toxin production. Toxins include alpha (hemolytic), beta (necrotizing), delta (cytotoxic and hemoltyic), epsilon, and iota. Types of C. perfingens are A, B, C, D, and E. This is a common and economically significant disease of sheep, goats, and cattle. Clinical signs and diagnosis. The beta toxin associated with overgrowth of this bacterium results in a fatal hemorrhagic enterocolitis within the first 72 hr of a young ruminant's life. Many animals may be found dead, with no clinical presentation. Affected animals are acutely anemic, dehydrated, anorexic, restless, and depressed and may display tremors or convulsions as well as abdominal pain. Feces may range from loose gray-brown to dark red and malodorous. Morbidity and mortality may be nearly 100%. A similar noncontagious but acutely fatal form of enterotoxemia in adult sheep, called struck, occurs in yearlings and adults. Struck is rare in the United States. The disease is also caused by the beta toxin of C. perfringens type C and is often associated with rapid dietary changes or shearing stresses in sheep. Although affected animals are usually found dead, clinical signs include uneasiness, depression, and convulsions. Mortality is usually less than 15%. Diagnosis is usually based on necropsy findings, although confirmation can be made by culture of the organism. Identification of the beta toxin in intestinal contents may be difficult because of instability of the toxin. Epizootiology and transmission. Clostridial organisms are ubiquitous in the environment as well as in the gastrointestinal tract and contaminated feeds. Confinement and poor sanitation predisposes to infection with C. perfringens. Transmission is by ingestion of contaminated material. Necropsy findings. Necropsy findings include a milk-filled abomasum, and hemorrhage in the distal small intestine and throughout the large intestine. Petechial hemorrhages of the serosal surfaces of many organs, especially the thymus, heart, and gastrointestinal tract, will be visible. Hydropericardium, hydroperitoneum, and hemorrhagic mesenteric lymph nodes will also be present. Pulmonary and brain edema may also be seen. Histologically, the gram-positive C. perfringens organisms may be visible in excess numbers along the mucosal surface of the swollen, congested, necrotic intestines. In cases of struck, necropsy findings include congestion and erosions of the mucosa of the gastrointestinal tract, serosal hemorrhages, and serous peritoneal and pericardial fluids. In late stages of the disease and especially if prompt necropsy is not performed, the organism will infiltrate the muscle fascial layers and produce serohemorrhagic and gaseous infiltration of perimysial and epimysial spaces. Pathogenesis. Hemorrhagic enterotoxemia is an acute, sporadic disease caused by the beta toxin of Clostridium perfringens type C. Neonates ingest the organism, which then proliferates and attaches to the gastrointestinal microvilli and elaborates primarily the beta toxins. The trypsin inhibitors present in colostrum prevent inactivation of the beta toxin. The toxins injure intestinal epithelial cells and then enter the blood, leading to acute toxemia. The intestinal injury may result in diarrhea, with small amounts of hemorrhage. Associated electrolyte and water loss result in dehydration, acidosis, and shock. Differential diagnosis. Differential diagnoses include other clostridial diseases such as blackleg and black disease, as well as coccidiosis, salmonellosis, anthrax, and acute poisoning. Prevention and control. A commercial toxoid is available and should be administered to the pregnant animals prior to parturition. An alternative includes administration of an antitoxin to the newborn lambs. The disease may become endemic once it is on the premises. Treatment. Treatment is difficult and usually unsuccessful. Antitoxin may be useful in milder cases, and the antitoxin and toxoid can also be administered during an outbreak. Research complications. This disease can be costly in losses of neonates and younger animals. ii. Clostridium perfringens type D infection (pulpy kidney disease) Etiology. Clostridium perfringens type D releases epsilon toxin that is proteolytically activated by trypsin. This disease caused by C. perfringens tends to be associated with sheep and is of less importance in goats and cattle. Clinical signs. The peracute condition in younger animals is characterized by sudden deaths, which are occasionally preceded by neurological signs such as incoordination, opisthotonus, and convulsions. Because the disease progresses so rapidly to death (within 1–2 hr), clinical signs are rarely observed. Hypersalivation, rapid respirations, hyperthermia, convulsions, and opisthotonus have been noted. In acute cases, hyperglycemia and glucosuria are considered almost pathognomonic. Clinical signs in chronic cases in older animals, such as adult goats, include soft stools, weight loss, anorexia, depression, and severe diarrhea, sometimes with mucus and blood. Mature affected sheep may be blind and anorectic and may head-press. Necropsy findings. Necropsy findings are similar to those seen with C. perfringens type C. Additionally, extremely necrotic, soft kidneys ("pulpy kidneys") are usually observed immediately following death. (This phenomenon is in contrast to what is normally associated with later stages of postmortem autolysis.) Focal encephalomalacia, and petechial hemorrhages on serosal surfaces of the brain, diaphragm, gastrointestinal tract, and heart are common findings. Diagnosis can be made from the typical clinical signs and necropsy findings as well as the observation of glucose in the urine at necropsy. Pathogenesis. The epsilon toxin causes neuronal death and shock, probably through vascular damage. The noncontagious, peracute form of enterotoxemia occurs in suckling, fast-growing animals, either nursing from their dams or on high-protein, high-energy concentrates. The largest, fastest-growing animals generally are predisposed to this condition; for example, lambs, fat ewe lambs, and usually singleton lambs tend to be most susceptible. The hyperglycemia and glucosuria seen in acute cases are due to epsilon toxin effects on liver glycogen metabolism. Differential diagnosis. Tetanus, enterotoxigenic E. coli, botulism, polioencephalomalacia, grain overload, and listeriosis are differentials. Prevention and control. Vaccination prevents the disease. Maternal antibodies last approximately 5 weeks postpartum; thus young animals should be vaccinated at about this time. Feeding regimens to young, fast-growing animals and feeding of concentrates to adults should be evaluated carefully. Treatment. Treatment consists of support (fluids, warmth), antitoxin administration, oral antibiotics, and diet adjustment. Etiology. Clostridium perfringens type D releases epsilon toxin that is proteolytically activated by trypsin. This disease caused by C. perfringens tends to be associated with sheep and is of less importance in goats and cattle. Clinical signs. The peracute condition in younger animals is characterized by sudden deaths, which are occasionally preceded by neurological signs such as incoordination, opisthotonus, and convulsions. Because the disease progresses so rapidly to death (within 1–2 hr), clinical signs are rarely observed. Hypersalivation, rapid respirations, hyperthermia, convulsions, and opisthotonus have been noted. In acute cases, hyperglycemia and glucosuria are considered almost pathognomonic. Clinical signs in chronic cases in older animals, such as adult goats, include soft stools, weight loss, anorexia, depression, and severe diarrhea, sometimes with mucus and blood. Mature affected sheep may be blind and anorectic and may head-press. Necropsy findings. Necropsy findings are similar to those seen with C. perfringens type C. Additionally, extremely necrotic, soft kidneys ("pulpy kidneys") are usually observed immediately following death. (This phenomenon is in contrast to what is normally associated with later stages of postmortem autolysis.) Focal encephalomalacia, and petechial hemorrhages on serosal surfaces of the brain, diaphragm, gastrointestinal tract, and heart are common findings. Diagnosis can be made from the typical clinical signs and necropsy findings as well as the observation of glucose in the urine at necropsy. Pathogenesis. The epsilon toxin causes neuronal death and shock, probably through vascular damage. The noncontagious, peracute form of enterotoxemia occurs in suckling, fast-growing animals, either nursing from their dams or on high-protein, high-energy concentrates. The largest, fastest-growing animals generally are predisposed to this condition; for example, lambs, fat ewe lambs, and usually singleton lambs tend to be most susceptible. The hyperglycemia and glucosuria seen in acute cases are due to epsilon toxin effects on liver glycogen metabolism. Differential diagnosis. Tetanus, enterotoxigenic E. coli, botulism, polioencephalomalacia, grain overload, and listeriosis are differentials. Prevention and control. Vaccination prevents the disease. Maternal antibodies last approximately 5 weeks postpartum; thus young animals should be vaccinated at about this time. Feeding regimens to young, fast-growing animals and feeding of concentrates to adults should be evaluated carefully. Treatment. Treatment consists of support (fluids, warmth), antitoxin administration, oral antibiotics, and diet adjustment. iii. Clostridium tetani infection (tetanus, lockjaw) Etiology. Clostridium tetani is a strictly anaerobic, motile, spore-forming, gram-positive rod that persists in soils and manure and within the gastrointestinal tract. At least 10 serotypes of C. tetani exist. Clinical signs. Infection by C. tetani is characterized by a sporadic, acute, and fatal neuropathy. After an incubation period of 4 days to 3 weeks, the animal exhibits bloat; muscular spasticity; prolapse of the third eyelid; rigidity and extension of the limbs, leading to a stiff gate; an inability to chew; and hyperthermia. Erect or drooped ears, retracted lips, drooling, hypersensitivity to external stimuli, and a "sawhorse" stance are frequent signs. The animal may convulse. Death occurs within 3–10 days, and mortality is nearly 100%, primarily from respiratory failure. Diagnosis is based on clinical signs. Muscle-related serum enzymes such as aspartate aminotransferase (AST), creatinine kinase (CK), and lactate dehydrogenase (LDH) might be elevated. ( Jensen and Swift, 1982 ). Serum cortisol may also be elevated, and stress hyperglycemia may be evident. Permanent lameness may result in survivors. Epizootiology and transmission. Clostridium tetani is a soil contaminant and is often found as part of the gut microflora of herbivores. The organisms sporulate and persist in the environment. All species of livestock are susceptible, but sheep and goats are more susceptible than cattle. Individual cases may occur, or herd outbreaks may follow castration, tail docking, ear tagging, or dehorning. Mouth wounds may also be sites of entry. Pathogenesis. Tetanus, or lockjaw, is caused by the toxins of C. tetani. All serovars produce the same exotoxin, which is a multiunit protein composed of tetanospasmin, which is neurotoxic, and tetanolysin, which is hemolytic. A nonspasmogenic toxin is also produced. Contamination of wounds results in anaerobic proliferation of the bacterium and liberation of the tetanospasmin, which diffuses through motor neurons in a retrograde direction to the spinal cord. The toxin inhibits the release of glycine and γ-aminobutyric acid from Renshaw cells; this results in hypertonia and muscular spasms. Proliferation of C. tetani in the gut of affected animals may also serve as a source and may produce clinical signs. The uterus is the most common site of infection in postparturient dairy cattle with retained placentas. Differential diagnoses. Early in the course of the infection, differential diagnoses include bloat, rabies, hypomagnesemic tetany, polioencephalomalacia, white muscle disease, enterotoxemia in lambs, and lead poisoning. Polyarthritis of cattle is a differential for the gait changes in that species. Necropsy findings. Findings are nonspecific except for the inflammatory reaction associated with the wound. Because of the low number of organisms necessary to cause neurotoxicosis, isolation of C. tetani from the wound may be difficult. Treatment. Treatment consists of cleaning the infected wound; administering tetanus antitoxin (e.g., at least 500 IU in an adult sheep or goat); vaccinating with tetanus toxoid; administering of antibiotics (penicillin, both parenterally [potassium penicillin intravenously and procaine penicillin intramuscularly] and flushed into the cleaned wound), a sedative or tranquilizer (e.g., acepromazine or chlorpromazine) and a muscle relaxant; and keeping the animal in a dark, quiet environment. Supportive fluids and glucose must be administered until the animal is capable of feeding. If the animal survives, revaccination should be done 14 days after the previous dose. Prevention and control. Like other ubiquitous clostridial diseases, tetanus is impossible to eradicate. The disease can be controlled and prevented by following good sanitation measures, aseptic surgical procedures, and vaccination programs. Tetanus toxoid vaccine is available and very effective for stimulating long-term immunity. Tetanus antitoxin can be administered (200 IU in lambs) as a preventive or in the face of disease as an adjunct to therapy. Both the toxoid and the antitoxin can be administered to an animal at the same time, but they should not be mixed in the syringe, and each should be administered at different sites, with a second toxoid dose administered 4 weeks later. Animals should be vaccinated 2 or 3 times during the first year of life. Does and ewes should receive booster vaccinations within 2 months of parturition to ensure colostral antibodies. Research complications. Unprotected, younger ruminants may be affected following routine flock or herd management procedures. Contaminated or inadequately managed open wounds or lesions in older animals may provide anaerobic incubation sites. Etiology. Clostridium tetani is a strictly anaerobic, motile, spore-forming, gram-positive rod that persists in soils and manure and within the gastrointestinal tract. At least 10 serotypes of C. tetani exist. Clinical signs. Infection by C. tetani is characterized by a sporadic, acute, and fatal neuropathy. After an incubation period of 4 days to 3 weeks, the animal exhibits bloat; muscular spasticity; prolapse of the third eyelid; rigidity and extension of the limbs, leading to a stiff gate; an inability to chew; and hyperthermia. Erect or drooped ears, retracted lips, drooling, hypersensitivity to external stimuli, and a "sawhorse" stance are frequent signs. The animal may convulse. Death occurs within 3–10 days, and mortality is nearly 100%, primarily from respiratory failure. Diagnosis is based on clinical signs. Muscle-related serum enzymes such as aspartate aminotransferase (AST), creatinine kinase (CK), and lactate dehydrogenase (LDH) might be elevated. ( Jensen and Swift, 1982 ). Serum cortisol may also be elevated, and stress hyperglycemia may be evident. Permanent lameness may result in survivors. Epizootiology and transmission. Clostridium tetani is a soil contaminant and is often found as part of the gut microflora of herbivores. The organisms sporulate and persist in the environment. All species of livestock are susceptible, but sheep and goats are more susceptible than cattle. Individual cases may occur, or herd outbreaks may follow castration, tail docking, ear tagging, or dehorning. Mouth wounds may also be sites of entry. Pathogenesis. Tetanus, or lockjaw, is caused by the toxins of C. tetani. All serovars produce the same exotoxin, which is a multiunit protein composed of tetanospasmin, which is neurotoxic, and tetanolysin, which is hemolytic. A nonspasmogenic toxin is also produced. Contamination of wounds results in anaerobic proliferation of the bacterium and liberation of the tetanospasmin, which diffuses through motor neurons in a retrograde direction to the spinal cord. The toxin inhibits the release of glycine and γ-aminobutyric acid from Renshaw cells; this results in hypertonia and muscular spasms. Proliferation of C. tetani in the gut of affected animals may also serve as a source and may produce clinical signs. The uterus is the most common site of infection in postparturient dairy cattle with retained placentas. Differential diagnoses. Early in the course of the infection, differential diagnoses include bloat, rabies, hypomagnesemic tetany, polioencephalomalacia, white muscle disease, enterotoxemia in lambs, and lead poisoning. Polyarthritis of cattle is a differential for the gait changes in that species. Necropsy findings. Findings are nonspecific except for the inflammatory reaction associated with the wound. Because of the low number of organisms necessary to cause neurotoxicosis, isolation of C. tetani from the wound may be difficult. Treatment. Treatment consists of cleaning the infected wound; administering tetanus antitoxin (e.g., at least 500 IU in an adult sheep or goat); vaccinating with tetanus toxoid; administering of antibiotics (penicillin, both parenterally [potassium penicillin intravenously and procaine penicillin intramuscularly] and flushed into the cleaned wound), a sedative or tranquilizer (e.g., acepromazine or chlorpromazine) and a muscle relaxant; and keeping the animal in a dark, quiet environment. Supportive fluids and glucose must be administered until the animal is capable of feeding. If the animal survives, revaccination should be done 14 days after the previous dose. Prevention and control. Like other ubiquitous clostridial diseases, tetanus is impossible to eradicate. The disease can be controlled and prevented by following good sanitation measures, aseptic surgical procedures, and vaccination programs. Tetanus toxoid vaccine is available and very effective for stimulating long-term immunity. Tetanus antitoxin can be administered (200 IU in lambs) as a preventive or in the face of disease as an adjunct to therapy. Both the toxoid and the antitoxin can be administered to an animal at the same time, but they should not be mixed in the syringe, and each should be administered at different sites, with a second toxoid dose administered 4 weeks later. Animals should be vaccinated 2 or 3 times during the first year of life. Does and ewes should receive booster vaccinations within 2 months of parturition to ensure colostral antibodies. Research complications. Unprotected, younger ruminants may be affected following routine flock or herd management procedures. Contaminated or inadequately managed open wounds or lesions in older animals may provide anaerobic incubation sites. iv. Clostridium novyi infection (bighead; black disease; bacillary hemoglobinuria, or red water) and C. chauvoei infection (blackleg) Etiology. Clostridium novyi, an anaerobic, motile, spore-forming, gram-positive bacteria, is the agent of bighead and black disease. Clostridium novyi type D (C. hemolyticum) is the cause of bacillary hemoglobinuria, or "red water." Clostridium chauvoei is the causative agent of blackleg. Clinical signs. Bighead is a disease of rams characterized by edema of the head and neck. The edema may migrate to ventral regions such as the throat. Additional clinical signs include swelling of the eyelids and nostrils. Most animals will die within 48–72 hours. Black disease, or infectious necrotic hepatitis, is a peracute, fatal disease associated with C. novyi. It is more common in cattle and sheep but may be seen in goats. The clinical course is 1–2 days in cattle and slightly shorter in sheep. Otherwise healthy-appearing adult animals are often affected. Clinical signs are rarely seen, because of the peracute nature of the disease. Occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency are observed prior to death. Bacillary hemoglobinuria is an acute disease seen primarily in cattle and characterized by fever and anorexia, in addition to the hemoglobinemia and hemoglobinuria indicated by the name. Animals that survive a few days will develop icterus. Mortality may be high. Blackleg, a disease similar to bighead, causes necrosis and emphysema of muscle masses, serohemorrhagic fluid accumulation around the infected area, and edema ( Jackson et al., 1995 ). Blackleg is more common in cattle than in sheep. The incubation period is 2–5 days and is followed by hyperthermia, muscular stiffness and pain, anorexia, and gangrenous myositis. The clinical course is short, 24–48 hr, and untreated animals invariably die. Blackleg in cattle can be associated with subcutaneous edema or crepitation; these do not usually occur in sheep. Most lesions are associated with muscles of the face, neck, perineum, thigh, and back. Epizootiology and transmission. Bighead is caused by the toxins of C. novyi, which enters through wounds often associated with horn injuries during fighting. The C. novyi type B organisms produce alpha and beta toxins, and the alpha toxins are mostly responsible for toxemia, tissue necrosis, and subsequent death. Clostridium novyi type D is endemic in the western United States. It is hypothesized that the C. chauvoei organisms enter through the gastrointestinal tract. Black disease and bacillary hemoglobinuria are associated with concurrent liver disease, often associated with Fasciola infections (liver flukes); it is sometimes seen as a sequela to liver biopsies. The diseases are more common in summer months, and fecal contamination of pastures, flooding, and infected carcasses are sources of the organism. Birds and wild animals may be vectors of the pathogen. Ingested spores are believed to develop in hepatic tissue damaged and anoxic from the fluke migrations. Necropsy. Diagnosis of black disease is usually based on postmortem lesions. Subcutaneous vessels will be engorged with blood, resulting in dried skin with a dark appearance. Carcasses putrefy quickly. In addition, hepatomegaly and endocardial hemorrhages are common, and hepatic damage from flukes may be so severe that diagnosis is difficult. Blood coagulates slowly in affected animals. Pathogenesis. The propagation of the clostridial organisms is self-promoted by the damage caused by the toxins and the increased local anaerobic environment created. Clostridium novyi proliferates in the soft tissues of the head and neck, and the resultant clostridial toxin causes increased capillary permeability and the liberation of serous fluids into the tissues. Mixed infections with related clostridial organisms may lead to increasing hemorrhage and necrosis in the affected tissues. Diagnosis is based on clinical signs. In black disease and bacillary hemoglobinuria disease, the ingested clostridial spores are absorbed, enter the liver, and cause hepatic necrosis. Associated toxemia causes subcutaneous vascular dilatation; increased pericardial, pleural, and peritoneal fluid; and endocardial hemorrhages. The toxins produced by C. novyi, identified as beta, eta, and theta, and each having enzymatic or lytic properties or both, also contribute to the hemolytic disease. Clostridium chauvoei spores proliferate in traumatized muscle areas damaged by transportation, rough handling, or injury. Differential diagnosis. Differential diagnoses include other clostridial diseases as well as photosensitization. Hemolytic diseases such as babesiosis, leptospirosis, and hemobartonellosis should be included as differentials. Treatment. For C. chauvoei infection (blackleg), early treatment with penicillin or tetracycline may be helpful. Treatment for black disease is not rewarding even if the animal is found before death. Carcasses from bacillary hemoglobinuria losses should be burned, buried deeply, or removed from the premises. Prevention and control. Vaccinating animals with multivalent clostridial vaccines can prevent these diseases. Subcutaneous administration of vaccine material is recommended over intramuscular. Vaccinations may be useful in an outbreak. Careful handling of ruminants during shipping and transfers will contribute to fewer muscular injuries. For bighead, mature rams penned together should be monitored for lesions, especially during breeding season. Control of fascioliasis is very important in prevention and control of black disease and in the optimal timing of vaccinations. Etiology. Clostridium novyi, an anaerobic, motile, spore-forming, gram-positive bacteria, is the agent of bighead and black disease. Clostridium novyi type D (C. hemolyticum) is the cause of bacillary hemoglobinuria, or "red water." Clostridium chauvoei is the causative agent of blackleg. Clinical signs. Bighead is a disease of rams characterized by edema of the head and neck. The edema may migrate to ventral regions such as the throat. Additional clinical signs include swelling of the eyelids and nostrils. Most animals will die within 48–72 hours. Black disease, or infectious necrotic hepatitis, is a peracute, fatal disease associated with C. novyi. It is more common in cattle and sheep but may be seen in goats. The clinical course is 1–2 days in cattle and slightly shorter in sheep. Otherwise healthy-appearing adult animals are often affected. Clinical signs are rarely seen, because of the peracute nature of the disease. Occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency are observed prior to death. Bacillary hemoglobinuria is an acute disease seen primarily in cattle and characterized by fever and anorexia, in addition to the hemoglobinemia and hemoglobinuria indicated by the name. Animals that survive a few days will develop icterus. Mortality may be high. Blackleg, a disease similar to bighead, causes necrosis and emphysema of muscle masses, serohemorrhagic fluid accumulation around the infected area, and edema ( Jackson et al., 1995 ). Blackleg is more common in cattle than in sheep. The incubation period is 2–5 days and is followed by hyperthermia, muscular stiffness and pain, anorexia, and gangrenous myositis. The clinical course is short, 24–48 hr, and untreated animals invariably die. Blackleg in cattle can be associated with subcutaneous edema or crepitation; these do not usually occur in sheep. Most lesions are associated with muscles of the face, neck, perineum, thigh, and back. Epizootiology and transmission. Bighead is caused by the toxins of C. novyi, which enters through wounds often associated with horn injuries during fighting. The C. novyi type B organisms produce alpha and beta toxins, and the alpha toxins are mostly responsible for toxemia, tissue necrosis, and subsequent death. Clostridium novyi type D is endemic in the western United States. It is hypothesized that the C. chauvoei organisms enter through the gastrointestinal tract. Black disease and bacillary hemoglobinuria are associated with concurrent liver disease, often associated with Fasciola infections (liver flukes); it is sometimes seen as a sequela to liver biopsies. The diseases are more common in summer months, and fecal contamination of pastures, flooding, and infected carcasses are sources of the organism. Birds and wild animals may be vectors of the pathogen. Ingested spores are believed to develop in hepatic tissue damaged and anoxic from the fluke migrations. Necropsy. Diagnosis of black disease is usually based on postmortem lesions. Subcutaneous vessels will be engorged with blood, resulting in dried skin with a dark appearance. Carcasses putrefy quickly. In addition, hepatomegaly and endocardial hemorrhages are common, and hepatic damage from flukes may be so severe that diagnosis is difficult. Blood coagulates slowly in affected animals. Pathogenesis. The propagation of the clostridial organisms is self-promoted by the damage caused by the toxins and the increased local anaerobic environment created. Clostridium novyi proliferates in the soft tissues of the head and neck, and the resultant clostridial toxin causes increased capillary permeability and the liberation of serous fluids into the tissues. Mixed infections with related clostridial organisms may lead to increasing hemorrhage and necrosis in the affected tissues. Diagnosis is based on clinical signs. In black disease and bacillary hemoglobinuria disease, the ingested clostridial spores are absorbed, enter the liver, and cause hepatic necrosis. Associated toxemia causes subcutaneous vascular dilatation; increased pericardial, pleural, and peritoneal fluid; and endocardial hemorrhages. The toxins produced by C. novyi, identified as beta, eta, and theta, and each having enzymatic or lytic properties or both, also contribute to the hemolytic disease. Clostridium chauvoei spores proliferate in traumatized muscle areas damaged by transportation, rough handling, or injury. Differential diagnosis. Differential diagnoses include other clostridial diseases as well as photosensitization. Hemolytic diseases such as babesiosis, leptospirosis, and hemobartonellosis should be included as differentials. Treatment. For C. chauvoei infection (blackleg), early treatment with penicillin or tetracycline may be helpful. Treatment for black disease is not rewarding even if the animal is found before death. Carcasses from bacillary hemoglobinuria losses should be burned, buried deeply, or removed from the premises. Prevention and control. Vaccinating animals with multivalent clostridial vaccines can prevent these diseases. Subcutaneous administration of vaccine material is recommended over intramuscular. Vaccinations may be useful in an outbreak. Careful handling of ruminants during shipping and transfers will contribute to fewer muscular injuries. For bighead, mature rams penned together should be monitored for lesions, especially during breeding season. Control of fascioliasis is very important in prevention and control of black disease and in the optimal timing of vaccinations. v. Clostridium septicum infection (malignant edema) Etiology. Clostridium septicum is the species usually associated with malignant edema, but mixed infections involving other clostridial species such as C. chauvoei, C. novyi, C. sordellii, and C. perfringens may occur. Clostridium spp. are motile (C. chauvoei, C. septicum) or nonmotile, anaerobic, spore-forming, gram-positive rods. Clinicial signs. Malignant edema, or gas gangrene, is an acute and often fatal bacterial disease caused by Clostridium spp. The incubation period is approximately 2–4 days. The affected area will be warm and will contain gaseous accumulations that can be palpated as crepitation of the subcutaneous tissue around the infected area. Regional lymphadenopathy and fever may occur. The animal becomes anorexic, severely depressed, and possibly hyperthermic. Edema and crepitation may be noted around the wound; death occurs within 12 hr to 2 days. Epizootiology and transmission. The organisms are ubiquitous in the environment and may survive in the soil for years. The disease is especially prevalent in animals that have had recent wounds such as those that have undergone castration, docking, ear notching, shearing, or dystocia. Necropsy findings. The tissue necrosis and hemorrhagic serous fluid accumulations resemble those of other clostridial diseases. Pathogenesis. In most cases, the clostridial organisms cause a spreading infection through the fascial planes around the area of the injury; vegetative organisms then produce potent exotoxins, which result in necrosis (alpha toxin) and/or hemolysis (beta toxin). Furthermore, the toxins enter the bloodstream and central nervous system, resulting in systemic collapse and high mortality. Necropsy. Spreading, crepitant lesions around wounds are suggestive of malignant edema. Affected tissues are inflamed and necrotic. Gas and serosanguineous fluids with foul odors infiltrate the tissue planes. Large rod-shaped bacteria may be observed on histopathology; confirmation is made through culture and identification. Intramuscular inoculation of guinea pigs causes a necrotizing myositis and death. Organisms can be cultured from guinea pig tissues. Treatment. Infected animals can be treated with large doses of penicillin and fenestration of the wound is recommended. Prevention and control. Proper preparation of surgical sites, correct sanitation of instruments and the housing environment, and attention to postoperative wounds will help prevent this disease. Multivalent clostridial vaccines are available. Research complications. Morbidity or loss of animals from lack of or unsuccessful vaccination and from contaminated surgical sites or wounds may be consequences of this disease. Etiology. Clostridium septicum is the species usually associated with malignant edema, but mixed infections involving other clostridial species such as C. chauvoei, C. novyi, C. sordellii, and C. perfringens may occur. Clostridium spp. are motile (C. chauvoei, C. septicum) or nonmotile, anaerobic, spore-forming, gram-positive rods. Clinicial signs. Malignant edema, or gas gangrene, is an acute and often fatal bacterial disease caused by Clostridium spp. The incubation period is approximately 2–4 days. The affected area will be warm and will contain gaseous accumulations that can be palpated as crepitation of the subcutaneous tissue around the infected area. Regional lymphadenopathy and fever may occur. The animal becomes anorexic, severely depressed, and possibly hyperthermic. Edema and crepitation may be noted around the wound; death occurs within 12 hr to 2 days. Epizootiology and transmission. The organisms are ubiquitous in the environment and may survive in the soil for years. The disease is especially prevalent in animals that have had recent wounds such as those that have undergone castration, docking, ear notching, shearing, or dystocia. Necropsy findings. The tissue necrosis and hemorrhagic serous fluid accumulations resemble those of other clostridial diseases. Pathogenesis. In most cases, the clostridial organisms cause a spreading infection through the fascial planes around the area of the injury; vegetative organisms then produce potent exotoxins, which result in necrosis (alpha toxin) and/or hemolysis (beta toxin). Furthermore, the toxins enter the bloodstream and central nervous system, resulting in systemic collapse and high mortality. Necropsy. Spreading, crepitant lesions around wounds are suggestive of malignant edema. Affected tissues are inflamed and necrotic. Gas and serosanguineous fluids with foul odors infiltrate the tissue planes. Large rod-shaped bacteria may be observed on histopathology; confirmation is made through culture and identification. Intramuscular inoculation of guinea pigs causes a necrotizing myositis and death. Organisms can be cultured from guinea pig tissues. Treatment. Infected animals can be treated with large doses of penicillin and fenestration of the wound is recommended. Prevention and control. Proper preparation of surgical sites, correct sanitation of instruments and the housing environment, and attention to postoperative wounds will help prevent this disease. Multivalent clostridial vaccines are available. Research complications. Morbidity or loss of animals from lack of or unsuccessful vaccination and from contaminated surgical sites or wounds may be consequences of this disease. i. Colibacillosis Etiology. Escherichia coli is a motile, aerobic, gram-negative, non-spore-forming coccobacillus commonly found in the environment and gastrointestinal tracts of ruminants. Escherichia coli organisms have three areas of surface antigenic complexes (O, somatic; K, envelope or pili; and H, flagellar), which are used to "group" or classify the serotypes. Colibacillosis is the common term for infections in younger animals caused by this bacteria. Clinical signs. Presentation of E. coli infections vary with the animal's age and the type of E. coli involved. Enterotoxigenic E. coli infection causes gastroenteritis and/or septicemia in lambs and calves. Colibacillosis generally develops within the first 72 hr of life when newborn animals are exposed to the organism. The enteric infection causes a semifluid, yellow to gray diarrhea. Occasionally blood streaking of the feces may be observed. The animal may demonstrate abdominal pain, evidenced by arching of the back and extension of the tail, classically described as "tucked up." Hyperthermia is rare. Severe acidosis, depression, and recumbancy ensue, and mortality may be as high as 75%. The septicemic form generally occurs between 2 and 6 weeks of age. Animals display an elevated body temperature and show signs suggestive of nervous system involvement such as incoordination, head pressing, circling, and the appearance of blindness. Opisthotonos, depression, and death follow. Occasionally, swollen, painful joints may be observed with septicemic colibacillosis. Blood cultures may be helpful in identifying the septicemic form. In ruminants, E. coli is is a less common cause of cystitis and pyelonephritis. The cystitis is characterized by dysuria and pollakiuria; gross hematuria and pyuria may be present. The infection may or may not be restricted to the bladder; in the later presentation, and in cases of pyelonephritis, a cow will be acutely depressed, have a fever and ruminal stasis, and be anorexic. In chronic cases, animals will be polyuric and undergo weight loss. Escherichia coli may also cause in utero disease in cattle, resulting in abortion or weakened offspring. Epizootiology and transmission. Escherichia coli is one of the most common gram-negative pathogens isolated from ruminant neonates. Zeman et al. (1989 ) classify E. coli infections into four groups: enterotoxigenic, enterohemorrhagic, enteropathogenic, and enteroinvasive. Enterotoxigenic E. coli (ETEC) attach to the enterocytes via pili, produce enterotoxins, and are the primary cause of colibacillosis in animals and humans. Fimbrial (pili) antigens associated with ovine disease include K99 and F41. Enterohemorrhagic E. coli (EHEC) attach and efface the microvillus, produce verotoxins, and occasionally cause disease in humans and animals. Enteropathogenic E. coli (EPEC) colonize and efface the microvillus but do not produce verotoxins. EPEC are associated with disease in humans and rabbits and cause a secretory diarrhea. Enteroinvasive E. coli (EIEC) invade the enterocytes of humans and cause a shigella-like disease. Overcrowding and poor sanitation contribute significantly to the development of this disease in young animals. The organism will be endemic in a contaminated environment and present on dams' udders. The bacteria rapidly proliferate in the neonates' small intestines. The bacteria and associated toxins cause a secretory diarrhea, resulting in the loss of water and electrolytes. If the bacteria infiltrate the intestinal barrier and enter the blood, septicemia results. Diagnosis of the enteric form can be made by observation of clinical signs, including diarrhea and staining of the tail and wool. Necropsy findings. Swollen, yellow to gray, fluid-filled small and large intestines, swollen and hemorrhagic mesenteric lymph nodes, and generalized tissue dehydration are common. Septicemic lambs may have serofibrinous fluid in the peritoneal, thoracic, and pericardial cavities; enlarged joints containing fibrinopurulent exudates; and congested and inflamed meninges. Isolation and serotyping of E. coli confirm the diagnosis. ELISA and latex agglutination tests are available diagnostic tools. Differential diagnosis. Differential diagnoses include the enterotoxemias caused by C. perfringens type A, B, or C; Campylobacter jejuni; Coccidia, rotavirus, coronavirus, Salmonella, and Cryptosporidia. Other contributing causes of abomasal tympany in young ruminants, such as dietary changes, copper deficiency, excessive intervals between feedings of milk replacer, or feeding large volumes should be considered. Prevention and control. The best preventive measures are maintenance of proper housing conditions, limiting overcrowding, and frequently sanitizing lambing areas. Attention to colostrum feeding techniques and colostral quality are important means of preventing disease. Treatment must include intravenous fluid hydration and reestablishment of acid-base and electrolyte abnormalities. Treatment. Antibiotics such as trimethoprim-sulfadiazine, enrofloxacin, cephalothin, amikacin, and apramycin may be helpful; oral antibiotics are not recommended. Vaccines are available for prevention of colibacillosis in cattle. Etiology. Escherichia coli is a motile, aerobic, gram-negative, non-spore-forming coccobacillus commonly found in the environment and gastrointestinal tracts of ruminants. Escherichia coli organisms have three areas of surface antigenic complexes (O, somatic; K, envelope or pili; and H, flagellar), which are used to "group" or classify the serotypes. Colibacillosis is the common term for infections in younger animals caused by this bacteria. Clinical signs. Presentation of E. coli infections vary with the animal's age and the type of E. coli involved. Enterotoxigenic E. coli infection causes gastroenteritis and/or septicemia in lambs and calves. Colibacillosis generally develops within the first 72 hr of life when newborn animals are exposed to the organism. The enteric infection causes a semifluid, yellow to gray diarrhea. Occasionally blood streaking of the feces may be observed. The animal may demonstrate abdominal pain, evidenced by arching of the back and extension of the tail, classically described as "tucked up." Hyperthermia is rare. Severe acidosis, depression, and recumbancy ensue, and mortality may be as high as 75%. The septicemic form generally occurs between 2 and 6 weeks of age. Animals display an elevated body temperature and show signs suggestive of nervous system involvement such as incoordination, head pressing, circling, and the appearance of blindness. Opisthotonos, depression, and death follow. Occasionally, swollen, painful joints may be observed with septicemic colibacillosis. Blood cultures may be helpful in identifying the septicemic form. In ruminants, E. coli is is a less common cause of cystitis and pyelonephritis. The cystitis is characterized by dysuria and pollakiuria; gross hematuria and pyuria may be present. The infection may or may not be restricted to the bladder; in the later presentation, and in cases of pyelonephritis, a cow will be acutely depressed, have a fever and ruminal stasis, and be anorexic. In chronic cases, animals will be polyuric and undergo weight loss. Escherichia coli may also cause in utero disease in cattle, resulting in abortion or weakened offspring. Epizootiology and transmission. Escherichia coli is one of the most common gram-negative pathogens isolated from ruminant neonates. Zeman et al. (1989 ) classify E. coli infections into four groups: enterotoxigenic, enterohemorrhagic, enteropathogenic, and enteroinvasive. Enterotoxigenic E. coli (ETEC) attach to the enterocytes via pili, produce enterotoxins, and are the primary cause of colibacillosis in animals and humans. Fimbrial (pili) antigens associated with ovine disease include K99 and F41. Enterohemorrhagic E. coli (EHEC) attach and efface the microvillus, produce verotoxins, and occasionally cause disease in humans and animals. Enteropathogenic E. coli (EPEC) colonize and efface the microvillus but do not produce verotoxins. EPEC are associated with disease in humans and rabbits and cause a secretory diarrhea. Enteroinvasive E. coli (EIEC) invade the enterocytes of humans and cause a shigella-like disease. Overcrowding and poor sanitation contribute significantly to the development of this disease in young animals. The organism will be endemic in a contaminated environment and present on dams' udders. The bacteria rapidly proliferate in the neonates' small intestines. The bacteria and associated toxins cause a secretory diarrhea, resulting in the loss of water and electrolytes. If the bacteria infiltrate the intestinal barrier and enter the blood, septicemia results. Diagnosis of the enteric form can be made by observation of clinical signs, including diarrhea and staining of the tail and wool. Necropsy findings. Swollen, yellow to gray, fluid-filled small and large intestines, swollen and hemorrhagic mesenteric lymph nodes, and generalized tissue dehydration are common. Septicemic lambs may have serofibrinous fluid in the peritoneal, thoracic, and pericardial cavities; enlarged joints containing fibrinopurulent exudates; and congested and inflamed meninges. Isolation and serotyping of E. coli confirm the diagnosis. ELISA and latex agglutination tests are available diagnostic tools. Differential diagnosis. Differential diagnoses include the enterotoxemias caused by C. perfringens type A, B, or C; Campylobacter jejuni; Coccidia, rotavirus, coronavirus, Salmonella, and Cryptosporidia. Other contributing causes of abomasal tympany in young ruminants, such as dietary changes, copper deficiency, excessive intervals between feedings of milk replacer, or feeding large volumes should be considered. Prevention and control. The best preventive measures are maintenance of proper housing conditions, limiting overcrowding, and frequently sanitizing lambing areas. Attention to colostrum feeding techniques and colostral quality are important means of preventing disease. Treatment must include intravenous fluid hydration and reestablishment of acid-base and electrolyte abnormalities. Treatment. Antibiotics such as trimethoprim-sulfadiazine, enrofloxacin, cephalothin, amikacin, and apramycin may be helpful; oral antibiotics are not recommended. Vaccines are available for prevention of colibacillosis in cattle. j. Corynebacterium pseudotuberculosis Infection (Caseous Lymphadenitis) Etiology. Corynebacterium pseudotuberculosis (previously C. ovis) are nonmotile, non-spore-forming, aerobic, short and curved, gram-positive coccobacilli. Caseous lymphadenitis (CLA) is such a common, chronic contagious disease of sheep and goats that any presentation of abscessing and draining lymph nodes should be presumed to be this disease until proven otherwise. The disease has been reported occasionally in cattle. Clinical signs and diagnosis. Abscessation of superficial lymph nodes, such as the superficial cervical, retropharyngeal, subiliacs (prefemoral), mammary, superficial inguinals, and popliteal nodes, and of deep nodes, such as mediastinal and mesenteric lymph nodes, is typical. Radiographs may be helpful in identifying affected central nodes. Peripheral lymph nodes may erode and drain caseous, "cheesy," yellow-green-tan secretions. The incubation period may be weeks to months. Over time, an infected animal may become exercise-intolerant, anorexic, and debilitated. Fever, increased respiratory rates, and pneumonia may also be common signs. Exotoxin-induced hemolytic crises may occur occasionally. Morbidity up to 15% is common, and morbid animals will often eventually succumb to the disease. Diagnosis is based on clinical lesions; ELISA serological testing is also available. Smears of the exudate or lymph nodes aspirates can be Gram-stained. Lymph node aspirates may also be sent for culturing. Epizootiology and transmission. The organism can survive for 6 months or more in the environment and enters via skin wounds, shearing, fighting, castration, and docking. Ingestion and aerosolization (leading to pulmonary abscesses) have been reported as alternative routes of entry. Necropsy findings. Disseminated superficial abscesses as well as lesions of the mediastinal and mesenteric lymph nodes will be identified. Cut surfaces of the affected lymph nodes may appear lamellated. Lungs, liver, spleen, and kidneys may also be affected. Cranioventral lung consolidation with hemorrhage, fibrin, and edema are seen histologically. Pathogenesis. Corynebacterium pseudotuberculosis produces an exotoxin (phospholipase D) that damages endothelial and blood cell membranes. This process enhances the organisms' ability to withstand phagocytosis. The infection spreads through the lymphatics to local lymph nodes. The necrotic lymph nodes seed local capillaries and hematogenously and lymphatically spread the organisms to other areas, especially the lungs. Differential diagnosis. Differentials include pathogens causing lymphadenopathy and abscessation. Treatment. Antibiotic therapy is not usually helpful. Abscesses can be surgically lanced and flushed with iodine-containing and/or hydrogen peroxide solutions. Abscessing lymph nodes can be removed entirely from valuable animals. During warmer months, an insect repellent should be applied to and around healing lesions. All materials used to treat animals should be disposed of properly. Because of the contagious nature of the disease, animals with draining and lanced lesions should be isolated from CLA-negative animals at least until healed. Commercial vaccines are available ( Piontkowski and Shivvers, 1998 ). Prevention and control. Minimizing contamination of the environment, using proper sanitation methods for facilities and instruments, segregating affected animals, and taking precautions to prevent injuries are all important. Research complications. This pathogen is a risk for animals undergoing routine management procedures or invasive research procedures, because of its persistence in the environment, its long clinical incubation period, and its poor response to antibiotics. Etiology. Corynebacterium pseudotuberculosis (previously C. ovis) are nonmotile, non-spore-forming, aerobic, short and curved, gram-positive coccobacilli. Caseous lymphadenitis (CLA) is such a common, chronic contagious disease of sheep and goats that any presentation of abscessing and draining lymph nodes should be presumed to be this disease until proven otherwise. The disease has been reported occasionally in cattle. Clinical signs and diagnosis. Abscessation of superficial lymph nodes, such as the superficial cervical, retropharyngeal, subiliacs (prefemoral), mammary, superficial inguinals, and popliteal nodes, and of deep nodes, such as mediastinal and mesenteric lymph nodes, is typical. Radiographs may be helpful in identifying affected central nodes. Peripheral lymph nodes may erode and drain caseous, "cheesy," yellow-green-tan secretions. The incubation period may be weeks to months. Over time, an infected animal may become exercise-intolerant, anorexic, and debilitated. Fever, increased respiratory rates, and pneumonia may also be common signs. Exotoxin-induced hemolytic crises may occur occasionally. Morbidity up to 15% is common, and morbid animals will often eventually succumb to the disease. Diagnosis is based on clinical lesions; ELISA serological testing is also available. Smears of the exudate or lymph nodes aspirates can be Gram-stained. Lymph node aspirates may also be sent for culturing. Epizootiology and transmission. The organism can survive for 6 months or more in the environment and enters via skin wounds, shearing, fighting, castration, and docking. Ingestion and aerosolization (leading to pulmonary abscesses) have been reported as alternative routes of entry. Necropsy findings. Disseminated superficial abscesses as well as lesions of the mediastinal and mesenteric lymph nodes will be identified. Cut surfaces of the affected lymph nodes may appear lamellated. Lungs, liver, spleen, and kidneys may also be affected. Cranioventral lung consolidation with hemorrhage, fibrin, and edema are seen histologically. Pathogenesis. Corynebacterium pseudotuberculosis produces an exotoxin (phospholipase D) that damages endothelial and blood cell membranes. This process enhances the organisms' ability to withstand phagocytosis. The infection spreads through the lymphatics to local lymph nodes. The necrotic lymph nodes seed local capillaries and hematogenously and lymphatically spread the organisms to other areas, especially the lungs. Differential diagnosis. Differentials include pathogens causing lymphadenopathy and abscessation. Treatment. Antibiotic therapy is not usually helpful. Abscesses can be surgically lanced and flushed with iodine-containing and/or hydrogen peroxide solutions. Abscessing lymph nodes can be removed entirely from valuable animals. During warmer months, an insect repellent should be applied to and around healing lesions. All materials used to treat animals should be disposed of properly. Because of the contagious nature of the disease, animals with draining and lanced lesions should be isolated from CLA-negative animals at least until healed. Commercial vaccines are available ( Piontkowski and Shivvers, 1998 ). Prevention and control. Minimizing contamination of the environment, using proper sanitation methods for facilities and instruments, segregating affected animals, and taking precautions to prevent injuries are all important. Research complications. This pathogen is a risk for animals undergoing routine management procedures or invasive research procedures, because of its persistence in the environment, its long clinical incubation period, and its poor response to antibiotics. Etiology. Corynebacterium pseudotuberculosis (previously C. ovis) are nonmotile, non-spore-forming, aerobic, short and curved, gram-positive coccobacilli. Caseous lymphadenitis (CLA) is such a common, chronic contagious disease of sheep and goats that any presentation of abscessing and draining lymph nodes should be presumed to be this disease until proven otherwise. The disease has been reported occasionally in cattle. Clinical signs and diagnosis. Abscessation of superficial lymph nodes, such as the superficial cervical, retropharyngeal, subiliacs (prefemoral), mammary, superficial inguinals, and popliteal nodes, and of deep nodes, such as mediastinal and mesenteric lymph nodes, is typical. Radiographs may be helpful in identifying affected central nodes. Peripheral lymph nodes may erode and drain caseous, "cheesy," yellow-green-tan secretions. The incubation period may be weeks to months. Over time, an infected animal may become exercise-intolerant, anorexic, and debilitated. Fever, increased respiratory rates, and pneumonia may also be common signs. Exotoxin-induced hemolytic crises may occur occasionally. Morbidity up to 15% is common, and morbid animals will often eventually succumb to the disease. Diagnosis is based on clinical lesions; ELISA serological testing is also available. Smears of the exudate or lymph nodes aspirates can be Gram-stained. Lymph node aspirates may also be sent for culturing. Epizootiology and transmission. The organism can survive for 6 months or more in the environment and enters via skin wounds, shearing, fighting, castration, and docking. Ingestion and aerosolization (leading to pulmonary abscesses) have been reported as alternative routes of entry. Necropsy findings. Disseminated superficial abscesses as well as lesions of the mediastinal and mesenteric lymph nodes will be identified. Cut surfaces of the affected lymph nodes may appear lamellated. Lungs, liver, spleen, and kidneys may also be affected. Cranioventral lung consolidation with hemorrhage, fibrin, and edema are seen histologically. Pathogenesis. Corynebacterium pseudotuberculosis produces an exotoxin (phospholipase D) that damages endothelial and blood cell membranes. This process enhances the organisms' ability to withstand phagocytosis. The infection spreads through the lymphatics to local lymph nodes. The necrotic lymph nodes seed local capillaries and hematogenously and lymphatically spread the organisms to other areas, especially the lungs. Differential diagnosis. Differentials include pathogens causing lymphadenopathy and abscessation. Treatment. Antibiotic therapy is not usually helpful. Abscesses can be surgically lanced and flushed with iodine-containing and/or hydrogen peroxide solutions. Abscessing lymph nodes can be removed entirely from valuable animals. During warmer months, an insect repellent should be applied to and around healing lesions. All materials used to treat animals should be disposed of properly. Because of the contagious nature of the disease, animals with draining and lanced lesions should be isolated from CLA-negative animals at least until healed. Commercial vaccines are available ( Piontkowski and Shivvers, 1998 ). Prevention and control. Minimizing contamination of the environment, using proper sanitation methods for facilities and instruments, segregating affected animals, and taking precautions to prevent injuries are all important. Research complications. This pathogen is a risk for animals undergoing routine management procedures or invasive research procedures, because of its persistence in the environment, its long clinical incubation period, and its poor response to antibiotics. k. Corynebacterium renale, C. cystitidis, and C. pilosum Infections (Pyelonephritis; Posthitis and Ulcerative Vulvovaginitis) Etiology. Corynebacterium renale, C. cystitidis, and C. pilosum are sometimes referred to as the C. renale group. These are piliated and nonmotile gram-positive rods and are distinguished biochemically. Corynebacterium renale causes pyelonephritis in cattle, and C. pilosum and C. cystitidis cause posthitis, also known as pizzle rot or sheath rot, in sheep and goats. In many references, all these clinical presentations are attributed to C. renale. Clinical signs and diagnosis. Acute pyelonephritis is characterized by fever, anorexia, polyuria, hematuria, pyuria, and arched back posture. Untreated infections usually become chronic, with weight loss, anorexia, and loss of production in dairy animals. Relapses are common, and some infections are severe and fatal. Diagnosis of pyelonephritis is based on urinalysis (proteinuria and hematuria) and rectal or vaginal palpation (assessing ureteral enlargement). Urine culturing may not be productive. In chronic cases, E. coli and other gram-negatives may be present. Posthitis and vulvovaginitis are characteriazed by ulcers, crusting, swelling and pain. The area may have a distinct malodor. Necrosis and scarring may be sequelae of more severe infections. Fly-strike may also be a complication. Diagnosis is based on clinical signs and on investigation of feeding regimens. Epizootiology and transmission. Ascending urinary tract infections with cystitis, ureteritis, and pyelonephritis are widespread problems, but incidence is relatively low. The vaginitis and posthitis contribute to the venereal transmission, but indirect transmission is possible because the organisms are stable in the environment and present on the wool or scabs shed from affected animals. Posthitis occurs in intact and castrated sheep and goats. Necropsy findings. Pyelonephritis, multifocal kidney abscessation, dilated and thickened ureters, cystitis, and purulent exudate in many sections of the urinary tract are common finding at gross necropsy. Pathogenesis. Corynebacterium renale is a normal inhabitant of bovine genitourinary tracts. The pilus mediates colonization. Conditions such as trauma, urinary tract obstruction, and anatomic anomalies may predispose to infection. In addition, more basic pH urine levels may block some immune defenses. Infections ascend through the urinary tract. The bacteria are urease-positive when tested in vitro, and the ammonia produced in vivo during an infection damages mucosal linings, with subsequent inflammation. Corynebacterium cystitidis and C. pilosum are normally found around the prepuce of sheep and goats. High-protein diets, resulting in higher urea excretion and more basic urine, are contributing factors. Posthitis and vulvovaginitis may develop within a week of change to the more concentrated or richer diet, such as pasture or the addition of high-protein forage. The ammonia produced irritates the preputial and vulvar skin, increasing the vulnerability to infection. Differential diagnosis. Urolithiasis is a primary consideration for these diseases. Contagious ecthyma should be considered for the crusting that is seen with posthitis and vulvovaginitis, although the lesions of contagious ecthyma are more likely to develop around the mouth. Ovine viral ulcerative dermatosis is also a differential for the lesions of posthitis and vulvovaginitis. Prevention and treatment. Because high-protein feed is often associated with posthitis and vulvovaginitis, feeding practices must be reconsidered. Clipping long wool and hair also is helpful. Treatment. Long-term (3 weeks) penicillin treatment is effective for pyelonephritis. Reduction of dietary protein, clipping and cleaning skin lesions, treating for or preventing fly-strike, and topical antibacterial treatments are effective for posthitis and vulvovaginitis; systemic therapy may be necessary for severe cases. Surgical debridement or correction of scarring may also be indicated in severe cases. Etiology. Corynebacterium renale, C. cystitidis, and C. pilosum are sometimes referred to as the C. renale group. These are piliated and nonmotile gram-positive rods and are distinguished biochemically. Corynebacterium renale causes pyelonephritis in cattle, and C. pilosum and C. cystitidis cause posthitis, also known as pizzle rot or sheath rot, in sheep and goats. In many references, all these clinical presentations are attributed to C. renale. Clinical signs and diagnosis. Acute pyelonephritis is characterized by fever, anorexia, polyuria, hematuria, pyuria, and arched back posture. Untreated infections usually become chronic, with weight loss, anorexia, and loss of production in dairy animals. Relapses are common, and some infections are severe and fatal. Diagnosis of pyelonephritis is based on urinalysis (proteinuria and hematuria) and rectal or vaginal palpation (assessing ureteral enlargement). Urine culturing may not be productive. In chronic cases, E. coli and other gram-negatives may be present. Posthitis and vulvovaginitis are characteriazed by ulcers, crusting, swelling and pain. The area may have a distinct malodor. Necrosis and scarring may be sequelae of more severe infections. Fly-strike may also be a complication. Diagnosis is based on clinical signs and on investigation of feeding regimens. Epizootiology and transmission. Ascending urinary tract infections with cystitis, ureteritis, and pyelonephritis are widespread problems, but incidence is relatively low. The vaginitis and posthitis contribute to the venereal transmission, but indirect transmission is possible because the organisms are stable in the environment and present on the wool or scabs shed from affected animals. Posthitis occurs in intact and castrated sheep and goats. Necropsy findings. Pyelonephritis, multifocal kidney abscessation, dilated and thickened ureters, cystitis, and purulent exudate in many sections of the urinary tract are common finding at gross necropsy. Pathogenesis. Corynebacterium renale is a normal inhabitant of bovine genitourinary tracts. The pilus mediates colonization. Conditions such as trauma, urinary tract obstruction, and anatomic anomalies may predispose to infection. In addition, more basic pH urine levels may block some immune defenses. Infections ascend through the urinary tract. The bacteria are urease-positive when tested in vitro, and the ammonia produced in vivo during an infection damages mucosal linings, with subsequent inflammation. Corynebacterium cystitidis and C. pilosum are normally found around the prepuce of sheep and goats. High-protein diets, resulting in higher urea excretion and more basic urine, are contributing factors. Posthitis and vulvovaginitis may develop within a week of change to the more concentrated or richer diet, such as pasture or the addition of high-protein forage. The ammonia produced irritates the preputial and vulvar skin, increasing the vulnerability to infection. Differential diagnosis. Urolithiasis is a primary consideration for these diseases. Contagious ecthyma should be considered for the crusting that is seen with posthitis and vulvovaginitis, although the lesions of contagious ecthyma are more likely to develop around the mouth. Ovine viral ulcerative dermatosis is also a differential for the lesions of posthitis and vulvovaginitis. Prevention and treatment. Because high-protein feed is often associated with posthitis and vulvovaginitis, feeding practices must be reconsidered. Clipping long wool and hair also is helpful. Treatment. Long-term (3 weeks) penicillin treatment is effective for pyelonephritis. Reduction of dietary protein, clipping and cleaning skin lesions, treating for or preventing fly-strike, and topical antibacterial treatments are effective for posthitis and vulvovaginitis; systemic therapy may be necessary for severe cases. Surgical debridement or correction of scarring may also be indicated in severe cases. Etiology. Corynebacterium renale, C. cystitidis, and C. pilosum are sometimes referred to as the C. renale group. These are piliated and nonmotile gram-positive rods and are distinguished biochemically. Corynebacterium renale causes pyelonephritis in cattle, and C. pilosum and C. cystitidis cause posthitis, also known as pizzle rot or sheath rot, in sheep and goats. In many references, all these clinical presentations are attributed to C. renale. Clinical signs and diagnosis. Acute pyelonephritis is characterized by fever, anorexia, polyuria, hematuria, pyuria, and arched back posture. Untreated infections usually become chronic, with weight loss, anorexia, and loss of production in dairy animals. Relapses are common, and some infections are severe and fatal. Diagnosis of pyelonephritis is based on urinalysis (proteinuria and hematuria) and rectal or vaginal palpation (assessing ureteral enlargement). Urine culturing may not be productive. In chronic cases, E. coli and other gram-negatives may be present. Posthitis and vulvovaginitis are characteriazed by ulcers, crusting, swelling and pain. The area may have a distinct malodor. Necrosis and scarring may be sequelae of more severe infections. Fly-strike may also be a complication. Diagnosis is based on clinical signs and on investigation of feeding regimens. Epizootiology and transmission. Ascending urinary tract infections with cystitis, ureteritis, and pyelonephritis are widespread problems, but incidence is relatively low. The vaginitis and posthitis contribute to the venereal transmission, but indirect transmission is possible because the organisms are stable in the environment and present on the wool or scabs shed from affected animals. Posthitis occurs in intact and castrated sheep and goats. Necropsy findings. Pyelonephritis, multifocal kidney abscessation, dilated and thickened ureters, cystitis, and purulent exudate in many sections of the urinary tract are common finding at gross necropsy. Pathogenesis. Corynebacterium renale is a normal inhabitant of bovine genitourinary tracts. The pilus mediates colonization. Conditions such as trauma, urinary tract obstruction, and anatomic anomalies may predispose to infection. In addition, more basic pH urine levels may block some immune defenses. Infections ascend through the urinary tract. The bacteria are urease-positive when tested in vitro, and the ammonia produced in vivo during an infection damages mucosal linings, with subsequent inflammation. Corynebacterium cystitidis and C. pilosum are normally found around the prepuce of sheep and goats. High-protein diets, resulting in higher urea excretion and more basic urine, are contributing factors. Posthitis and vulvovaginitis may develop within a week of change to the more concentrated or richer diet, such as pasture or the addition of high-protein forage. The ammonia produced irritates the preputial and vulvar skin, increasing the vulnerability to infection. Differential diagnosis. Urolithiasis is a primary consideration for these diseases. Contagious ecthyma should be considered for the crusting that is seen with posthitis and vulvovaginitis, although the lesions of contagious ecthyma are more likely to develop around the mouth. Ovine viral ulcerative dermatosis is also a differential for the lesions of posthitis and vulvovaginitis. Prevention and treatment. Because high-protein feed is often associated with posthitis and vulvovaginitis, feeding practices must be reconsidered. Clipping long wool and hair also is helpful. Treatment. Long-term (3 weeks) penicillin treatment is effective for pyelonephritis. Reduction of dietary protein, clipping and cleaning skin lesions, treating for or preventing fly-strike, and topical antibacterial treatments are effective for posthitis and vulvovaginitis; systemic therapy may be necessary for severe cases. Surgical debridement or correction of scarring may also be indicated in severe cases. l. Erysipelas Etiology. Erysipelothrix rhusiopathiae is a nonmotile, non-spore-forming, gram-positive rod that resides in alkaline soils. Clinical signs. Erysipelothrix causes sporadic but chronic polyarthritis in lambs less than 3 months of age. In older goats, erysipelas has been associated with joint infections. Epizootiology and transmission. The disease may follow wound inoculation associated with castration, docking, or improper disinfection of the umbilicus. Following wound contamination and a 1- to 5-day incubation period, the lamb exhibits a fever and stiffness and lameness in one or more limbs. Joints, especially the stifle, hock, elbow, and carpus, are tender but not greatly enlarged. Necropsy findings. Thickened articular capsules, mild increases in normal-appearing joint fluid and erosions of the articular cartilage are usually found. The joint capsule is infiltrated with mononuclear cells, but bacteria are difficult to find. Diagnosis is based on clinical signs of polyarthritis, and confirmation is made by culturing the organism from the joints. Differential diagnosis. Differential diagnoses include polyarthritis caused by chlamydia or other bacteria and stiffness caused by white muscle disease. Other bacteria causing septic joints include Areanobacterium pyogenes and Fusobacterium necrophorum. Caprine arthritis encephalitis (CAE) should also be considered. Prevention and control. Proper sanitation and prevention of wound contamination are important in preventing the infection in lambs. Screening of goat herds for CAE is recommended. Treatment. Erysipelas is sensitive to penicillin antibiotic therapy. Etiology. Erysipelothrix rhusiopathiae is a nonmotile, non-spore-forming, gram-positive rod that resides in alkaline soils. Clinical signs. Erysipelothrix causes sporadic but chronic polyarthritis in lambs less than 3 months of age. In older goats, erysipelas has been associated with joint infections. Epizootiology and transmission. The disease may follow wound inoculation associated with castration, docking, or improper disinfection of the umbilicus. Following wound contamination and a 1- to 5-day incubation period, the lamb exhibits a fever and stiffness and lameness in one or more limbs. Joints, especially the stifle, hock, elbow, and carpus, are tender but not greatly enlarged. Necropsy findings. Thickened articular capsules, mild increases in normal-appearing joint fluid and erosions of the articular cartilage are usually found. The joint capsule is infiltrated with mononuclear cells, but bacteria are difficult to find. Diagnosis is based on clinical signs of polyarthritis, and confirmation is made by culturing the organism from the joints. Differential diagnosis. Differential diagnoses include polyarthritis caused by chlamydia or other bacteria and stiffness caused by white muscle disease. Other bacteria causing septic joints include Areanobacterium pyogenes and Fusobacterium necrophorum. Caprine arthritis encephalitis (CAE) should also be considered. Prevention and control. Proper sanitation and prevention of wound contamination are important in preventing the infection in lambs. Screening of goat herds for CAE is recommended. Treatment. Erysipelas is sensitive to penicillin antibiotic therapy. Etiology. Erysipelothrix rhusiopathiae is a nonmotile, non-spore-forming, gram-positive rod that resides in alkaline soils. Clinical signs. Erysipelothrix causes sporadic but chronic polyarthritis in lambs less than 3 months of age. In older goats, erysipelas has been associated with joint infections. Epizootiology and transmission. The disease may follow wound inoculation associated with castration, docking, or improper disinfection of the umbilicus. Following wound contamination and a 1- to 5-day incubation period, the lamb exhibits a fever and stiffness and lameness in one or more limbs. Joints, especially the stifle, hock, elbow, and carpus, are tender but not greatly enlarged. Necropsy findings. Thickened articular capsules, mild increases in normal-appearing joint fluid and erosions of the articular cartilage are usually found. The joint capsule is infiltrated with mononuclear cells, but bacteria are difficult to find. Diagnosis is based on clinical signs of polyarthritis, and confirmation is made by culturing the organism from the joints. Differential diagnosis. Differential diagnoses include polyarthritis caused by chlamydia or other bacteria and stiffness caused by white muscle disease. Other bacteria causing septic joints include Areanobacterium pyogenes and Fusobacterium necrophorum. Caprine arthritis encephalitis (CAE) should also be considered. Prevention and control. Proper sanitation and prevention of wound contamination are important in preventing the infection in lambs. Screening of goat herds for CAE is recommended. Treatment. Erysipelas is sensitive to penicillin antibiotic therapy. m. Dermatophilosis (Cutaneous Streptothricosis, Lumpy Wool, Strawberry Foot Rot) Etiology. Dermatophilus congolensis is an aerobic, gram-positive, filamentous bacterium with branching hyphae. Dermatophilosis is a chronic bacterial skin disease characterized by crustiness and exudates accumulating at the base of the hair or wool fibers ( Scanlan et al., 1984 ). Clinical signs. Animals will be painful but will not be pruritic. Two forms of the disease exist in sheep: mycotic dermatitis (also known as lumpy wool) and strawberry foot rot. Mycotic dermatitis is characterized by crusts and wool matting, with exudates over the back and sides of adult animals and about the face of lambs. Strawberry foot rot is rare in the United States but is characterized by crusts and inflammation between the carpi and/or tarsi and the coronary bands. Animals will be lame. In goats and cattle, similar clinical signs of crusty, suppurative dermatitis are seen; the disease is often referred to as cutaneous streptothricosis in these species. Lesions in younger goats are seen along the tips of the ears and under the tail. Diagnosis is based on clinical signs as well as the typical microscopic appearance on stained skin scrapings, cultures, and serology. Epizootiology and transmission. The disease occurs worldwide, and the Dermatophilus organism is believed to be a saprophyte. Transmission occurs by direct or indirect contact and is aggravated by prolonged wet wool or hair associated with inclement weather. Biting insects may aid in transmission. Necropsy findings. Lymphadenopathy as well as liver and splenic changes may be observed. Histopathologically, superficial epidermal layers are necrotic and crusted with serum, white blood cells, and wool or hair. Dermal layers are hyperemic and edematous and may be infiltrated with mononuclear cells. Pathogenesis. Lesions typically begin around the muzzle and hooves and the dorsal midline. Prevention and control. Potash alum and aluminum sulfate have been used as wool dusts in sheep to prevent dermatophilosis. Minimizing moist conditions is helpful in controlling and preventing the disease. In addition, controlling external parasites or other factors that cause skin lesions is important. Lesions will resolve during dry periods. Treatment. Animals can be treated with antibiotics such as penicillin and oxytetracycline. Treating the animals with povidone-iodine shampoos or chlorhexidine solutions is also useful in clearing the disease. Etiology. Dermatophilus congolensis is an aerobic, gram-positive, filamentous bacterium with branching hyphae. Dermatophilosis is a chronic bacterial skin disease characterized by crustiness and exudates accumulating at the base of the hair or wool fibers ( Scanlan et al., 1984 ). Clinical signs. Animals will be painful but will not be pruritic. Two forms of the disease exist in sheep: mycotic dermatitis (also known as lumpy wool) and strawberry foot rot. Mycotic dermatitis is characterized by crusts and wool matting, with exudates over the back and sides of adult animals and about the face of lambs. Strawberry foot rot is rare in the United States but is characterized by crusts and inflammation between the carpi and/or tarsi and the coronary bands. Animals will be lame. In goats and cattle, similar clinical signs of crusty, suppurative dermatitis are seen; the disease is often referred to as cutaneous streptothricosis in these species. Lesions in younger goats are seen along the tips of the ears and under the tail. Diagnosis is based on clinical signs as well as the typical microscopic appearance on stained skin scrapings, cultures, and serology. Epizootiology and transmission. The disease occurs worldwide, and the Dermatophilus organism is believed to be a saprophyte. Transmission occurs by direct or indirect contact and is aggravated by prolonged wet wool or hair associated with inclement weather. Biting insects may aid in transmission. Necropsy findings. Lymphadenopathy as well as liver and splenic changes may be observed. Histopathologically, superficial epidermal layers are necrotic and crusted with serum, white blood cells, and wool or hair. Dermal layers are hyperemic and edematous and may be infiltrated with mononuclear cells. Pathogenesis. Lesions typically begin around the muzzle and hooves and the dorsal midline. Prevention and control. Potash alum and aluminum sulfate have been used as wool dusts in sheep to prevent dermatophilosis. Minimizing moist conditions is helpful in controlling and preventing the disease. In addition, controlling external parasites or other factors that cause skin lesions is important. Lesions will resolve during dry periods. Treatment. Animals can be treated with antibiotics such as penicillin and oxytetracycline. Treating the animals with povidone-iodine shampoos or chlorhexidine solutions is also useful in clearing the disease. Etiology. Dermatophilus congolensis is an aerobic, gram-positive, filamentous bacterium with branching hyphae. Dermatophilosis is a chronic bacterial skin disease characterized by crustiness and exudates accumulating at the base of the hair or wool fibers ( Scanlan et al., 1984 ). Clinical signs. Animals will be painful but will not be pruritic. Two forms of the disease exist in sheep: mycotic dermatitis (also known as lumpy wool) and strawberry foot rot. Mycotic dermatitis is characterized by crusts and wool matting, with exudates over the back and sides of adult animals and about the face of lambs. Strawberry foot rot is rare in the United States but is characterized by crusts and inflammation between the carpi and/or tarsi and the coronary bands. Animals will be lame. In goats and cattle, similar clinical signs of crusty, suppurative dermatitis are seen; the disease is often referred to as cutaneous streptothricosis in these species. Lesions in younger goats are seen along the tips of the ears and under the tail. Diagnosis is based on clinical signs as well as the typical microscopic appearance on stained skin scrapings, cultures, and serology. Epizootiology and transmission. The disease occurs worldwide, and the Dermatophilus organism is believed to be a saprophyte. Transmission occurs by direct or indirect contact and is aggravated by prolonged wet wool or hair associated with inclement weather. Biting insects may aid in transmission. Necropsy findings. Lymphadenopathy as well as liver and splenic changes may be observed. Histopathologically, superficial epidermal layers are necrotic and crusted with serum, white blood cells, and wool or hair. Dermal layers are hyperemic and edematous and may be infiltrated with mononuclear cells. Pathogenesis. Lesions typically begin around the muzzle and hooves and the dorsal midline. Prevention and control. Potash alum and aluminum sulfate have been used as wool dusts in sheep to prevent dermatophilosis. Minimizing moist conditions is helpful in controlling and preventing the disease. In addition, controlling external parasites or other factors that cause skin lesions is important. Lesions will resolve during dry periods. Treatment. Animals can be treated with antibiotics such as penicillin and oxytetracycline. Treating the animals with povidone-iodine shampoos or chlorhexidine solutions is also useful in clearing the disease. n. Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum Infection (Virulent Foot Rot; Contagious Foot Rot of Sheep and Goats; Foot Scald) Etiology. Two bacteria, Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum, work synergistically in causing contagious foot rot in sheep and goats. Other organisms may be involved as secondary invaders. Both Dichelobacter and Fusobacterium are nonmotile, non-spore-forming, anaerobic, gram-negative bacilli. Foot rot is a contagious, acute or chronic dermatitis involving the hoof and underlying tissues ( Bulgin, 1986 ). It is the leading cause of lameness in sheep. At least 20 serotypes of Dichelobacter are known. Arcanobacterium pyogenes may also contribute to the pathogenicity or to foot abscesses in goats. Foot scald, an interdigital dermatitis, is caused primarily by D. nodosus alone. Clinical signs. Varying degrees of lameness are observed in all ages of animals within 2–3 weeks of exposure to the organisms. Severely infected animals will show generalized signs of weight loss, decreased productivity, and anorexia associated with an inability to move. The interdigital skin and hooves will be moist, with a distinct necrotic odor. Morbidity may reach 70% in susceptible animals. Diagnosis is based on clinical signs. Smears and cultures confirm the definitive agents. Clinical signs of the milder disease, foot scald, include mild lameness, redness and swelling, and little to no odor. Epizootiology and transmission. Fusobacterium necrophorum is ubiquitous in soil and manure, in the gastrointestinal tract, and on the skin and hooves of domestic animals. In contrast, Dichelobacter contaminates the soil and manure but rarely remains in the environment for more than about 2 weeks. Some animals may be chronic carriers. Overcrowded, warm, and moist environments are key elements in transmission. Outbreaks are likely in the spring season. Shipping trailers and contaminated pens or yards should be considered also as likely sources of the bacteria. Pathogenesis. Both organisms are transmitted to the susceptible animal by direct or indirect contact. The organisms enter the hoof through injuries or through sites where Strongyloides papillosus larvae have penetrated. Fusobacterium necrophorum initiates the colonization and is followed by D. nodosus. The latter attaches and releases proteases; these cause necrosis of the epidermal layers and separation of the hoof from the underlying dermis. The pathogenicity of the serotypes of D. nodosus is correlated with the production of these proteases and numbers of pili. Additionally, F. necrophorum causes a severe, damaging inflammatory reaction. Differential diagnosis. Foot abscesses, tetanus, selenium/vitamin E deficiencies, copper deficiency, strawberry foot rot, bluetongue virus infection (manifested with myopathy and coronitis), and trauma are among the many differentials that must be considered. Treatment. Affected animals are best treated by manually trimming the necrotic debris from the hooves, followed by application of local antibiotics and foot wraps. Systemic antibiotics such as penicillin, oxytetracycline, and erythromycin may be used. Goats have improved dramatically when given a single dose of penicillin (40,000 U/kg) ( Smith and Sherman, 1994 ). Footbaths containing 10% zinc sulfate, 20% copper sulfate, or 10% formalin (not legal in all states) can be used for treatment as well as for prevention of the disease. Affected animals should be separated from the flock. Vaccination has been shown to be effective as part of the treatment regimen. Some breeds of sheep and some breeds and lines of goats are resistant to infection. Individual sheep may recover without treatment or are resistant to infection. Prevention and control. Prevention and control programs involve scrutiny of herd and flock management; quarantine of incoming animals; vaccination; segregation of affected animals; careful and regular hoof trimming; discarding trimmings from known or suspected infected hooves; maintaining animals in good body condition; avoiding muddy pens and holding areas; and culling individuals with chronic and nonresponsive infections. Dichelobacter nodosus bacterins are commercially available; cross protection between serotypes varies. Biannual vaccinination in wet areas may be essential. Some breeds may develop vaccination site lumps. Footbaths of 10% zinc sulfate, 10% formalin (where allowed by state regulations), or 10% copper sulfate are also considered very effective preventive measures. Goats are less sensitive than sheep to the copper in the footbaths. Research complications. Treating and controlling foot rot is costly in terms of time, initial handling and treatments and their follow-up, housing space, and medications. Etiology. Two bacteria, Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum, work synergistically in causing contagious foot rot in sheep and goats. Other organisms may be involved as secondary invaders. Both Dichelobacter and Fusobacterium are nonmotile, non-spore-forming, anaerobic, gram-negative bacilli. Foot rot is a contagious, acute or chronic dermatitis involving the hoof and underlying tissues ( Bulgin, 1986 ). It is the leading cause of lameness in sheep. At least 20 serotypes of Dichelobacter are known. Arcanobacterium pyogenes may also contribute to the pathogenicity or to foot abscesses in goats. Foot scald, an interdigital dermatitis, is caused primarily by D. nodosus alone. Clinical signs. Varying degrees of lameness are observed in all ages of animals within 2–3 weeks of exposure to the organisms. Severely infected animals will show generalized signs of weight loss, decreased productivity, and anorexia associated with an inability to move. The interdigital skin and hooves will be moist, with a distinct necrotic odor. Morbidity may reach 70% in susceptible animals. Diagnosis is based on clinical signs. Smears and cultures confirm the definitive agents. Clinical signs of the milder disease, foot scald, include mild lameness, redness and swelling, and little to no odor. Epizootiology and transmission. Fusobacterium necrophorum is ubiquitous in soil and manure, in the gastrointestinal tract, and on the skin and hooves of domestic animals. In contrast, Dichelobacter contaminates the soil and manure but rarely remains in the environment for more than about 2 weeks. Some animals may be chronic carriers. Overcrowded, warm, and moist environments are key elements in transmission. Outbreaks are likely in the spring season. Shipping trailers and contaminated pens or yards should be considered also as likely sources of the bacteria. Pathogenesis. Both organisms are transmitted to the susceptible animal by direct or indirect contact. The organisms enter the hoof through injuries or through sites where Strongyloides papillosus larvae have penetrated. Fusobacterium necrophorum initiates the colonization and is followed by D. nodosus. The latter attaches and releases proteases; these cause necrosis of the epidermal layers and separation of the hoof from the underlying dermis. The pathogenicity of the serotypes of D. nodosus is correlated with the production of these proteases and numbers of pili. Additionally, F. necrophorum causes a severe, damaging inflammatory reaction. Differential diagnosis. Foot abscesses, tetanus, selenium/vitamin E deficiencies, copper deficiency, strawberry foot rot, bluetongue virus infection (manifested with myopathy and coronitis), and trauma are among the many differentials that must be considered. Treatment. Affected animals are best treated by manually trimming the necrotic debris from the hooves, followed by application of local antibiotics and foot wraps. Systemic antibiotics such as penicillin, oxytetracycline, and erythromycin may be used. Goats have improved dramatically when given a single dose of penicillin (40,000 U/kg) ( Smith and Sherman, 1994 ). Footbaths containing 10% zinc sulfate, 20% copper sulfate, or 10% formalin (not legal in all states) can be used for treatment as well as for prevention of the disease. Affected animals should be separated from the flock. Vaccination has been shown to be effective as part of the treatment regimen. Some breeds of sheep and some breeds and lines of goats are resistant to infection. Individual sheep may recover without treatment or are resistant to infection. Prevention and control. Prevention and control programs involve scrutiny of herd and flock management; quarantine of incoming animals; vaccination; segregation of affected animals; careful and regular hoof trimming; discarding trimmings from known or suspected infected hooves; maintaining animals in good body condition; avoiding muddy pens and holding areas; and culling individuals with chronic and nonresponsive infections. Dichelobacter nodosus bacterins are commercially available; cross protection between serotypes varies. Biannual vaccinination in wet areas may be essential. Some breeds may develop vaccination site lumps. Footbaths of 10% zinc sulfate, 10% formalin (where allowed by state regulations), or 10% copper sulfate are also considered very effective preventive measures. Goats are less sensitive than sheep to the copper in the footbaths. Research complications. Treating and controlling foot rot is costly in terms of time, initial handling and treatments and their follow-up, housing space, and medications. Etiology. Two bacteria, Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum, work synergistically in causing contagious foot rot in sheep and goats. Other organisms may be involved as secondary invaders. Both Dichelobacter and Fusobacterium are nonmotile, non-spore-forming, anaerobic, gram-negative bacilli. Foot rot is a contagious, acute or chronic dermatitis involving the hoof and underlying tissues ( Bulgin, 1986 ). It is the leading cause of lameness in sheep. At least 20 serotypes of Dichelobacter are known. Arcanobacterium pyogenes may also contribute to the pathogenicity or to foot abscesses in goats. Foot scald, an interdigital dermatitis, is caused primarily by D. nodosus alone. Clinical signs. Varying degrees of lameness are observed in all ages of animals within 2–3 weeks of exposure to the organisms. Severely infected animals will show generalized signs of weight loss, decreased productivity, and anorexia associated with an inability to move. The interdigital skin and hooves will be moist, with a distinct necrotic odor. Morbidity may reach 70% in susceptible animals. Diagnosis is based on clinical signs. Smears and cultures confirm the definitive agents. Clinical signs of the milder disease, foot scald, include mild lameness, redness and swelling, and little to no odor. Epizootiology and transmission. Fusobacterium necrophorum is ubiquitous in soil and manure, in the gastrointestinal tract, and on the skin and hooves of domestic animals. In contrast, Dichelobacter contaminates the soil and manure but rarely remains in the environment for more than about 2 weeks. Some animals may be chronic carriers. Overcrowded, warm, and moist environments are key elements in transmission. Outbreaks are likely in the spring season. Shipping trailers and contaminated pens or yards should be considered also as likely sources of the bacteria. Pathogenesis. Both organisms are transmitted to the susceptible animal by direct or indirect contact. The organisms enter the hoof through injuries or through sites where Strongyloides papillosus larvae have penetrated. Fusobacterium necrophorum initiates the colonization and is followed by D. nodosus. The latter attaches and releases proteases; these cause necrosis of the epidermal layers and separation of the hoof from the underlying dermis. The pathogenicity of the serotypes of D. nodosus is correlated with the production of these proteases and numbers of pili. Additionally, F. necrophorum causes a severe, damaging inflammatory reaction. Differential diagnosis. Foot abscesses, tetanus, selenium/vitamin E deficiencies, copper deficiency, strawberry foot rot, bluetongue virus infection (manifested with myopathy and coronitis), and trauma are among the many differentials that must be considered. Treatment. Affected animals are best treated by manually trimming the necrotic debris from the hooves, followed by application of local antibiotics and foot wraps. Systemic antibiotics such as penicillin, oxytetracycline, and erythromycin may be used. Goats have improved dramatically when given a single dose of penicillin (40,000 U/kg) ( Smith and Sherman, 1994 ). Footbaths containing 10% zinc sulfate, 20% copper sulfate, or 10% formalin (not legal in all states) can be used for treatment as well as for prevention of the disease. Affected animals should be separated from the flock. Vaccination has been shown to be effective as part of the treatment regimen. Some breeds of sheep and some breeds and lines of goats are resistant to infection. Individual sheep may recover without treatment or are resistant to infection. Prevention and control. Prevention and control programs involve scrutiny of herd and flock management; quarantine of incoming animals; vaccination; segregation of affected animals; careful and regular hoof trimming; discarding trimmings from known or suspected infected hooves; maintaining animals in good body condition; avoiding muddy pens and holding areas; and culling individuals with chronic and nonresponsive infections. Dichelobacter nodosus bacterins are commercially available; cross protection between serotypes varies. Biannual vaccinination in wet areas may be essential. Some breeds may develop vaccination site lumps. Footbaths of 10% zinc sulfate, 10% formalin (where allowed by state regulations), or 10% copper sulfate are also considered very effective preventive measures. Goats are less sensitive than sheep to the copper in the footbaths. Research complications. Treating and controlling foot rot is costly in terms of time, initial handling and treatments and their follow-up, housing space, and medications. o. Fusobacterium necrophorum and Bacteroides melaninogenicus Infection (Foot Rot of Cattle, Interdigital Necrobacillosis of Cattle) Etiology. Interdigital necrobacillosis of cattle is caused by the synergistic infection of traumatized interdigital tissues by Fusobacterium necrophorum and Bacteroides melaninogenicus. Like F. necrophorum, B. melaninogenicus is a nonmotile, anaerobic, gram-negative bacterium. Dichelobacter nodosus, the agent of interdigital dermatitis, may be present in some cases. This is a common cause of lameness in cattle. Clinical signs. Clinical signs include mild to moderate lameness of sudden onset. Hindlimbs are more commonly affected, and cattle will often flex the pastern and bear weight only on the toe. The interdigital space will be swollen, as will be the coronet and bulb areas. Characteristic malodors will be noted, but there will be little purulent discharge. In more severe cases, animals will have elevated body temperature and loss of appetite. The lesions progress to fissures with necrosis until healing occurs. The diagnosis is by the odor and appearance. Anaerobic culturing confirms the organisms involved. Epizootiology and transmission. Cases may be sporadic, or epizootics may occur. Bos taurus dairy breeds and animals with wide interdigital spaces are more commonly affected. The factors here are comparable to those present in foot rot of smaller ruminants. Necropsy findings. Findings at necropsy include dermatitis and necrosis of the skin and subcutaneous tissues. Although necropsy would rarely be performed, secondary osteomyelitis may be noted in severe cases by sectioning limbs. Pathogenesis. The bacteria enter through the skin of the interdigital area after trauma to the interdigital skin, from hardened mud, or from softening of the skin due to, for example, constant wet conditions in pens. Colonization leads to cellulitis. In addition, F. necrophorum releases a leukocidal exotoxin that reduces phagocytosis and causes the necrosis, whereas the tissues and tendons are damaged by the proteases and collagenases produced by B. melaninogenicus. Zinc deficiency may play a role in the pathogenesis in some situations. Differential diagnoses. The most common differentials for sudden lameness include hairy heel warts and subsolar abcesses. Bluetongue virus should also be considered. Grain engorgement and secondary infection from cracks caused by selenium toxicosis should also be considered. The exotic foot-and-mouth disease virus would be considered in areas where that pathogen is found. Prevention and control. As with foot rot in smaller ruminants, management of the area and herd are important. Paddocks and pens should be kept dry, well drained, and free of material that will damage feet. Footbaths and chlortetracycline in the feed have been shown to control incidence. Affected animals should be segregated during treatment. Chronically affected or severely lame animals should be culled. New cattle should be quarantined and evaluated. Treatment. Successful treatment regimens that result in healing within a week include cleaning the feet and trimming necrotic tissue; parenteral antimicrobials, such as oxytetracycline or procaine penicillin, or sulfonomethazine in the drinking water or tetracyclines in feed; and footbaths (such as 10% zinc sulfate, 2.5% formalin, or 5% copper sulfate) twice a day. In severe cases, more aggressive therapy such as bandaging the feet or wiring the digits together may be needed. Animals can recover without treatment but will be lame for several weeks. Acquired immunity is reported to be poor. Research complications. Research complications are comparable to those noted for foot rot in smaller ruminants. Etiology. Interdigital necrobacillosis of cattle is caused by the synergistic infection of traumatized interdigital tissues by Fusobacterium necrophorum and Bacteroides melaninogenicus. Like F. necrophorum, B. melaninogenicus is a nonmotile, anaerobic, gram-negative bacterium. Dichelobacter nodosus, the agent of interdigital dermatitis, may be present in some cases. This is a common cause of lameness in cattle. Clinical signs. Clinical signs include mild to moderate lameness of sudden onset. Hindlimbs are more commonly affected, and cattle will often flex the pastern and bear weight only on the toe. The interdigital space will be swollen, as will be the coronet and bulb areas. Characteristic malodors will be noted, but there will be little purulent discharge. In more severe cases, animals will have elevated body temperature and loss of appetite. The lesions progress to fissures with necrosis until healing occurs. The diagnosis is by the odor and appearance. Anaerobic culturing confirms the organisms involved. Epizootiology and transmission. Cases may be sporadic, or epizootics may occur. Bos taurus dairy breeds and animals with wide interdigital spaces are more commonly affected. The factors here are comparable to those present in foot rot of smaller ruminants. Necropsy findings. Findings at necropsy include dermatitis and necrosis of the skin and subcutaneous tissues. Although necropsy would rarely be performed, secondary osteomyelitis may be noted in severe cases by sectioning limbs. Pathogenesis. The bacteria enter through the skin of the interdigital area after trauma to the interdigital skin, from hardened mud, or from softening of the skin due to, for example, constant wet conditions in pens. Colonization leads to cellulitis. In addition, F. necrophorum releases a leukocidal exotoxin that reduces phagocytosis and causes the necrosis, whereas the tissues and tendons are damaged by the proteases and collagenases produced by B. melaninogenicus. Zinc deficiency may play a role in the pathogenesis in some situations. Differential diagnoses. The most common differentials for sudden lameness include hairy heel warts and subsolar abcesses. Bluetongue virus should also be considered. Grain engorgement and secondary infection from cracks caused by selenium toxicosis should also be considered. The exotic foot-and-mouth disease virus would be considered in areas where that pathogen is found. Prevention and control. As with foot rot in smaller ruminants, management of the area and herd are important. Paddocks and pens should be kept dry, well drained, and free of material that will damage feet. Footbaths and chlortetracycline in the feed have been shown to control incidence. Affected animals should be segregated during treatment. Chronically affected or severely lame animals should be culled. New cattle should be quarantined and evaluated. Treatment. Successful treatment regimens that result in healing within a week include cleaning the feet and trimming necrotic tissue; parenteral antimicrobials, such as oxytetracycline or procaine penicillin, or sulfonomethazine in the drinking water or tetracyclines in feed; and footbaths (such as 10% zinc sulfate, 2.5% formalin, or 5% copper sulfate) twice a day. In severe cases, more aggressive therapy such as bandaging the feet or wiring the digits together may be needed. Animals can recover without treatment but will be lame for several weeks. Acquired immunity is reported to be poor. Research complications. Research complications are comparable to those noted for foot rot in smaller ruminants. Etiology. Interdigital necrobacillosis of cattle is caused by the synergistic infection of traumatized interdigital tissues by Fusobacterium necrophorum and Bacteroides melaninogenicus. Like F. necrophorum, B. melaninogenicus is a nonmotile, anaerobic, gram-negative bacterium. Dichelobacter nodosus, the agent of interdigital dermatitis, may be present in some cases. This is a common cause of lameness in cattle. Clinical signs. Clinical signs include mild to moderate lameness of sudden onset. Hindlimbs are more commonly affected, and cattle will often flex the pastern and bear weight only on the toe. The interdigital space will be swollen, as will be the coronet and bulb areas. Characteristic malodors will be noted, but there will be little purulent discharge. In more severe cases, animals will have elevated body temperature and loss of appetite. The lesions progress to fissures with necrosis until healing occurs. The diagnosis is by the odor and appearance. Anaerobic culturing confirms the organisms involved. Epizootiology and transmission. Cases may be sporadic, or epizootics may occur. Bos taurus dairy breeds and animals with wide interdigital spaces are more commonly affected. The factors here are comparable to those present in foot rot of smaller ruminants. Necropsy findings. Findings at necropsy include dermatitis and necrosis of the skin and subcutaneous tissues. Although necropsy would rarely be performed, secondary osteomyelitis may be noted in severe cases by sectioning limbs. Pathogenesis. The bacteria enter through the skin of the interdigital area after trauma to the interdigital skin, from hardened mud, or from softening of the skin due to, for example, constant wet conditions in pens. Colonization leads to cellulitis. In addition, F. necrophorum releases a leukocidal exotoxin that reduces phagocytosis and causes the necrosis, whereas the tissues and tendons are damaged by the proteases and collagenases produced by B. melaninogenicus. Zinc deficiency may play a role in the pathogenesis in some situations. Differential diagnoses. The most common differentials for sudden lameness include hairy heel warts and subsolar abcesses. Bluetongue virus should also be considered. Grain engorgement and secondary infection from cracks caused by selenium toxicosis should also be considered. The exotic foot-and-mouth disease virus would be considered in areas where that pathogen is found. Prevention and control. As with foot rot in smaller ruminants, management of the area and herd are important. Paddocks and pens should be kept dry, well drained, and free of material that will damage feet. Footbaths and chlortetracycline in the feed have been shown to control incidence. Affected animals should be segregated during treatment. Chronically affected or severely lame animals should be culled. New cattle should be quarantined and evaluated. Treatment. Successful treatment regimens that result in healing within a week include cleaning the feet and trimming necrotic tissue; parenteral antimicrobials, such as oxytetracycline or procaine penicillin, or sulfonomethazine in the drinking water or tetracyclines in feed; and footbaths (such as 10% zinc sulfate, 2.5% formalin, or 5% copper sulfate) twice a day. In severe cases, more aggressive therapy such as bandaging the feet or wiring the digits together may be needed. Animals can recover without treatment but will be lame for several weeks. Acquired immunity is reported to be poor. Research complications. Research complications are comparable to those noted for foot rot in smaller ruminants. p. Fusobacterium necrophorum infection (Foot Abscesses) Fusobacterium necrophorum is also associated with foot abscesses, the infection of the deeper structures of the foot, in sheep and goats. Only one claw of the affected hoof may be involved. The animals will be three-legged lame, and the affected hoof will be hot. Pockets of purulent material may be in the heel or toe. q. Heel Warts (Bovine Digital Dermatitis, Interdigital Papillomatosis, Papillomatous Digital Dermatitis, Foot Warts, Heel Warts, Hairy Foot Warts, Mortellaro's Disease) Etiology. Bacteria such as Fusobacterium spp., Bacteroides spp., and Dichelobacter nodosus have been isolated from bovine heel lesions. Spirochete-like organisms have also been shown in the lesions of cows with papillomatous digital dermatitis (PDD), in the United States and Europe; these have culturing requirements similar to those of Treponema species. Clinical signs. All lesions occur on the haired, digital skin. One or all feet may be affected. Most lesions occur on the plantar surface of the hindfoot (near the heel bulbs and/or extending from the interdigital space), but the palmar and dorsal aspect of the interdigital spaces may also be involved. Progression of lesions, typically over 2–3 weeks, includes erect hairs, loss of hair, and thickening skin. Moist plaques begin as red and remain red or turn gray or black. Exudate or blood may be present on the plaque. Plaques enlarge and "hairs" protrude from the roughened surface. Lesioned areas are painful when touched. The lesions may or may not be malodorous. Epizootiology and transmission. Facility conditions and herd management are considered contributing factors. The following have been examined as contributing factors: nutrition, particularly zinc deficiency; poorly drained, low-oxygen, organic material underfoot; poor ventilation; rough flooring; damp and dirty bedding areas; and overcrowding. These interdigital lesions occur commonly in young stock and in dairy facilities throughout the world. The disease is seen only in cattle. Pathogenesis. The organisms noted above, combined with poor facility and herd management, are critical in the pathogenesis. Differential diagnosis. Differentials for lameness will include sole abscesses, laminitis, and trauma. Prevention and control. Each facility and management condition noted above should be addressed in conjunction with appropriate antibiotic and/or antiseptic treatment regimens. All equipment used for hoof trimming must be cleaned and disinfected after every use. Trucks and trailers should also be sanitized between groups of animals. Treatment. Antibiotic and antiseptic regimens have been used successfully for this problem. Antibiotics include parenteral cephalosporins and pencillins, as well as topical tetracyclines with bandaging. Antiseptic or antibiotic solutions in footbaths include tetracyclines, zinc sulfate, lincomycin, spectinomycin, copper sulfate, and formalin. The footbaths must be well maintained, minimizing contamination by feces and other materials. Tandem arrangements, such as the cleaning footbaths and then the medicated footbaths, and preventing dilution from precipitation are useful. Other treatments such as surgical debridement, cryotherapy, and caustic topical solutions have been successful. Research complications. Infectious, contagious PPD is one of the major causes of lameness among heifers and dairy cattle and is a costly problem to treat. The outbreaks are generally worse in younger animals in chronically infected herds. The immune response is not well understood, and it may be temporary in older animals. Etiology. Bacteria such as Fusobacterium spp., Bacteroides spp., and Dichelobacter nodosus have been isolated from bovine heel lesions. Spirochete-like organisms have also been shown in the lesions of cows with papillomatous digital dermatitis (PDD), in the United States and Europe; these have culturing requirements similar to those of Treponema species. Clinical signs. All lesions occur on the haired, digital skin. One or all feet may be affected. Most lesions occur on the plantar surface of the hindfoot (near the heel bulbs and/or extending from the interdigital space), but the palmar and dorsal aspect of the interdigital spaces may also be involved. Progression of lesions, typically over 2–3 weeks, includes erect hairs, loss of hair, and thickening skin. Moist plaques begin as red and remain red or turn gray or black. Exudate or blood may be present on the plaque. Plaques enlarge and "hairs" protrude from the roughened surface. Lesioned areas are painful when touched. The lesions may or may not be malodorous. Epizootiology and transmission. Facility conditions and herd management are considered contributing factors. The following have been examined as contributing factors: nutrition, particularly zinc deficiency; poorly drained, low-oxygen, organic material underfoot; poor ventilation; rough flooring; damp and dirty bedding areas; and overcrowding. These interdigital lesions occur commonly in young stock and in dairy facilities throughout the world. The disease is seen only in cattle. Pathogenesis. The organisms noted above, combined with poor facility and herd management, are critical in the pathogenesis. Differential diagnosis. Differentials for lameness will include sole abscesses, laminitis, and trauma. Prevention and control. Each facility and management condition noted above should be addressed in conjunction with appropriate antibiotic and/or antiseptic treatment regimens. All equipment used for hoof trimming must be cleaned and disinfected after every use. Trucks and trailers should also be sanitized between groups of animals. Treatment. Antibiotic and antiseptic regimens have been used successfully for this problem. Antibiotics include parenteral cephalosporins and pencillins, as well as topical tetracyclines with bandaging. Antiseptic or antibiotic solutions in footbaths include tetracyclines, zinc sulfate, lincomycin, spectinomycin, copper sulfate, and formalin. The footbaths must be well maintained, minimizing contamination by feces and other materials. Tandem arrangements, such as the cleaning footbaths and then the medicated footbaths, and preventing dilution from precipitation are useful. Other treatments such as surgical debridement, cryotherapy, and caustic topical solutions have been successful. Research complications. Infectious, contagious PPD is one of the major causes of lameness among heifers and dairy cattle and is a costly problem to treat. The outbreaks are generally worse in younger animals in chronically infected herds. The immune response is not well understood, and it may be temporary in older animals. Etiology. Bacteria such as Fusobacterium spp., Bacteroides spp., and Dichelobacter nodosus have been isolated from bovine heel lesions. Spirochete-like organisms have also been shown in the lesions of cows with papillomatous digital dermatitis (PDD), in the United States and Europe; these have culturing requirements similar to those of Treponema species. Clinical signs. All lesions occur on the haired, digital skin. One or all feet may be affected. Most lesions occur on the plantar surface of the hindfoot (near the heel bulbs and/or extending from the interdigital space), but the palmar and dorsal aspect of the interdigital spaces may also be involved. Progression of lesions, typically over 2–3 weeks, includes erect hairs, loss of hair, and thickening skin. Moist plaques begin as red and remain red or turn gray or black. Exudate or blood may be present on the plaque. Plaques enlarge and "hairs" protrude from the roughened surface. Lesioned areas are painful when touched. The lesions may or may not be malodorous. Epizootiology and transmission. Facility conditions and herd management are considered contributing factors. The following have been examined as contributing factors: nutrition, particularly zinc deficiency; poorly drained, low-oxygen, organic material underfoot; poor ventilation; rough flooring; damp and dirty bedding areas; and overcrowding. These interdigital lesions occur commonly in young stock and in dairy facilities throughout the world. The disease is seen only in cattle. Pathogenesis. The organisms noted above, combined with poor facility and herd management, are critical in the pathogenesis. Differential diagnosis. Differentials for lameness will include sole abscesses, laminitis, and trauma. Prevention and control. Each facility and management condition noted above should be addressed in conjunction with appropriate antibiotic and/or antiseptic treatment regimens. All equipment used for hoof trimming must be cleaned and disinfected after every use. Trucks and trailers should also be sanitized between groups of animals. Treatment. Antibiotic and antiseptic regimens have been used successfully for this problem. Antibiotics include parenteral cephalosporins and pencillins, as well as topical tetracyclines with bandaging. Antiseptic or antibiotic solutions in footbaths include tetracyclines, zinc sulfate, lincomycin, spectinomycin, copper sulfate, and formalin. The footbaths must be well maintained, minimizing contamination by feces and other materials. Tandem arrangements, such as the cleaning footbaths and then the medicated footbaths, and preventing dilution from precipitation are useful. Other treatments such as surgical debridement, cryotherapy, and caustic topical solutions have been successful. Research complications. Infectious, contagious PPD is one of the major causes of lameness among heifers and dairy cattle and is a costly problem to treat. The outbreaks are generally worse in younger animals in chronically infected herds. The immune response is not well understood, and it may be temporary in older animals. r. Haemophilus somnus infection (Thromboembolic Meningoencephalitis) Etiology. Haemophilus somnus is a pleomorphic, nonencapsulated, gram-negative bacterium. Diseases caused by this organism include thromboembolic meningoencephalitis (TEME), septicemia, arthritis, and reproductive failures due to genital tract infections in males and females. Haemophilus somnus is a also major contributor to the bovine respiratory disease complex. Haemophilus spp. have been associated with respiratory disease in sheep and goats. Clinical signs. The neurologic presentation may be preceded by 1–2 weeks of dry, harsh coughing. Neurologic signs include depression, ataxia, falling, conscious proprioceptive deficits; signs such as head tilt from otitis interna or otitis media, opisthotonus, and convulsions may be seen as the brain stem is affected. High fever, extreme morbidity, and death within 36 hr may occur. Respiratory tract infections are usually part of the complex with infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza 3, Mycoplasma, and Pasteurella, and the synergism among these contributes to the signs of bovine respiratory disease complex (BRDC). In acute neurologic as well as chronic pneumonic infections, polyarthritis may develop. Abortion, vulvitis, vaginitis, endometritis, placentitis, and failure to conceive are manifestations of reproductive tract disease. In all cases, asymptomatic infections may also occur. Diagnosis based on culture findings is difficult because H. somnus is part of the normal nasopharyngeal flora. Paired serum samples are recommended; single titers in some animals seem to be high because of passive immunity, previous vaccination, or previous exposure. In cases of abortion, other causes should be eliminated from consideration. Epizootiology and transmission. Because the organism is considered part of the normal flora of cattle and can be isolated from numerous tissues, the distinction between the normal flora and the status of chronic carrier is not clear. Outbreaks are associated with younger cattle in feedlots in western United States, but stresses of travel and coinfection with other respiratory pathogens are involved in some cases. Adult cattle have also been affected. Vaccination for viral respiratory pathogens may increase susceptibility. Transmission is by respiratory and genital tract secretions. The organism does not persist in the environment. Necropsy findings. Pathognomonic central nervous system lesions include multifocal red-brown foci of necrosis and inflammation on and within the brain and the meninges. Many thrombi with bacterial colonies will be seen in these affected areas. Ocular lesions may also be seen, including conjunctivitis, retinal hemorrhages, and edema. Usually animals with neurological disease will not have respiratory tract lesions. The respiratory tract lesions include bronchopneumonia and suppurative pleuritis. When combined with Pasteurella infection, the pathology becomes more severe. Aborted fetuses will not show lesions, but necrotizing placentitis will be evident histologically. Pure cultures of H. somnus may be possible from these tissues. Pathogenesis. Inhalation of contaminated respiratory secretions from carrier animals is the primary means of transmission. The anatomical location of bacterial residence within the carriers has not been identified. After gaining access by way of the respiratory tract, the bacteria proliferate, and a bacteremia develops. The bacteria are phagocytosed by neutrophils but are not killed. The thrombosis formation is due to the adherence by the nonphagocytosed organisms to vascular endothelial cells, degeneration and desquamation of these cells, and exposure of subendothelial collagen, with subsequent initiation of the intrinsic coagulation pathway. Antigen-antibody complex formation, resulting in vasculitis, is also correlated with high levels of agglutinating antibodies. Differential diagnosis. Differentials in all ruminants include other pathogens associated with neurological disease and respiratory disease such as Pasteurella hemolytica, P. multocida, and P. aeruginosa. In smaller ruminants, Corynebacterium pseudotuberculosis should be considered. Prevention and control. Stressed animals or those exposed to known carriers can be treated prophylactically with tetracycline administered parenterally or orally (in the feed or water). The late-stage polyarthritis is resistant to antibiotic therapy, because of failure of the antibiotic to reach the site of infection. Planning vaccination programs carefully will decrease chances of outbreaks. For example, avoiding vaccinating animals for infectious bovine rhinotrachetitis and bovine viral diarrhea during times of stress to the cattle is worthwhile. Killed whole-cell bacterins are commercially available; these have been shown to be effective in controlling the respiratory disease presentation. Control of other clinical aspects of the H. somnus disease by these bacterins has not been well described. Treatment. Rapid treatment at the first signs of neurologic disease is important in an outbreak. Haemophilus somnus is susceptible to several antibiotics, such as Oxytetracycline and penicillin, and these are often used in sequence until the cattle are recovered. Etiology. Haemophilus somnus is a pleomorphic, nonencapsulated, gram-negative bacterium. Diseases caused by this organism include thromboembolic meningoencephalitis (TEME), septicemia, arthritis, and reproductive failures due to genital tract infections in males and females. Haemophilus somnus is a also major contributor to the bovine respiratory disease complex. Haemophilus spp. have been associated with respiratory disease in sheep and goats. Clinical signs. The neurologic presentation may be preceded by 1–2 weeks of dry, harsh coughing. Neurologic signs include depression, ataxia, falling, conscious proprioceptive deficits; signs such as head tilt from otitis interna or otitis media, opisthotonus, and convulsions may be seen as the brain stem is affected. High fever, extreme morbidity, and death within 36 hr may occur. Respiratory tract infections are usually part of the complex with infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza 3, Mycoplasma, and Pasteurella, and the synergism among these contributes to the signs of bovine respiratory disease complex (BRDC). In acute neurologic as well as chronic pneumonic infections, polyarthritis may develop. Abortion, vulvitis, vaginitis, endometritis, placentitis, and failure to conceive are manifestations of reproductive tract disease. In all cases, asymptomatic infections may also occur. Diagnosis based on culture findings is difficult because H. somnus is part of the normal nasopharyngeal flora. Paired serum samples are recommended; single titers in some animals seem to be high because of passive immunity, previous vaccination, or previous exposure. In cases of abortion, other causes should be eliminated from consideration. Epizootiology and transmission. Because the organism is considered part of the normal flora of cattle and can be isolated from numerous tissues, the distinction between the normal flora and the status of chronic carrier is not clear. Outbreaks are associated with younger cattle in feedlots in western United States, but stresses of travel and coinfection with other respiratory pathogens are involved in some cases. Adult cattle have also been affected. Vaccination for viral respiratory pathogens may increase susceptibility. Transmission is by respiratory and genital tract secretions. The organism does not persist in the environment. Necropsy findings. Pathognomonic central nervous system lesions include multifocal red-brown foci of necrosis and inflammation on and within the brain and the meninges. Many thrombi with bacterial colonies will be seen in these affected areas. Ocular lesions may also be seen, including conjunctivitis, retinal hemorrhages, and edema. Usually animals with neurological disease will not have respiratory tract lesions. The respiratory tract lesions include bronchopneumonia and suppurative pleuritis. When combined with Pasteurella infection, the pathology becomes more severe. Aborted fetuses will not show lesions, but necrotizing placentitis will be evident histologically. Pure cultures of H. somnus may be possible from these tissues. Pathogenesis. Inhalation of contaminated respiratory secretions from carrier animals is the primary means of transmission. The anatomical location of bacterial residence within the carriers has not been identified. After gaining access by way of the respiratory tract, the bacteria proliferate, and a bacteremia develops. The bacteria are phagocytosed by neutrophils but are not killed. The thrombosis formation is due to the adherence by the nonphagocytosed organisms to vascular endothelial cells, degeneration and desquamation of these cells, and exposure of subendothelial collagen, with subsequent initiation of the intrinsic coagulation pathway. Antigen-antibody complex formation, resulting in vasculitis, is also correlated with high levels of agglutinating antibodies. Differential diagnosis. Differentials in all ruminants include other pathogens associated with neurological disease and respiratory disease such as Pasteurella hemolytica, P. multocida, and P. aeruginosa. In smaller ruminants, Corynebacterium pseudotuberculosis should be considered. Prevention and control. Stressed animals or those exposed to known carriers can be treated prophylactically with tetracycline administered parenterally or orally (in the feed or water). The late-stage polyarthritis is resistant to antibiotic therapy, because of failure of the antibiotic to reach the site of infection. Planning vaccination programs carefully will decrease chances of outbreaks. For example, avoiding vaccinating animals for infectious bovine rhinotrachetitis and bovine viral diarrhea during times of stress to the cattle is worthwhile. Killed whole-cell bacterins are commercially available; these have been shown to be effective in controlling the respiratory disease presentation. Control of other clinical aspects of the H. somnus disease by these bacterins has not been well described. Treatment. Rapid treatment at the first signs of neurologic disease is important in an outbreak. Haemophilus somnus is susceptible to several antibiotics, such as Oxytetracycline and penicillin, and these are often used in sequence until the cattle are recovered. Etiology. Haemophilus somnus is a pleomorphic, nonencapsulated, gram-negative bacterium. Diseases caused by this organism include thromboembolic meningoencephalitis (TEME), septicemia, arthritis, and reproductive failures due to genital tract infections in males and females. Haemophilus somnus is a also major contributor to the bovine respiratory disease complex. Haemophilus spp. have been associated with respiratory disease in sheep and goats. Clinical signs. The neurologic presentation may be preceded by 1–2 weeks of dry, harsh coughing. Neurologic signs include depression, ataxia, falling, conscious proprioceptive deficits; signs such as head tilt from otitis interna or otitis media, opisthotonus, and convulsions may be seen as the brain stem is affected. High fever, extreme morbidity, and death within 36 hr may occur. Respiratory tract infections are usually part of the complex with infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza 3, Mycoplasma, and Pasteurella, and the synergism among these contributes to the signs of bovine respiratory disease complex (BRDC). In acute neurologic as well as chronic pneumonic infections, polyarthritis may develop. Abortion, vulvitis, vaginitis, endometritis, placentitis, and failure to conceive are manifestations of reproductive tract disease. In all cases, asymptomatic infections may also occur. Diagnosis based on culture findings is difficult because H. somnus is part of the normal nasopharyngeal flora. Paired serum samples are recommended; single titers in some animals seem to be high because of passive immunity, previous vaccination, or previous exposure. In cases of abortion, other causes should be eliminated from consideration. Epizootiology and transmission. Because the organism is considered part of the normal flora of cattle and can be isolated from numerous tissues, the distinction between the normal flora and the status of chronic carrier is not clear. Outbreaks are associated with younger cattle in feedlots in western United States, but stresses of travel and coinfection with other respiratory pathogens are involved in some cases. Adult cattle have also been affected. Vaccination for viral respiratory pathogens may increase susceptibility. Transmission is by respiratory and genital tract secretions. The organism does not persist in the environment. Necropsy findings. Pathognomonic central nervous system lesions include multifocal red-brown foci of necrosis and inflammation on and within the brain and the meninges. Many thrombi with bacterial colonies will be seen in these affected areas. Ocular lesions may also be seen, including conjunctivitis, retinal hemorrhages, and edema. Usually animals with neurological disease will not have respiratory tract lesions. The respiratory tract lesions include bronchopneumonia and suppurative pleuritis. When combined with Pasteurella infection, the pathology becomes more severe. Aborted fetuses will not show lesions, but necrotizing placentitis will be evident histologically. Pure cultures of H. somnus may be possible from these tissues. Pathogenesis. Inhalation of contaminated respiratory secretions from carrier animals is the primary means of transmission. The anatomical location of bacterial residence within the carriers has not been identified. After gaining access by way of the respiratory tract, the bacteria proliferate, and a bacteremia develops. The bacteria are phagocytosed by neutrophils but are not killed. The thrombosis formation is due to the adherence by the nonphagocytosed organisms to vascular endothelial cells, degeneration and desquamation of these cells, and exposure of subendothelial collagen, with subsequent initiation of the intrinsic coagulation pathway. Antigen-antibody complex formation, resulting in vasculitis, is also correlated with high levels of agglutinating antibodies. Differential diagnosis. Differentials in all ruminants include other pathogens associated with neurological disease and respiratory disease such as Pasteurella hemolytica, P. multocida, and P. aeruginosa. In smaller ruminants, Corynebacterium pseudotuberculosis should be considered. Prevention and control. Stressed animals or those exposed to known carriers can be treated prophylactically with tetracycline administered parenterally or orally (in the feed or water). The late-stage polyarthritis is resistant to antibiotic therapy, because of failure of the antibiotic to reach the site of infection. Planning vaccination programs carefully will decrease chances of outbreaks. For example, avoiding vaccinating animals for infectious bovine rhinotrachetitis and bovine viral diarrhea during times of stress to the cattle is worthwhile. Killed whole-cell bacterins are commercially available; these have been shown to be effective in controlling the respiratory disease presentation. Control of other clinical aspects of the H. somnus disease by these bacterins has not been well described. Treatment. Rapid treatment at the first signs of neurologic disease is important in an outbreak. Haemophilus somnus is susceptible to several antibiotics, such as Oxytetracycline and penicillin, and these are often used in sequence until the cattle are recovered. s. Leptospirosis Etiology. Seven different species of the spirochete genus Leptospira are now recognized, and pathogenic serovars exist within each species; previously pathogenic leptospires were all classified as members of the species L. interrogans. The serovars pomona, icterohaemorrhagiae, grippotyphosa, interrogans, and hardjo are recognized pathogens. Leptospira hardjo and L. pomona are the serovars most commonly diagnosed in cattle, with L. hardjo causing endemic infection. Leptospira hardjo is also the major sheep serovar. Goats are susceptible to several serovars. Clinical signs. Leptospirosis is a contagious but uncommon disease in sheep and goats. The disease may cause abortion, anemia, hemoglobinuria, and icterus and is often associated with a concurrent fever. After a 4- to 10-day incubation period, the organism enters the bloodstream and causes bacteremia, fever, and red-cell hemolysis. Leptospiremia may last up to 7 days. Immune stimulation is apparently rapid, and antibodies are detectable at the end of the first week of infection; cross-serovar protection does not occur. During active bacteremia, hemolysis may result in hemoglobin levels of 50% below normal. Hyperthermia, hemoglobinuria, icterus, and anemia may be observed during this phase, and ewes in late gestation may abort. Abortion usually occurs only once. Mortality rates of above 50% have been reported in infected ewes and lambs ( Jensen and Swift, 1982 ). Subclinical infection is more common in nonpregnant and nonlactating animals. Sheep infected with leptospirosis may display a hemolytic crisis associated with IgM acting as a cold-reacting hemagglutinin. Acute and chronic infections in cattle are more common than infections in sheep and goats. Acute forms in cattle display signs similar to those in sheep. Acute infection in calves may progress to meningitis and death. Lactating cows will have severe drops in production. Chronic cases may lead to abortion, with retained placenta, and weakened calves or animals that carry the infection. Infertility may also be a sequela. Epizootiology and transmission. Leptospires are a large genus, and leptospirosis is a complicated disease to prevent, treat, and control. The organism survives well in the environment, especially in moist, warm, stagnant water. Cattle, swine, and other domestic and wild animals are potential carriers of serovars common to particular regions. Wild animals often serve as maintenance hosts, but domestic livestock may be reservoirs also. Organisms are shed in urine, in uterine discharges, and through milk. Animals become carriers when they are infected with a host-adapted serovar; sporadic clinical disease is more commonly associated with exposure to a non-host-adapted serovar ( Heath and Johnson, 1994 ). Infection may occur via oral ingestion of contaminated feed and water, via placental fluids, or through the mucous membranes of the susceptible animal. Placental or venereal transmission may occur. As the organisms are cleared from the bloodstream, they chronically infect the renal convoluted tubules and the reproductive tract (and occasionally the cerebrospinal fluid or vitreous humor). Chronically infected animals may shed the organism in the urine for 60 days or longer. Necropsy. Diagnosis is confirmed by identification of leptospires in fetal tissues. The leptospires are visible in silver- or fluorescent antibody-stained sections of liver or kidney. Leptospires may also be seen under dark-field or phase-contrast microscopy of fetal stomach contents. Fetal and maternal serology, and diagnostic tests such as the microscopic agglutination test, are useful; interpretation is complicated because of cross reaction of antibodies to many serovars. Differential diagnosis. More than one serovar may cause infection in one animal, and each serovar should be considered as a separate pathogen. Because of the associated anemia, differential diagnoses should include copper toxicity and parasites, in addition to other abortifacient diseases. Prevention and control. Polyvalent vaccines, tailored to common serovars regionally, are available and effective for preventing leptospirosis in cattle. Immunity is serovar specific. Because serological titers tend to diminish rapidly (40–50 days in sheep [ Jensen and Swift, 1982 ]), frequent vaccination may be necessary. Other prevention measures such as species-specific housing, control of wild rodents, and proper sanitation should be instituted. Treatment. Antibiotic treatment is aimed at treating ill animals and trying to clear the carrier state. Treatment methods for acute leptospirosis include oxytetracycline for 3–6 days. Addition of oxytetracycline or chlortetracycline to the feed for 1 week may be helpful. These antibiotics are considered best for removal of the carrier state of some serovars. Vaccination and antibiotic therapy can be combined in an outbreak. Research complications. Leptospirosis is zoonotic and may be associated with flulike symptoms, meningitis, or hepatorenal failure in humans. Etiology. Seven different species of the spirochete genus Leptospira are now recognized, and pathogenic serovars exist within each species; previously pathogenic leptospires were all classified as members of the species L. interrogans. The serovars pomona, icterohaemorrhagiae, grippotyphosa, interrogans, and hardjo are recognized pathogens. Leptospira hardjo and L. pomona are the serovars most commonly diagnosed in cattle, with L. hardjo causing endemic infection. Leptospira hardjo is also the major sheep serovar. Goats are susceptible to several serovars. Clinical signs. Leptospirosis is a contagious but uncommon disease in sheep and goats. The disease may cause abortion, anemia, hemoglobinuria, and icterus and is often associated with a concurrent fever. After a 4- to 10-day incubation period, the organism enters the bloodstream and causes bacteremia, fever, and red-cell hemolysis. Leptospiremia may last up to 7 days. Immune stimulation is apparently rapid, and antibodies are detectable at the end of the first week of infection; cross-serovar protection does not occur. During active bacteremia, hemolysis may result in hemoglobin levels of 50% below normal. Hyperthermia, hemoglobinuria, icterus, and anemia may be observed during this phase, and ewes in late gestation may abort. Abortion usually occurs only once. Mortality rates of above 50% have been reported in infected ewes and lambs ( Jensen and Swift, 1982 ). Subclinical infection is more common in nonpregnant and nonlactating animals. Sheep infected with leptospirosis may display a hemolytic crisis associated with IgM acting as a cold-reacting hemagglutinin. Acute and chronic infections in cattle are more common than infections in sheep and goats. Acute forms in cattle display signs similar to those in sheep. Acute infection in calves may progress to meningitis and death. Lactating cows will have severe drops in production. Chronic cases may lead to abortion, with retained placenta, and weakened calves or animals that carry the infection. Infertility may also be a sequela. Epizootiology and transmission. Leptospires are a large genus, and leptospirosis is a complicated disease to prevent, treat, and control. The organism survives well in the environment, especially in moist, warm, stagnant water. Cattle, swine, and other domestic and wild animals are potential carriers of serovars common to particular regions. Wild animals often serve as maintenance hosts, but domestic livestock may be reservoirs also. Organisms are shed in urine, in uterine discharges, and through milk. Animals become carriers when they are infected with a host-adapted serovar; sporadic clinical disease is more commonly associated with exposure to a non-host-adapted serovar ( Heath and Johnson, 1994 ). Infection may occur via oral ingestion of contaminated feed and water, via placental fluids, or through the mucous membranes of the susceptible animal. Placental or venereal transmission may occur. As the organisms are cleared from the bloodstream, they chronically infect the renal convoluted tubules and the reproductive tract (and occasionally the cerebrospinal fluid or vitreous humor). Chronically infected animals may shed the organism in the urine for 60 days or longer. Necropsy. Diagnosis is confirmed by identification of leptospires in fetal tissues. The leptospires are visible in silver- or fluorescent antibody-stained sections of liver or kidney. Leptospires may also be seen under dark-field or phase-contrast microscopy of fetal stomach contents. Fetal and maternal serology, and diagnostic tests such as the microscopic agglutination test, are useful; interpretation is complicated because of cross reaction of antibodies to many serovars. Differential diagnosis. More than one serovar may cause infection in one animal, and each serovar should be considered as a separate pathogen. Because of the associated anemia, differential diagnoses should include copper toxicity and parasites, in addition to other abortifacient diseases. Prevention and control. Polyvalent vaccines, tailored to common serovars regionally, are available and effective for preventing leptospirosis in cattle. Immunity is serovar specific. Because serological titers tend to diminish rapidly (40–50 days in sheep [ Jensen and Swift, 1982 ]), frequent vaccination may be necessary. Other prevention measures such as species-specific housing, control of wild rodents, and proper sanitation should be instituted. Treatment. Antibiotic treatment is aimed at treating ill animals and trying to clear the carrier state. Treatment methods for acute leptospirosis include oxytetracycline for 3–6 days. Addition of oxytetracycline or chlortetracycline to the feed for 1 week may be helpful. These antibiotics are considered best for removal of the carrier state of some serovars. Vaccination and antibiotic therapy can be combined in an outbreak. Research complications. Leptospirosis is zoonotic and may be associated with flulike symptoms, meningitis, or hepatorenal failure in humans. Etiology. Seven different species of the spirochete genus Leptospira are now recognized, and pathogenic serovars exist within each species; previously pathogenic leptospires were all classified as members of the species L. interrogans. The serovars pomona, icterohaemorrhagiae, grippotyphosa, interrogans, and hardjo are recognized pathogens. Leptospira hardjo and L. pomona are the serovars most commonly diagnosed in cattle, with L. hardjo causing endemic infection. Leptospira hardjo is also the major sheep serovar. Goats are susceptible to several serovars. Clinical signs. Leptospirosis is a contagious but uncommon disease in sheep and goats. The disease may cause abortion, anemia, hemoglobinuria, and icterus and is often associated with a concurrent fever. After a 4- to 10-day incubation period, the organism enters the bloodstream and causes bacteremia, fever, and red-cell hemolysis. Leptospiremia may last up to 7 days. Immune stimulation is apparently rapid, and antibodies are detectable at the end of the first week of infection; cross-serovar protection does not occur. During active bacteremia, hemolysis may result in hemoglobin levels of 50% below normal. Hyperthermia, hemoglobinuria, icterus, and anemia may be observed during this phase, and ewes in late gestation may abort. Abortion usually occurs only once. Mortality rates of above 50% have been reported in infected ewes and lambs ( Jensen and Swift, 1982 ). Subclinical infection is more common in nonpregnant and nonlactating animals. Sheep infected with leptospirosis may display a hemolytic crisis associated with IgM acting as a cold-reacting hemagglutinin. Acute and chronic infections in cattle are more common than infections in sheep and goats. Acute forms in cattle display signs similar to those in sheep. Acute infection in calves may progress to meningitis and death. Lactating cows will have severe drops in production. Chronic cases may lead to abortion, with retained placenta, and weakened calves or animals that carry the infection. Infertility may also be a sequela. Epizootiology and transmission. Leptospires are a large genus, and leptospirosis is a complicated disease to prevent, treat, and control. The organism survives well in the environment, especially in moist, warm, stagnant water. Cattle, swine, and other domestic and wild animals are potential carriers of serovars common to particular regions. Wild animals often serve as maintenance hosts, but domestic livestock may be reservoirs also. Organisms are shed in urine, in uterine discharges, and through milk. Animals become carriers when they are infected with a host-adapted serovar; sporadic clinical disease is more commonly associated with exposure to a non-host-adapted serovar ( Heath and Johnson, 1994 ). Infection may occur via oral ingestion of contaminated feed and water, via placental fluids, or through the mucous membranes of the susceptible animal. Placental or venereal transmission may occur. As the organisms are cleared from the bloodstream, they chronically infect the renal convoluted tubules and the reproductive tract (and occasionally the cerebrospinal fluid or vitreous humor). Chronically infected animals may shed the organism in the urine for 60 days or longer. Necropsy. Diagnosis is confirmed by identification of leptospires in fetal tissues. The leptospires are visible in silver- or fluorescent antibody-stained sections of liver or kidney. Leptospires may also be seen under dark-field or phase-contrast microscopy of fetal stomach contents. Fetal and maternal serology, and diagnostic tests such as the microscopic agglutination test, are useful; interpretation is complicated because of cross reaction of antibodies to many serovars. Differential diagnosis. More than one serovar may cause infection in one animal, and each serovar should be considered as a separate pathogen. Because of the associated anemia, differential diagnoses should include copper toxicity and parasites, in addition to other abortifacient diseases. Prevention and control. Polyvalent vaccines, tailored to common serovars regionally, are available and effective for preventing leptospirosis in cattle. Immunity is serovar specific. Because serological titers tend to diminish rapidly (40–50 days in sheep [ Jensen and Swift, 1982 ]), frequent vaccination may be necessary. Other prevention measures such as species-specific housing, control of wild rodents, and proper sanitation should be instituted. Treatment. Antibiotic treatment is aimed at treating ill animals and trying to clear the carrier state. Treatment methods for acute leptospirosis include oxytetracycline for 3–6 days. Addition of oxytetracycline or chlortetracycline to the feed for 1 week may be helpful. These antibiotics are considered best for removal of the carrier state of some serovars. Vaccination and antibiotic therapy can be combined in an outbreak. Research complications. Leptospirosis is zoonotic and may be associated with flulike symptoms, meningitis, or hepatorenal failure in humans. t. Listeria (Circling Disease, Silage Disease) Etiology. Listeria monocytogenes is a pleomorphic, motile, non-spore-forming, β-hemolytic, gram-positive bacillus that inhabits the soil for long periods of time and has been often found in fermented feedstuffs such as spoiled silage. Of the 16 known serovars, several produce clinical signs in ruminants. Listeria ivanovii (associated with abortions in sheep) is serovar 5. Clinical signs. Listeriosis is an acute, sporadic, noncontagious disease associated with neurological signs or abortions in sheep and other ruminants. The overall case rate is low. The disease may present as an isolated case or with multiple animals affected. Three forms of disease are described: encephalitis, placentitis with abortion, and septicemia with hepatitis and pneumonia. The encephalitic form is most common in sheep; septicemic forms may occur in neonatal lambs ( Scarratt, 1987 ). Clinically, the encephalitic form begins with depression, anorexia, and mild hyperthermia after an incubation period of 2–3 weeks. As the disease progresses, animals exhibit nasal discharges and conjunctivitis and begin to walk in circles, as if disoriented. Facial paralytic lesions, including drooping of an ear or eyelid, dilation of a nostril, or strabismus occur unilaterally on the affected side as the result of dysfunction of some or all the cranial nerves V-XII. The neck will by flexed away from the affected side. Facial muscle twitching, protrusion of the tongue, dysphagia, hypersalivation, and nasal discharges may be noted. The hypersalivation may lead to metabolic acidosis in advanced cases in cattle. Anorexia, prostration, coma, and death follow. The placental form usually results in last-trimester abortions in ewes and does, which typically survive this form of the disease. The affected females may be asymptomatic or may show severe clinical signs such as fever and depression, with subsequent retained placenta or endometritis. Abortion usually occurs within 2 weeks of Listeria infection. In cattle, abortion occurs during the last 2 months of gestation and has been induced experimentally 6–8 days after exposure. Cows present with the range of clinical signs seen in smaller-ruminant dams. There is no long-term effect on the fertility of affected dams. Epizootiology and transmission. The organism is transmitted by oral ingestion of contaminated feeds and water or possibly by inhalation. By the oral route, the organism enters through breaks in the oral cavity and ascends to the brain stem by way of nerves. When severe outbreaks occur, feedstuffs should be assessed for spoilage. Listeria organisms can be shed by asymptomatic carriers, especially at the end of pregnancy and at lambing. Diagnosis and necropsy findings. Diagnosis is usually made from clinical signs. Culture confirms the diagnosis (cold enrichment at 20° C is preferable but not essential for isolation). Impression smears will show the pleomorphic gram-positive characterisitics of the pathogen. Tissue fluorescent antibody techniques may also be utilized. Gross lesions are not observed with the encephalitic form. Microscopic lesions include thrombosis, neutrophilic or mononuclear foci in areas of inflammation, and neuritis. The pons, medulla, and anterior spinal cord are primarily affected in the encephalitic form. Microabscesses of the midbrain are characteristic of Listeria encephalitis in sheep. Aborted fetuses that are intact may show fibrinous polyserositis, with excessive serous fluids; small, necrotic foci of the liver; and small abomasal erosions. Necrotic lesions of the fetal spleen and lungs may also be seen. In goats, Listeria-induced neurological lesions occur only in the brain stem. Placentitis, focal bronchopneumonia, hepatitis, splenitis, and nephritis may be seen with other forms. Pathogenesis. With the encephalitic form, the organism penetrates mucosal abrasions and enters the trigeminal or hypoglossal nerves. The Listeria organisms then migrate along the nerves and associated lymphatics to the brain stem (medulla and pons). In the septicemic form, the organism penetrates tissues of the gastrointestinal tract and enters the bloodstream, to be distributed to the liver, spleen, lungs, kidneys, and placenta. After infection, organisms are shed in all body secretions (infected milk is an important risk factor for zoonosis). A toxin produced by Listeria monocytogenes is correlated with pathogenicity, but the mechanism of the pathogenesis of this molecule has not been elucidated. Differential diagnoses. Rabies, bacterial meningitis, brain abscess, lead toxicity, and otitis media must be considered as differentials. In sheep, the differentials include organisms that cause abortion, and neurological signs, such as enterotoxemia due to Clostridium perfringens type D. In goats, the major differentials include caprine arthritis encephalitis viral infection and chlamydial and mycoplasmal infections. In both species, scrapie is a differential. In cattle, aberrant parasite migration or Hemophilus somnus infection must also be considered. Prevention and control. Affected dams should be segregated and treated. Other animals in the group may be treated with oxytetracycline as needed. Aborted tissues should be removed immediately. Proper storage of fermented feeds minimizes this source of contamination. When silage spoils, the pH increases, producing a suitable growth environment for the organism. Commercial vaccines are not available in the United States. Treatment. Affected animals can be treated aggressively with penicillin, ampicillin, oxytetracycline, or erythromycin. Exceptionally high levels of penicillin are required for treating affected cattle. Severely affected animals should receive appropriate fluid support and other nursing care. Treatment is less successful, and mortality is especially high in sheep. Recovered animals tend to resist reinfection. Research complications. In addition to the loss of fetal animals, stress to the dams, and risks to other animals, any aborted tissue by a ruminant should be regarded as a potential zoonotic risk. Listeria can cause mild to severe flulike symptoms in humans and may be a particular risk for pregnant women and for older or immune-compromised individuals. Listeriosis in humans is a reportable disease. Etiology. Listeria monocytogenes is a pleomorphic, motile, non-spore-forming, β-hemolytic, gram-positive bacillus that inhabits the soil for long periods of time and has been often found in fermented feedstuffs such as spoiled silage. Of the 16 known serovars, several produce clinical signs in ruminants. Listeria ivanovii (associated with abortions in sheep) is serovar 5. Clinical signs. Listeriosis is an acute, sporadic, noncontagious disease associated with neurological signs or abortions in sheep and other ruminants. The overall case rate is low. The disease may present as an isolated case or with multiple animals affected. Three forms of disease are described: encephalitis, placentitis with abortion, and septicemia with hepatitis and pneumonia. The encephalitic form is most common in sheep; septicemic forms may occur in neonatal lambs ( Scarratt, 1987 ). Clinically, the encephalitic form begins with depression, anorexia, and mild hyperthermia after an incubation period of 2–3 weeks. As the disease progresses, animals exhibit nasal discharges and conjunctivitis and begin to walk in circles, as if disoriented. Facial paralytic lesions, including drooping of an ear or eyelid, dilation of a nostril, or strabismus occur unilaterally on the affected side as the result of dysfunction of some or all the cranial nerves V-XII. The neck will by flexed away from the affected side. Facial muscle twitching, protrusion of the tongue, dysphagia, hypersalivation, and nasal discharges may be noted. The hypersalivation may lead to metabolic acidosis in advanced cases in cattle. Anorexia, prostration, coma, and death follow. The placental form usually results in last-trimester abortions in ewes and does, which typically survive this form of the disease. The affected females may be asymptomatic or may show severe clinical signs such as fever and depression, with subsequent retained placenta or endometritis. Abortion usually occurs within 2 weeks of Listeria infection. In cattle, abortion occurs during the last 2 months of gestation and has been induced experimentally 6–8 days after exposure. Cows present with the range of clinical signs seen in smaller-ruminant dams. There is no long-term effect on the fertility of affected dams. Epizootiology and transmission. The organism is transmitted by oral ingestion of contaminated feeds and water or possibly by inhalation. By the oral route, the organism enters through breaks in the oral cavity and ascends to the brain stem by way of nerves. When severe outbreaks occur, feedstuffs should be assessed for spoilage. Listeria organisms can be shed by asymptomatic carriers, especially at the end of pregnancy and at lambing. Diagnosis and necropsy findings. Diagnosis is usually made from clinical signs. Culture confirms the diagnosis (cold enrichment at 20° C is preferable but not essential for isolation). Impression smears will show the pleomorphic gram-positive characterisitics of the pathogen. Tissue fluorescent antibody techniques may also be utilized. Gross lesions are not observed with the encephalitic form. Microscopic lesions include thrombosis, neutrophilic or mononuclear foci in areas of inflammation, and neuritis. The pons, medulla, and anterior spinal cord are primarily affected in the encephalitic form. Microabscesses of the midbrain are characteristic of Listeria encephalitis in sheep. Aborted fetuses that are intact may show fibrinous polyserositis, with excessive serous fluids; small, necrotic foci of the liver; and small abomasal erosions. Necrotic lesions of the fetal spleen and lungs may also be seen. In goats, Listeria-induced neurological lesions occur only in the brain stem. Placentitis, focal bronchopneumonia, hepatitis, splenitis, and nephritis may be seen with other forms. Pathogenesis. With the encephalitic form, the organism penetrates mucosal abrasions and enters the trigeminal or hypoglossal nerves. The Listeria organisms then migrate along the nerves and associated lymphatics to the brain stem (medulla and pons). In the septicemic form, the organism penetrates tissues of the gastrointestinal tract and enters the bloodstream, to be distributed to the liver, spleen, lungs, kidneys, and placenta. After infection, organisms are shed in all body secretions (infected milk is an important risk factor for zoonosis). A toxin produced by Listeria monocytogenes is correlated with pathogenicity, but the mechanism of the pathogenesis of this molecule has not been elucidated. Differential diagnoses. Rabies, bacterial meningitis, brain abscess, lead toxicity, and otitis media must be considered as differentials. In sheep, the differentials include organisms that cause abortion, and neurological signs, such as enterotoxemia due to Clostridium perfringens type D. In goats, the major differentials include caprine arthritis encephalitis viral infection and chlamydial and mycoplasmal infections. In both species, scrapie is a differential. In cattle, aberrant parasite migration or Hemophilus somnus infection must also be considered. Prevention and control. Affected dams should be segregated and treated. Other animals in the group may be treated with oxytetracycline as needed. Aborted tissues should be removed immediately. Proper storage of fermented feeds minimizes this source of contamination. When silage spoils, the pH increases, producing a suitable growth environment for the organism. Commercial vaccines are not available in the United States. Treatment. Affected animals can be treated aggressively with penicillin, ampicillin, oxytetracycline, or erythromycin. Exceptionally high levels of penicillin are required for treating affected cattle. Severely affected animals should receive appropriate fluid support and other nursing care. Treatment is less successful, and mortality is especially high in sheep. Recovered animals tend to resist reinfection. Research complications. In addition to the loss of fetal animals, stress to the dams, and risks to other animals, any aborted tissue by a ruminant should be regarded as a potential zoonotic risk. Listeria can cause mild to severe flulike symptoms in humans and may be a particular risk for pregnant women and for older or immune-compromised individuals. Listeriosis in humans is a reportable disease. Etiology. Listeria monocytogenes is a pleomorphic, motile, non-spore-forming, β-hemolytic, gram-positive bacillus that inhabits the soil for long periods of time and has been often found in fermented feedstuffs such as spoiled silage. Of the 16 known serovars, several produce clinical signs in ruminants. Listeria ivanovii (associated with abortions in sheep) is serovar 5. Clinical signs. Listeriosis is an acute, sporadic, noncontagious disease associated with neurological signs or abortions in sheep and other ruminants. The overall case rate is low. The disease may present as an isolated case or with multiple animals affected. Three forms of disease are described: encephalitis, placentitis with abortion, and septicemia with hepatitis and pneumonia. The encephalitic form is most common in sheep; septicemic forms may occur in neonatal lambs ( Scarratt, 1987 ). Clinically, the encephalitic form begins with depression, anorexia, and mild hyperthermia after an incubation period of 2–3 weeks. As the disease progresses, animals exhibit nasal discharges and conjunctivitis and begin to walk in circles, as if disoriented. Facial paralytic lesions, including drooping of an ear or eyelid, dilation of a nostril, or strabismus occur unilaterally on the affected side as the result of dysfunction of some or all the cranial nerves V-XII. The neck will by flexed away from the affected side. Facial muscle twitching, protrusion of the tongue, dysphagia, hypersalivation, and nasal discharges may be noted. The hypersalivation may lead to metabolic acidosis in advanced cases in cattle. Anorexia, prostration, coma, and death follow. The placental form usually results in last-trimester abortions in ewes and does, which typically survive this form of the disease. The affected females may be asymptomatic or may show severe clinical signs such as fever and depression, with subsequent retained placenta or endometritis. Abortion usually occurs within 2 weeks of Listeria infection. In cattle, abortion occurs during the last 2 months of gestation and has been induced experimentally 6–8 days after exposure. Cows present with the range of clinical signs seen in smaller-ruminant dams. There is no long-term effect on the fertility of affected dams. Epizootiology and transmission. The organism is transmitted by oral ingestion of contaminated feeds and water or possibly by inhalation. By the oral route, the organism enters through breaks in the oral cavity and ascends to the brain stem by way of nerves. When severe outbreaks occur, feedstuffs should be assessed for spoilage. Listeria organisms can be shed by asymptomatic carriers, especially at the end of pregnancy and at lambing. Diagnosis and necropsy findings. Diagnosis is usually made from clinical signs. Culture confirms the diagnosis (cold enrichment at 20° C is preferable but not essential for isolation). Impression smears will show the pleomorphic gram-positive characterisitics of the pathogen. Tissue fluorescent antibody techniques may also be utilized. Gross lesions are not observed with the encephalitic form. Microscopic lesions include thrombosis, neutrophilic or mononuclear foci in areas of inflammation, and neuritis. The pons, medulla, and anterior spinal cord are primarily affected in the encephalitic form. Microabscesses of the midbrain are characteristic of Listeria encephalitis in sheep. Aborted fetuses that are intact may show fibrinous polyserositis, with excessive serous fluids; small, necrotic foci of the liver; and small abomasal erosions. Necrotic lesions of the fetal spleen and lungs may also be seen. In goats, Listeria-induced neurological lesions occur only in the brain stem. Placentitis, focal bronchopneumonia, hepatitis, splenitis, and nephritis may be seen with other forms. Pathogenesis. With the encephalitic form, the organism penetrates mucosal abrasions and enters the trigeminal or hypoglossal nerves. The Listeria organisms then migrate along the nerves and associated lymphatics to the brain stem (medulla and pons). In the septicemic form, the organism penetrates tissues of the gastrointestinal tract and enters the bloodstream, to be distributed to the liver, spleen, lungs, kidneys, and placenta. After infection, organisms are shed in all body secretions (infected milk is an important risk factor for zoonosis). A toxin produced by Listeria monocytogenes is correlated with pathogenicity, but the mechanism of the pathogenesis of this molecule has not been elucidated. Differential diagnoses. Rabies, bacterial meningitis, brain abscess, lead toxicity, and otitis media must be considered as differentials. In sheep, the differentials include organisms that cause abortion, and neurological signs, such as enterotoxemia due to Clostridium perfringens type D. In goats, the major differentials include caprine arthritis encephalitis viral infection and chlamydial and mycoplasmal infections. In both species, scrapie is a differential. In cattle, aberrant parasite migration or Hemophilus somnus infection must also be considered. Prevention and control. Affected dams should be segregated and treated. Other animals in the group may be treated with oxytetracycline as needed. Aborted tissues should be removed immediately. Proper storage of fermented feeds minimizes this source of contamination. When silage spoils, the pH increases, producing a suitable growth environment for the organism. Commercial vaccines are not available in the United States. Treatment. Affected animals can be treated aggressively with penicillin, ampicillin, oxytetracycline, or erythromycin. Exceptionally high levels of penicillin are required for treating affected cattle. Severely affected animals should receive appropriate fluid support and other nursing care. Treatment is less successful, and mortality is especially high in sheep. Recovered animals tend to resist reinfection. Research complications. In addition to the loss of fetal animals, stress to the dams, and risks to other animals, any aborted tissue by a ruminant should be regarded as a potential zoonotic risk. Listeria can cause mild to severe flulike symptoms in humans and may be a particular risk for pregnant women and for older or immune-compromised individuals. Listeriosis in humans is a reportable disease. u. Lyme Disease (Borrelia burgdorferi Infection, Borreliosis) Etiology. Lyme disease is caused by the spirochete Borrelia burgdorferi. Clinical signs and diagnosis. Reports in ruminants indicate seroconversion to B. burgdorferi, but there are few definitive correlations to the arthritis that is present. Diagnosis requires culturing from the affected joints and diagnostic elimination of other causes of lameness and arthritis. Epizootiology and transmission. The organism is present throughout much of the Northern Hemisphere and has been reported in many mammals and also in birds. Ticks of the Ixodes ricinus complex are the major vectors of the spirochete and must be attached for 24 hr for successful transmission. Pathogenesis. The Ixodes ticks have three life stages: larval, nymphal, and adult. Feeding occurs once during each stage, and wild animals are the source of blood meals. The larval stages feed from rodents, such as the white-footed deer mouse, Peromyscus leucopus, from which they acquire the spirochete. The nymphal stage is that which usually infects other animals. The adult ticks are usually found on deer. Differential diagnosis. Seroconversion to B. burgdorferi does not necessarily confirm the cause of arthritis. Other causes of arthritis and lameness in ruminants include trauma, caprine arthritis encephalitis virus, Mycoplasma spp., Chlamydia psittaci, Erysipelothrix spp., Arcanobacterium pyogenes, Brucella spp., and rickets. Prevention and control. Control of the tick vector is the most important factor in preventing the possibility of exposure or disease. Treatment. Antibiotic therapy, with tetracycline, penicillin, amoxicillin, and cephalosporins, is used for diagnosed or suspected Lyme arthritis. Research complications. Lyme disease is zoonotic, and the Ixodes ticks transmit the disease to humans. Etiology. Lyme disease is caused by the spirochete Borrelia burgdorferi. Clinical signs and diagnosis. Reports in ruminants indicate seroconversion to B. burgdorferi, but there are few definitive correlations to the arthritis that is present. Diagnosis requires culturing from the affected joints and diagnostic elimination of other causes of lameness and arthritis. Epizootiology and transmission. The organism is present throughout much of the Northern Hemisphere and has been reported in many mammals and also in birds. Ticks of the Ixodes ricinus complex are the major vectors of the spirochete and must be attached for 24 hr for successful transmission. Pathogenesis. The Ixodes ticks have three life stages: larval, nymphal, and adult. Feeding occurs once during each stage, and wild animals are the source of blood meals. The larval stages feed from rodents, such as the white-footed deer mouse, Peromyscus leucopus, from which they acquire the spirochete. The nymphal stage is that which usually infects other animals. The adult ticks are usually found on deer. Differential diagnosis. Seroconversion to B. burgdorferi does not necessarily confirm the cause of arthritis. Other causes of arthritis and lameness in ruminants include trauma, caprine arthritis encephalitis virus, Mycoplasma spp., Chlamydia psittaci, Erysipelothrix spp., Arcanobacterium pyogenes, Brucella spp., and rickets. Prevention and control. Control of the tick vector is the most important factor in preventing the possibility of exposure or disease. Treatment. Antibiotic therapy, with tetracycline, penicillin, amoxicillin, and cephalosporins, is used for diagnosed or suspected Lyme arthritis. Research complications. Lyme disease is zoonotic, and the Ixodes ticks transmit the disease to humans. Etiology. Lyme disease is caused by the spirochete Borrelia burgdorferi. Clinical signs and diagnosis. Reports in ruminants indicate seroconversion to B. burgdorferi, but there are few definitive correlations to the arthritis that is present. Diagnosis requires culturing from the affected joints and diagnostic elimination of other causes of lameness and arthritis. Epizootiology and transmission. The organism is present throughout much of the Northern Hemisphere and has been reported in many mammals and also in birds. Ticks of the Ixodes ricinus complex are the major vectors of the spirochete and must be attached for 24 hr for successful transmission. Pathogenesis. The Ixodes ticks have three life stages: larval, nymphal, and adult. Feeding occurs once during each stage, and wild animals are the source of blood meals. The larval stages feed from rodents, such as the white-footed deer mouse, Peromyscus leucopus, from which they acquire the spirochete. The nymphal stage is that which usually infects other animals. The adult ticks are usually found on deer. Differential diagnosis. Seroconversion to B. burgdorferi does not necessarily confirm the cause of arthritis. Other causes of arthritis and lameness in ruminants include trauma, caprine arthritis encephalitis virus, Mycoplasma spp., Chlamydia psittaci, Erysipelothrix spp., Arcanobacterium pyogenes, Brucella spp., and rickets. Prevention and control. Control of the tick vector is the most important factor in preventing the possibility of exposure or disease. Treatment. Antibiotic therapy, with tetracycline, penicillin, amoxicillin, and cephalosporins, is used for diagnosed or suspected Lyme arthritis. Research complications. Lyme disease is zoonotic, and the Ixodes ticks transmit the disease to humans. v. Mastitis i. Ovine mastitis Mastitis in ewes may be acute, subclinical, or chronic. Acute mastitis often results in anorexia, fever, abnormal milk, and swelling of the mammary gland. Pasteurella haemolytica is the most common cause of acute mastitis. Additional isolates may include, in order of prevalence, Staphylococcus aureus, Actinomyces (Corynebacterium) spp., and Histophilus ovis. Escherichia coli and Pseudomonas aeruginosa have also been found to cause acute mastitis. As many as six serotypes of Pasteurella haemolytica have been isolated from the mammary glands of mastitic ewes. Furthermore, intramammary inoculation of these organisms isolated from ovine and bovine pulmonary lesions has resulted in clinical mastitis in ewes ( Watkins and Jones, 1992 ). Subclinical mastitis is detected only indirectly, by counting somatic cells. The most common isolate from ewes with subclinical mastitis is coagulase-negative staphylococci. Other isolates include Actinomyces bovis, Streptococcus uberis, S. dysgalactiae, Micrococcus spp., Bacillus spp., and fecal streptococci. Most of these organisms are commonly found in the environment. Diffuse chronic mastitis, or hardbag, results from interstitial accumulations of lymphocytes in the udder. Both glands are usually affected, but no inflammation is present. Serological evidence suggests that diffuse chronic mastitis is caused by the retrovirus that causes ovine progressive pneumonia (OPP or maedi/visna virus). Other bacterial agents or Mycoplasma have not usually been isolated from udders with this type of mastitis. Acute mastitis occurs in approximately 5% of lactating ewes annually, and it usually occurs either soon after lambing or when lambs are 3–4 months old ( Lasgard and Vaabenoe, 1993 ). Subclinical mastitis occurs in 4–50% of lactating ewes ( Kirk and Glenn, 1996 ). Subclinical mastitis is more common in ewes from high-milk-producing breeds. Skin or teat lesions and dermatitis increase the prevalence of disease. Acute mastitis can be diagnosed in ewes with associated systemic signs of disease by physical examination of the udder and inspection of the milk. Subclinical mastitis is often suggested by somatic cell counts elevated above 1 × 10 6 cells/ml. When high somatic cell counts are identified, subclinical mastitis can be diagnosed by milk culture. The California mastitis test may also be helpful as an indicator of mastitis. Manual palpation of a hard, indurated udder as well as serological testing for the maedi/visna virus is helpful in confirming the diagnosis of diffuse chronic mastitis. Treatment for acute bacterial mastitis should include aggressive application of broad-spectrum antibiotics (intramammary and systemic) and supportive therapy such as fluids and anti-inflammatory drugs. It is may be helpful to milk out the infected udder frequently; oxytocin injections preceding milking will improve gland evacuation. Because somatic cell counting is often not routinely performed, treatment of subclinical mastitis is seldom done. There is currently no treatment available for diffuse chronic mastitis. ii. Caprine mastitis Lactating goats are subject to inflammation of mammary gland, or mastitis. The primary causative organisms are Staphylococcus epidermidis and other coagulase-negative Staphylococcus spp. Clinical signs of mastitis include abnormal coloration or composition of milk, mammary gland redness, heat and pain, enlargement of the mammary gland, discoloration of the mammary gland, and systemic signs of septicemia. Large abscesses may be present in the affected gland. Staphylococcus aureus is also associated with caprine mastitis, and toxemia may be part of the clinical picture. This organism produces a necrotizing alpha toxin that can result in gangrenous mastitis. Caprine mastitis may be clinical or subclinical, and the first indication of mastitis may be weak, depressed, or thin kids. Diagnosis is based on careful culture of mastitic milk. Treatment includes frequent stripping, intramammary antibiotics, and nonsteroidal anti-inflammatory drugs. Oxytocin (5–10 U) may help milk letdown for frequent strippings. Bovine mastitis products can be used in the goat; however, care should be taken not to insert the mastitis tube tip fully, because damage to the protective keratin layer lining the teat canal may occur. In severe acute systemic cases, steroids, fluids, and systemic antibiotics may be necessary. Other less common causes of mastitis in goats include Streptococcus spp. (S. agalactiae, S. dysgalactiae, S. uberis, and zooepidemicus). Gram-negative causes of caprine mastitis include Escherichia coli, Klebsiella pneumoniae, Pasteurella spp., Pseudomonas, and Proteus mirabilis. Corynebacterium pseudotuberculosis can cause mammary gland abscessation, whereas Mycoplasma mycoides may cause agalactia and systemic disease. "Hard udder" can be caused by caprine arthritis encephalitis virus (CAEV). Brucellosis and listeriosis can cause a subclinical interstitial mastitis ( Smith and Sherman, 1994 ). iii. Bovine mastitis Mastitis is the disease of greatest economic importance for the dairy cattle industry. The majority of the impact will be on the production and overall health of the cows, but low-incidence herds also diminish the risk of calves' ingesting or being exposed to pathogens. The most common bovine mastitis pathogens include Staphylococcus aureus and Streptococcus agalactiae, S. dysgalactiae, and S. uberis; coliform agents such as Escherichia coli, Enterobacter aerogenes, Serratia marcescens, and Klebsiella pneumoniae; mycoplasmal species such as Mycoplasma bovis, M. bovigenitalium, M. californicum, M. canadensis, and M. alkalescens; and Salmonella spp. such as S. typhimurium, S. newport, S. enteritidis, S. dublin, and S. muenster. Many of these agents such as Staphylococcus spp., Salmonella spp., and the coliforms can cause both acute and chronic mastitis, as well as severe systemic disease, including fever and anorexia. These must be regarded as herd and environmental pathogens in terms of treatment and prevention. The pathogenesis of staphylococcal infections is comparable to that in goats. Staphylococcus agalactiae can be cleared from udders because it does not invade other tissues, is an obligate resident of the glands, and is susceptible to penicillin. In contrast, S. uberis and S. dysgalactiae are environmental organisms and can be highly resistant to pencillin. Mycoplasma bovis is the more common of the mycoplasmal pathogens and can cause severe infections. Transmission of the mycoplasmas is not well defined but may be related to their presence in other organ systems. Treatments for mycoplasmal mastitis are not successful; culling is recommended. There are many interrelated factors associated with prevention and control of mastitis in a herd, including herd health and dry cow management, order of animals milked, milking procedures, milking equipment, condition of the teats, and the condition of the environment. Management of the overall herd includes aspects such as vaccination programs, nutrition, isolation of incoming animals, and quarantine and treatment of or culling diseased individuals. Culturing or testing newly freshened cows and monitoring the bulk milk tank serve as indicators of subclinical mastitis. Herd management will diminish teat lesions. Bacterin vaccines are available for preventing and controlling coliform mastitis and S. aureus mastitis. At the time of dry-off, all cows must be treated by intramammary route. Some infections can be successfully cleared during this time. Younger, disease-free animals should be milked first; any animals with diagnosed problems should be milked after the rest of the herd and/or segregated during treatment. Milkers' hands easily serve as a means of pathogen transmission, and wearing rubber gloves is recommended. Teat and udder cleaning practices include washing and drying with single-service paper or cloth towels or pre-and postmilking dipping. Milking equipment must be maintained to provide proper vacuum levels and pumping rates, and liners should be the appropriate size. Facilities that provide clean and dry areas for the animals to rest, feed, and move will diminish teat injuries and reduce exposures to mastitis pathogens. In that regard, inorganic bedding such as clean sand harbors few pathogens in contrast to shavings and sawdust. i. Ovine mastitis Mastitis in ewes may be acute, subclinical, or chronic. Acute mastitis often results in anorexia, fever, abnormal milk, and swelling of the mammary gland. Pasteurella haemolytica is the most common cause of acute mastitis. Additional isolates may include, in order of prevalence, Staphylococcus aureus, Actinomyces (Corynebacterium) spp., and Histophilus ovis. Escherichia coli and Pseudomonas aeruginosa have also been found to cause acute mastitis. As many as six serotypes of Pasteurella haemolytica have been isolated from the mammary glands of mastitic ewes. Furthermore, intramammary inoculation of these organisms isolated from ovine and bovine pulmonary lesions has resulted in clinical mastitis in ewes ( Watkins and Jones, 1992 ). Subclinical mastitis is detected only indirectly, by counting somatic cells. The most common isolate from ewes with subclinical mastitis is coagulase-negative staphylococci. Other isolates include Actinomyces bovis, Streptococcus uberis, S. dysgalactiae, Micrococcus spp., Bacillus spp., and fecal streptococci. Most of these organisms are commonly found in the environment. Diffuse chronic mastitis, or hardbag, results from interstitial accumulations of lymphocytes in the udder. Both glands are usually affected, but no inflammation is present. Serological evidence suggests that diffuse chronic mastitis is caused by the retrovirus that causes ovine progressive pneumonia (OPP or maedi/visna virus). Other bacterial agents or Mycoplasma have not usually been isolated from udders with this type of mastitis. Acute mastitis occurs in approximately 5% of lactating ewes annually, and it usually occurs either soon after lambing or when lambs are 3–4 months old ( Lasgard and Vaabenoe, 1993 ). Subclinical mastitis occurs in 4–50% of lactating ewes ( Kirk and Glenn, 1996 ). Subclinical mastitis is more common in ewes from high-milk-producing breeds. Skin or teat lesions and dermatitis increase the prevalence of disease. Acute mastitis can be diagnosed in ewes with associated systemic signs of disease by physical examination of the udder and inspection of the milk. Subclinical mastitis is often suggested by somatic cell counts elevated above 1 × 10 6 cells/ml. When high somatic cell counts are identified, subclinical mastitis can be diagnosed by milk culture. The California mastitis test may also be helpful as an indicator of mastitis. Manual palpation of a hard, indurated udder as well as serological testing for the maedi/visna virus is helpful in confirming the diagnosis of diffuse chronic mastitis. Treatment for acute bacterial mastitis should include aggressive application of broad-spectrum antibiotics (intramammary and systemic) and supportive therapy such as fluids and anti-inflammatory drugs. It is may be helpful to milk out the infected udder frequently; oxytocin injections preceding milking will improve gland evacuation. Because somatic cell counting is often not routinely performed, treatment of subclinical mastitis is seldom done. There is currently no treatment available for diffuse chronic mastitis. ii. Caprine mastitis Lactating goats are subject to inflammation of mammary gland, or mastitis. The primary causative organisms are Staphylococcus epidermidis and other coagulase-negative Staphylococcus spp. Clinical signs of mastitis include abnormal coloration or composition of milk, mammary gland redness, heat and pain, enlargement of the mammary gland, discoloration of the mammary gland, and systemic signs of septicemia. Large abscesses may be present in the affected gland. Staphylococcus aureus is also associated with caprine mastitis, and toxemia may be part of the clinical picture. This organism produces a necrotizing alpha toxin that can result in gangrenous mastitis. Caprine mastitis may be clinical or subclinical, and the first indication of mastitis may be weak, depressed, or thin kids. Diagnosis is based on careful culture of mastitic milk. Treatment includes frequent stripping, intramammary antibiotics, and nonsteroidal anti-inflammatory drugs. Oxytocin (5–10 U) may help milk letdown for frequent strippings. Bovine mastitis products can be used in the goat; however, care should be taken not to insert the mastitis tube tip fully, because damage to the protective keratin layer lining the teat canal may occur. In severe acute systemic cases, steroids, fluids, and systemic antibiotics may be necessary. Other less common causes of mastitis in goats include Streptococcus spp. (S. agalactiae, S. dysgalactiae, S. uberis, and zooepidemicus). Gram-negative causes of caprine mastitis include Escherichia coli, Klebsiella pneumoniae, Pasteurella spp., Pseudomonas, and Proteus mirabilis. Corynebacterium pseudotuberculosis can cause mammary gland abscessation, whereas Mycoplasma mycoides may cause agalactia and systemic disease. "Hard udder" can be caused by caprine arthritis encephalitis virus (CAEV). Brucellosis and listeriosis can cause a subclinical interstitial mastitis ( Smith and Sherman, 1994 ). iii. Bovine mastitis Mastitis is the disease of greatest economic importance for the dairy cattle industry. The majority of the impact will be on the production and overall health of the cows, but low-incidence herds also diminish the risk of calves' ingesting or being exposed to pathogens. The most common bovine mastitis pathogens include Staphylococcus aureus and Streptococcus agalactiae, S. dysgalactiae, and S. uberis; coliform agents such as Escherichia coli, Enterobacter aerogenes, Serratia marcescens, and Klebsiella pneumoniae; mycoplasmal species such as Mycoplasma bovis, M. bovigenitalium, M. californicum, M. canadensis, and M. alkalescens; and Salmonella spp. such as S. typhimurium, S. newport, S. enteritidis, S. dublin, and S. muenster. Many of these agents such as Staphylococcus spp., Salmonella spp., and the coliforms can cause both acute and chronic mastitis, as well as severe systemic disease, including fever and anorexia. These must be regarded as herd and environmental pathogens in terms of treatment and prevention. The pathogenesis of staphylococcal infections is comparable to that in goats. Staphylococcus agalactiae can be cleared from udders because it does not invade other tissues, is an obligate resident of the glands, and is susceptible to penicillin. In contrast, S. uberis and S. dysgalactiae are environmental organisms and can be highly resistant to pencillin. Mycoplasma bovis is the more common of the mycoplasmal pathogens and can cause severe infections. Transmission of the mycoplasmas is not well defined but may be related to their presence in other organ systems. Treatments for mycoplasmal mastitis are not successful; culling is recommended. There are many interrelated factors associated with prevention and control of mastitis in a herd, including herd health and dry cow management, order of animals milked, milking procedures, milking equipment, condition of the teats, and the condition of the environment. Management of the overall herd includes aspects such as vaccination programs, nutrition, isolation of incoming animals, and quarantine and treatment of or culling diseased individuals. Culturing or testing newly freshened cows and monitoring the bulk milk tank serve as indicators of subclinical mastitis. Herd management will diminish teat lesions. Bacterin vaccines are available for preventing and controlling coliform mastitis and S. aureus mastitis. At the time of dry-off, all cows must be treated by intramammary route. Some infections can be successfully cleared during this time. Younger, disease-free animals should be milked first; any animals with diagnosed problems should be milked after the rest of the herd and/or segregated during treatment. Milkers' hands easily serve as a means of pathogen transmission, and wearing rubber gloves is recommended. Teat and udder cleaning practices include washing and drying with single-service paper or cloth towels or pre-and postmilking dipping. Milking equipment must be maintained to provide proper vacuum levels and pumping rates, and liners should be the appropriate size. Facilities that provide clean and dry areas for the animals to rest, feed, and move will diminish teat injuries and reduce exposures to mastitis pathogens. In that regard, inorganic bedding such as clean sand harbors few pathogens in contrast to shavings and sawdust. i. Ovine mastitis Mastitis in ewes may be acute, subclinical, or chronic. Acute mastitis often results in anorexia, fever, abnormal milk, and swelling of the mammary gland. Pasteurella haemolytica is the most common cause of acute mastitis. Additional isolates may include, in order of prevalence, Staphylococcus aureus, Actinomyces (Corynebacterium) spp., and Histophilus ovis. Escherichia coli and Pseudomonas aeruginosa have also been found to cause acute mastitis. As many as six serotypes of Pasteurella haemolytica have been isolated from the mammary glands of mastitic ewes. Furthermore, intramammary inoculation of these organisms isolated from ovine and bovine pulmonary lesions has resulted in clinical mastitis in ewes ( Watkins and Jones, 1992 ). Subclinical mastitis is detected only indirectly, by counting somatic cells. The most common isolate from ewes with subclinical mastitis is coagulase-negative staphylococci. Other isolates include Actinomyces bovis, Streptococcus uberis, S. dysgalactiae, Micrococcus spp., Bacillus spp., and fecal streptococci. Most of these organisms are commonly found in the environment. Diffuse chronic mastitis, or hardbag, results from interstitial accumulations of lymphocytes in the udder. Both glands are usually affected, but no inflammation is present. Serological evidence suggests that diffuse chronic mastitis is caused by the retrovirus that causes ovine progressive pneumonia (OPP or maedi/visna virus). Other bacterial agents or Mycoplasma have not usually been isolated from udders with this type of mastitis. Acute mastitis occurs in approximately 5% of lactating ewes annually, and it usually occurs either soon after lambing or when lambs are 3–4 months old ( Lasgard and Vaabenoe, 1993 ). Subclinical mastitis occurs in 4–50% of lactating ewes ( Kirk and Glenn, 1996 ). Subclinical mastitis is more common in ewes from high-milk-producing breeds. Skin or teat lesions and dermatitis increase the prevalence of disease. Acute mastitis can be diagnosed in ewes with associated systemic signs of disease by physical examination of the udder and inspection of the milk. Subclinical mastitis is often suggested by somatic cell counts elevated above 1 × 10 6 cells/ml. When high somatic cell counts are identified, subclinical mastitis can be diagnosed by milk culture. The California mastitis test may also be helpful as an indicator of mastitis. Manual palpation of a hard, indurated udder as well as serological testing for the maedi/visna virus is helpful in confirming the diagnosis of diffuse chronic mastitis. Treatment for acute bacterial mastitis should include aggressive application of broad-spectrum antibiotics (intramammary and systemic) and supportive therapy such as fluids and anti-inflammatory drugs. It is may be helpful to milk out the infected udder frequently; oxytocin injections preceding milking will improve gland evacuation. Because somatic cell counting is often not routinely performed, treatment of subclinical mastitis is seldom done. There is currently no treatment available for diffuse chronic mastitis. ii. Caprine mastitis Lactating goats are subject to inflammation of mammary gland, or mastitis. The primary causative organisms are Staphylococcus epidermidis and other coagulase-negative Staphylococcus spp. Clinical signs of mastitis include abnormal coloration or composition of milk, mammary gland redness, heat and pain, enlargement of the mammary gland, discoloration of the mammary gland, and systemic signs of septicemia. Large abscesses may be present in the affected gland. Staphylococcus aureus is also associated with caprine mastitis, and toxemia may be part of the clinical picture. This organism produces a necrotizing alpha toxin that can result in gangrenous mastitis. Caprine mastitis may be clinical or subclinical, and the first indication of mastitis may be weak, depressed, or thin kids. Diagnosis is based on careful culture of mastitic milk. Treatment includes frequent stripping, intramammary antibiotics, and nonsteroidal anti-inflammatory drugs. Oxytocin (5–10 U) may help milk letdown for frequent strippings. Bovine mastitis products can be used in the goat; however, care should be taken not to insert the mastitis tube tip fully, because damage to the protective keratin layer lining the teat canal may occur. In severe acute systemic cases, steroids, fluids, and systemic antibiotics may be necessary. Other less common causes of mastitis in goats include Streptococcus spp. (S. agalactiae, S. dysgalactiae, S. uberis, and zooepidemicus). Gram-negative causes of caprine mastitis include Escherichia coli, Klebsiella pneumoniae, Pasteurella spp., Pseudomonas, and Proteus mirabilis. Corynebacterium pseudotuberculosis can cause mammary gland abscessation, whereas Mycoplasma mycoides may cause agalactia and systemic disease. "Hard udder" can be caused by caprine arthritis encephalitis virus (CAEV). Brucellosis and listeriosis can cause a subclinical interstitial mastitis ( Smith and Sherman, 1994 ). iii. Bovine mastitis Mastitis is the disease of greatest economic importance for the dairy cattle industry. The majority of the impact will be on the production and overall health of the cows, but low-incidence herds also diminish the risk of calves' ingesting or being exposed to pathogens. The most common bovine mastitis pathogens include Staphylococcus aureus and Streptococcus agalactiae, S. dysgalactiae, and S. uberis; coliform agents such as Escherichia coli, Enterobacter aerogenes, Serratia marcescens, and Klebsiella pneumoniae; mycoplasmal species such as Mycoplasma bovis, M. bovigenitalium, M. californicum, M. canadensis, and M. alkalescens; and Salmonella spp. such as S. typhimurium, S. newport, S. enteritidis, S. dublin, and S. muenster. Many of these agents such as Staphylococcus spp., Salmonella spp., and the coliforms can cause both acute and chronic mastitis, as well as severe systemic disease, including fever and anorexia. These must be regarded as herd and environmental pathogens in terms of treatment and prevention. The pathogenesis of staphylococcal infections is comparable to that in goats. Staphylococcus agalactiae can be cleared from udders because it does not invade other tissues, is an obligate resident of the glands, and is susceptible to penicillin. In contrast, S. uberis and S. dysgalactiae are environmental organisms and can be highly resistant to pencillin. Mycoplasma bovis is the more common of the mycoplasmal pathogens and can cause severe infections. Transmission of the mycoplasmas is not well defined but may be related to their presence in other organ systems. Treatments for mycoplasmal mastitis are not successful; culling is recommended. There are many interrelated factors associated with prevention and control of mastitis in a herd, including herd health and dry cow management, order of animals milked, milking procedures, milking equipment, condition of the teats, and the condition of the environment. Management of the overall herd includes aspects such as vaccination programs, nutrition, isolation of incoming animals, and quarantine and treatment of or culling diseased individuals. Culturing or testing newly freshened cows and monitoring the bulk milk tank serve as indicators of subclinical mastitis. Herd management will diminish teat lesions. Bacterin vaccines are available for preventing and controlling coliform mastitis and S. aureus mastitis. At the time of dry-off, all cows must be treated by intramammary route. Some infections can be successfully cleared during this time. Younger, disease-free animals should be milked first; any animals with diagnosed problems should be milked after the rest of the herd and/or segregated during treatment. Milkers' hands easily serve as a means of pathogen transmission, and wearing rubber gloves is recommended. Teat and udder cleaning practices include washing and drying with single-service paper or cloth towels or pre-and postmilking dipping. Milking equipment must be maintained to provide proper vacuum levels and pumping rates, and liners should be the appropriate size. Facilities that provide clean and dry areas for the animals to rest, feed, and move will diminish teat injuries and reduce exposures to mastitis pathogens. In that regard, inorganic bedding such as clean sand harbors few pathogens in contrast to shavings and sawdust. w. Moraxella bovis Infection (Infectious Bovine Keratoconjunctivitis, Pinkeye) Etiology. Moraxella bovis, a gram-negative coccobacillus, is the most common cause of infectious bovine keratoconjunctivitis (IBK) in cattle. This organism is not a cause of keratoconjunctivitis in sheep and goats. The disease includes conjunctivitis and ulcerative keratitis. The pathogenic M. bovis strain is piliated, and at least seven serotypes exist. Clinical signs. Lacrimation, photophobia, and blepharospasm are seen initially. Conjunctival injection and chemosis develop within a day of exposure, and then keratitis with corneal edema and ulcers. Anterior uveitis may be a sequela within a few days, and thicker mucopurulent ocular discharge may be seen. Corneal vascularization begins by 10 days after onset. Reepithelialization of the corneal ulcers occurs by 2–3 weeks after onset. Diagnosis is usually based on clinical signs, but culturing is helpful and fluorescein staining is useful for demonstrating corneal ulceration. Epizootiology and transmission. The disease is more severe in younger cattle. The clinical signs of IBK tend to be more severe in cattle that are also infected with infectious bovine rhinotracheitis (IBR) virus or those that have been vaccinated recently with modified live IBR vaccine. The bacteria are shed in nasal secretions and cattle with no clinical symptoms may be carriers. Transmission is by fomites, flies, aerosols, and direct contact. Incidence in winter months is very low. Nonhemolytic strains are associated with the winter epidemics, and hemolytic strains are associated with summer epidemics. Necropsy findings. Necropsy is not typically performed on these cases. Corneal edema, ulceration, hypopyon, and uveitis would be noted, depending on the stage of infection. Pathogenesis. The pili of M. bovis bind to receptors of corneal epithelium. The virulent strains of the bacteria then release the enzymes that damage the corneal epithelial cells. Other factors contributing to infection include ultraviolet light and trauma from dust and plant materials. Differential diagnoses. Infectious bovine rhinotrachetitis virus causes conjunctivitis, but the central corneal ulceration that is characteristic of IBK is not seen with M. bovis infections. Mycoplasma, Listeria, Branhamella (Neisseria), and adenovirus may be cultured from affected bovine eyes but none has been shown to produce the corneal lesions when inoculated into susceptible animals. Prevention and control. Cattle should not be immunized intranasally with modified live infectious bovine rhinotracheitis vaccine during IBK outbreaks; this will likely exacerbate the infection. New animals should be quarantined and treated prophylactically before introduction to herds. The available vaccines, containing. M. bovis pili or killed M. bovis, help decrease incidence and severity of disease; these preparations are not completely effective, because the M. bovis strain may not be homologous to that used for the vaccine preparation. Other preventive measures include 10% permethrin-impregnated bilateral ear tags, pour-on avermectins, or dust bags or face rubbers containing insecticide (such as 5% coumaphos) to control flies throughout the season and premises; mowing of high pasture grass to minimize ocular trauma; provision of shade; control of dust and sources of other mechanical trauma; and segregation of animals by age. Treatment. Cattle can recover without treatment, but younger animals should be treated as soon as the infection is detected. Antibiotic treatments include topical, subconjunctival administration and intramuscular dosing. Several standard topical antibiotics have been shown to be effective, including oxytetracycline, gentamicin, and triple antibiotic combinations. These should be administered twice per day. Subconjunctival injections of antibiotics, such as penicillin G, provide higher corneal levels of drug; these are typically administered only once or twice in severe cases. Intramuscular doses of long-acting oxytetracycline, given on alternate days, are effective in larger herds, and 2 doses 72 hr apart eliminate carriers. Third-eyelid flaps, temporary tarsorrhaphy, or eye patches may be useful in certain cases. Research complications. This pathogen does present a complication due to the carrier status of some animals, the likelihood of herd outbreaks, the severity of disease in younger animals, and the morbidity, possible progression to uveitis, and time and treatment costs associated with infections. The overall condition of the cattle will be affected for several weeks, and permanent visual impairment or loss, as well as ocular disfigurement, may occur. Etiology. Moraxella bovis, a gram-negative coccobacillus, is the most common cause of infectious bovine keratoconjunctivitis (IBK) in cattle. This organism is not a cause of keratoconjunctivitis in sheep and goats. The disease includes conjunctivitis and ulcerative keratitis. The pathogenic M. bovis strain is piliated, and at least seven serotypes exist. Clinical signs. Lacrimation, photophobia, and blepharospasm are seen initially. Conjunctival injection and chemosis develop within a day of exposure, and then keratitis with corneal edema and ulcers. Anterior uveitis may be a sequela within a few days, and thicker mucopurulent ocular discharge may be seen. Corneal vascularization begins by 10 days after onset. Reepithelialization of the corneal ulcers occurs by 2–3 weeks after onset. Diagnosis is usually based on clinical signs, but culturing is helpful and fluorescein staining is useful for demonstrating corneal ulceration. Epizootiology and transmission. The disease is more severe in younger cattle. The clinical signs of IBK tend to be more severe in cattle that are also infected with infectious bovine rhinotracheitis (IBR) virus or those that have been vaccinated recently with modified live IBR vaccine. The bacteria are shed in nasal secretions and cattle with no clinical symptoms may be carriers. Transmission is by fomites, flies, aerosols, and direct contact. Incidence in winter months is very low. Nonhemolytic strains are associated with the winter epidemics, and hemolytic strains are associated with summer epidemics. Necropsy findings. Necropsy is not typically performed on these cases. Corneal edema, ulceration, hypopyon, and uveitis would be noted, depending on the stage of infection. Pathogenesis. The pili of M. bovis bind to receptors of corneal epithelium. The virulent strains of the bacteria then release the enzymes that damage the corneal epithelial cells. Other factors contributing to infection include ultraviolet light and trauma from dust and plant materials. Differential diagnoses. Infectious bovine rhinotrachetitis virus causes conjunctivitis, but the central corneal ulceration that is characteristic of IBK is not seen with M. bovis infections. Mycoplasma, Listeria, Branhamella (Neisseria), and adenovirus may be cultured from affected bovine eyes but none has been shown to produce the corneal lesions when inoculated into susceptible animals. Prevention and control. Cattle should not be immunized intranasally with modified live infectious bovine rhinotracheitis vaccine during IBK outbreaks; this will likely exacerbate the infection. New animals should be quarantined and treated prophylactically before introduction to herds. The available vaccines, containing. M. bovis pili or killed M. bovis, help decrease incidence and severity of disease; these preparations are not completely effective, because the M. bovis strain may not be homologous to that used for the vaccine preparation. Other preventive measures include 10% permethrin-impregnated bilateral ear tags, pour-on avermectins, or dust bags or face rubbers containing insecticide (such as 5% coumaphos) to control flies throughout the season and premises; mowing of high pasture grass to minimize ocular trauma; provision of shade; control of dust and sources of other mechanical trauma; and segregation of animals by age. Treatment. Cattle can recover without treatment, but younger animals should be treated as soon as the infection is detected. Antibiotic treatments include topical, subconjunctival administration and intramuscular dosing. Several standard topical antibiotics have been shown to be effective, including oxytetracycline, gentamicin, and triple antibiotic combinations. These should be administered twice per day. Subconjunctival injections of antibiotics, such as penicillin G, provide higher corneal levels of drug; these are typically administered only once or twice in severe cases. Intramuscular doses of long-acting oxytetracycline, given on alternate days, are effective in larger herds, and 2 doses 72 hr apart eliminate carriers. Third-eyelid flaps, temporary tarsorrhaphy, or eye patches may be useful in certain cases. Research complications. This pathogen does present a complication due to the carrier status of some animals, the likelihood of herd outbreaks, the severity of disease in younger animals, and the morbidity, possible progression to uveitis, and time and treatment costs associated with infections. The overall condition of the cattle will be affected for several weeks, and permanent visual impairment or loss, as well as ocular disfigurement, may occur. Etiology. Moraxella bovis, a gram-negative coccobacillus, is the most common cause of infectious bovine keratoconjunctivitis (IBK) in cattle. This organism is not a cause of keratoconjunctivitis in sheep and goats. The disease includes conjunctivitis and ulcerative keratitis. The pathogenic M. bovis strain is piliated, and at least seven serotypes exist. Clinical signs. Lacrimation, photophobia, and blepharospasm are seen initially. Conjunctival injection and chemosis develop within a day of exposure, and then keratitis with corneal edema and ulcers. Anterior uveitis may be a sequela within a few days, and thicker mucopurulent ocular discharge may be seen. Corneal vascularization begins by 10 days after onset. Reepithelialization of the corneal ulcers occurs by 2–3 weeks after onset. Diagnosis is usually based on clinical signs, but culturing is helpful and fluorescein staining is useful for demonstrating corneal ulceration. Epizootiology and transmission. The disease is more severe in younger cattle. The clinical signs of IBK tend to be more severe in cattle that are also infected with infectious bovine rhinotracheitis (IBR) virus or those that have been vaccinated recently with modified live IBR vaccine. The bacteria are shed in nasal secretions and cattle with no clinical symptoms may be carriers. Transmission is by fomites, flies, aerosols, and direct contact. Incidence in winter months is very low. Nonhemolytic strains are associated with the winter epidemics, and hemolytic strains are associated with summer epidemics. Necropsy findings. Necropsy is not typically performed on these cases. Corneal edema, ulceration, hypopyon, and uveitis would be noted, depending on the stage of infection. Pathogenesis. The pili of M. bovis bind to receptors of corneal epithelium. The virulent strains of the bacteria then release the enzymes that damage the corneal epithelial cells. Other factors contributing to infection include ultraviolet light and trauma from dust and plant materials. Differential diagnoses. Infectious bovine rhinotrachetitis virus causes conjunctivitis, but the central corneal ulceration that is characteristic of IBK is not seen with M. bovis infections. Mycoplasma, Listeria, Branhamella (Neisseria), and adenovirus may be cultured from affected bovine eyes but none has been shown to produce the corneal lesions when inoculated into susceptible animals. Prevention and control. Cattle should not be immunized intranasally with modified live infectious bovine rhinotracheitis vaccine during IBK outbreaks; this will likely exacerbate the infection. New animals should be quarantined and treated prophylactically before introduction to herds. The available vaccines, containing. M. bovis pili or killed M. bovis, help decrease incidence and severity of disease; these preparations are not completely effective, because the M. bovis strain may not be homologous to that used for the vaccine preparation. Other preventive measures include 10% permethrin-impregnated bilateral ear tags, pour-on avermectins, or dust bags or face rubbers containing insecticide (such as 5% coumaphos) to control flies throughout the season and premises; mowing of high pasture grass to minimize ocular trauma; provision of shade; control of dust and sources of other mechanical trauma; and segregation of animals by age. Treatment. Cattle can recover without treatment, but younger animals should be treated as soon as the infection is detected. Antibiotic treatments include topical, subconjunctival administration and intramuscular dosing. Several standard topical antibiotics have been shown to be effective, including oxytetracycline, gentamicin, and triple antibiotic combinations. These should be administered twice per day. Subconjunctival injections of antibiotics, such as penicillin G, provide higher corneal levels of drug; these are typically administered only once or twice in severe cases. Intramuscular doses of long-acting oxytetracycline, given on alternate days, are effective in larger herds, and 2 doses 72 hr apart eliminate carriers. Third-eyelid flaps, temporary tarsorrhaphy, or eye patches may be useful in certain cases. Research complications. This pathogen does present a complication due to the carrier status of some animals, the likelihood of herd outbreaks, the severity of disease in younger animals, and the morbidity, possible progression to uveitis, and time and treatment costs associated with infections. The overall condition of the cattle will be affected for several weeks, and permanent visual impairment or loss, as well as ocular disfigurement, may occur. x. Mycobacterial Diseases Mycobacterium bovis Infection (Tuberculosis) Etiology. Mycobacteria are aerobic, nonmotile, non-spore-forming, acid-fast pleomorphic bacteria. Most cases of tuberculosis in sheep are related to Mycobacterium bovis or M. avium. Cases in goats have been attributed to M. bovis, M. avium, or M. tuberculosis. Mycobacterium bovis, or the bovine tubercle bacillus, is the cause in cattle but has been isolated from many domestic and wild mammals. Other agents of mammalian tuberculosis include M. microti and M. africanum. Clinical signs. Tuberculosis is a sporadic, chronic, contagious disease of ruminants and is zoonotic. The infection is often asymptomatic later in the illness, and it may be diagnosed only at necropsy. The respiratory system (M. bovis) or the digestive system (M. avium) is the primary site of infection; other tissues such as mammary tissue and reproductive tract may be infrequently involved. Locations of the characteristic tubercles will determine whether clinical signs are seen. Respiratory signs may include dyspnea, coughing, and pneumonia. Digestive tract signs include diarrhea, bloat, or constipation; diarrhea is most common. Lymphadenopathy occurs in advanced cases. Fever and generalized disease may be seen after calving. Infected goats lose weight and develop a persistent cough. Epizootiology and transmission. Although M. bovis can be killed by sunlight, it otherwise survives a long time in the environment and in cattle feces. Animals acquire the infection from the environment or from other animals via aerosols, from contaminated feed and water, and from secretions such as milk, semen, genital discharges, urine, and feces. Clinically normal animals may serve as carriers. The bacilli stimulate an initial neutrophilic tissue response. Neutrophils become necrotic and are phagocytosed by macrophages, forming giant epithelioid cells called Langhans' giant cells. An outer lymphocytic zone is formed, and fibrotic encapsulation creates the classical caseous nodules. Vascular erosion and hematogenous migration of the organisms may lead to lesions throughout the body. Necropsy findings. Yellow primary tubercles (granulomas) with central areas of caseous necrosis and calcification are present in the lungs. Caseous nodules are also associated with gastrointestinal organs and mesenteric lymph nodes. Prevention and control. Significant progress has been made in eradication programs in the United States during the past several decades, but during the 1990s, infected animals continued to be found in domestic cattle herds and particularly in captive deer herds in hunting preserves. The intradermal tuberculin test, using purified protein derivative (PPD), is usually used as a diagnostic indicator in live animals. This test should be performed annually on bovine and caprine dairy herds (and bison herds); the official tests are the caudal fold, comparative cervical, and single cervical tests. Notification to state officials is required following identification of intradermal-positive animals. Great care must be exercised in any handling of tissue or necropsies of reactors, and state animal health officials should be consulted regarding disposal of materials and cleaning of premises following depopulation of positive animals. Treatment. No treatment is recommended, and treatment is usually not allowed, because of the zoonotic potential, chronicity of the disease, and the treatment costs. Slaughter is preferred, to prevent potential transmission to humans. Research complications. The pathogen is zoonotic. Paratuberculosis, or Johne's disease (Mycobacterium paratuberculosis) Etiology. Mycobacterium paratuberculosis, the causative agent of Johne's disease, is a fastidious, non-spore-forming, acid-fast, gram-positive rod. The organism is actually a subspecies of M. avium, but M. paratuberculosis does not produce the siderophore mycobactin (an iron-binding molecule) of M. avium. Clinical signs and diagnosis. Johne's disease is a chronic, contagious, granulomatous disease of adult ruminants and is characterized by unthriftiness, weight loss, and intermittent diarrhea. In sheep and goats, chronic wasting is usually seen, occasionally with pasty feces or diarrhea. In cattle, chronic diarrhea and rapid weight loss are the most common clinical signs of the disease. Usually older adult animals are infected, but over time in an infected herd, younger animals will become infected when sufficient doses of organisms are ingested. Although clinical signs are nonspecific, Johne's disease should be considered if the affected diarrheic animals have a good appetite and are on a good anthelmintic program. The disease is diagnosed based on clinical signs and laboratory analyses, although none of the tests is more than 50% sensitive. In addition, the sensitivity of the serological tests differs between species. The standard is the fecal culture that takes 8–12 weeks. The enzyme-linked immunosorbent assay (ELISA) is now considered the most reliable serological test, but false negatives do occur. Other serological tests such as agar gel immunodiffusion (AGID) and complement fixation are useful. Herd screening may be done using the AGID or ELISA serological tests. Identification of the organism on culture, or the presence of acid-fast organisms on mucosal or mesenteric lymph node smears or from rectal biopsies, helps confirm the diagnosis. Some animals serologically negative for Johne's disease, however, have been found to be positive on fecal culture. Commercial AGID tests approved for use in cattle may be useful in diagnosing Johne's disease in sheep ( Dubash et al., 1996 ). Serological tests cross-react with other species of Mycobacterium, especially M. avium. Epizootiology and transmission. The organism is prevalent in the environment and is transmitted to young animals by direct or indirect contact. Although vertical transmission has been reported, the organism more commonly enters the gastrointestinal tract and penetrates the mucosa of the distal small intestine, primarily the ileum. Chronic carriers may intermittently shed the organisms. Pathogenesis. Mycobacterium paratuberculosis is an obligate parasite that grows only in macrophages of infected animals. Nursing infected dams are a primary source of infection of neonates. If the organism is not cleared, it proliferates slowly in the tissue, leading to inflammatory reactions that progress through neutrophilic to mononuclear stages. The organism may penetrate the lymphatics and proliferate in mesenteric lymph nodes. After an incubation period of a year or more, some of the carriers will progress to clinical disease manifested by fibrotic and hyperplastic changes in the ileum, leading to the classic thickening in the region. Gut changes result in intermittent diarrhea, with subsequent dehydration, electrolyte imbalances, and malnutrition, although this clinical sign is more common in cattle than in sheep or goats. Necropsy and diagnosis. The ileum from infected cattle is grossly thickened; this is not seen in sheep and goats. Ileal and ileocecal lymph nodes provide the best samples for histology and acid-fast staining. Differential diagnosis. Diseases causing chronic wasting and poor coat and body condition of all ruminants should be considered. These include chronic salmonellosis, peritonitis, severe parasitism, winter dysentery, and pyelonephritis. Deer can be infected, and the lesions can be confused with those of tuberculosis. Treatment. Treatment is not worthwhile. Prevention and control. Prevention is the most effective method to manage this pathogen. Efforts should be focused on eliminating the disease through test and slaughter. Neonates should not be reared by infected dams. Some states have Johne's disease eradication programs. Facilities and pastures where animals testing positive for Johne' disease were maintained should be thoroughly cleaned and kept vacant for a year after culling. Other considerations. Mycobacterium paratuberculosis is being investigated as a factor in the development of Crohn's disease in humans. Mycobacterium bovis Infection (Tuberculosis) Etiology. Mycobacteria are aerobic, nonmotile, non-spore-forming, acid-fast pleomorphic bacteria. Most cases of tuberculosis in sheep are related to Mycobacterium bovis or M. avium. Cases in goats have been attributed to M. bovis, M. avium, or M. tuberculosis. Mycobacterium bovis, or the bovine tubercle bacillus, is the cause in cattle but has been isolated from many domestic and wild mammals. Other agents of mammalian tuberculosis include M. microti and M. africanum. Clinical signs. Tuberculosis is a sporadic, chronic, contagious disease of ruminants and is zoonotic. The infection is often asymptomatic later in the illness, and it may be diagnosed only at necropsy. The respiratory system (M. bovis) or the digestive system (M. avium) is the primary site of infection; other tissues such as mammary tissue and reproductive tract may be infrequently involved. Locations of the characteristic tubercles will determine whether clinical signs are seen. Respiratory signs may include dyspnea, coughing, and pneumonia. Digestive tract signs include diarrhea, bloat, or constipation; diarrhea is most common. Lymphadenopathy occurs in advanced cases. Fever and generalized disease may be seen after calving. Infected goats lose weight and develop a persistent cough. Epizootiology and transmission. Although M. bovis can be killed by sunlight, it otherwise survives a long time in the environment and in cattle feces. Animals acquire the infection from the environment or from other animals via aerosols, from contaminated feed and water, and from secretions such as milk, semen, genital discharges, urine, and feces. Clinically normal animals may serve as carriers. The bacilli stimulate an initial neutrophilic tissue response. Neutrophils become necrotic and are phagocytosed by macrophages, forming giant epithelioid cells called Langhans' giant cells. An outer lymphocytic zone is formed, and fibrotic encapsulation creates the classical caseous nodules. Vascular erosion and hematogenous migration of the organisms may lead to lesions throughout the body. Necropsy findings. Yellow primary tubercles (granulomas) with central areas of caseous necrosis and calcification are present in the lungs. Caseous nodules are also associated with gastrointestinal organs and mesenteric lymph nodes. Prevention and control. Significant progress has been made in eradication programs in the United States during the past several decades, but during the 1990s, infected animals continued to be found in domestic cattle herds and particularly in captive deer herds in hunting preserves. The intradermal tuberculin test, using purified protein derivative (PPD), is usually used as a diagnostic indicator in live animals. This test should be performed annually on bovine and caprine dairy herds (and bison herds); the official tests are the caudal fold, comparative cervical, and single cervical tests. Notification to state officials is required following identification of intradermal-positive animals. Great care must be exercised in any handling of tissue or necropsies of reactors, and state animal health officials should be consulted regarding disposal of materials and cleaning of premises following depopulation of positive animals. Treatment. No treatment is recommended, and treatment is usually not allowed, because of the zoonotic potential, chronicity of the disease, and the treatment costs. Slaughter is preferred, to prevent potential transmission to humans. Research complications. The pathogen is zoonotic. Paratuberculosis, or Johne's disease (Mycobacterium paratuberculosis) Etiology. Mycobacterium paratuberculosis, the causative agent of Johne's disease, is a fastidious, non-spore-forming, acid-fast, gram-positive rod. The organism is actually a subspecies of M. avium, but M. paratuberculosis does not produce the siderophore mycobactin (an iron-binding molecule) of M. avium. Clinical signs and diagnosis. Johne's disease is a chronic, contagious, granulomatous disease of adult ruminants and is characterized by unthriftiness, weight loss, and intermittent diarrhea. In sheep and goats, chronic wasting is usually seen, occasionally with pasty feces or diarrhea. In cattle, chronic diarrhea and rapid weight loss are the most common clinical signs of the disease. Usually older adult animals are infected, but over time in an infected herd, younger animals will become infected when sufficient doses of organisms are ingested. Although clinical signs are nonspecific, Johne's disease should be considered if the affected diarrheic animals have a good appetite and are on a good anthelmintic program. The disease is diagnosed based on clinical signs and laboratory analyses, although none of the tests is more than 50% sensitive. In addition, the sensitivity of the serological tests differs between species. The standard is the fecal culture that takes 8–12 weeks. The enzyme-linked immunosorbent assay (ELISA) is now considered the most reliable serological test, but false negatives do occur. Other serological tests such as agar gel immunodiffusion (AGID) and complement fixation are useful. Herd screening may be done using the AGID or ELISA serological tests. Identification of the organism on culture, or the presence of acid-fast organisms on mucosal or mesenteric lymph node smears or from rectal biopsies, helps confirm the diagnosis. Some animals serologically negative for Johne's disease, however, have been found to be positive on fecal culture. Commercial AGID tests approved for use in cattle may be useful in diagnosing Johne's disease in sheep ( Dubash et al., 1996 ). Serological tests cross-react with other species of Mycobacterium, especially M. avium. Epizootiology and transmission. The organism is prevalent in the environment and is transmitted to young animals by direct or indirect contact. Although vertical transmission has been reported, the organism more commonly enters the gastrointestinal tract and penetrates the mucosa of the distal small intestine, primarily the ileum. Chronic carriers may intermittently shed the organisms. Pathogenesis. Mycobacterium paratuberculosis is an obligate parasite that grows only in macrophages of infected animals. Nursing infected dams are a primary source of infection of neonates. If the organism is not cleared, it proliferates slowly in the tissue, leading to inflammatory reactions that progress through neutrophilic to mononuclear stages. The organism may penetrate the lymphatics and proliferate in mesenteric lymph nodes. After an incubation period of a year or more, some of the carriers will progress to clinical disease manifested by fibrotic and hyperplastic changes in the ileum, leading to the classic thickening in the region. Gut changes result in intermittent diarrhea, with subsequent dehydration, electrolyte imbalances, and malnutrition, although this clinical sign is more common in cattle than in sheep or goats. Necropsy and diagnosis. The ileum from infected cattle is grossly thickened; this is not seen in sheep and goats. Ileal and ileocecal lymph nodes provide the best samples for histology and acid-fast staining. Differential diagnosis. Diseases causing chronic wasting and poor coat and body condition of all ruminants should be considered. These include chronic salmonellosis, peritonitis, severe parasitism, winter dysentery, and pyelonephritis. Deer can be infected, and the lesions can be confused with those of tuberculosis. Treatment. Treatment is not worthwhile. Prevention and control. Prevention is the most effective method to manage this pathogen. Efforts should be focused on eliminating the disease through test and slaughter. Neonates should not be reared by infected dams. Some states have Johne's disease eradication programs. Facilities and pastures where animals testing positive for Johne' disease were maintained should be thoroughly cleaned and kept vacant for a year after culling. Other considerations. Mycobacterium paratuberculosis is being investigated as a factor in the development of Crohn's disease in humans. Etiology. Mycobacteria are aerobic, nonmotile, non-spore-forming, acid-fast pleomorphic bacteria. Most cases of tuberculosis in sheep are related to Mycobacterium bovis or M. avium. Cases in goats have been attributed to M. bovis, M. avium, or M. tuberculosis. Mycobacterium bovis, or the bovine tubercle bacillus, is the cause in cattle but has been isolated from many domestic and wild mammals. Other agents of mammalian tuberculosis include M. microti and M. africanum. Clinical signs. Tuberculosis is a sporadic, chronic, contagious disease of ruminants and is zoonotic. The infection is often asymptomatic later in the illness, and it may be diagnosed only at necropsy. The respiratory system (M. bovis) or the digestive system (M. avium) is the primary site of infection; other tissues such as mammary tissue and reproductive tract may be infrequently involved. Locations of the characteristic tubercles will determine whether clinical signs are seen. Respiratory signs may include dyspnea, coughing, and pneumonia. Digestive tract signs include diarrhea, bloat, or constipation; diarrhea is most common. Lymphadenopathy occurs in advanced cases. Fever and generalized disease may be seen after calving. Infected goats lose weight and develop a persistent cough. Epizootiology and transmission. Although M. bovis can be killed by sunlight, it otherwise survives a long time in the environment and in cattle feces. Animals acquire the infection from the environment or from other animals via aerosols, from contaminated feed and water, and from secretions such as milk, semen, genital discharges, urine, and feces. Clinically normal animals may serve as carriers. The bacilli stimulate an initial neutrophilic tissue response. Neutrophils become necrotic and are phagocytosed by macrophages, forming giant epithelioid cells called Langhans' giant cells. An outer lymphocytic zone is formed, and fibrotic encapsulation creates the classical caseous nodules. Vascular erosion and hematogenous migration of the organisms may lead to lesions throughout the body. Necropsy findings. Yellow primary tubercles (granulomas) with central areas of caseous necrosis and calcification are present in the lungs. Caseous nodules are also associated with gastrointestinal organs and mesenteric lymph nodes. Prevention and control. Significant progress has been made in eradication programs in the United States during the past several decades, but during the 1990s, infected animals continued to be found in domestic cattle herds and particularly in captive deer herds in hunting preserves. The intradermal tuberculin test, using purified protein derivative (PPD), is usually used as a diagnostic indicator in live animals. This test should be performed annually on bovine and caprine dairy herds (and bison herds); the official tests are the caudal fold, comparative cervical, and single cervical tests. Notification to state officials is required following identification of intradermal-positive animals. Great care must be exercised in any handling of tissue or necropsies of reactors, and state animal health officials should be consulted regarding disposal of materials and cleaning of premises following depopulation of positive animals. Treatment. No treatment is recommended, and treatment is usually not allowed, because of the zoonotic potential, chronicity of the disease, and the treatment costs. Slaughter is preferred, to prevent potential transmission to humans. Research complications. The pathogen is zoonotic. Paratuberculosis, or Johne's disease (Mycobacterium paratuberculosis) Etiology. Mycobacterium paratuberculosis, the causative agent of Johne's disease, is a fastidious, non-spore-forming, acid-fast, gram-positive rod. The organism is actually a subspecies of M. avium, but M. paratuberculosis does not produce the siderophore mycobactin (an iron-binding molecule) of M. avium. Clinical signs and diagnosis. Johne's disease is a chronic, contagious, granulomatous disease of adult ruminants and is characterized by unthriftiness, weight loss, and intermittent diarrhea. In sheep and goats, chronic wasting is usually seen, occasionally with pasty feces or diarrhea. In cattle, chronic diarrhea and rapid weight loss are the most common clinical signs of the disease. Usually older adult animals are infected, but over time in an infected herd, younger animals will become infected when sufficient doses of organisms are ingested. Although clinical signs are nonspecific, Johne's disease should be considered if the affected diarrheic animals have a good appetite and are on a good anthelmintic program. The disease is diagnosed based on clinical signs and laboratory analyses, although none of the tests is more than 50% sensitive. In addition, the sensitivity of the serological tests differs between species. The standard is the fecal culture that takes 8–12 weeks. The enzyme-linked immunosorbent assay (ELISA) is now considered the most reliable serological test, but false negatives do occur. Other serological tests such as agar gel immunodiffusion (AGID) and complement fixation are useful. Herd screening may be done using the AGID or ELISA serological tests. Identification of the organism on culture, or the presence of acid-fast organisms on mucosal or mesenteric lymph node smears or from rectal biopsies, helps confirm the diagnosis. Some animals serologically negative for Johne's disease, however, have been found to be positive on fecal culture. Commercial AGID tests approved for use in cattle may be useful in diagnosing Johne's disease in sheep ( Dubash et al., 1996 ). Serological tests cross-react with other species of Mycobacterium, especially M. avium. Epizootiology and transmission. The organism is prevalent in the environment and is transmitted to young animals by direct or indirect contact. Although vertical transmission has been reported, the organism more commonly enters the gastrointestinal tract and penetrates the mucosa of the distal small intestine, primarily the ileum. Chronic carriers may intermittently shed the organisms. Pathogenesis. Mycobacterium paratuberculosis is an obligate parasite that grows only in macrophages of infected animals. Nursing infected dams are a primary source of infection of neonates. If the organism is not cleared, it proliferates slowly in the tissue, leading to inflammatory reactions that progress through neutrophilic to mononuclear stages. The organism may penetrate the lymphatics and proliferate in mesenteric lymph nodes. After an incubation period of a year or more, some of the carriers will progress to clinical disease manifested by fibrotic and hyperplastic changes in the ileum, leading to the classic thickening in the region. Gut changes result in intermittent diarrhea, with subsequent dehydration, electrolyte imbalances, and malnutrition, although this clinical sign is more common in cattle than in sheep or goats. Necropsy and diagnosis. The ileum from infected cattle is grossly thickened; this is not seen in sheep and goats. Ileal and ileocecal lymph nodes provide the best samples for histology and acid-fast staining. Differential diagnosis. Diseases causing chronic wasting and poor coat and body condition of all ruminants should be considered. These include chronic salmonellosis, peritonitis, severe parasitism, winter dysentery, and pyelonephritis. Deer can be infected, and the lesions can be confused with those of tuberculosis. Treatment. Treatment is not worthwhile. Prevention and control. Prevention is the most effective method to manage this pathogen. Efforts should be focused on eliminating the disease through test and slaughter. Neonates should not be reared by infected dams. Some states have Johne's disease eradication programs. Facilities and pastures where animals testing positive for Johne' disease were maintained should be thoroughly cleaned and kept vacant for a year after culling. Other considerations. Mycobacterium paratuberculosis is being investigated as a factor in the development of Crohn's disease in humans. y. Navel Ill (Omphalitis, Omphalophlebitis, Omphaloarteritis, Joint Ill) Etiology. The most common organism causing infection of the umbilicus is Arcanobacterium (formerly Actinomyces, Corynebacterium) pyogenes; other bacteria may be present. Arcanobacterium spp. are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Other environmental contaminants are also associated with this disease, such as Escherichia coli, Enterococcus spp., Proteus, Streptococcus spp., and Staplylococcus spp. Clinical signs and diagnosis. Navel ill is an acute localized inflammation and infection of the external umbilicus. Animals present with fever and painful enlargement of the umbilicus. Animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, and hematuria. Other common severe sequelae include septicemia, pneumonia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, uveitis, endocarditis, and diarrhea. Epizootiology and transmission. Many cases occur in neonates, and most cases occur within the first 3 months of age. Cleanliness of the birthing and housing environment and successful transfer of passive immunity are important factors in the occurrence of the disease. Dystocia resulting in weak neonates can be a factor predisposing to the development of the disease. Navel ill is diagnosed by typical clinical signs. The presence of microabscesses and palpation of the umbilical area for firm intra-abdominal structures extending from the umbilicus are abnormal. Assessment of colostral immunoglobulin transfer may contribute to determination of the prognosis. Navel ill should always be considered for young ruminants with fever of unknown origin during the first week of life and for slightly older lambs, kids, or calves that are not thriving. Arthrocentesis of affected joints and culture of the fluid for identification of the pathogen are also diagnostic options and essential for effective antimicrobial selection. Differential diagnosis. The major differential is an umbilical hernia, which will typically not be painful or infected and can often be reduced. Mycoplasmal arthritis is a differential in kids. In the past, Erysipelothrix rhusopathiae was a common navel ill pathogen in sheep. Treatment. Omphalitis can be treated with a 10 to 14 day course of broad-spectrum antibiotics such as ampicillin, amoxicillin, penicillin, ceftiofur, florfenicol, and erythromycin. If an isolated abscess is palpable, it should be surgically opened and repeatedly flushed with iodine solutions. Surgical reduction of the infected umbilicus is indicated if intra-abdominal structures are involved. The prognosis for recovery is good if systemic involvement has not occurred. Prevention and control. The disease is best prevented and controlled by providing clean birthing environments, ensuring adequate colostral immunity, thoroughly dipping the umbilicus of newborns in tincture of iodine or strong iodine solution (Lugol's), monitoring for dystocias, and maintaining young growing animals in noncontaminated environments. Research complications. The disease can be costly to treat, and the toll taken on young animals due to the consequences of systemic infection may detract from their research value. Etiology. The most common organism causing infection of the umbilicus is Arcanobacterium (formerly Actinomyces, Corynebacterium) pyogenes; other bacteria may be present. Arcanobacterium spp. are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Other environmental contaminants are also associated with this disease, such as Escherichia coli, Enterococcus spp., Proteus, Streptococcus spp., and Staplylococcus spp. Clinical signs and diagnosis. Navel ill is an acute localized inflammation and infection of the external umbilicus. Animals present with fever and painful enlargement of the umbilicus. Animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, and hematuria. Other common severe sequelae include septicemia, pneumonia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, uveitis, endocarditis, and diarrhea. Epizootiology and transmission. Many cases occur in neonates, and most cases occur within the first 3 months of age. Cleanliness of the birthing and housing environment and successful transfer of passive immunity are important factors in the occurrence of the disease. Dystocia resulting in weak neonates can be a factor predisposing to the development of the disease. Navel ill is diagnosed by typical clinical signs. The presence of microabscesses and palpation of the umbilical area for firm intra-abdominal structures extending from the umbilicus are abnormal. Assessment of colostral immunoglobulin transfer may contribute to determination of the prognosis. Navel ill should always be considered for young ruminants with fever of unknown origin during the first week of life and for slightly older lambs, kids, or calves that are not thriving. Arthrocentesis of affected joints and culture of the fluid for identification of the pathogen are also diagnostic options and essential for effective antimicrobial selection. Differential diagnosis. The major differential is an umbilical hernia, which will typically not be painful or infected and can often be reduced. Mycoplasmal arthritis is a differential in kids. In the past, Erysipelothrix rhusopathiae was a common navel ill pathogen in sheep. Treatment. Omphalitis can be treated with a 10 to 14 day course of broad-spectrum antibiotics such as ampicillin, amoxicillin, penicillin, ceftiofur, florfenicol, and erythromycin. If an isolated abscess is palpable, it should be surgically opened and repeatedly flushed with iodine solutions. Surgical reduction of the infected umbilicus is indicated if intra-abdominal structures are involved. The prognosis for recovery is good if systemic involvement has not occurred. Prevention and control. The disease is best prevented and controlled by providing clean birthing environments, ensuring adequate colostral immunity, thoroughly dipping the umbilicus of newborns in tincture of iodine or strong iodine solution (Lugol's), monitoring for dystocias, and maintaining young growing animals in noncontaminated environments. Research complications. The disease can be costly to treat, and the toll taken on young animals due to the consequences of systemic infection may detract from their research value. Etiology. The most common organism causing infection of the umbilicus is Arcanobacterium (formerly Actinomyces, Corynebacterium) pyogenes; other bacteria may be present. Arcanobacterium spp. are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Other environmental contaminants are also associated with this disease, such as Escherichia coli, Enterococcus spp., Proteus, Streptococcus spp., and Staplylococcus spp. Clinical signs and diagnosis. Navel ill is an acute localized inflammation and infection of the external umbilicus. Animals present with fever and painful enlargement of the umbilicus. Animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, and hematuria. Other common severe sequelae include septicemia, pneumonia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, uveitis, endocarditis, and diarrhea. Epizootiology and transmission. Many cases occur in neonates, and most cases occur within the first 3 months of age. Cleanliness of the birthing and housing environment and successful transfer of passive immunity are important factors in the occurrence of the disease. Dystocia resulting in weak neonates can be a factor predisposing to the development of the disease. Navel ill is diagnosed by typical clinical signs. The presence of microabscesses and palpation of the umbilical area for firm intra-abdominal structures extending from the umbilicus are abnormal. Assessment of colostral immunoglobulin transfer may contribute to determination of the prognosis. Navel ill should always be considered for young ruminants with fever of unknown origin during the first week of life and for slightly older lambs, kids, or calves that are not thriving. Arthrocentesis of affected joints and culture of the fluid for identification of the pathogen are also diagnostic options and essential for effective antimicrobial selection. Differential diagnosis. The major differential is an umbilical hernia, which will typically not be painful or infected and can often be reduced. Mycoplasmal arthritis is a differential in kids. In the past, Erysipelothrix rhusopathiae was a common navel ill pathogen in sheep. Treatment. Omphalitis can be treated with a 10 to 14 day course of broad-spectrum antibiotics such as ampicillin, amoxicillin, penicillin, ceftiofur, florfenicol, and erythromycin. If an isolated abscess is palpable, it should be surgically opened and repeatedly flushed with iodine solutions. Surgical reduction of the infected umbilicus is indicated if intra-abdominal structures are involved. The prognosis for recovery is good if systemic involvement has not occurred. Prevention and control. The disease is best prevented and controlled by providing clean birthing environments, ensuring adequate colostral immunity, thoroughly dipping the umbilicus of newborns in tincture of iodine or strong iodine solution (Lugol's), monitoring for dystocias, and maintaining young growing animals in noncontaminated environments. Research complications. The disease can be costly to treat, and the toll taken on young animals due to the consequences of systemic infection may detract from their research value. z. Pasteurellosis (Shipping Fever, Hemorrhagic Septicemia, Enzootic Pneumonia) Etiology. Pasteurella hemolytica and P. multocida are aerobic, nonmotile, non-spore-forming, bipolar, gram-negative rods. Biotype A serotypes are associated with pneumonia and septicemia in all ruminants ( Ellis, 1984 ). Serotype 1 of P. hemolytica is considered a major cause of pulmonary lesions of bovine bronchopneumonia and fibrinous bronchopneumonia. Clinical signs. Pasteurellosis is an acute bacterial disease characterized by bronchopneumonia, septicemia, and sudden death. The organism invades the mucosa of the gastrointestinal tract or respiratory tract and causes localized areas of necrosis, hemorrhage, and thrombosis. The lungs and liver are frequent areas of formation of microabscesses. Acute rhinitis or pharyngitis often precedes the respiratory form. The organism also may invade the bloodstream, causing disseminated septicemia. Clinically, the lambs may exhibit nasal discharge of mucopurulent to hemorrhagic exudate, hyperthermia, coughing, dyspnea, anorexia, and depression. With the respiratory form, auscultation of the thorax suggests dullness and consolidation of anteroventral lobes; this will be confirmed by radiographs. The disease is diagnosed by clinical signs, blood cultures from septicemic animals, blood smears showing bipolar organisms, and history of predisposing stressors. In cultures, P. hemolytica is distinguished from P. multocida by hemolysis on blood agar; only P. multocida produces indole. Epizootiology and transmission. The organism is ubiquitous in the environment and in the respiratory tracts of these animals. Younger ruminants, between 2 and 12 months of age, are especially prone to infection during times of stress, such as weaning, transportation, dietary changes, weather changes, and overcrowding. The pneumonic form appears as a complex associated with concurrent infections such as parainfluenza 3, adenovirus type 6, respiratory syncytial virus, mycoplasmas, chlamydia, Pasteurella multocida and Bordetella parapertussis ( Martin, 1996 ; Brogden et al., 1998 ). The organism is transmitted between animals by direct and indirect contact, through inhalation or ingestion. Necropsy findings. Necropsy lesions include areas of necrosis and hemorrhage in the small intestines and multifocal 1 mm lesions distributed on the surfaces of the lungs and liver. With the pneumonic form, serofibrinous exudates fill the alveoli; ventral lung lobes are consolidated and are congested and purple-gray in color. Fibrinous pleuritis, pericarditis, and hematogenously induced arthritis also may be evident. Pathogenesis. A leukotoxin is considered to be a key factor in the pathogenesis of the P. hemolytica infection. Macrophages and neutrophils are lysed by the toxin as they arrive at the lung, and the enzymes released by the neutrophils cause additional damage to the tissue. Treatment. Treatment may include the use of antibiotics such as penicillin, ampicillin, tylosin, sulfonamides, or oxytetracycline. Newer antibiotics, such as ceftiofur, tilmicosin, spectinomycin, and florfenicol, are very effective and approved for use in cattle. In outbreaks, cultures from fresh necropsies are helpful for determining sensitivities useful for the remaining group. Prevention and control. The incidence of disease can be decreased by minimizing the degree of stress; by improving management, such as nutrition and control of parasitism; and, in cattle and sheep, by vaccinating for viral respiratory infections such as parainfluenza. Early Pasteurella hemolytica bacterin vaccines for use in cattle are not considered effective, but newer products based on immunizing against the leukotoxin and some bacterial capsule surface antigens are effective. Pasteurella multocida bacterins and live streptomycin-dependent mutant vaccines are available. In young animals, passive immunity is protective. Preventive measures also include maintaining good ventilation in enclosures and barns. New animals to the flock or herds should be quarantined for at least 2 weeks before introduction. Etiology. Pasteurella hemolytica and P. multocida are aerobic, nonmotile, non-spore-forming, bipolar, gram-negative rods. Biotype A serotypes are associated with pneumonia and septicemia in all ruminants ( Ellis, 1984 ). Serotype 1 of P. hemolytica is considered a major cause of pulmonary lesions of bovine bronchopneumonia and fibrinous bronchopneumonia. Clinical signs. Pasteurellosis is an acute bacterial disease characterized by bronchopneumonia, septicemia, and sudden death. The organism invades the mucosa of the gastrointestinal tract or respiratory tract and causes localized areas of necrosis, hemorrhage, and thrombosis. The lungs and liver are frequent areas of formation of microabscesses. Acute rhinitis or pharyngitis often precedes the respiratory form. The organism also may invade the bloodstream, causing disseminated septicemia. Clinically, the lambs may exhibit nasal discharge of mucopurulent to hemorrhagic exudate, hyperthermia, coughing, dyspnea, anorexia, and depression. With the respiratory form, auscultation of the thorax suggests dullness and consolidation of anteroventral lobes; this will be confirmed by radiographs. The disease is diagnosed by clinical signs, blood cultures from septicemic animals, blood smears showing bipolar organisms, and history of predisposing stressors. In cultures, P. hemolytica is distinguished from P. multocida by hemolysis on blood agar; only P. multocida produces indole. Epizootiology and transmission. The organism is ubiquitous in the environment and in the respiratory tracts of these animals. Younger ruminants, between 2 and 12 months of age, are especially prone to infection during times of stress, such as weaning, transportation, dietary changes, weather changes, and overcrowding. The pneumonic form appears as a complex associated with concurrent infections such as parainfluenza 3, adenovirus type 6, respiratory syncytial virus, mycoplasmas, chlamydia, Pasteurella multocida and Bordetella parapertussis ( Martin, 1996 ; Brogden et al., 1998 ). The organism is transmitted between animals by direct and indirect contact, through inhalation or ingestion. Necropsy findings. Necropsy lesions include areas of necrosis and hemorrhage in the small intestines and multifocal 1 mm lesions distributed on the surfaces of the lungs and liver. With the pneumonic form, serofibrinous exudates fill the alveoli; ventral lung lobes are consolidated and are congested and purple-gray in color. Fibrinous pleuritis, pericarditis, and hematogenously induced arthritis also may be evident. Pathogenesis. A leukotoxin is considered to be a key factor in the pathogenesis of the P. hemolytica infection. Macrophages and neutrophils are lysed by the toxin as they arrive at the lung, and the enzymes released by the neutrophils cause additional damage to the tissue. Treatment. Treatment may include the use of antibiotics such as penicillin, ampicillin, tylosin, sulfonamides, or oxytetracycline. Newer antibiotics, such as ceftiofur, tilmicosin, spectinomycin, and florfenicol, are very effective and approved for use in cattle. In outbreaks, cultures from fresh necropsies are helpful for determining sensitivities useful for the remaining group. Prevention and control. The incidence of disease can be decreased by minimizing the degree of stress; by improving management, such as nutrition and control of parasitism; and, in cattle and sheep, by vaccinating for viral respiratory infections such as parainfluenza. Early Pasteurella hemolytica bacterin vaccines for use in cattle are not considered effective, but newer products based on immunizing against the leukotoxin and some bacterial capsule surface antigens are effective. Pasteurella multocida bacterins and live streptomycin-dependent mutant vaccines are available. In young animals, passive immunity is protective. Preventive measures also include maintaining good ventilation in enclosures and barns. New animals to the flock or herds should be quarantined for at least 2 weeks before introduction. Etiology. Pasteurella hemolytica and P. multocida are aerobic, nonmotile, non-spore-forming, bipolar, gram-negative rods. Biotype A serotypes are associated with pneumonia and septicemia in all ruminants ( Ellis, 1984 ). Serotype 1 of P. hemolytica is considered a major cause of pulmonary lesions of bovine bronchopneumonia and fibrinous bronchopneumonia. Clinical signs. Pasteurellosis is an acute bacterial disease characterized by bronchopneumonia, septicemia, and sudden death. The organism invades the mucosa of the gastrointestinal tract or respiratory tract and causes localized areas of necrosis, hemorrhage, and thrombosis. The lungs and liver are frequent areas of formation of microabscesses. Acute rhinitis or pharyngitis often precedes the respiratory form. The organism also may invade the bloodstream, causing disseminated septicemia. Clinically, the lambs may exhibit nasal discharge of mucopurulent to hemorrhagic exudate, hyperthermia, coughing, dyspnea, anorexia, and depression. With the respiratory form, auscultation of the thorax suggests dullness and consolidation of anteroventral lobes; this will be confirmed by radiographs. The disease is diagnosed by clinical signs, blood cultures from septicemic animals, blood smears showing bipolar organisms, and history of predisposing stressors. In cultures, P. hemolytica is distinguished from P. multocida by hemolysis on blood agar; only P. multocida produces indole. Epizootiology and transmission. The organism is ubiquitous in the environment and in the respiratory tracts of these animals. Younger ruminants, between 2 and 12 months of age, are especially prone to infection during times of stress, such as weaning, transportation, dietary changes, weather changes, and overcrowding. The pneumonic form appears as a complex associated with concurrent infections such as parainfluenza 3, adenovirus type 6, respiratory syncytial virus, mycoplasmas, chlamydia, Pasteurella multocida and Bordetella parapertussis ( Martin, 1996 ; Brogden et al., 1998 ). The organism is transmitted between animals by direct and indirect contact, through inhalation or ingestion. Necropsy findings. Necropsy lesions include areas of necrosis and hemorrhage in the small intestines and multifocal 1 mm lesions distributed on the surfaces of the lungs and liver. With the pneumonic form, serofibrinous exudates fill the alveoli; ventral lung lobes are consolidated and are congested and purple-gray in color. Fibrinous pleuritis, pericarditis, and hematogenously induced arthritis also may be evident. Pathogenesis. A leukotoxin is considered to be a key factor in the pathogenesis of the P. hemolytica infection. Macrophages and neutrophils are lysed by the toxin as they arrive at the lung, and the enzymes released by the neutrophils cause additional damage to the tissue. Treatment. Treatment may include the use of antibiotics such as penicillin, ampicillin, tylosin, sulfonamides, or oxytetracycline. Newer antibiotics, such as ceftiofur, tilmicosin, spectinomycin, and florfenicol, are very effective and approved for use in cattle. In outbreaks, cultures from fresh necropsies are helpful for determining sensitivities useful for the remaining group. Prevention and control. The incidence of disease can be decreased by minimizing the degree of stress; by improving management, such as nutrition and control of parasitism; and, in cattle and sheep, by vaccinating for viral respiratory infections such as parainfluenza. Early Pasteurella hemolytica bacterin vaccines for use in cattle are not considered effective, but newer products based on immunizing against the leukotoxin and some bacterial capsule surface antigens are effective. Pasteurella multocida bacterins and live streptomycin-dependent mutant vaccines are available. In young animals, passive immunity is protective. Preventive measures also include maintaining good ventilation in enclosures and barns. New animals to the flock or herds should be quarantined for at least 2 weeks before introduction. aa. Salmonellosis Etiology. Salmonella typhimurium is a motile, aerobic to facultatively anaerobic, non-spore-forming, gram-negative bacillus and is the organism associated with enteric disease and some abortions in ruminants. It is a common inhabitant of the gastrointestinal tract of ruminants. Current nomenclature categorizes S. typhimurium as a serovar within the species S. enteritidis (the other two species are S. typhi and S. choleraesuis). Salmonella typhimurium, S. dublin, and S. newport are the common species seen in bovine cases. Salmonella typhimurium, S. dublin, S. anatum, and S. montevideo are seen in ovine and caprine cases, although a host-adapted species has not been identified in the goat. Ovine abortions due to various Salmonella species are not reported in the United States but are enzootic in other countries. Salmonella serotypes have been associated with aborted fetuses in all ruminant species. Clinical signs and diagnosis. Salmonellosis causes acute gastroenteritis, dysentery, and septicemia ( Anderson and Blanchard, 1989 ). Clinically, the animals become anorexic and hyperthermic. Diarrhea or dysentery develops; feces may contain mucus and/or blood and have a putrid odor. Animals become severely depressed and weak, losing a high percentage of their body weight. Animals may die in 1–5 days because of dehydration associated with dysenteric fluid loss, septicemia, shock, and acidosis. Morbidity may be 25%, and mortality may be high. Septicemia may result in subsequent meningitis, polyarthritis, and pneumonia. Chronically infected animals may have intermittent diarrhea. In goats, salmonellosis may be recognized as diarrhea and septicemia in neonates, as enteritis in preweaned kids and mature goats, and, rarely, as abortion. Adult cases may be sporadic, with intermittent bouts of diarrhea, subacute or even chronic. Morbidity and mortality will be highest in neonates, and some may simply be found dead. The older animals generally tend to fare better during the disease. Abdominal distension with profuse yellow feces is common. Kids become severely depressed, anorexic, febrile (with temperatures as high as 106°–107°F), dehydrated, acidotic, recumbent, and comatose. Salmonella abortions may occur throughout gestation. There may not be any other clinical signs, or abortion may be seen with diarrhea, fever, and vulvar discharges. Hemorrhage, placental necrosis, and edema will be present. Metritis and placental retention may occur. Some mortality of dams may occur. Diagnosis is based on clinical signs and can be confirmed by culturing fresh feces or at necropsy. Because of intermittent shedding of organisms, culture may be difficult; repeated cultures are recommended. Leukopenia and a degenerative shift to the left are not uncommon hematological findings. Epizootiology and transmission. Stresses associated with recent shipping, overcrowding, and inclement weather may predispose the animal to enteric infection. Birds and rodents may be natural reservoirs of Salmonella in external housing environments. Transmission is fecal-oral. After ingestion, the organisms may proliferate throughout the gastrointestinal tract and may penetrate the mucosa of the intestines, invade the Peyer's patches and lymphatics, and migrate to the spleen, liver, and other organs. Animals that survive may become chronic carriers and shedders of the organisms, and this has been demonstrated experimentally ( Arora, 1983 ). Fecal-oral transmission is also associated with Salmonella abortion; veneral transmission has not been reported. Necropsy findings and diagnosis. Animals will have noticeable perineal staining. Intestines (particularly the ileum, cecum, and colon) may contain mucoid feces with or without hemorrhages. Petechial hemorrhages and areas of necrosis may be noticed on the surface of the liver, heart, and mesenteric lymph nodes. The wall of the intestines, gallbladder, and mesenteric lymph nodes will be edematous, and a pseudodiphtheritic membrane lining the distal small intestines and colon may be observed. This membrane is not normally seen in the goat ( Smith and Sherman, 1994 ). Splenomegaly may be present. Aborted fetuses will often be autolysed. Placentitis, placental necrosis, and hemorrhage are commonly seen. Serologic evidence of recent infection can be demonstrated in the dam. Salmonella can be isolated from the aborted tissues. Pathogenesis. After ingestion, the organism proliferates in the intestine. Damage to the intestines and the resulting diarrhea are due to the bacterial production of cytoxin and endotoxin. Although the Salmonella organisms will be taken up by phagocytic cells involved in the inflammatory response, they survive and multiply further. Septicemia is a common sequela, with the bacteria localizing throughout the body. In latently infected animals, it is often shed from the gallbladder and mesenteric lymph nodes. Younger animals may be susceptible because of immature immunity and intestinal flora and higher intestinal pH. Carriers may develop clinical disease when stressed. Differential diagnoses. In young animals, differentials include other enteropathogens: Escherichia coli, rotavirus and coronavirus, clostridia, cryptosporidia, and other coccidial forms. These pathogens may also be present in the affected animals. Differentials in adults include bovine viral diarrheas and winter dysentery in cattle and parasitemia and enterotoxemia in all ruminants. Prevention and control. Affected animals should be isolated during herd outbreaks. Samples for culture should include herd-mates, water and feed sources, recently arrived livestock (other species), and area wildlife, including birds and rodents. Repeated cultures, culling of animals, intensive cleaning, and disinfection of facilities are all important during outbreaks. The bacteria survive for about a week in moist cow manure. Vaccination using the commercially available killed bacterin or autologous bacterins may be useful in outbreaks involving pregnant cattle, although the J-5 bacterin is now considered better. Treatment. Nursing care includes rehydration and correction of acid-base abnormalities. Antibiotic therapy may be useful in cases with septicemia, but it is controversial because it may induce carrier animals. Gentamicin, trimethoprim-sulfadiazine, ampicillin, enrofloxacin, and amikacin antibiotics may be successful. Research complications. Salmonellosis is zoonotic, and some serotypes of the organism have caused fatalities even in immunocompetent humans. Attempts should be made to identify and cull carrier animals. Etiology. Salmonella typhimurium is a motile, aerobic to facultatively anaerobic, non-spore-forming, gram-negative bacillus and is the organism associated with enteric disease and some abortions in ruminants. It is a common inhabitant of the gastrointestinal tract of ruminants. Current nomenclature categorizes S. typhimurium as a serovar within the species S. enteritidis (the other two species are S. typhi and S. choleraesuis). Salmonella typhimurium, S. dublin, and S. newport are the common species seen in bovine cases. Salmonella typhimurium, S. dublin, S. anatum, and S. montevideo are seen in ovine and caprine cases, although a host-adapted species has not been identified in the goat. Ovine abortions due to various Salmonella species are not reported in the United States but are enzootic in other countries. Salmonella serotypes have been associated with aborted fetuses in all ruminant species. Clinical signs and diagnosis. Salmonellosis causes acute gastroenteritis, dysentery, and septicemia ( Anderson and Blanchard, 1989 ). Clinically, the animals become anorexic and hyperthermic. Diarrhea or dysentery develops; feces may contain mucus and/or blood and have a putrid odor. Animals become severely depressed and weak, losing a high percentage of their body weight. Animals may die in 1–5 days because of dehydration associated with dysenteric fluid loss, septicemia, shock, and acidosis. Morbidity may be 25%, and mortality may be high. Septicemia may result in subsequent meningitis, polyarthritis, and pneumonia. Chronically infected animals may have intermittent diarrhea. In goats, salmonellosis may be recognized as diarrhea and septicemia in neonates, as enteritis in preweaned kids and mature goats, and, rarely, as abortion. Adult cases may be sporadic, with intermittent bouts of diarrhea, subacute or even chronic. Morbidity and mortality will be highest in neonates, and some may simply be found dead. The older animals generally tend to fare better during the disease. Abdominal distension with profuse yellow feces is common. Kids become severely depressed, anorexic, febrile (with temperatures as high as 106°–107°F), dehydrated, acidotic, recumbent, and comatose. Salmonella abortions may occur throughout gestation. There may not be any other clinical signs, or abortion may be seen with diarrhea, fever, and vulvar discharges. Hemorrhage, placental necrosis, and edema will be present. Metritis and placental retention may occur. Some mortality of dams may occur. Diagnosis is based on clinical signs and can be confirmed by culturing fresh feces or at necropsy. Because of intermittent shedding of organisms, culture may be difficult; repeated cultures are recommended. Leukopenia and a degenerative shift to the left are not uncommon hematological findings. Epizootiology and transmission. Stresses associated with recent shipping, overcrowding, and inclement weather may predispose the animal to enteric infection. Birds and rodents may be natural reservoirs of Salmonella in external housing environments. Transmission is fecal-oral. After ingestion, the organisms may proliferate throughout the gastrointestinal tract and may penetrate the mucosa of the intestines, invade the Peyer's patches and lymphatics, and migrate to the spleen, liver, and other organs. Animals that survive may become chronic carriers and shedders of the organisms, and this has been demonstrated experimentally ( Arora, 1983 ). Fecal-oral transmission is also associated with Salmonella abortion; veneral transmission has not been reported. Necropsy findings and diagnosis. Animals will have noticeable perineal staining. Intestines (particularly the ileum, cecum, and colon) may contain mucoid feces with or without hemorrhages. Petechial hemorrhages and areas of necrosis may be noticed on the surface of the liver, heart, and mesenteric lymph nodes. The wall of the intestines, gallbladder, and mesenteric lymph nodes will be edematous, and a pseudodiphtheritic membrane lining the distal small intestines and colon may be observed. This membrane is not normally seen in the goat ( Smith and Sherman, 1994 ). Splenomegaly may be present. Aborted fetuses will often be autolysed. Placentitis, placental necrosis, and hemorrhage are commonly seen. Serologic evidence of recent infection can be demonstrated in the dam. Salmonella can be isolated from the aborted tissues. Pathogenesis. After ingestion, the organism proliferates in the intestine. Damage to the intestines and the resulting diarrhea are due to the bacterial production of cytoxin and endotoxin. Although the Salmonella organisms will be taken up by phagocytic cells involved in the inflammatory response, they survive and multiply further. Septicemia is a common sequela, with the bacteria localizing throughout the body. In latently infected animals, it is often shed from the gallbladder and mesenteric lymph nodes. Younger animals may be susceptible because of immature immunity and intestinal flora and higher intestinal pH. Carriers may develop clinical disease when stressed. Differential diagnoses. In young animals, differentials include other enteropathogens: Escherichia coli, rotavirus and coronavirus, clostridia, cryptosporidia, and other coccidial forms. These pathogens may also be present in the affected animals. Differentials in adults include bovine viral diarrheas and winter dysentery in cattle and parasitemia and enterotoxemia in all ruminants. Prevention and control. Affected animals should be isolated during herd outbreaks. Samples for culture should include herd-mates, water and feed sources, recently arrived livestock (other species), and area wildlife, including birds and rodents. Repeated cultures, culling of animals, intensive cleaning, and disinfection of facilities are all important during outbreaks. The bacteria survive for about a week in moist cow manure. Vaccination using the commercially available killed bacterin or autologous bacterins may be useful in outbreaks involving pregnant cattle, although the J-5 bacterin is now considered better. Treatment. Nursing care includes rehydration and correction of acid-base abnormalities. Antibiotic therapy may be useful in cases with septicemia, but it is controversial because it may induce carrier animals. Gentamicin, trimethoprim-sulfadiazine, ampicillin, enrofloxacin, and amikacin antibiotics may be successful. Research complications. Salmonellosis is zoonotic, and some serotypes of the organism have caused fatalities even in immunocompetent humans. Attempts should be made to identify and cull carrier animals. Etiology. Salmonella typhimurium is a motile, aerobic to facultatively anaerobic, non-spore-forming, gram-negative bacillus and is the organism associated with enteric disease and some abortions in ruminants. It is a common inhabitant of the gastrointestinal tract of ruminants. Current nomenclature categorizes S. typhimurium as a serovar within the species S. enteritidis (the other two species are S. typhi and S. choleraesuis). Salmonella typhimurium, S. dublin, and S. newport are the common species seen in bovine cases. Salmonella typhimurium, S. dublin, S. anatum, and S. montevideo are seen in ovine and caprine cases, although a host-adapted species has not been identified in the goat. Ovine abortions due to various Salmonella species are not reported in the United States but are enzootic in other countries. Salmonella serotypes have been associated with aborted fetuses in all ruminant species. Clinical signs and diagnosis. Salmonellosis causes acute gastroenteritis, dysentery, and septicemia ( Anderson and Blanchard, 1989 ). Clinically, the animals become anorexic and hyperthermic. Diarrhea or dysentery develops; feces may contain mucus and/or blood and have a putrid odor. Animals become severely depressed and weak, losing a high percentage of their body weight. Animals may die in 1–5 days because of dehydration associated with dysenteric fluid loss, septicemia, shock, and acidosis. Morbidity may be 25%, and mortality may be high. Septicemia may result in subsequent meningitis, polyarthritis, and pneumonia. Chronically infected animals may have intermittent diarrhea. In goats, salmonellosis may be recognized as diarrhea and septicemia in neonates, as enteritis in preweaned kids and mature goats, and, rarely, as abortion. Adult cases may be sporadic, with intermittent bouts of diarrhea, subacute or even chronic. Morbidity and mortality will be highest in neonates, and some may simply be found dead. The older animals generally tend to fare better during the disease. Abdominal distension with profuse yellow feces is common. Kids become severely depressed, anorexic, febrile (with temperatures as high as 106°–107°F), dehydrated, acidotic, recumbent, and comatose. Salmonella abortions may occur throughout gestation. There may not be any other clinical signs, or abortion may be seen with diarrhea, fever, and vulvar discharges. Hemorrhage, placental necrosis, and edema will be present. Metritis and placental retention may occur. Some mortality of dams may occur. Diagnosis is based on clinical signs and can be confirmed by culturing fresh feces or at necropsy. Because of intermittent shedding of organisms, culture may be difficult; repeated cultures are recommended. Leukopenia and a degenerative shift to the left are not uncommon hematological findings. Epizootiology and transmission. Stresses associated with recent shipping, overcrowding, and inclement weather may predispose the animal to enteric infection. Birds and rodents may be natural reservoirs of Salmonella in external housing environments. Transmission is fecal-oral. After ingestion, the organisms may proliferate throughout the gastrointestinal tract and may penetrate the mucosa of the intestines, invade the Peyer's patches and lymphatics, and migrate to the spleen, liver, and other organs. Animals that survive may become chronic carriers and shedders of the organisms, and this has been demonstrated experimentally ( Arora, 1983 ). Fecal-oral transmission is also associated with Salmonella abortion; veneral transmission has not been reported. Necropsy findings and diagnosis. Animals will have noticeable perineal staining. Intestines (particularly the ileum, cecum, and colon) may contain mucoid feces with or without hemorrhages. Petechial hemorrhages and areas of necrosis may be noticed on the surface of the liver, heart, and mesenteric lymph nodes. The wall of the intestines, gallbladder, and mesenteric lymph nodes will be edematous, and a pseudodiphtheritic membrane lining the distal small intestines and colon may be observed. This membrane is not normally seen in the goat ( Smith and Sherman, 1994 ). Splenomegaly may be present. Aborted fetuses will often be autolysed. Placentitis, placental necrosis, and hemorrhage are commonly seen. Serologic evidence of recent infection can be demonstrated in the dam. Salmonella can be isolated from the aborted tissues. Pathogenesis. After ingestion, the organism proliferates in the intestine. Damage to the intestines and the resulting diarrhea are due to the bacterial production of cytoxin and endotoxin. Although the Salmonella organisms will be taken up by phagocytic cells involved in the inflammatory response, they survive and multiply further. Septicemia is a common sequela, with the bacteria localizing throughout the body. In latently infected animals, it is often shed from the gallbladder and mesenteric lymph nodes. Younger animals may be susceptible because of immature immunity and intestinal flora and higher intestinal pH. Carriers may develop clinical disease when stressed. Differential diagnoses. In young animals, differentials include other enteropathogens: Escherichia coli, rotavirus and coronavirus, clostridia, cryptosporidia, and other coccidial forms. These pathogens may also be present in the affected animals. Differentials in adults include bovine viral diarrheas and winter dysentery in cattle and parasitemia and enterotoxemia in all ruminants. Prevention and control. Affected animals should be isolated during herd outbreaks. Samples for culture should include herd-mates, water and feed sources, recently arrived livestock (other species), and area wildlife, including birds and rodents. Repeated cultures, culling of animals, intensive cleaning, and disinfection of facilities are all important during outbreaks. The bacteria survive for about a week in moist cow manure. Vaccination using the commercially available killed bacterin or autologous bacterins may be useful in outbreaks involving pregnant cattle, although the J-5 bacterin is now considered better. Treatment. Nursing care includes rehydration and correction of acid-base abnormalities. Antibiotic therapy may be useful in cases with septicemia, but it is controversial because it may induce carrier animals. Gentamicin, trimethoprim-sulfadiazine, ampicillin, enrofloxacin, and amikacin antibiotics may be successful. Research complications. Salmonellosis is zoonotic, and some serotypes of the organism have caused fatalities even in immunocompetent humans. Attempts should be made to identify and cull carrier animals. bb. Spirochete-Associated Abortion in Cattle (Epizootic Foothill Abortion) Etiology. Spirochete-like organisms are associated with this disease; it is now recognized that the agent is not a chlamydial organism. The disease has been reported only in the foothills bordering the central valley of California. Clinical signs. Cows that become infected with the causative agent before 6 months of gestation abort or give birth to weak calves without any clinical sign of infection. Cows infected after 6 months of gestation give birth to normal calves. Affected cows rarely abort in subsequent pregnancies. Epizootiology and transmission. The tick vector is Ornithodorus coriaceus. Necropsy. Fetuses show several pathological changes, including enlargement of the cervical lymph nodes, spleen, and liver. The calf's thymus will be small, and histologically there will be losses of thymic cortical lymphocytes. Histologic changes in lymph nodes and spleen include vasculitis, necrosis, and histiocytosis. Treatment. Chlortetracycline treatment has been effective in controlling this disease. Etiology. Spirochete-like organisms are associated with this disease; it is now recognized that the agent is not a chlamydial organism. The disease has been reported only in the foothills bordering the central valley of California. Clinical signs. Cows that become infected with the causative agent before 6 months of gestation abort or give birth to weak calves without any clinical sign of infection. Cows infected after 6 months of gestation give birth to normal calves. Affected cows rarely abort in subsequent pregnancies. Epizootiology and transmission. The tick vector is Ornithodorus coriaceus. Necropsy. Fetuses show several pathological changes, including enlargement of the cervical lymph nodes, spleen, and liver. The calf's thymus will be small, and histologically there will be losses of thymic cortical lymphocytes. Histologic changes in lymph nodes and spleen include vasculitis, necrosis, and histiocytosis. Treatment. Chlortetracycline treatment has been effective in controlling this disease. Etiology. Spirochete-like organisms are associated with this disease; it is now recognized that the agent is not a chlamydial organism. The disease has been reported only in the foothills bordering the central valley of California. Clinical signs. Cows that become infected with the causative agent before 6 months of gestation abort or give birth to weak calves without any clinical sign of infection. Cows infected after 6 months of gestation give birth to normal calves. Affected cows rarely abort in subsequent pregnancies. Epizootiology and transmission. The tick vector is Ornithodorus coriaceus. Necropsy. Fetuses show several pathological changes, including enlargement of the cervical lymph nodes, spleen, and liver. The calf's thymus will be small, and histologically there will be losses of thymic cortical lymphocytes. Histologic changes in lymph nodes and spleen include vasculitis, necrosis, and histiocytosis. Treatment. Chlortetracycline treatment has been effective in controlling this disease. cc. Tularemia Etiology. Tularemia is caused by Pasteurella (Francisella) tularensis a nonmotile, non-spore-forming, aerobic, gram-negative, rod-shaped bacterium. Type A is more virulent than type B. Clinical Signs. Although tularemia is a disease of livestock, pets, and wild animals, sheep are most commonly affected. The disease is characterized by hyperthermia, muscular stiffness, and lymphadenopathy. Infected animals move stiffly, are depressed, and are hyperthermic. Anemia and diarrhea may develop, and infected lymph nodes enlarge and may ulcerate. Mortality may reach 40%. Animals that recover will have immunity of long duration. Epizootiology and transmission. The disease is most commonly transmitted by ticks or biting flies. The wood tick, Dermacentor andersoni, is an important vector in transmitting the disease in the western United States, and, as natural hosts, wild rodents and rabbits tend to be reservoirs of the pathogen. Pathogenesis. The organisms, entering the tick bite wound, move via lymphatics to lymph nodes and subsequently to the bloodstream, where they cause septicemia. The organisms can also be transmitted orally through contaminated water. Necropsy findings. Ticks may also be present on the carcasses. Suppurative, necrotic lymph nodes are typical. Lungs will be congested and edematous. Diagnosis is confirmed by prompt culturing of the organism from lymph nodes, spleen, or liver where granulomatous lesions form; P. tularensis does not survive for long periods in carcasses. Serological findings may also be helpful. Treatment. Infected animals can be treated with oxytetracycline, aminoglycosides, or cephalosporins. Differential diagnosis. When tick infestations are heavy, P. tularensis should be suspected. Pasteurella haemolytica (sheep), Haemophilus somnus (cattle), and Mycoplasma mycoides (goats), and anthrax (all ruminant species) should be considered as differentials. Control and prevention. Eliminating the tick vectors can prevent tularemia. Animals should be provided with fresh water frequently. The organism can survive in freezing conditions and in water and mud for long periods of time. Caretakers, veterinarians, and researchers should take special precautions before handling the tissues of infected sheep, because this is a method of zoonotic spread. Research complications. The disease is zoonotic, and transmission to people may result from tick bites or from handling contaminated tissues. Although not a major disease of concern in sheep, researchers using potentially infected animals from western range states of the United States should be aware of it. The organism is antigenically related to Brucella spp. Etiology. Tularemia is caused by Pasteurella (Francisella) tularensis a nonmotile, non-spore-forming, aerobic, gram-negative, rod-shaped bacterium. Type A is more virulent than type B. Clinical Signs. Although tularemia is a disease of livestock, pets, and wild animals, sheep are most commonly affected. The disease is characterized by hyperthermia, muscular stiffness, and lymphadenopathy. Infected animals move stiffly, are depressed, and are hyperthermic. Anemia and diarrhea may develop, and infected lymph nodes enlarge and may ulcerate. Mortality may reach 40%. Animals that recover will have immunity of long duration. Epizootiology and transmission. The disease is most commonly transmitted by ticks or biting flies. The wood tick, Dermacentor andersoni, is an important vector in transmitting the disease in the western United States, and, as natural hosts, wild rodents and rabbits tend to be reservoirs of the pathogen. Pathogenesis. The organisms, entering the tick bite wound, move via lymphatics to lymph nodes and subsequently to the bloodstream, where they cause septicemia. The organisms can also be transmitted orally through contaminated water. Necropsy findings. Ticks may also be present on the carcasses. Suppurative, necrotic lymph nodes are typical. Lungs will be congested and edematous. Diagnosis is confirmed by prompt culturing of the organism from lymph nodes, spleen, or liver where granulomatous lesions form; P. tularensis does not survive for long periods in carcasses. Serological findings may also be helpful. Treatment. Infected animals can be treated with oxytetracycline, aminoglycosides, or cephalosporins. Differential diagnosis. When tick infestations are heavy, P. tularensis should be suspected. Pasteurella haemolytica (sheep), Haemophilus somnus (cattle), and Mycoplasma mycoides (goats), and anthrax (all ruminant species) should be considered as differentials. Control and prevention. Eliminating the tick vectors can prevent tularemia. Animals should be provided with fresh water frequently. The organism can survive in freezing conditions and in water and mud for long periods of time. Caretakers, veterinarians, and researchers should take special precautions before handling the tissues of infected sheep, because this is a method of zoonotic spread. Research complications. The disease is zoonotic, and transmission to people may result from tick bites or from handling contaminated tissues. Although not a major disease of concern in sheep, researchers using potentially infected animals from western range states of the United States should be aware of it. The organism is antigenically related to Brucella spp. Etiology. Tularemia is caused by Pasteurella (Francisella) tularensis a nonmotile, non-spore-forming, aerobic, gram-negative, rod-shaped bacterium. Type A is more virulent than type B. Clinical Signs. Although tularemia is a disease of livestock, pets, and wild animals, sheep are most commonly affected. The disease is characterized by hyperthermia, muscular stiffness, and lymphadenopathy. Infected animals move stiffly, are depressed, and are hyperthermic. Anemia and diarrhea may develop, and infected lymph nodes enlarge and may ulcerate. Mortality may reach 40%. Animals that recover will have immunity of long duration. Epizootiology and transmission. The disease is most commonly transmitted by ticks or biting flies. The wood tick, Dermacentor andersoni, is an important vector in transmitting the disease in the western United States, and, as natural hosts, wild rodents and rabbits tend to be reservoirs of the pathogen. Pathogenesis. The organisms, entering the tick bite wound, move via lymphatics to lymph nodes and subsequently to the bloodstream, where they cause septicemia. The organisms can also be transmitted orally through contaminated water. Necropsy findings. Ticks may also be present on the carcasses. Suppurative, necrotic lymph nodes are typical. Lungs will be congested and edematous. Diagnosis is confirmed by prompt culturing of the organism from lymph nodes, spleen, or liver where granulomatous lesions form; P. tularensis does not survive for long periods in carcasses. Serological findings may also be helpful. Treatment. Infected animals can be treated with oxytetracycline, aminoglycosides, or cephalosporins. Differential diagnosis. When tick infestations are heavy, P. tularensis should be suspected. Pasteurella haemolytica (sheep), Haemophilus somnus (cattle), and Mycoplasma mycoides (goats), and anthrax (all ruminant species) should be considered as differentials. Control and prevention. Eliminating the tick vectors can prevent tularemia. Animals should be provided with fresh water frequently. The organism can survive in freezing conditions and in water and mud for long periods of time. Caretakers, veterinarians, and researchers should take special precautions before handling the tissues of infected sheep, because this is a method of zoonotic spread. Research complications. The disease is zoonotic, and transmission to people may result from tick bites or from handling contaminated tissues. Although not a major disease of concern in sheep, researchers using potentially infected animals from western range states of the United States should be aware of it. The organism is antigenically related to Brucella spp. dd. Yersinia Etiology. Yersiniosis is caused by infections with Yersinia enterocolitica, a gram-negative, aerobic, and facultative anaerobe of the family Enterobacteriaceae. There are 50 serotypes reported for Y. enterocolitica. Yersinia pseudotuberculosis infections have also been seen in ruminants. Enteric infections predominate in the diseases caused by these bacteria. Clinical signs and diagnosis. Clinical disease may be seen rarely in many groups of ruminants. Goats of 1–6 months old suffer from the enteric form of the disease, which is characterized by sudden death or the acute onset of watery diarrhea lasting 1 or more days. Spontaneous abortions and weak neonates are also clinical manifestations of infection. Lactating does may have mastitis that becomes chronically hemorrhagic. Bacteremia results in internal abscesses, abortion, and acute deaths. Yersinia pseudotuberculosis has been associated with laboratory goat epizootics ( Obwolo, 1976 ). Diarrhea in pastured sheep, stressed by other factors, has also been reported. Diagnosis is based on culture and serology. Epizootiology and transmission. The bacteria are carried by wild birds and rodents, and transmission is by ingestion of contaminated feed and water. Necropsy findings. Edema of mesenteric lymph nodes is the most common postmortem finding. Liver abscesses, micro-absecesses in the intestines, and granuloma formation have also been reported. Placentas are white, with opaque white foci found on cotyledons. Histologically, suppurative placentitis and suppurative pneumonia are found in the fetal tissue. Pathogenesis. After ingestion, the bacteria cause an enteric infection, and bacteremia follows. Differential diagnoses. Other causes of abortions, including abortion storms, acute deaths, enteritis, neonatal deaths, and white foci on cotyledons, should be considered. In young animals, differentials include coccidiosis and nematode parasitism. Corynebacterium pseudotuberculosis and tuberculosis are differentials for the internal abscesses. Prevention and control. Control measure are not well defined, because the epidemiology of the disease is poorly understood ( Smith and Sherman, 1994 ). Tissues from affected goats must be handled and disposed of properly. Areas housing affected goats must be thoroughly sanitized. Treatment. In case of an abortion storm, treatment of goats with tetracycline has been useful. Other broad-spectrum antibiotics may also be useful. Research complications. Yersinia is zoonotic. The risk of severe enteric disease is considered particularly great for immunocompromised persons. Etiology. Yersiniosis is caused by infections with Yersinia enterocolitica, a gram-negative, aerobic, and facultative anaerobe of the family Enterobacteriaceae. There are 50 serotypes reported for Y. enterocolitica. Yersinia pseudotuberculosis infections have also been seen in ruminants. Enteric infections predominate in the diseases caused by these bacteria. Clinical signs and diagnosis. Clinical disease may be seen rarely in many groups of ruminants. Goats of 1–6 months old suffer from the enteric form of the disease, which is characterized by sudden death or the acute onset of watery diarrhea lasting 1 or more days. Spontaneous abortions and weak neonates are also clinical manifestations of infection. Lactating does may have mastitis that becomes chronically hemorrhagic. Bacteremia results in internal abscesses, abortion, and acute deaths. Yersinia pseudotuberculosis has been associated with laboratory goat epizootics ( Obwolo, 1976 ). Diarrhea in pastured sheep, stressed by other factors, has also been reported. Diagnosis is based on culture and serology. Epizootiology and transmission. The bacteria are carried by wild birds and rodents, and transmission is by ingestion of contaminated feed and water. Necropsy findings. Edema of mesenteric lymph nodes is the most common postmortem finding. Liver abscesses, micro-absecesses in the intestines, and granuloma formation have also been reported. Placentas are white, with opaque white foci found on cotyledons. Histologically, suppurative placentitis and suppurative pneumonia are found in the fetal tissue. Pathogenesis. After ingestion, the bacteria cause an enteric infection, and bacteremia follows. Differential diagnoses. Other causes of abortions, including abortion storms, acute deaths, enteritis, neonatal deaths, and white foci on cotyledons, should be considered. In young animals, differentials include coccidiosis and nematode parasitism. Corynebacterium pseudotuberculosis and tuberculosis are differentials for the internal abscesses. Prevention and control. Control measure are not well defined, because the epidemiology of the disease is poorly understood ( Smith and Sherman, 1994 ). Tissues from affected goats must be handled and disposed of properly. Areas housing affected goats must be thoroughly sanitized. Treatment. In case of an abortion storm, treatment of goats with tetracycline has been useful. Other broad-spectrum antibiotics may also be useful. Research complications. Yersinia is zoonotic. The risk of severe enteric disease is considered particularly great for immunocompromised persons. Etiology. Yersiniosis is caused by infections with Yersinia enterocolitica, a gram-negative, aerobic, and facultative anaerobe of the family Enterobacteriaceae. There are 50 serotypes reported for Y. enterocolitica. Yersinia pseudotuberculosis infections have also been seen in ruminants. Enteric infections predominate in the diseases caused by these bacteria. Clinical signs and diagnosis. Clinical disease may be seen rarely in many groups of ruminants. Goats of 1–6 months old suffer from the enteric form of the disease, which is characterized by sudden death or the acute onset of watery diarrhea lasting 1 or more days. Spontaneous abortions and weak neonates are also clinical manifestations of infection. Lactating does may have mastitis that becomes chronically hemorrhagic. Bacteremia results in internal abscesses, abortion, and acute deaths. Yersinia pseudotuberculosis has been associated with laboratory goat epizootics ( Obwolo, 1976 ). Diarrhea in pastured sheep, stressed by other factors, has also been reported. Diagnosis is based on culture and serology. Epizootiology and transmission. The bacteria are carried by wild birds and rodents, and transmission is by ingestion of contaminated feed and water. Necropsy findings. Edema of mesenteric lymph nodes is the most common postmortem finding. Liver abscesses, micro-absecesses in the intestines, and granuloma formation have also been reported. Placentas are white, with opaque white foci found on cotyledons. Histologically, suppurative placentitis and suppurative pneumonia are found in the fetal tissue. Pathogenesis. After ingestion, the bacteria cause an enteric infection, and bacteremia follows. Differential diagnoses. Other causes of abortions, including abortion storms, acute deaths, enteritis, neonatal deaths, and white foci on cotyledons, should be considered. In young animals, differentials include coccidiosis and nematode parasitism. Corynebacterium pseudotuberculosis and tuberculosis are differentials for the internal abscesses. Prevention and control. Control measure are not well defined, because the epidemiology of the disease is poorly understood ( Smith and Sherman, 1994 ). Tissues from affected goats must be handled and disposed of properly. Areas housing affected goats must be thoroughly sanitized. Treatment. In case of an abortion storm, treatment of goats with tetracycline has been useful. Other broad-spectrum antibiotics may also be useful. Research complications. Yersinia is zoonotic. The risk of severe enteric disease is considered particularly great for immunocompromised persons. ee. Mycoplasmal Diseases i. Mycoplasma bovigenitalium and M. bovis infections Etiology. Mycoplasma bovigenitalium and M. bovis are associated sporadically with bovine infertility and abortions. This pathogen has also been reported associated with similar clinical signs in sheep and goats. Clinical signs and diagnosis. Infertility is more commonly caused by M. bovigenitalium infections, and granular vulvovaginitis and endometritis will be present. Granular vulvovaginitis is characterized by raised papules on the mucous membranes and mucopurulent exudate. Abortions and mastitis are associated with M. bovis infections. Calves that are born may be weak. It is rare to have a definitive diagnosis of an abortion due to Mycoplasma. After consideration of other causes of abortion and evaluation of tissues for placentitis or fetal inflammation, diagnosis is confirmed by isolation of Mycoplasma from the genital tract or aborted tissues. Epidemiology and transmission. Mycoplasmal species are considered ubiquitous, are carried in the genital tracts of males and females, and are transmitted during natural breeding or through contaminated insemination materials. Aerosols also serve as a means of transmission. In addition, transmission occurs by passage through the birth canal, by direct contact, and by contamination from urine of infected animals. Pathophysiology. Experimental infections of M. bovis have resulted in placentitis and fetal pneumonia. Differential diagnoses. Acholeplasma, Ureaplasma, and Haemophilus somnus are differentials for granular vulvovaginitis. Treatment. Fluoroquinolone antibiotics may be useful for treating Mycoplasma-induced reproductive diseases. ii. Mycoplasma ovipneumoniae (ovine mycoplasmal pneumonia) Etiology. Mycoplasma ovipneumoniae causes acute or chronic pneumonia in lambs. Clinical signs. Mycoplasmas induce serious diseases in sheep, causing pneumonia, conjunctivitis, and genitourinary disease. The disease may be coincidental with pasteurellosis. Respiratory distress, coughing, and nasal discharge are observed in infected animals. Bronchoalveolar lavage followed by culture is the best method for diagnosis (mycoplasmas are fastidious organisms requiring special handling techniques). Mycoplasmas are isolated from the genitourinary tract of sheep. Vulvovaginitis and reproductive problems are associated conditions. Treatment. Tylosin, quinolones, oxytetracycline, and gentamicin are good choices for therapy. Prevention. No vaccine is available. iii. Mycoplasma mycoides biotype F38 (contagious caprine pleuropneumonia, caprine pneumonia, pleuritis, and pleuropneumonia) Etiology. Mycoplasma mycoides biotype F38 is the agent of contagious caprine pleuropneumonia and is found worldwide. In the United States, caprine pneumonia is also caused by M. ovipneumoniae, M. mycoides subsp. capri, and M. mycoides subsp. mycoides (large colony type). Clinical signs. Contagious caprine pleuropneumonia is characterized by severe dyspnea, nasal discharge, cough, and fever ( McMartin et al., 1980 ). Infections with other Mycoplasma species also have similar clinical signs. Septicemia without respiratory involvement may also be a presentation. Epizootiology and transmission. This disease is highly contagious, with high morbidity and mortality. Transmission is by aerosols. Mycoplasma mycoides subsp. mycoides has become a serious cause of morbidity and mortality of goat kids in the United States. Necropsy. Large amounts of pale straw-colored fluid and fibrinous pneumonia and pleurisy are typical. Some lung consolidation may be present. Meningitis, fibrinous pericarditis, and fibrinopurulent arthritis may also be found. Diagnosis is usually made at necropsy by culture of the organism from lungs and other internal organs. Differential dagnosis. In the United States, the principal differential for M. mycoides subsp. mycoides is caprine arthritis encephalitis. Treatment. Tylosin and oxytetracycline are effective. Some infections are slow to resolve. Prevention and control. Vaccines are available in some areas. Infected herds are quarantined. New goats should be quarantined before introduction to the herd. Research complications. The worldwide distribution of the F38 biotype, as well as the aerosol transmission and high morbidity and mortality characteristics of mycoplasmal infectious, make these infections economically important diseases. Considerable attention is presently given to this genus as a source of morbidity and mortality in goats. iv. Mycoplasma conjunctivae (mycoplasmal keratoconjunctivitis) Etiology. Mycoplasma conjunctivae causes infectious conjunctivitis, or pinkeye, in sheep and goats with associated hyperemia, edema, lacrimation, and corneal lesions. Mycoplasma mycoides subsp. mycoides, M. agalactiae, M. arginini, and Acholeplasma oculusi have also been associated with keratoconjunctivitis in these species. Respiratory disease and other infections, such as mastitis, may also be observed. Clinical signs and diagnosis. All ages of animals may be affected. Initially, lacrimation, conjunctival vessel injection, and then keratitis and neovascularization are seen. Sometimes uveitis is evident. Although the presentation is usually unilateral, bilateral involvement is possible. Recurring infections are common. Culturing provides the better diagnostic information, and cultures will be positive even after clinical signs have diminished. Epizootiology and transmission. The infection is passed easily between animals by direct contact. Animals can become reinfected, and carrier animals may be a factor in outbreaks. Necropsy. It is unlikely that animals would die or be euthanized and undergo necropsy for this problem. Conjunctival scrapings would include neutrophils during earlier stages and lymphocytes during later stages. Epithelial cell cytoplasm should be examined for organisms. Differential diagnosis. The primary differential in sheep and goats is Chlamydia, as well as Branhamella, Rickettsia (Colesiota) conjunctivae, and infectious bovine rhinotracheitis in goats only. It is important to consider these differentials if arthritis, pneumonia, or mastitis is present in the group or the individual. Treatment. Animals do recover spontaneously within about 10 weeks. Tetracycline ointments and powders are also used. Third-eyelid flaps may be necessary if corneal ulceration develops. Prevention and control. New animals should be quarantined and, if necessary treated, before introduction to the flock or herd. i. Mycoplasma bovigenitalium and M. bovis infections Etiology. Mycoplasma bovigenitalium and M. bovis are associated sporadically with bovine infertility and abortions. This pathogen has also been reported associated with similar clinical signs in sheep and goats. Clinical signs and diagnosis. Infertility is more commonly caused by M. bovigenitalium infections, and granular vulvovaginitis and endometritis will be present. Granular vulvovaginitis is characterized by raised papules on the mucous membranes and mucopurulent exudate. Abortions and mastitis are associated with M. bovis infections. Calves that are born may be weak. It is rare to have a definitive diagnosis of an abortion due to Mycoplasma. After consideration of other causes of abortion and evaluation of tissues for placentitis or fetal inflammation, diagnosis is confirmed by isolation of Mycoplasma from the genital tract or aborted tissues. Epidemiology and transmission. Mycoplasmal species are considered ubiquitous, are carried in the genital tracts of males and females, and are transmitted during natural breeding or through contaminated insemination materials. Aerosols also serve as a means of transmission. In addition, transmission occurs by passage through the birth canal, by direct contact, and by contamination from urine of infected animals. Pathophysiology. Experimental infections of M. bovis have resulted in placentitis and fetal pneumonia. Differential diagnoses. Acholeplasma, Ureaplasma, and Haemophilus somnus are differentials for granular vulvovaginitis. Treatment. Fluoroquinolone antibiotics may be useful for treating Mycoplasma-induced reproductive diseases. Etiology. Mycoplasma bovigenitalium and M. bovis are associated sporadically with bovine infertility and abortions. This pathogen has also been reported associated with similar clinical signs in sheep and goats. Clinical signs and diagnosis. Infertility is more commonly caused by M. bovigenitalium infections, and granular vulvovaginitis and endometritis will be present. Granular vulvovaginitis is characterized by raised papules on the mucous membranes and mucopurulent exudate. Abortions and mastitis are associated with M. bovis infections. Calves that are born may be weak. It is rare to have a definitive diagnosis of an abortion due to Mycoplasma. After consideration of other causes of abortion and evaluation of tissues for placentitis or fetal inflammation, diagnosis is confirmed by isolation of Mycoplasma from the genital tract or aborted tissues. Epidemiology and transmission. Mycoplasmal species are considered ubiquitous, are carried in the genital tracts of males and females, and are transmitted during natural breeding or through contaminated insemination materials. Aerosols also serve as a means of transmission. In addition, transmission occurs by passage through the birth canal, by direct contact, and by contamination from urine of infected animals. Pathophysiology. Experimental infections of M. bovis have resulted in placentitis and fetal pneumonia. Differential diagnoses. Acholeplasma, Ureaplasma, and Haemophilus somnus are differentials for granular vulvovaginitis. Treatment. Fluoroquinolone antibiotics may be useful for treating Mycoplasma-induced reproductive diseases. ii. Mycoplasma ovipneumoniae (ovine mycoplasmal pneumonia) Etiology. Mycoplasma ovipneumoniae causes acute or chronic pneumonia in lambs. Clinical signs. Mycoplasmas induce serious diseases in sheep, causing pneumonia, conjunctivitis, and genitourinary disease. The disease may be coincidental with pasteurellosis. Respiratory distress, coughing, and nasal discharge are observed in infected animals. Bronchoalveolar lavage followed by culture is the best method for diagnosis (mycoplasmas are fastidious organisms requiring special handling techniques). Mycoplasmas are isolated from the genitourinary tract of sheep. Vulvovaginitis and reproductive problems are associated conditions. Treatment. Tylosin, quinolones, oxytetracycline, and gentamicin are good choices for therapy. Prevention. No vaccine is available. Clinical signs. Mycoplasmas induce serious diseases in sheep, causing pneumonia, conjunctivitis, and genitourinary disease. The disease may be coincidental with pasteurellosis. Respiratory distress, coughing, and nasal discharge are observed in infected animals. Bronchoalveolar lavage followed by culture is the best method for diagnosis (mycoplasmas are fastidious organisms requiring special handling techniques). Mycoplasmas are isolated from the genitourinary tract of sheep. Vulvovaginitis and reproductive problems are associated conditions. Treatment. Tylosin, quinolones, oxytetracycline, and gentamicin are good choices for therapy. Prevention. No vaccine is available. iii. Mycoplasma mycoides biotype F38 (contagious caprine pleuropneumonia, caprine pneumonia, pleuritis, and pleuropneumonia) Etiology. Mycoplasma mycoides biotype F38 is the agent of contagious caprine pleuropneumonia and is found worldwide. In the United States, caprine pneumonia is also caused by M. ovipneumoniae, M. mycoides subsp. capri, and M. mycoides subsp. mycoides (large colony type). Clinical signs. Contagious caprine pleuropneumonia is characterized by severe dyspnea, nasal discharge, cough, and fever ( McMartin et al., 1980 ). Infections with other Mycoplasma species also have similar clinical signs. Septicemia without respiratory involvement may also be a presentation. Epizootiology and transmission. This disease is highly contagious, with high morbidity and mortality. Transmission is by aerosols. Mycoplasma mycoides subsp. mycoides has become a serious cause of morbidity and mortality of goat kids in the United States. Necropsy. Large amounts of pale straw-colored fluid and fibrinous pneumonia and pleurisy are typical. Some lung consolidation may be present. Meningitis, fibrinous pericarditis, and fibrinopurulent arthritis may also be found. Diagnosis is usually made at necropsy by culture of the organism from lungs and other internal organs. Differential dagnosis. In the United States, the principal differential for M. mycoides subsp. mycoides is caprine arthritis encephalitis. Treatment. Tylosin and oxytetracycline are effective. Some infections are slow to resolve. Prevention and control. Vaccines are available in some areas. Infected herds are quarantined. New goats should be quarantined before introduction to the herd. Research complications. The worldwide distribution of the F38 biotype, as well as the aerosol transmission and high morbidity and mortality characteristics of mycoplasmal infectious, make these infections economically important diseases. Considerable attention is presently given to this genus as a source of morbidity and mortality in goats. Etiology. Mycoplasma mycoides biotype F38 is the agent of contagious caprine pleuropneumonia and is found worldwide. In the United States, caprine pneumonia is also caused by M. ovipneumoniae, M. mycoides subsp. capri, and M. mycoides subsp. mycoides (large colony type). Clinical signs. Contagious caprine pleuropneumonia is characterized by severe dyspnea, nasal discharge, cough, and fever ( McMartin et al., 1980 ). Infections with other Mycoplasma species also have similar clinical signs. Septicemia without respiratory involvement may also be a presentation. Epizootiology and transmission. This disease is highly contagious, with high morbidity and mortality. Transmission is by aerosols. Mycoplasma mycoides subsp. mycoides has become a serious cause of morbidity and mortality of goat kids in the United States. Necropsy. Large amounts of pale straw-colored fluid and fibrinous pneumonia and pleurisy are typical. Some lung consolidation may be present. Meningitis, fibrinous pericarditis, and fibrinopurulent arthritis may also be found. Diagnosis is usually made at necropsy by culture of the organism from lungs and other internal organs. Differential dagnosis. In the United States, the principal differential for M. mycoides subsp. mycoides is caprine arthritis encephalitis. Treatment. Tylosin and oxytetracycline are effective. Some infections are slow to resolve. Prevention and control. Vaccines are available in some areas. Infected herds are quarantined. New goats should be quarantined before introduction to the herd. Research complications. The worldwide distribution of the F38 biotype, as well as the aerosol transmission and high morbidity and mortality characteristics of mycoplasmal infectious, make these infections economically important diseases. Considerable attention is presently given to this genus as a source of morbidity and mortality in goats. iv. Mycoplasma conjunctivae (mycoplasmal keratoconjunctivitis) Etiology. Mycoplasma conjunctivae causes infectious conjunctivitis, or pinkeye, in sheep and goats with associated hyperemia, edema, lacrimation, and corneal lesions. Mycoplasma mycoides subsp. mycoides, M. agalactiae, M. arginini, and Acholeplasma oculusi have also been associated with keratoconjunctivitis in these species. Respiratory disease and other infections, such as mastitis, may also be observed. Clinical signs and diagnosis. All ages of animals may be affected. Initially, lacrimation, conjunctival vessel injection, and then keratitis and neovascularization are seen. Sometimes uveitis is evident. Although the presentation is usually unilateral, bilateral involvement is possible. Recurring infections are common. Culturing provides the better diagnostic information, and cultures will be positive even after clinical signs have diminished. Epizootiology and transmission. The infection is passed easily between animals by direct contact. Animals can become reinfected, and carrier animals may be a factor in outbreaks. Necropsy. It is unlikely that animals would die or be euthanized and undergo necropsy for this problem. Conjunctival scrapings would include neutrophils during earlier stages and lymphocytes during later stages. Epithelial cell cytoplasm should be examined for organisms. Differential diagnosis. The primary differential in sheep and goats is Chlamydia, as well as Branhamella, Rickettsia (Colesiota) conjunctivae, and infectious bovine rhinotracheitis in goats only. It is important to consider these differentials if arthritis, pneumonia, or mastitis is present in the group or the individual. Treatment. Animals do recover spontaneously within about 10 weeks. Tetracycline ointments and powders are also used. Third-eyelid flaps may be necessary if corneal ulceration develops. Prevention and control. New animals should be quarantined and, if necessary treated, before introduction to the flock or herd. Etiology. Mycoplasma conjunctivae causes infectious conjunctivitis, or pinkeye, in sheep and goats with associated hyperemia, edema, lacrimation, and corneal lesions. Mycoplasma mycoides subsp. mycoides, M. agalactiae, M. arginini, and Acholeplasma oculusi have also been associated with keratoconjunctivitis in these species. Respiratory disease and other infections, such as mastitis, may also be observed. Clinical signs and diagnosis. All ages of animals may be affected. Initially, lacrimation, conjunctival vessel injection, and then keratitis and neovascularization are seen. Sometimes uveitis is evident. Although the presentation is usually unilateral, bilateral involvement is possible. Recurring infections are common. Culturing provides the better diagnostic information, and cultures will be positive even after clinical signs have diminished. Epizootiology and transmission. The infection is passed easily between animals by direct contact. Animals can become reinfected, and carrier animals may be a factor in outbreaks. Necropsy. It is unlikely that animals would die or be euthanized and undergo necropsy for this problem. Conjunctival scrapings would include neutrophils during earlier stages and lymphocytes during later stages. Epithelial cell cytoplasm should be examined for organisms. Differential diagnosis. The primary differential in sheep and goats is Chlamydia, as well as Branhamella, Rickettsia (Colesiota) conjunctivae, and infectious bovine rhinotracheitis in goats only. It is important to consider these differentials if arthritis, pneumonia, or mastitis is present in the group or the individual. Treatment. Animals do recover spontaneously within about 10 weeks. Tetracycline ointments and powders are also used. Third-eyelid flaps may be necessary if corneal ulceration develops. Prevention and control. New animals should be quarantined and, if necessary treated, before introduction to the flock or herd. ff. Rickettsial Diseases i. Eperythrozoonosis (Eperythrozoon, Haemobartonella) Etiology. Eperythrozoonosis is a rare, sporadic, noncontagious, blood-borne disease in ruminants worldwide caused by the rickettsial agent Eperythrozoon. Host-specific species of importance are E. ovis, the causative species in sheep and goats, and E. wenyoni, E. tegnodes, and E. tuomii, the causative agents in cattle. Although the disease is of minor importance, it can cause severe anemia and debilitation in affected animals. Haemobartonella bovis is also rare, and is usually found only in association with other rickettsial diseases. Clinical signs and diagnosis. The disease is more severe in sheep. Following an incubation period of 1–3 weeks, infected animals exhibit episodic hyperthermia, weakness, and anemia. Losses may be greater in younger lambs. Cattle are usually latently infected but may have swollen and tender teats and legs. Fever, anemia, and depression will be present if the cattle are stressed by another systemic disease. Diagnosis is based on clinical evidence of anemia and is confirmed by observing the rickettsiae on the surface of red blood cells in a blood smear. Epizootiology and transmission. The rickettsial organisms are transmitted typically to young sheep by biting insects, ticks, contaminated needles or blood-contaminated surgical instruments. Necropsy findings. Necropsy findings include splenic enlargement and tissue icterus. Pathogenesis. The organism invades and destroys red blood cells. It is believed that intravascular hemolysis and erythrophagocytosis contribute to the macrocytic anemia. As with other red blood cell parasites, splenectomy aggravates the disease. Differential diagnosis. Clontridium novyi type D, babesiosis, and leptospirosis are the primary differentials. Prevention and control. Following strict sanitation practices for surgical procedures and controlling external parasites prevent the disease. Treatment. Treatment is not usually recommended, but Oxytetracycline has been used. Sheep will develop immunity if supported nutritionally during the disease. Research complications. Splenectomized animals are the experimental models used to study these diseases. ii. Q fever, or query fever (Coxiella burnetii) Etiology. Coxiella burnetii is a small, gram-negative, obligate intracellular rickettsial organism that causes query fever and is regarded as a major cause of late abortion in sheep. Clinical signs. Infection of ruminants with C. burnetii is usually asymptomatic. Experimental inoculation in other mammals has resulted in transient hyperthermia, mild respiratory disease, and mastitis. Abortions, stillbirths, and births of weak lambs are also seen. Epizootiology and transmission. Coxiella burnetii is extremely resistant to environmental changes as well as to disinfectants; persistence in the environment for a year or longer is possible. The organism is associated with either a free-living or an arthropod-borne cycle. Coxiella burnetii is found in a variety of tick species, such as ixodid or argasid, where it replicates and is excreted in the feces. Once introduced into a mammal, Coxiella may be maintained without a tick intermediate. The organism is especially concentrated in placental tissues, replicates in trophoblasts, and will be in reproductive fluids. Additionally, the organism is shed in milk, urine, feces, and oronasal secretions. Necropsy findings. No specific lesion will be seen in aborted or stillborn fetuses, but necrotizing placentitis will be a finding in cases of abortion. The placenta will contain white chalky plaques and a red-brown exudate. The disease can be diagnosed by identifying the rickettsial organisms in smears of placental secretions. The organism has been found in the placentas of clinically normal animals. The organism stains red with modified Ziehl-Neelsen and Macchiavello stains and purple with Giemsa stain. Differential diagnosis. Because of the organisms' similarity to Chlamydia, confirmation must be made by culture techniques, immunofluorescent procedures, ELISA, and complement fixation tests. Treatment. Coxiella can be treated with oxytetracyclines. A vaccine is not commercially available. Prevention and control. Any aborting animals should be segregated from other animals, and other pregnant animals should be treated prophylactically with tetracycline. Serologic screening of ruminant sources should be performed routinely. Barrier housing, a review of ventilation exhaust, and defined handling procedures are often required. All placentas and all aborted tissues should be handled and disposed of carefully. Q fever has been reported in many mammalian species, including cats. Research complications. Coxiella burnetii–hee animals are particularly important in studies involving fetuses and placentation. Because of its zoonotic potential, C. burnetii presents a unique problem in the animal research facility environment. A single organism has been shown to cause disease. Some of the greatest concerns are the risk to immunocompromised individuals, pregnant women, and other animals, and the presence of carrier animals or those that may shed the organism in placentas, for example. i. Eperythrozoonosis (Eperythrozoon, Haemobartonella) Etiology. Eperythrozoonosis is a rare, sporadic, noncontagious, blood-borne disease in ruminants worldwide caused by the rickettsial agent Eperythrozoon. Host-specific species of importance are E. ovis, the causative species in sheep and goats, and E. wenyoni, E. tegnodes, and E. tuomii, the causative agents in cattle. Although the disease is of minor importance, it can cause severe anemia and debilitation in affected animals. Haemobartonella bovis is also rare, and is usually found only in association with other rickettsial diseases. Clinical signs and diagnosis. The disease is more severe in sheep. Following an incubation period of 1–3 weeks, infected animals exhibit episodic hyperthermia, weakness, and anemia. Losses may be greater in younger lambs. Cattle are usually latently infected but may have swollen and tender teats and legs. Fever, anemia, and depression will be present if the cattle are stressed by another systemic disease. Diagnosis is based on clinical evidence of anemia and is confirmed by observing the rickettsiae on the surface of red blood cells in a blood smear. Epizootiology and transmission. The rickettsial organisms are transmitted typically to young sheep by biting insects, ticks, contaminated needles or blood-contaminated surgical instruments. Necropsy findings. Necropsy findings include splenic enlargement and tissue icterus. Pathogenesis. The organism invades and destroys red blood cells. It is believed that intravascular hemolysis and erythrophagocytosis contribute to the macrocytic anemia. As with other red blood cell parasites, splenectomy aggravates the disease. Differential diagnosis. Clontridium novyi type D, babesiosis, and leptospirosis are the primary differentials. Prevention and control. Following strict sanitation practices for surgical procedures and controlling external parasites prevent the disease. Treatment. Treatment is not usually recommended, but Oxytetracycline has been used. Sheep will develop immunity if supported nutritionally during the disease. Research complications. Splenectomized animals are the experimental models used to study these diseases. Etiology. Eperythrozoonosis is a rare, sporadic, noncontagious, blood-borne disease in ruminants worldwide caused by the rickettsial agent Eperythrozoon. Host-specific species of importance are E. ovis, the causative species in sheep and goats, and E. wenyoni, E. tegnodes, and E. tuomii, the causative agents in cattle. Although the disease is of minor importance, it can cause severe anemia and debilitation in affected animals. Haemobartonella bovis is also rare, and is usually found only in association with other rickettsial diseases. Clinical signs and diagnosis. The disease is more severe in sheep. Following an incubation period of 1–3 weeks, infected animals exhibit episodic hyperthermia, weakness, and anemia. Losses may be greater in younger lambs. Cattle are usually latently infected but may have swollen and tender teats and legs. Fever, anemia, and depression will be present if the cattle are stressed by another systemic disease. Diagnosis is based on clinical evidence of anemia and is confirmed by observing the rickettsiae on the surface of red blood cells in a blood smear. Epizootiology and transmission. The rickettsial organisms are transmitted typically to young sheep by biting insects, ticks, contaminated needles or blood-contaminated surgical instruments. Necropsy findings. Necropsy findings include splenic enlargement and tissue icterus. Pathogenesis. The organism invades and destroys red blood cells. It is believed that intravascular hemolysis and erythrophagocytosis contribute to the macrocytic anemia. As with other red blood cell parasites, splenectomy aggravates the disease. Differential diagnosis. Clontridium novyi type D, babesiosis, and leptospirosis are the primary differentials. Prevention and control. Following strict sanitation practices for surgical procedures and controlling external parasites prevent the disease. Treatment. Treatment is not usually recommended, but Oxytetracycline has been used. Sheep will develop immunity if supported nutritionally during the disease. Research complications. Splenectomized animals are the experimental models used to study these diseases. ii. Q fever, or query fever (Coxiella burnetii) Etiology. Coxiella burnetii is a small, gram-negative, obligate intracellular rickettsial organism that causes query fever and is regarded as a major cause of late abortion in sheep. Clinical signs. Infection of ruminants with C. burnetii is usually asymptomatic. Experimental inoculation in other mammals has resulted in transient hyperthermia, mild respiratory disease, and mastitis. Abortions, stillbirths, and births of weak lambs are also seen. Epizootiology and transmission. Coxiella burnetii is extremely resistant to environmental changes as well as to disinfectants; persistence in the environment for a year or longer is possible. The organism is associated with either a free-living or an arthropod-borne cycle. Coxiella burnetii is found in a variety of tick species, such as ixodid or argasid, where it replicates and is excreted in the feces. Once introduced into a mammal, Coxiella may be maintained without a tick intermediate. The organism is especially concentrated in placental tissues, replicates in trophoblasts, and will be in reproductive fluids. Additionally, the organism is shed in milk, urine, feces, and oronasal secretions. Necropsy findings. No specific lesion will be seen in aborted or stillborn fetuses, but necrotizing placentitis will be a finding in cases of abortion. The placenta will contain white chalky plaques and a red-brown exudate. The disease can be diagnosed by identifying the rickettsial organisms in smears of placental secretions. The organism has been found in the placentas of clinically normal animals. The organism stains red with modified Ziehl-Neelsen and Macchiavello stains and purple with Giemsa stain. Differential diagnosis. Because of the organisms' similarity to Chlamydia, confirmation must be made by culture techniques, immunofluorescent procedures, ELISA, and complement fixation tests. Treatment. Coxiella can be treated with oxytetracyclines. A vaccine is not commercially available. Prevention and control. Any aborting animals should be segregated from other animals, and other pregnant animals should be treated prophylactically with tetracycline. Serologic screening of ruminant sources should be performed routinely. Barrier housing, a review of ventilation exhaust, and defined handling procedures are often required. All placentas and all aborted tissues should be handled and disposed of carefully. Q fever has been reported in many mammalian species, including cats. Research complications. Coxiella burnetii–hee animals are particularly important in studies involving fetuses and placentation. Because of its zoonotic potential, C. burnetii presents a unique problem in the animal research facility environment. A single organism has been shown to cause disease. Some of the greatest concerns are the risk to immunocompromised individuals, pregnant women, and other animals, and the presence of carrier animals or those that may shed the organism in placentas, for example. Etiology. Coxiella burnetii is a small, gram-negative, obligate intracellular rickettsial organism that causes query fever and is regarded as a major cause of late abortion in sheep. Clinical signs. Infection of ruminants with C. burnetii is usually asymptomatic. Experimental inoculation in other mammals has resulted in transient hyperthermia, mild respiratory disease, and mastitis. Abortions, stillbirths, and births of weak lambs are also seen. Epizootiology and transmission. Coxiella burnetii is extremely resistant to environmental changes as well as to disinfectants; persistence in the environment for a year or longer is possible. The organism is associated with either a free-living or an arthropod-borne cycle. Coxiella burnetii is found in a variety of tick species, such as ixodid or argasid, where it replicates and is excreted in the feces. Once introduced into a mammal, Coxiella may be maintained without a tick intermediate. The organism is especially concentrated in placental tissues, replicates in trophoblasts, and will be in reproductive fluids. Additionally, the organism is shed in milk, urine, feces, and oronasal secretions. Necropsy findings. No specific lesion will be seen in aborted or stillborn fetuses, but necrotizing placentitis will be a finding in cases of abortion. The placenta will contain white chalky plaques and a red-brown exudate. The disease can be diagnosed by identifying the rickettsial organisms in smears of placental secretions. The organism has been found in the placentas of clinically normal animals. The organism stains red with modified Ziehl-Neelsen and Macchiavello stains and purple with Giemsa stain. Differential diagnosis. Because of the organisms' similarity to Chlamydia, confirmation must be made by culture techniques, immunofluorescent procedures, ELISA, and complement fixation tests. Treatment. Coxiella can be treated with oxytetracyclines. A vaccine is not commercially available. Prevention and control. Any aborting animals should be segregated from other animals, and other pregnant animals should be treated prophylactically with tetracycline. Serologic screening of ruminant sources should be performed routinely. Barrier housing, a review of ventilation exhaust, and defined handling procedures are often required. All placentas and all aborted tissues should be handled and disposed of carefully. Q fever has been reported in many mammalian species, including cats. Research complications. Coxiella burnetii–hee animals are particularly important in studies involving fetuses and placentation. Because of its zoonotic potential, C. burnetii presents a unique problem in the animal research facility environment. A single organism has been shown to cause disease. Some of the greatest concerns are the risk to immunocompromised individuals, pregnant women, and other animals, and the presence of carrier animals or those that may shed the organism in placentas, for example. 2. Viral Diseases a. Adenovirus Infections Etiology. The ruminant adenoviruses are DNA viruses that cause respiratory and reproductive tract diseases. Nine antigenic types of the bovine adenovirus have been identified, with type 3 associated with respiratory disease. Two of the ovine and two of the caprine antigenic types have been identified. Clinical Signs. Signs of infection range from subclinical to severe, including pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, weak calf syndrome, and abortion. Respiratory tract and intestinal tract diseases may be concurrent. Infections caused by this virus are often found associated with other viral and bacterial infections. Epizootiology and transmission. The virus is believed to be widespread, but prevalence and characteristics of infection have not been characterized. Transmission of adenoviruses in other species (e.g., canine) is by aerosols or fecal-oral routes. Necropsy findings. Lesions found after experimental infections include atelectasis, edema, and consolidation of the lungs. b. Bluetongue Virus Infection (Reoviridae) Etiology. The bluetongue virus is an RNA virus in the Orbivirus genus and Reoviridae family. Five serotypes (2, 10, 11, 13, and 17) have been identified in the United States, where it is seen mostly in western states. Bluetongue is an acute arthropod-borne viral disease of ruminants, characterized by stomatitis, depression, coronary band lesions, and congenital abnormalities ( Bulgin, 1986 ). Clinical signs and diagnosis. Sheep are the most likely to show clinical signs. Clinical disease is less common in goats and cattle. Early in the infection, animals will spike a fever and will develop hyperemia and congestion of tissues of the mouth, lips, and ears. The virus name, bluetongue, is associated with the typical cyanotic membranes. The fever may subside, but tissue lesions erode, causing ulcers. Increased salivary discharges and anorexia are often related to ulcers of the dental pad, lips, gums, and tongue, although salivation and lacrimation may precede apparent ulceration. Chorioretinitis and conjunctivitis are also common signs in cattle and sheep. Lameness may be observed associated with coronitis and is evident in the rear legs. Skin lesions such as drying and cracking of the nose, alopecia, and mammary glands are also observed. Secondary bacterial pneumonia may also occur. Animals may also develop severe diarrhea and become recumbent. Sudden deaths due to cardiomyopathy may occur at any time during the disease. Hematologically, animals will be leukopenic. The course of the disease is about 2 weeks, and mortality may reach 80%. If animals are pregnant, the virus crosses the placenta and causes central nervous system lesions. Abortions may occur at any stage of gestation in cattle. Prolonged gestation may result from cerebellar hypoplasia and lack of normal sequence to induce parturition. Cerebellar hypoplasia will also be present in young born of the infected dams, as well as hydrocephalus, cataracts, gingival hyperplasia, or arthrogryposis. Diagnosis is suspected with the characteristic clinical signs and exposure to viral vectors. Virus isolation is the best diagnostic approach if blood is collected during the febrile stage of the disease or brains from aborted fetuses. Fluorescent antibody tests, ELISA, virus neutralization tests, PCR, and agar gel immunodiffusion (AGID) tests are also used to confirm the diagnosis. Epizootiology and transmission. Severe outbreaks have occurred in other countries during this century. Screening for this disease has limited the strains present in the United States. The disease is most common in outdoor-housed animals primarily in the western United States. The virus is primarily transmitted by biting midges, Culicoides. Culicoides variipennis is the most common vector in the United States. A combination of factors associated with viral strain, available and susceptible hosts, environmental conditions (such as damp areas where flies breed), and vector presence are factors in the severity of outbreaks. The disease is rarely transmitted by animal-to-animal contact or by infected animal products. Virus-contaminated semen, transplacental transfer, and carriage on transferred embyros are other possible means of transmission. Necropsy findings. At necropsy, erosive lesions may be observed around the mouth, tongue, palate, esophagus, and pillars of the rumen. Ulceration or hyperemia of the coronary bands may also be seen. Many of the internal organs will contain petechial and ecchymotic hemorrhages of the surfaces, and hemorrhage may be seen at the base of the pulmonary artery. Pathogenesis. The virus multiplies in the hemocoel and salivary glands of the fly and is excreted in transmissible form in the insect's saliva. After entering the host, the virus causes prolonged viremia. The incubation period is 6–14 days. The virus migrates to and attacks the vascular endothelium. The resulting vasculitis accounts for the lesions of the skin, mouth, tongue, esophagus, and rumen and the edema often found in many tissues. Ballooning degeneration of affected tissues, followed by necrosis and ulceration, occurs. The effects on fetuses appear to be due to generalized infections of developing organs. Differential diagnosis. Differentials include other infectious vesicular diseases such as foot-and-mouth disease, contagious ecthyma, bovine viral diarrhea virus-mucosal disease, infectious bovine rhinotracheitis, bovine papular stomatitis, and malignant catarrhal fever. Rinderpest is a differential in countries where it is endemic. Photosensitization should be considered. Foot rot is a differential for the lameness and coronitis. Differentials for the manifestations such as arthrogryposis include border disease virus and genetic predispositions of some breeds such as Charolais cattle and Merino sheep. Prevention and control. Cellular and humoral immunity are necessary for protection from infection. The bluetongue virus is insidious because the genome is capable of reassortment, and some vaccines will not have the antigenic components represented in the local infection. In addition, there is little to no cross protection between strains. Modified live vaccines are available in some parts of the United States but should not be used in pregnant animals. Vaccinating lambs and rams in an outbreak is worthwhile, for example, but vaccinating late-gestation ewes may cause birth defects or abortions. Congenital defects are more common from vaccine use than from naturally occurring infection. Minimizing exposure to the vector in endemic areas will decrease the incidence of the disease. Treatment. Supportive care and nursing care are helpful, including gruels or softer feeds, easily accessed water, and shaded resting places. Nonsteroidal anti-inflammatory drugs are often administered. For the cases of secondary bacterial pneumonia and some cases of bluetongue conjunctivitis, antibiotics may be administered. Research complications. This is a reportable disease because clinical signs resemble foot-and-mouth disease and other exotic vesicular diseases. c. Bovine Lymphosarcoma (Bovine Leukemia Virus Infection, Bovine Leukosis) Etiology. Bovine lymphosarcoma refers to lymphoproliferative diseases in young cattle that are not associated with bovine leukemia virus (BLV) infection, and those in older cattle that are associated with BLV. BLV is a B lymphocyte-associated retrovirus ( Johnson and Kaneene, 1993 a,b,c). Clinical signs. Forms of bovine lymphosarcoma that are not associated with BLV infection are calf, or juvenile; thymic, or adolescent (animals 6 months to 2 years old); and cutaneous (any age). The calf form is rare and characterized by generalized lymphadenopathy. Onset may be sudden, and the disease is usually fatal within a few weeks. Signs include lymphadenopathy, anemia, weight loss, and weakness. Some animals may be paralyzed because of spinal cord compression from subperiosteal infiltration of neoplastic cells. The adolescent form is also rare, the course rapid, and the prognosis poor. The disease is seen most often in beef breeds such as Hereford cattle and is characterized by space-occupying masses in the neck or thorax. These masses are also often present in the brisket. Secondary effects of the masses are loss of condition, dysphagia, rumen tympany, and fatal bloat. The cutaneous presentation has a longer course and may wax and wane. The masses are found at the anus, vulva, escutcheon, shoulder, and flank; they are painful when palpated, raised, and often ulcerated. The animals are anemic, and neoplastic involvement may affect cardiac function. Generalized or limited lymphadenopathy may be apparent. Only the adult, or enzootic, form of bovine lymphosarcoma is associated with BLV infection. Many animals do not develop any malignancies or clinical signs of infection and simply remain permanently infected. Some cows manifest disease only during the periparturient period. Malignant lymphoma is the more common, whereas leukosis, due to B-lymphocyte proliferation, is rare. Clinical signs are loss of condition and a drop in production of dairy cattle, anorexia, diarrhea, ataxia, paresis, and other signs dependent on the location of the neoplastic tissue. Tumors are associated with lymphoid tissues. Common sites also include the abomasum, spinal canal, and uterus. Cardiac tumors develop at the right atrial or left ventricular myocardium, and associated beat and rate abnormalities may be auscultated. The common ocular manifestation of the disease is exophthalmos due to retrobulbar masses. Many internal organs may be involved, and tumors may be palpable per rectum. Secondary infections will be due to immunosuppression and the weakened state of the animal. Sheep have acquired BLV infection naturally and have been used as experimental models; in both situations, this species is susceptible to tumor and leukemia development. Goats seroconvert but do not develop the clinical syndromes. Diagnosis is based on the animal's age, clinical signs, serology, hematology findings according to the form, aspirates or biopsies of masses, and necropsy findings. Kits are available for running AGID, for which the BLV antigens gp-51 and gp-24 are used; antibodies may be detected within weeks after exposure and may also help in predicting disease in clinically normal cattle. ELISA and PCR diagnostic aids will also be helpful. Epizootiology and transmission. This disease is present worldwide. It is estimated that at least 50% of the cattle in the United States are infected with BLV. As few as 1% of these animals develop lymphosarcoma, but the adult form of the disease described here is the most common bovine neoplastic disease in the United States. Larger herds tend to have higher rates. Genetic predisposition may be involved; in addition to the presence of BLV, the type of bovine lymphocyte antigen (BoLA) may be correlated to resistance or susceptibility and to the course of the disease. Transmission is believed to be by inhalation of BLV in secretions; in colostrum; horizontally by contaminated equipment not sanitized between cattle; and by rectum (e.g., mucosal irritation during per-rectum exams or procedures). Natural-service bulls may transmit the infection to cows. Cows infected with BLV may transmit the infection to their calves in utero. Tabanid and other flies also serve as vectors, but these represent a minor means of transmission. Necropsy findings. Neoplastic infiltration of many organs and tissues are found in the calf form and the cutaneous forms. Tumors may be local or widely distributed in the enzootic form. Definitive diagnosis of neoplastic tissue specimens is by histology. Pathogenesis. As with other retroviruses, the BLV integrates viral DNA into host target cell DNA by means of the reverse transcriptase enzyme, creating a provirus. Prevention and control. There is no vaccine for this disease. Development and maintenance of a BLV-free herd, or controlling infection within a herd, requires financial and programmatic commitments: BLV-positive and BLV-negative animals maintained separately; serologic testing (such as at least every 6 months) and separating positive animals; and washing and then disinfecting instruments, needles (or using sterile single-use products), and equipment for ear tagging and dehorning and other such equipment between animals. A fresh rectal exam sleeve and lubricant should be used for each animal examined. Otherwise serologically positive cows may have undetectable antibodies during the periparturient period. Embryo transfer recipients should be negative, and the virus will not be transferred by the embryonic stage. Calves should be fed colostrum from serologically negative cows. Treatment. Treatment regimens of corticosteroids and cancer chemotherapeutic agents provide only short-term improvement. In cases where ova, embryos, or semen need to be collected, supportive care for the affected animals is essential. Research complications. The United States and several countries, some in Europe, have official programs for eradication of enzootic bovine leukosis. d. Bovine Herpes Mammillitis (Bovine Herpesvirus 2 Bovine Ulcerative Mammillitis) Etiology. Bovine herpesvirus 2 causes bovine herpes mammillitis, a widespread disease characterized by teat and udder lesions, as well as oral and skin lesions. Clinical signs and diagnosis. Lesions begin suddenly with teat swelling; the tissue will be edematous and tender when touched. The udder lesions may extend to the perineum. The lesions progress to vesicles, then to ulcers; these may take 10 weeks to heal. Lesions rarely may also develop focally around the mouth and generally on the skin of the udder. Secondary mastitis may occur, because of bacteria associated with the scabs. Diagnosis is by clinical signs and serologically. Epizootiology and transmission. The virus is reported to be widespread. Occurrence is often seasonal, and biting insects may be vectors. Transmission with successful infection requires deep penetration of the skin. Transmission may be by contaminated milkers' hands, contaminated equipment, and other fomites. Differential diagnosis. Differential diagnoses include other diseases that cause lesions on teats such as pseudocowpox, papillomatosis, and vesicular stomatitis. Other vesicular diseases may be considered, but other more severe clinical signs might be associated with those. Prevention and control. Established milking hygiene practices are important control measures: having milkers wash their hands with germicidal solutions or wear gloves, cleaning equipment between animals, and separating affected animals. Treatment. There is no treatment, and affected animals should be separated from the herd and milked last. Lesions can be cleaned and treated with topical antibacterials. e. Bovine Viral Diarrhea Virus Etiology. The bovine viral diarrhea virus (BVDV) is a pestivirus of the Flaviviridae family. The Flaviviridae include hog cholera virus and border disease virus of sheep. The virus contains a single strand of positive-sense RNA. A broad range of disease and immune effects is produced by BVDV only in cattle. In addition, this virus is important in the etiology of bovine pneumonias. Bovine viral diarrhea/mucosal disease (BVD/MD) is one of the most important viral diseases and one of the most complex diseases of cattle. Strains of BVDV are characterized as cytopathic (CP) and noncytopathic (NCP), based on cell-culture growth characteristics. The virus has also been categorized as type 1 and type 2 isolates. Heterologous strains exist that may confound even sound vaccination programs. Clinical signs and diagnosis. Signs of BVDV infections may be subclinical but also include abortions, congenital abnormalities, reduced fertility, persistent infection (PI) with gradual debilitation, and acute and fatal disease. The presence of antibodies, whether from passive transfer or immunizations, does not necessarily guarantee protection from the various forms of the disease. An acute form of the disease, caused by type 2 BVDV, occurs in cattle without sufficient immunity. After an incubation period of 5–7 days, clinical signs include fever, anorexia, oculonasal discharge, oral erosions (including on the hard palate), diarrhea, and decreased milk production. The disease course may be shorter with hemorrhagic syndrome and death within 2 days. Clinical signs of BVDV in calves also include severe enteritis and pneumonia. When susceptible cows are infected in utero from gestational days 50–100, or gestational cows are vaccinated with a modified live vaccine, abortion or stillbirth result. Congenital defects caused by BVDV during gestational days 90–170 include impaired immunity (thymic atrophy), cerebellar hypoplasia, ocular defects, alopecia or hypotrichosis, dysmyelinogenesis, hydranencephaly, hydrocephalus, and intrauterine growth retardation. Typical signs of cerebellar dysfunction will be evident in calves, such as wide-based stance, weakness, opisthotonus, hyperflexion, hypermetria, nystagmus, or strabismus. Some severely affected calves will not be able to stand. Ophthalmic effects include retinal degeneration and microphthalmia. Fetuses can also be infected in utero, normal at birth, immunotolerant to the virus, and persistently infected (PI). The term mucosal disease is commonly associated with this form of the infection. Many PI animals do not survive to maturity, however, and many have weakened immune systems. The PI animals are important because they shed virus and will probably show the clinical signs of mucosal disease (MD) caused by a CP BVDV strain derived from an NCP BVDV strain. These MD clinical signs include fever, anorexia, and profuse diarrhea that may include blood and fibrin casts, and oral and pharyngeal erosions, as well as erosion at the interdigital spaces and on the teats and vulva. Many other associated clinical signs include anemia, bloat, lameness, or corneal opacities and discharges. Secondary effects of hemorrhage and dehydration also contribute to the morbidity and mortality. Animals that do not succumb to the disease will be chronically unthrifty, debilitated, and infection-prone. Diagnosis in affected calves is based on herd health history, clinical signs, and antibodies to BVDV in precolostral serum. Viral culturing from blood may be useful. In older animals, oral lesions, serology, detection of viral antigen, and virus isolation contribute to the diagnosis. Leukopenia, and especially lymphopenia, are seen. Serology must be interpreted with the awareness of the possibility of PI immunotolerant animals. Vaccination against the disease carries its own set of side effects and potential problems, especially when using modified live vaccines, whether against CP or NCP strains. The condition of the animals is also a variable. Epizootiology and transmission. BVDV is present throughout the world. Transmission occurs easily by direct contact between cattle, from feed contaminated with secretions or feces, and by aborted fetuses and placentas. PI females transmit the virus to their fetuses. Semen also is a source of virus. Necropsy findings. In affected calves, histopathologic findings include necrosis of external germinal cells, focal hemorrhages, and folial edema. Later in the disease, large cavities develop in the cerebellum, and atrophy of the cerebellar folia and thin neuropil are evident. Older calves may have areas of intestinal necrosis. In cases where oral erosions occur, erosions will be found extending throughout the gastrointestinal tract to the cecum. The respiratory tract lesions will often be complicated by secondary bacterial pneumonia. When the hemorrhagic syndrome develops, petechiation and mucosal bleeding will be present. Pathogenesis. The CP and NCP strains are thought to be related mutations of the BVDV; the CP short-lived isolates are believed to arise from the NCP strains. The NCP strains are those present in the PI animals, and the strains are maintained in cattle populations. CP and NCP isolates vary in virulence, and classification of these types is based on viral surface proteins. Considerable antigenic variation also exists between strains and types. Other viral infections, such as bovine respiratory syncytial virus and infectious bovine rhinotracheitis, may also be present in the same animals. The pathology caused by BVDV is due to its ability to infect epithelial cells and impair the functioning of immune cell populations through out the bovine system. In type 2 BVDV hemorrhagic syndrome, death results from viral-induced thrombocytopenia. In fetuses, the virus infects developing germinal cells of the cerebellum. The Purkinje's cells in the granular layer are killed, and necrosis and inflammation follow. The immune effects are the result of the virus's interfering with neutrophil and macrophage functions and of lymphocyte blastogenesis. All of these predispose the affected animals to bacterial infections with Pasteurella haemolytica. BVDV damages dividing cells in fetal organ systems, resulting in abortions and congenital effects. Differential diagnosis. Many differentials must be considered for the clinical manifestations of BVDV infections. Differentials for enteritis of calves include viral infections, Cryptosporidia, Escherichia coli, Salmonella, and Coccidia. Salmonella, winter dysentery, Johne's disease, intestinal parasites, malignant catarrhal fever (MCF), and copper deficiency are differentials for the diarrhea seen in the disease in adult animals. Respiratory tract pathogens such as bovine respiratory syncytial virus, Pasteurella, Haemophilus, and Mycoplasma must be considered for the respiratory tract manifestations. Oral lesions are also produced by MCF, vesicular stomatitis, bluetongue, and papular stomatitis. Infectious bovine herpesvirus 1, leptospirosis, brucellosis, trichomoniasis, and mycosis should be considered in cases of abortion. Prevention and control. Combined with sound management in a typical cattle herd, vaccination is the best way to prevent BVDV and should be integrated into the herd health program, timed appropriately preceding breeding, gestation, or stressful events. Vaccine preparations for BVDV are modified live virus (MLV) or killed virus. Each has advantages and disadvantages. The former induces rapid immunity (within 1 week) after a single dose, provides longer duration of immunity against several strains, and induces serum neutralizing antibodies. MLV vaccines are not recommended for use in pregnant cattle, may induce mucosal disease, and may be immunosuppressive at the time of vaccination. The immunosuppression is detrimental if cattle are concurrently exposed to field-strain virus because it will facilitate infection and possible clinical disease. The MLV strains may cross the placenta, resulting in fetal infections. The killed vaccines are safer in pregnant animals but require booster doses after the initial immunization, may need to be given 2–3 times per year, and do not induce cell-mediated immunity. Passive immunity may protect most calves for up to 6–8 months of age. Subsequent vaccination with MLV may provide lifelong immunity, but this is not guaranteed. Annual boosters are recommended to protect against vaccine breaks. The virus persists in the environment for 2 weeks and is susceptible to the disfectants chlorhexidine, hypochlorite, iodophors, and aldehydes. Maintenance of a closed herd to prevent any possibility of the introduction of the virus is difficult. Isolation of new animals, avoidance of the purchase of pregnant cows, scrutiny of records from source farms, use of semen tested bulls, minimization of stress, testing of embryo-recipient cows, and maintainenance of populations of ruminants (smaller or wild species) separately on the premises will minimize viral exposure. Other management strategies may require a program for testing and culling PI cattle. This can be expensive but may be a worthwhile investment to remove the virus shedders from a herd. Treatment. No specific treatment is available. Supportive care and treatment with antibiotics to prevent secondary infection are recommended. Animals that survive the infection should be evaluated a month after recovery to determine their status as PI or virus-free. f. Cache Valley Virus Etiology. Cache Valley virus (CVV), of the arbovirus genus of the Bunyaviridae family, is a cause of congenital defects in lambs. Clinical signs and diagnosis. Teratogenic effects of in utero CVV infection in fetal and newborn lambs include arthrogryposis, microencephaly, hydranencephaly, porencephaly, cerebellar hypoplasia, and micromyelia. Stillbirths and mummified fetuses are seen. Lambs will be born weak and will act abnormally. Diagnosis is by evidence of seroconversion in precolostral blood samples or fetal fluids, as the result of in utero infection. Epizootiology and transmission. The virus is present in the western United States, although it has been isolated in a few Midwestern states. Although considered a disease of sheep, virus has been isolated from cattle and from wild ruminants and antibodies found in white-tailed deer. Transmission is by arthropods during the first trimester of pregnancy. g. Caprine Arthritis Encephalitis Virus Etiology. Caprine arthritis encephalitis virus (CAEV) occurs worldwide, with a high prevalence in the United States. Caprine arthritis encephalitis (CAE) is considered the most important viral disease of goats. The CAEV is in the Lentivirus genus of the Retroviridae family. It causes chronic arthritis in adults and encephalitis in young. CAEV is in the same viral genus as the ovine progressive pneumonia virus (OPPV). Clinical signs and diagnosis. The most common presentation in goats is an insidious, progressive arthritis in animals 6 months of age and older. Animals become stiff, have difficulty getting up, and may be clinically lame in one or both forelimbs. Carpal joints are so swollen and painful that the animal prefers to eat, drink, and walk on its "knees." In dairy goats, milk production decreases, and udders may become firmer. This retrovirus also causes neurological clinical signs in young kids 2–6 months old. Kids may be bright and alert, afebrile, and able to eat normally even when recumbent. Some kids may initially show unilateral weakness in a rear limb, which progresses to hemiplegia or tetraplegia. Mild to severe lower motor neuron deficits may be noted, but spinal reflexes are intact. Clinical signs may also include head tilt, blindness, ataxia, and facial nerve paralysis. Older animals in the group may experience interstitial pneumonia or chronic arthritis. The pneumonia is similar to the pneumonia in sheep caused by OPPV; the course is gradual but progressive, and animals will eventually lose weight and have respiratory distress. Some animals in a herd may not develop any clinical signs. Diagnosis is based on clinical signs, postmortem lesions, and positive serology for viral antibodies to CAEV. An agar gel immunodiffusion (AGID) test identifies antibodies to the virus and is used for diagnosis. Kids acquire an anti-CAEV antibody in colostrum, and this passive immunity may be interpreted as indicative of infection with the virus. The antibody does not prevent viral transmission. Epizootiology and transmission. The virus is prevalent in most industrialized countries. The common means of transmission, from adults to kids, is in the colostrum and milk in spite of the presence of anti-CAEV antibody in the colostrum. Transmission may occur among adult goats by contact. Intrauterine transmission is believed to be rare. Transmission to sheep has occurred only experimentally; there is no documented case of natural transmission. Necropsy findings. Necropsy and histopathology reveal a striking synovial hyperplasia of the joints with infiltrates of lymphocytes, macrophages, and plasma cells. Other histologic lesions include demyelination in the brain and spinal cord, with multifocal invasion of lymphocytes, macrophages, and plasma cells. In severe cases of mastitis, the udder may appear to be composed of lymphoid tissue. Pathogenesis. The virus infects cells of the mononuclear system, resulting in the formation of non-neutralizing antibody to viral core proteins and envelope proteins. Immune complex formation in synovial, mammary gland, and neurological tissue is thought to result in the clinical changes observed. Most commonly, the carpal joint is affected, followed by the stifle, hock, and hip. The infection is lifelong. Differential diagnosis. The differential diagnosis for the neurologic form of CAEV should include copper deficiency, enzootic pneumonia, white muscle disease, listeriosis, and spinal cord disease or injury. The differential diagnosis for CAEV arthritis should include chlamydia and mycoplasma. Prevention and control. Herds can be screened for CAE by testing serologically, using an AGID or an enzyme-linked immunosorbent assay (ELISA) test. The ELISA is purported to be more sensitive, whereas the AGID is more specific. Individual animals show great variation in development of antibody. Because CAE is highly prevalent in the United States, and because seronegative animals can shed organisms in the milk, retesting herds at least annually may be necessary. Recently, an immuno-precipitation test for CAE has been developed that has high sensitivity and specificity. Control measures include management practices such as test and cull, prevention of milk transmission, and isolation of affected animals. Parturition must be monitored, and kids must be removed immediately and fed heat-treated colostrum (56° C for 1 hr). CAEV-negative goats should be separated from CAEV-positive goats. Treatment. There is no treatment for CAEV. h. Infectious Bovine Rhinotracheitis Virus (Infectious Bovine Rhinotracheitis-Infectious Pustular Vulvovaginitis) Etiology. The infectious bovine rhinotracheitis virus (IBRV) is also referred to as bovine herpesvirus 1 (BHV-1) and is an alphaherpesvirus. IBRV causes or contributes to several bovine syndromes, including respiratory and reproductive tract diseases. It is one of the primary pathogens in the bovine respiratory disease complex. Strains include BHV-1.1 (associated with respiratory disease), BHV 1.2 (associated with respiratory and genital diseases), and BHV 1.4 (associated with neurological diseases), which has been reclassified as bovine herpesvirus 5. Clinical signs and diagnosis. Diseases caused by the virus include conjunctivitis, rhinotracheitis, pustular vulvovaginitis, balanoposthitis, abortion, encephalomyelitis, and mastitis. The respiratory form is known as infectious bovine rhinotracheitis, and clinical signs may range from mild to severe, the latter particularly when there are additional respiratory viral infections or secondary bacterial infections. The mortality rate in more mature cattle is low, however, unless there is secondary bacterial pneumonia. Fever, anorexia, restlessness, hyperemia of the muzzle, gray pustules on the muzzle (that later form plaques), nasal discharge (that may progress from serous to mucopurulent), hyperpnea, coughing, salivation, conjunctivitis with excessive epiphora, and decreased production in dairy animals are typical signs. Open-mouth breathing may be seen if the larynx or nasopharygneal areas are blocked by mucopurulent discharges. Neonatal calves may develop respiratory as well as general systemic disease. In these cases, in addition to the symptoms already noted, the soft palate may become necrotic, and gastrointestinal tract ulceration occurs. Young calves are most susceptible to the encephalitic form; signs include dull attitude, head pressing, vocalizations, nystagmus, head tilt, blindness, convulsions, and coma, as well as some signs, such as discharges, seen with respiratory tract presentations. This form is usually fatal within 5 days. Abortion may occur simultaneously with the conjunctival or respiratory tract diseases, when the respiratory infection appears to be mild, or may be delayed by as much as 3 months after the respiratory tract disease signs. Infectious pustular vulvovaginitis is most commonly seen in dairy cows, and clinical signs may be mild and not noticed. Otherwise, signs are fever, depression, anorexia, swelling of the vulvar labia, vulvar discharge, and vestibular mucosa reddened by pustules. The cow will often carry her tail elevated away from these lesions. These soon coalesce, and a fibrous membrane covers the ulcerated area. If uncomplicated, the infection lasts about 4–5 days, and lesions heal in 2 weeks. Younger infected bulls may develop balanoposthitis with edema, swelling, and pain such that the animals will not service cows. Epizootiology and transmission. IBRV is widely distributed throughout the world, and adult animals are the reservoirs of infection. The disease is more common in intensive calf-rearing situations and in grouped or stressed cattle. Transmission is primarily by secretions, such as nasal, during and after clinical signs of disease. Modified live vaccines are capable of causing latent infections. Necropsy findings. Fibrinonecrotic rhinotracheitis is considered pathognomic for IBRV respiratory tract infections. There will be adherent necrotic lesions in the respiratory, ocular, and reproductive mucosa. When there are secondary bacterial infections, such as Pasteurella bronchopneumonia, findings will include congested tracheal mucosa and petechial and ecchymotic hemorrhages in that tissue. Lesions from the encephalitic form include lymphocytic meningoencephalitis and will be found throughout the gray matter (neuronal degeneration, perivascular cuffing) and white matter (myelitis, demyelination). Intranuclear inclusion bodies are not a common finding with this herpesvirus. Pathogenesis. In the encephalitic form, the virus first grows in nasal mucosa and produces plaques. These resolve within 11 days, and the encephalitis develops after the virus spreads centripetally to the brain stem by the trigeminal nerve dendrites. Latent infections are also established in neural tissue. Differential diagnosis. The severe oral erosions seen with BVDV infections are rare with infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV). The conjunctivitis of IBR may initially be mistaken for that of a Moraxella bovis (pinkeye) infection; the IBR will be peripheral, and there will not be corneal ulceration. Bovine viral diarrhea virus and IBRV are the most common viral causes of bovine abortion. Differentials for balanoposthitis include trauma from service. Prevention and control. Vaccination options include inactivated, attenuated, modified live, and genetically altered preparations. Some are in combination with parainfluenza 3 (PI-3) virus. The MLV preparations are administered intranasally; these are advantageous in calves for inducing mucosal immunity even when serologic passive immunity is already present and adequate. Some newer vaccines, with gene deletion, allow for serologic differentiation between antibody responses from infection or immunization. Bulls with the venereal form of the infection will transmit the virus in semen; intranasal vaccine may be used to provide some immunity. Treatment. Uncomplicated mild infections will resolve over a few weeks; palliative treatments, such as cleaning ocular discharges and supplying softened food, are helpful in recovery. Antibiotics are usually administered because of the high likelihood of secondary bacterial pneumonia. The encephalitic animals may need to be treated with anticonvulsants. i. Parainfluenza 3 (PI-3) Etiology. Parainfluenza 3, an RNA virus of the family Paramyxoviridae, causes mild respiratory disease of ruminants when it is the sole pathogen. The viral infection often predisposes the respiratory system to severe disease associated with concurrent viral or bacterial pathogens. Viral strains are reported to vary in virulence. Serotypes seen in the smaller ruminants are distinct from those isolated from cattle. Clinical signs and diagnosis. Infections ranging from asymptomatic to mild signs of upper respiratory tract disease are associated with this virus by itself; infections are almost never fatal. Clinical signs include ocular and nasal discharges, cough, fever, and increased respiratory rate and breath sounds. In pregnant animals, exposure to PI-3 can result in abortions. Clinical signs become apparent or more severe when additional viral pathogens are present, such as bovine viral diarrhea virus, or a secondary bacterial infection, such as Pasteurella haemolytica infection, is involved. Greater morbidity and mortality will be sequelae of the bacterial infections. Viral isolation or direct immunofluorescence antibody (IFA) from nasal swabs can be used for definitive diagnosis. Epizootiology and transmission. The virus is considered ubiquitous in cattle and is a common infection in sheep. Presently it is assumed that the virus is widespread in goats, but firm evidence is lacking. Necropsy findings. For an infection of PI-3 only, findings will be negligible. Some congestion of respiratory mucosa, swelling of respiratory tract-associated lymph nodes, and mild pneumonitis may be noted grossly and histologically. Intranuclear and intracytoplasmic inclusion bodies may be present in the mucosal epithelial cells. Findings will be similar but not as severe as those caused by bovine respiratory syncytial virus. Immunohistochemistry may also be used. Pathogenesis. PI-3 infects the epithelial mucosa of the respiratory tract; however, the disease is often asymptomatic when uncomplicated. Differential diagnosis. Differentials, particularly in cattle, include infections with other respiratory tract viruses of ruminants: IBRV, BVDV, bovine respiratory syncytial virus, and type 3 bovine adenovirus. Prevention and control. Immunization, management, and nutrition are important for this respiratory pathogen, as for others. In cattle, modified live vaccines for intramuscular (IM), subcutaneous (SC), or intranasal (IN) administration are available. The IM and SC routes provide immune protection within 1 week after administration but will not provide protection in the presence of passively acquired antibodies. It is contraindicated for pregnant animals because it will cause abortion. The IN route immunizes in the presence of passively acquired antibodies, provides immunity within 3 days of administration, and stimulates the production of interferon. Other vaccine formulations, about which less information is reported, include inactivated or chemically altered live-virus preparations; both are administered IM, and followup immunizations are needed within 4 weeks. Booster vaccinations are recommended for all preparations within 2–6 months after the initial immunization. All presently marketed vaccine products come in combination with other bovine respiratory viruses as multivaccine products. The humoral immunity protects against PI-3 abortions. There is no approved PI-3 vaccine for sheep and goats. The use of the cattle formulation in these smaller ruminants is not recommended. Sound management of housing, sanitation, nutrition, and preventive medicine programs are all equally important components in prevention and control. Treatment. Uncomplicated disease is not treated. j. Respiratory Syncytial Viruses of Ruminants Etiology. The respiratory syncytial viruses are pneumoviruses of the Paramyxoviridae family and are common causes of severe disease in ruminants, especially calves and yearling cattle. Two serotypes of the bovine respiratory syncytial virus (BRSV) have been described for cattle; these may be similar or identical to the virus seen in sheep and goats. Clinical findings and diagnosis. Infections may be subclinical or develop into severe illness. Clinical signs include fever, hyperpnea, spontaneous or easily induced cough, nasal discharge, and conjunctivitis. Interstitial pneumonia usually develops, and harsh respiratory sounds are evident on auscultation. Development of emphysema indicates a poor prognosis, and death may occur in the severe cases of the viral infection. Secondary bacterial pneumonia, especially with Pasteurella haemolytica, with morbidity and mortality, is also a common sequela. Abortions have been assciated with BRSV outbreaks. Diagnosis is based on virus isolation and serology (acute and convalescent). Nasal swabs for virus isolation should be taken when animals have fever and before onset of respiratory disease. Epizootiology and transmission. These viruses are considered ubiquitous in domestic cattle and are transmitted by aerosols. Necropsy findings. Gross lesions include consolidation of anteroventral lung lobes. Edema and emphysema are present. As the name indicates, syncytia, which may have inclusions, form in areas of the lungs infected with the virus. Necrotizing bronchiolitis, bronchiolitis obliterans, and hyaline membrane formation will be evident microscopically. Pathogenesis. The severe form of the disease, which often follows a mild preliminary infection, is thought to be caused by immune-mediated factors during the process of infection in the lung. Virulence may vary greatly among viral strains. Differential diagnosis. Differentials should include other ruminant respiratory tract viruses. Prevention and control. Vaccination should be part of the standard health program, and all animals should be vaccinated regularly. Vaccinations should be administered within 1–2 months of stressful events, such as weaning, shipping, and introduction to new surroundings. Currently available vaccines include an inactivated preparation and a modified live virus preparation administered intramuscularly or subcutaneously; immunity develops well in yearling animals, and colostral antibodies develop when cows are vaccinated during late gestation. Passive immunity from colostrum provides at least partial protection to calves in herds where disease is prevalent. But this immunity suppresses the mucosal IgA response and serum antibody responses. The basis for successful immune protection is the mucosal memory IgA, but this is difficult to achieve with present vaccine formulations. The virus is easily inactivated in the environment. Preventive measures in preweaning animals should include preconditioning to minimize weaning stress. Treatment. Recovery can be spontaneous; however, antibiotics and supportive therapy are useful to prevent or control secondary bacterial pneumonia. In severe cases, antihistamines and corticosteriods may also be necessary. Use of vaccine during natural infection is not productive and may result in severe disease. k. Ulcerative Dermatosis (Ovine Venereal Disease, Balanoposthitis) Etiology. Ulcerative dermatosis is a contagious disease of sheep only. It is caused by a poxvirus similar to but distinct from the causative agent of contagious ecthyma ( "Current Veterinary Therapy," 1993 ). Clinical signs and diagnosis. Lesions include ulcers and crusts associated with the skin and mucous membranes of the genitalia, face, and feet ( Bulgin, 1986 ). Genital lesions are much more common than the facial or coronal lesions. Discomfort may be associated with the lesions. Paraphimosis occasionally occurs. These lesions are painful; during breeding season, animals will avoid coitus. Morbidity is low to moderate, and mortality negligible if the flock is otherwise healthy. Diagnosis is based on clinical signs. Epizootiology and transmission. Endemic to the western United States, ulcerative dermatosis is transmitted through direct contact with abraded skin of the prepuce, vulva, face, and feet. Necropsy findings. Necropsy would rarely be necessary to diagnose an outbreak in a healthy flock. Findings will be similar to those described for contagious ecthyma. Pathogenesis. Following an incubation period of 2–5 days, the virus replicates in the epidermal cells and leads to necrosis and pustule formation. Pustules rapidly break, forming weeping ulcers. The ulcers scab over and eventually form a fibrotic scar. The disease usually resolves in 2–6 weeks. Rarely, the disease will persist for many months to more than a year. Differential diagnosis. The main differential is contagious ecthyma, which is grossly and histopathologically associated with epithelial hyperplasia. This is also a feature of ulcerative dermatosis. Prevention and control. No vaccine is available. Affected animals, especially males, should not be used for breeding. Treatment. Affected animals should be separated from the rest of the flock. Treatment is supportive, including antiseptic ointments and astringents. Research complications. Breeding and maintenance of the flocks' condition, because of the pain associated with eating, will be compromised during an outbreak. l. Border Disease Etiology. Border disease, also known as hairy shaker disease (or "fuzzies" in the southwestern United States), is a disease of sheep caused by a virus closely related to the bovine viral diarrhea virus (BVDV), a pestivirus of the Togaviridae family. Goats are also affected. The virus causes few pathogenic effects in cattle. Clinical signs and diagnosis. Border disease in ewes causes early embryonic death, abortion of macerated or mummified fetuses, or birth of lambs with developmental abnormalities. Lambs infected in utero that survive until parturition may be born weak and often exhibit a number of congenital defects such as tremor, hirsutism (sometimes darkly pigmented over the shoulders and head), hypothyroidism, central nervous system defects, and joint abnormalities, including arthrogryposis. Later, survivors may be more susceptible to diseases and may develop persistent, sometimes fatal, diarrhea. The virus infection produces similar clinical manifestations in goats, except that the hair changes are not seen. Diagnosis includes the typical signs described above, as well as serological evidence of viral infection. Virus isolation confirms the diagnosis. Epizootiology and transmission. The virus is present worldwide, and reports of disease are sporadic. Disease has occurred when no contact with cattle has occurred. Persistently infected animals, such as lambs, are shedding reservoirs of the virus in urine, feces, and saliva throughout their lives. Necropsy findings. Lesions include placentitis, and characteristic joint and hair-coat changes in the fetus. Histologically, axonal swelling, neuronal vacuolation, dysmyelination, and focal microgliosis are observed in central nervous system structures. Pathogenesis. The virus entering the ewe via the gastrointestinal or respiratory tracts penetrates the mucous membranes and causes maternal and fetal viremia. Infection during the first 45 days of gestation causes embryonic death. In lambs infected between 45 and 80 days, the virus activates follicular development, diminishes the myelination of neurons, and causes dysfunction of the thyroid gland. Infection after 80 days of gestation results in lambs that are born persistently infected. Infected lambs have high perinatal mortality; survivors have diminished signs over time but, as noted, continue to shed the virus. Prevention and control. Border disease can be prevented by vaccinating breeding ewes with killed-BVDV vaccine. Congenitally affected lambs should be maintained separately and disposed of as soon as humanely possible. New animals to the flock should be screened serologically. If cattle are housed nearby, vaccination programs for BVDV should be maintained. Treatment. There is no treatment other than supportive care for affected animals. m. Contagious Ecthyma (Contagious Pustular Dermatitis, Sore Mouth, Orf) Etiology. Contagious ecthyma, also known as contagious pustular dermatitis, sore mouth, or orf, is an acute dermatitis of sheep and goats caused by a parapoxvirus. This disease occurs worldwide and is zoonotic. Naturally occurring disease has also been reported in other species such as musk ox and reindeer. Other parapoxviruses infect the mucous membranes and skin of cattle, causing the diseases bovine pustular dermatitis and pseudocowpox. Clinical signs and diagnosis. The disease is characterized by the presence of papules, vesicles, or pustules and subsequently scabs of the skin of the face, genitals of both sexes, and coronary bands of the feet. Lesions develop most frequently at mucocutaneous junctions and are found most commonly at the commissures of the mouth. Orf is usually found in young animals less than 1 year of age. Younger lambs and kids will have difficulty nursing and become weak. Lesions may also develop on udders of nursing dams, which may resist suckling by offspring to nurse, leading to secondary mastitis. The scabs may appear nodular and raised above the surface of the surrounding skin. Morbidity in a susceptible group of animals may exceed 90%. Mortality is low, but the course of the disease may last up to 6 weeks. Diagnosis is based on characteristic lesions. Biopsies may reveal eosinophilic cytoplasmic inclusions and proliferative lesions under the skin. Electron microscopy will reveal the virus itself. Disease is confirmed by virus isolation. Epizootiology and transmission. All ages of sheep and goats are susceptible. Seasonal occurrences immediately after lambing and after entry into a feedlot are common; stress likely plays a role in susceptibility to this viral disease. Older animals develop immunity that usually prevents reinfection for at least 1 or more years. Resistant animals may be present in some flocks or herds. The virus is very resistant to environmental conditions and may contaminate small-ruminant facilities, pens, feedlots, and the like for many years as the result of scabs that have been shed from infected animals. Transmission occurs through superficial lesions such as punctures from grass awns, scrapes, shearing, and other common injuries. Necropsy findings. Necropsy findings include ballooning degeneration of epidermal and dermal layers, edema, granulomatous inflammation, vesiculation, and cellular hyperplasia. Secondary bacterial infection may also be evident. Pathogenesis. The virus is typical of the Poxviridae, resembling sheep poxvirus (not found in the United States) and vaccinia virus and replicating in the cytoplasm of epithelial cells. Following an incubation period of 2–14 days, papules and vesicles develop around the margins of the lips, nostrils, eyelids, gums, tongue, or teats; skin of the genitalia; or coronary band of the feet. The vesicles form pustules that rupture and finally scab over. Differential diagnosis. Ulcerative dermatosis and bluetongue virus should be considered in both sheep and goats. An important differential in goats is staphylococcal dermatitis. Prevention and control. Individuals handling infected animals should be advised of precautions beforehand, should wear gloves, and should separate work clothing and other personal protective equipment. Clippers, ear tagging devices, and other similar equipment should always be cleaned and disinfected after each use. Colostral antibodies may not be protective. Vaccinating lambs and kids with commercial vaccine best prevents the disease. Dried scabs from previous outbreaks may also be used by rubbing the material into scarified skin on the inner thigh or axilla. Animals newly introduced to infected premises should be vaccinated upon arrival. Precautions must be taken when vaccinating animals, because the vaccine may induce orf in the animal handlers; it is not recommended to vaccinate animals in flocks already free of the disease. Affected dairy goats should be milked last, using disposable towels for cleaning teat ends. Treatment. Affected animals should be isolated and provided supportive care, especially tube feeding for young animals whose mouths are too sore to nurse. Treatment should also address secondary bacterial infections of the orf lesions, including systemic antibiotics for more severe infections. Treatment for myiasis may also be necessary. The viral infection is self-limiting, with recovery in about 4 weeks. Research complications. Carrier animals may be a factor in flock or herd outbreaks. Contagious ecthyma is a zoonotic disease, and human-to-human transmission can also occur. The virus typically enters through abrasions on the hands and results in a large (several centimeters) nodule that is described as being extremely painful and lasting for as many as 6 weeks. Lesions heal without scarring. n. Foot-and-Mouth Disease Etiology. Foot-and-mouth disease (FMD) is caused by the foot-and-mouth disease virus, a Picornavirus in the Aphthovirus genus. The disease is also referred to as aftosa or aphthous fever. Seven immunologically distinct types of the virus have been identified, with 60 subtypes within those 7. Epidemics of the disease have occurred worldwide. North and Central America have been free of the virus since the mid-1950s. This is a reportable disease in the United States; clinical signs are very similar to other vesicular diseases. Cattle (and swine) are primarily affected, but disease can occur in sheep and is usually subclinical in goats. Clinical signs and diagnosis. In addition to vesicle formation around and in the mouth, hooves, and teats, fever, anorexia, weakness, and salivation occur. Vesicles may be as large as 10 cm, rupture after 2 days, and subsequently erode. Secondary bacterial infections often occur at the erosions. Anorexia is likely due to the pain associated with the oral lesions. High morbidity and low mortality, except for the high mortality in young cattle, are typical. Diagnosis must be based on ELISA, virus neutralization, fluorescent antibody tests, and complement fixation. Epizootiology and transmission. Domestic and wild ruminants and several other species, such as swine, rats, bears, and llamas are hosts. Asymptomatic goats can serve as virus reservoirs for more susceptible cohoused species such as cattle. Greater mortality occurs in younger animals. The United States, Great Britain, Canada, Japan, New Zealand, and Australia are FMD-free, whereas the disease is endemic in most of South America, parts of Europe, and throughout Asia and Africa. The virus is very contagious and is spread primarily by the inhalation of aerosols, which can be carried over long distances. Transmission may also occur by fomites, such as shoes, clothing, and equipment. Human hands, soiled bedding, and animal products such as frozen or partially cooked meat and meat products, hides, semen, and pasteurized milk also serve as sources of virus. Necropsy findings. Vesicles, erosions, and ulcers are present in the oral cavity as well as on the rumen pillars and mammary alveolar epithelium. Myocardial and skeletal muscle degeneration (Zenker's) is most common (and accounts for the greater mortality) in younger animals. Histological findings include lack of inclusion bodies. Vesicular lesions include intracellular and extracellular edema, cellular degeneration, and separation of the basal epithelium. Pathogenesis. The incubation period is 2–8 days. The virus replicates in the pharynx and digestive tract in the cells of the stratum spinosum, and viremia and spread of virus to many tissues occur before clinical signs develop. Virus shedding begins about 24 hr before clinical signs are apparent. Vesicles result from the separation of the superficial epithelium from the basal epithelium. Fluid fills the basal epithelium, and erosions develop when the epithelium sloughs. Persistent infection also occurs, and virus can be found for months or years in the pharnyx; the mechanisms for the persistence are not known. Differential diagnosis. Vesicular stomatitis is the principal differential. Other differentials include contagious ecthyma (orf), rinderpest, bluetongue, malignant catarrhal fever, bovine papular stomatitis, bovine herpes mammillitis, and infectious bovine rhinotracheitis virus infection. Prevention and control. Movement of animals and animal products from endemic areas is regulated. Quarantine and slaughter are practiced in outbreaks in endemic areas. Quarantine and vaccination are also used in endemic areas, but vaccines must be type-specific and repeated 2 or 3 times per year to be effective and will provide only partial protection. Autogenous vaccines are best in an outbreak. Passive immunity protects calves for up to 5 months after birth. The virus is inactivated by extremes of pH, sunlight, high temperatures, sodium hydroxide, sodium carbonate, and acetic acid. Treatment. Nursing care and antibiotic therapy to minimize secondary reactions help with recovery. Humoral immunity is considered the more important immune mechanism, with cell-mediated immunity of less importance. Research complications. Rare cases in humans have been reported. Importation into the United States of animal products from endemic areas is prohibited. o. Malignant Catarrhal Fever Etiology. Malignant catarrhal fever (MCF) is a severe disease primarily of cattle. The agents of MCF are viruses of the Gammaherpesvirinae subfamily. Alcelaphine herpesvirus 1 and 2 and ovine herpesvirus 2 are known strains. The alcelaphine strains are seen in Africa. The ovine strain is seen in North America. The alcelaphine and ovine strains differ in incubation times and duration of illness. Disease may occur sporadically or as outbreaks. Clinical signs and diagnosis. Signs range from subclinical to recrudescing latent infections to the lethal disease seen in susceptible species, such as cattle. Sudden death may also occur in cattle. Presentations of the disease may be categorized as alimentary, encephalitis, or skin forms; all three may occur in an animal. Corneal edema starting at the limbus and progressing centripetally is a nearly pathognomonic sign; photophobia, severe keratoconjunctivitis, and ocular involvement may follow. Other signs include prolonged fever, oral mucosal erosions, salivation, lacrimation, purulent nasal discharge, encephalitis, and pronounced lymphadenopathy. As the disease progresses, cattle may shed horns and hooves. In North America, cattle will also have severe diarrhea. The course of the disease may extend to 1 week. Recovery is usually prolonged, and some permanent debilitation may occur. The disease is fatal in severely affected individuals. History of exposure, as well as the clinical signs and lesions, contributes to the diagnosis. Serology, PCR-based assays, viral isolation, and cell-culture assays, such as cytopathic effects on thyroid cell cultures, are also used. Because of the susceptibility of rabbits, inoculation of this species may be used. In less severe outbreaks or individual animal disease, definitive diagnosis may never be made. Epizootiology and transmission. Most ruminant species are susceptible to MCF. Sheep are sources of infection for cattle, which are dead-end hosts. Other ruminants, including goats, may harbor the virus. Both the African and North American strains are transmissible to rabbits; these animals develop a fatal lymphoproliferative disease. The virus is shed from the nasopharynx. Infection of lambs is horizontal from direct contact. Other sources of the virus include water troughs, placental tissues, contaminated fomites, aerosols, birds, and caretakers. Necropsy. Gross findings at necropsy include necrotic and ulcerated nasal and oral mucosa; thickened, edematous, ulcerated, and hemorrhagic areas of the intestinal tract; swollen, friable, and hemorrhagic lymph nodes and other lymphatic tissues; and erosion of affected mucosal surfaces. Lymph nodes should be submitted for histological examination. Histological findings include nonsuppurative vasculitis and encephalitis; large numbers of lymphocytes and lymphoblasts will be present without evidence of virus. Pathogenesis. The incubation period may be up to 3 months. Vascular endothelium and all epithelial surfaces will be affected. The virus is believed to cause proliferation of cytotoxic T lymphocytes with natural killer cell activities, and the resulting lesions are due to an autoimmune type of phenomenon. Differential diagnoses. The differentials for this disease are bovine viral diarrhea/mucosal disease, bovine respiratory disease complex, infectious bovine rhinotracheitis, bluetongue, vesicular stomatitis, and foot-and-mouth disease. Causes of encephalitis, such as bovine spongiform encephalopathy and rabies, should be considered. In Africa, rinderpest is also a differential. Other differentials are arsenic toxicity and chlorinated naphthalene toxicity. Prevention and control. No vaccine is available at this time. In North America, sheep, as well as cattle that have been either exposed or that have survived the disease, are reservoirs for outbreaks in other cattle. If there is concern regarding presence of the virus, animals should be screened serologically; once an animal has been infected, it remains infected indefinitely. Lambs can be free of the infection if removed from the flock at weaning. The virus is very fragile outside of host's cells and will not survive in the environment for more than a few hours. Treatment. Affected and any exposed animals should be isolated from healthy animals. There is no specific treatment for MCF; supportive treatment may improve recovery rates. Corticosteroids may be useful. p. Ovine Progressive Pneumonia (Maedi/Visna) Etiology. An RNA virus in the lentivirus group of the Retroviridae family causes ovine progressive pneumonia (OPP), or maedi/visna. Maedi refers to the progressive pneumonia presentation of the disease; visna refers to the central nervous system disease, which is reported predominantly in Iceland. Visna has been reported in goats but may have been due to caprine arthritis encephalitis infection. Clinical signs and diagnosis. OPP is a viral disease of adult sheep characterized by weakness, unthriftiness, weight loss, and pneumonia ( Pepin et al., 1998 ; de la Concha Bermejillo, 1997 ). Clinically, animals exhibit signs of progressive pulmonary disease after an extremely long incubation period of up to 2 years. Respiratory rate and dyspnea gradually increase as the disease progresses. The animal continues to eat throughout the disease; however, animals progressively lose weight and become weak. Additionally, mastitis is a common clinical feature. Thoracic auscultation reveals consolidation of ventral lung lobes; and hematological findings indicate anemia and leukocytosis. The rare neurological signs include flexion of fetlock and pastern joints, tremors of facial muscles, progressive paresis and paralysis, depression, and prostration. Death occurs in weeks to months. The disease can be serologically diagnosed with agar gel immunodiffusion (AGID) tests, virus isolation, serum neutralization, complement fixation, and enzyme-linked immunosorbent assay (ELISA) tests. Epizootiology and transmission. Sixty-eight percent of sheep in some states have been infected with the virus ( Radostits et al., 1994 ). It is transmitted horizontally via inhalation of aerosolized virus particles and vertically between the infected dam and fetus. In addition, transmission through the milk or colostrum is considered common ( Knowles, 1997 ). Necropsy findings. Lesions are observed in lungs, mammary glands, joints, and the brain. Pulmonary adhesions, ventral lung lobe consolidation, bronchial lymph node enlargement, mastitis, and degenerative arthritis are visualized grossly. Meningeal edema, thickening of the choroid plexus, and foci of leukoencephalomalacia are seen in the central nervous system (CNS). Histologically, interalveolar septal thickening, lymphoid hyperplasia, histiocyte and fibrocyte proliferation, and squamous epithelial changes are seen in the lungs. Meningitis, lymphoid hyperplasia, demyelination, and glial fibrosis are seen in the CNS. Pathogenesis. The virus has a predilection for the lungs, mediastinal lymph nodes, udder, spleen, joints, and rarely the brain. After initial infection, the virus integrates into the DNA of mature monocytes and persists as a provirus. Later in the animal's life, infected monocytes mature as lung (and other tissue) macrophages and establish active infection. The virus induces lymphoproliferative disease, histiocyte and fibrocyte proliferation in the alveolar septa, and squamous metaplasia. Pulmonary alveolar and vascular changes impinge on oxygen and carbon dioxide exchange and lead to serious hypoxia and pulmonary hypertension. Secondary bacterial pneumonia may contribute to the animal's death. Differential diagnosis. Pulmonary adenomatosis is the differential diagnosis. Prevention and control. Isolating or removing infected animals can prevent the disease. Facilities and equipment should also be disinfected. Treatment. Treatment is unsuccessful. q. Poxviruses of Ruminants i. Ovine viral dermatosis. Ovine viral dermatosis is a venereal disease of sheep caused by a parapoxvirus distinct from contagious ecthyma. The disease resolves within 2 weeks in healthy animals, but lesions are painful and resemble those of Corynebacterium renale posthitis/vulvovaginitis. Symptomatic treatment may be necessary in some cases. There is no vaccine. Animals should not be used for breeding while clinical signs are present. ii. Proliferative stomatitis (bovine papular stomatitis) Etiology. A parapoxvirus is the causative agent of bovine papular stomatitis. This virus is considered to be closely related to the parapoxvirus that causes contagious ecthyma and pseudocowpox. It is also a zoonotic disease. The disease is not considered of major consequence, but high morbidity and mortality may be seen in severe outbreaks. In addition, lesions are comparable in appearance to those seen with vesicular stomatitis, bovine viral diarrhea virus, and foot-and-mouth disease. The disease occurs worldwide. Clinical signs and diagnosis. Raised red papules or erosions or shallow ulcers on the muzzle, nose, oral mucosa (including the hard palate), esophagus, and rumen of younger cattle are the most common findings. In some outbreaks, the papules will be associated with ulcerative esophagitis, salivation, diarrhea, and subsequent weight loss. Lesions persist or may come and go over a span of several months. Morbidity among herds may be 100%. Mortalities are rare. Bovine papular stomatitis is associated with "rat tail" in feedlot cattle. Animals continue to eat and usually do not show a fever. No lesion is seen on the feet. The infection may also be asymptomatic. Diagnosis is based on clinical signs, histological findings, and viral isolation. Epizootiology and transmission. Cattle less than 1 year of age are most commonly affected, and disease is rare in older cattle. Transmission is by animal-to-animal contact. Necropsy findings. Raised papules may be found around the muzzle and mouth and involve the mucosa of the esophagus and rumen. Histologically, epithelial cells will show hydropic degeneration and hyperplasia of the lamina propria. Eosinophilic inclusions will be in the cytoplasm of infected epithelial cells. Pathogenesis. Following exposure to the virus, erythematous macules most commonly appear on the nares, followed by the mouth. These become raised papules within a day, regressing after days to weeks; the lesions that remain will be persistent yellow, red, or brown spots. Some infections may recur or persist, with animals showing lesions intermittently or continuously over several months. Differential diagnosis. Pseudocowpox, vesicular stomatitis, foot-and-mouth disease, and bovine viral diarrhea virus infection are the differentials for this disease. The differential for the "rat tail" clinical sign is Sarcocystis infection. Prevention and control. There is no vaccine available for bovine papular stomatitis. Because of the similarity of this virus to the parapoxvirus of contagious ecthyma, it is important to be aware of the persistence in the environment and susceptibility of younger cattle. Vaccination using the local strain, and the skin scarification technique for orf, have been protective. Handlers should wear gloves and protective clothing. Treatment. Cattle usually will not require extensive nursing care, but lesions with secondary bacterial infections should be treated with antibiotics. Research complications. Handlers may develop lesions on their hands at sites of contact with lesions of cattle. iii. Pseudocowpox Etiology. Pseudocowpox is a worldwide cattle disease caused by a parapoxvirus related to the causative agents of contagious ecthyma and bovine papular stomatitis (see Sections III,A,2,m and III,A,2,q,ii). Lesions are confined to the teats. This is also a zoonotic disease. Clinical signs and diagnosis. Minor lesions are usually confined to the teats. These are distinctive because of the ring- or horseshoe-shaped scab that develops after 10 days. Additional lesions sometimes develop on the udder, the medial aspect of the thighs, and the scrotum. The teat lesions may predispose to mastitis. Pathogenesis. The virus is spread by contaminated hands, equipment, and fomites. Differential diagnosis. Differentials include bovine herpes mammillitis and papillomatosis. Prevention and control. Milking hygiene is helpful in control. Treatment. Lesions should be treated symptomatically, and affected animals milked last. Research complications. Like other related poxviruses, this virus causes nodular lesions on humans. r. Pulmonary Adenomatosis (Jaagsiekte) Etiology. Pulmonary adenomatosis is a rare but progressive wasting disease of sheep, with worldwide distribution. Pulmonary adenomatosis is caused by a type D retrovirus antigenically related to the Mason-Pfizer monkey virus. Jaagsiekte was the designation when the disease was described originally in South Africa. Clinical signs and diagnosis. Typical clinical signs include progressive respiratory signs such as dyspnea, rapid respiration, and wasting. The disease is diagnosed by these chronic clinical signs and histology. Epizootiology and transmission. The disease is transmitted by aerosols. Body fluids of viremic animals, such as milk, blood, saliva, tears, semen, and bronchial secretions, will contain the virus or cells carrying the virus. Necropsy. The adenomas and adenocarcinomas will be small firm lesions distributed throughout the lungs. The adenocarcinomas metastasize to regional lymph nodes. Pathogenesis. As with ovine progressive pneumonia (OPP), the incubation period is up to 2 years long. Adenocarcinomatous lesions arising from type II alveolar epithelial cells may be discrete or confluent and involve all lung lobes. Differential diagnosis. This disease occurs coincidentally with or is a differential diagnosis for OPP. Treatment. No treatment is effective. s. Papillomatosis (Warts, Verrucae) Etiology. Cutaneous papillomatosis is a very common disease in cattle and is much less common among sheep and goats. The disease is a viral-induced proliferation of the epithelium of the neck, face, back, and legs. These tumors are caused by a papillomavirus (DNA virus) of the Papovaviridae family, and the viruses are host-specific and often body site-specific. Most are benign, although some forms in cattle and one form in goats can become malignant. In cattle, the site specificity of the papillomavirus strains are particularly well recognized. Designations of the currently recognized bovine papillomavirus (BPV) types are BPV-1 through BPV-5. Clinical signs and diagnosis. The papillomas may last up to 12 months and are seen more frequently in younger animals. Lesions have typical wart appearances and may be single or multiple, small (1 mm) or very large (500 mm). The infections will generally be benign, but pain will be evident when warts develop on occlusal surfaces or within the gastrointestinal tract. In addition, when infections are severe, weight loss may occur. When warts occur on teats, secondary mastitis may develop. In cattle, BPV-1 and BPV-2 cause fibropapillomas on teats and penises or on head, neck, and dewlap, respectively. BPV-3 causes flat warts that occur in all body locations, BPV-4 causes warts in the gastrointestinal tract, and BPV-5 causes small white warts (called rice-grain warts) on teats. Warts caused by BPV-3 and BPV-5 do not regress spontaneously. Prognosis in cattle is poor only when papillomatosis involves more than 20% of the body surface. In sheep, warts are the verrucous type. The disease is of little consequence unless the warts develop in an area that causes discomfort or incapacitation such as between the digits, on the lips, or over the joints. In adult sheep, warts may transform to squamous cell carcinoma. In goats, the disease is rare, and the warts are also of the verrucous type and occasionally may develop into squamous cell carcinoma. Warts on goat udders tend to be persistent. Diagnosis is made by observing the typical proliferative lesions. Epizootiology and transmission. Older animals are less sensitive to papillomatosis than young animals, although immunosu-pressed animals of any age may develop warts as the result of harbored latent infections. The virus is transmitted by direct and indirect (fomite) contact, entering through surface wounds and sites such as tattoos. Pathogenesis. The incubation period ranges from 1 to 6 months. The virus induces epidermal and fibrous tissue proliferation, often described as cauliflower-like skin tumors. The disease is generally self-limiting. Differential diagnosis. In sheep and goats, differentials include contagious ecthyma, ulcerative dermatosis, strawberry foot rot, and sheep and goat pox. Prevention and control. Commercial vaccines (available only for cattle) or autogenous vaccines must be used with a recognition that papovavirus strains are host-specific and that immunity from infection or vaccination is viral-type-specific. Autogenous vaccines are generally considered more effective. Some vaccine preparations are effective at prevention but not treatment of outbreaks. Viricidal products are recommended for disinfection of contaminated environments. Minimizing cutaneous injuries and sanitizing equipment (tattoo devices, dehorners, ear taggers, etc.) in a virucidal solution between uses are also recommended preventive and control measures. Halters, brushes, and other items may also be sources of virus. Treatment. Warts will often spontaneously resolve as immunity develops. In severe cases or with flockwide or herdwide problems, affected animals should be isolated from nonaffected animals, and premises disinfected. Warts can be surgically excised and autogenous vaccines can be made and administered to help prevent disease spread. Cryosurgery with liquid nitrogen or dry ice has also proven to be successful for wart removal. Topical agents such as podophyllin (various formulations) and dimethyl sulfoxide may be applied to individual lesions once daily until regression. t. Pseudorabies (Mad Itch, Aujeszky's Disease) Etiology. Pseudorabies is an acute encephalitic disease caused by a neurotropic alphaherpesvirus, the porcine herpesvirus 1. One serotype is recognized, but strain differences exist. The disease has worldwide distribution. It is a primarily a clinical disease of cattle, with less frequent reports (but no less severe clinical manifestations) in sheep and goats. Clinical signs and diagnosis. A range of clinical signs is seen during the rapid course of this usually fatal disease. At the site of virus inoculation or in other locations, abrasions, swelling, intense pruritus, and alopecia are seen. Pruritus will not be asymmetric. Animals will also become hyperthermic and will vocalize frantically. Other neurological signs range from hoof stamping, kicking at the pruritic area, salivation, tongue chewing, head pressing and circling, to paresthesia or hyperesthesia, ataxia, and conscious proprioceptive deficits. Nystagmus and strabismus are also seen. Animals will be fearful or depressed, and aggression is sometimes seen. Recumbency and coma precede death. Diagnostic evidence includes clinical findings; virus isolation from nasal or pharyngeal secretions or postmortem tissues; and histological findings at necropsy. Serology of affected animals is not productive, because of the rapid course. If swine are housed nearby, or if swine were transported in the same vehicles as affected animals, serological evaluations are worthwhile from those animals. Epizootiology and transmission. Swine are the primary hosts for pseudorabies virus, but they are usually asymptomatic and serve as reservoirs for the virus. The infection can remain latent in the trigeminal ganglion of pigs and recrudesce during stressful conditions. Other animals are dead-end hosts. The unprotected virus will survive only a few weeks in the environment but may remain viable in meat (including carcasses) or saliva and will survive outside the host, in favorable conditions, in the summer for several weeks and the winter for several months. Transmission is by oral, intranasal, intradermal, or subcutaneous introduction of the virus. When the virus is inhaled, the clinical signs of pruritus are less likely to be seen. Transmission can also be by inadvertent exposure (e.g., contaminated syringes) of ruminants to the modified live vaccines developed for use in swine. Spread between infected ruminants is a less likely means of transmission, because of the relatively short period of virus shedding. Transport vehicles used for swine may also be sources of the virus. Raccoons are believed to be vectors of the virus. Horses are resistant to infection. Necropsy findings. There is no pathognomonic gross lesion. Definitive histologic findings include severe, focal, nonsuppurative encephalitis and myelitis. Eosinophilic intranuclear inclusion bodies (Cowdry type A) may be present in some affected neurons. Methods such as immunofluorescence and immunoperoxidase staining can be used to show presence of the porcine herpesvirus 1. Pathogenesis. The incubation period is 90–156 hr and duration of the illness is 8–72 hr. The longest duration is seen in animals with pruritus around the head. Differential diagnoses. Differentials for the neurologic signs of pseudorabies infection include rabies, polioencephalomalacia, salt poisoning, meningitis, lead poisoning, hypomagnesemia, and enterotoxemia. Those for the intense pruritus include psoroptic mange and scrapie in sheep, sarcoptic mange, and pediculosis. Prevention and control. Pseudorabies is a reportable disease in the United States, where a nationwide eradication program exists; states are rated regarding status. Effective disinfectants include sodium hypochlorite (10% solution), formalin, peracetic acid, tamed iodines, and quaternary ammonium compounds. Five minutes of contact time is required, and then surfaces must be rinsed. Other disinfectant methods for viral killing include 6 hr of formaldehyde fumigation, or 360 min of ultraviolet light. Transport vehicles should be cleaned and disinfected between species. Serological screening for pseudorabies of swine housed near ruminants is essential. Treatment. There is no treatment, and most affected animals die. Research complications. Swine housed close to research ruminants should be serologically screened prior to purchase, and all transport vehicles should be cleaned and disinfected between loads of large animals. Humans have been reported to seroconvert. The porcine herpesvirus 1 shares antigens with the infectious bovine rhinotracheitis virus. u. Rabies (Hydrophobia) Etiology. Rabies is a sporadic but fatal, acute viral disease affecting the central nervous system. The rabies virus is a neurotropic RNA virus of the Lyssavirus genus and the Rhabdoviridae family. Sheep, goats, and cattle are susceptible. The zoonotic potential of this virus must be kept in mind at all times when handling moribund animals with neurological signs characteristic of the disease. Rabies is endemic in many areas of the world and within areas of the Unites States. This is a reportable disease in North America. Clinical findings and diagnosis. Animals generally progress through three phases: prodromal, excitatory, and paralytic. Many signs in the different species during these stages are nonspecific, and forms of the disease are also referred to as dumb or furious. During the short prodromal phase, animals are hyperthermic and apprehensive. Animals progress to the excitatory phase, during which they refuse to eat or drink and are active and aggressive. Repeated vocalizations, tenesmus, sexual excitement, and salivation occur during this phase. The final paralytic stage, with recumbency and death, occurs over several hours to days. This paralytic stage is common in cattle, and animals may simply be found dead. The clinical course is usually 1–4 days. Diagnosis is based on clinical signs, with a progressive and fatal course. Confirmation presently is made with the fluorescent antibody technique on brain tissue. Epizootiology and transmission. The rabies virus is transmitted via a bite wound inflicted by a rabid animal. Cats, dogs, raccoons, skunks, foxes, wild canids, and bats are the common disease vectors in North America. Virus is also transmitted in milk and aerosols. Necropsy findings. Few lesions are seen at necropsy. Many secondary lesions from manic behaviors during the course of disease may be evident. Histological findings will include nonsuppurative encephalitis. Negri bodies in the cytoplasm of neurons of the hippocampus and in Purkinje's cells are pathognomonic histologic findings. Pathogenesis. After exposure, the incubation period is variable, from 2 weeks to several months, depending on the distance that the virus has to travel to reach the central nervous system. The rabies virus proliferates locally, gains access to neurons by attaching to acetylcholine receptors, via a viral surface glycoprotein, migrates along sensory nerves to the spinal cord and brain, and then descends via cranial nerves (trigeminal, facial, olfactory, glossopharyngeal) to oral and nasal cavity structures (i.e., salivary glands). The fatal outcome is currently believed to be multifactorial, related to anorexia, respiratory paralysis, and effects on the pituitary. Differential diagnosis. Rabies should be included on the differential list when clinical signs of neurologic disease are evident. Other differentials for ruminants include herpesvirus encephalitis, thromboemobolic meningoencephalitis, nervous ketosis, grass tetany, and nervous cocciodiosis. Prevention and control. Vaccines approved for use cattle and sheep are commercially available and contain inactivated virus; there is not one available in the United States for goats. Ruminants in endemic areas, such as the East Coast of the United States, should be routinely vaccinated. Any animals housed outside that may be exposed to rabid animals should be vaccinated. Vaccination programs generally begin at 3 months of age, with a booster at 1 year of age and then annual or triennial boosters. Awareness of the current rabies case reports for the region and wildlife reservoirs, however, is important. Monitoring for and exclusion of wildlife from large-animal facilities are worthwhile preventive measures. The virus is fragile and unstable outside of a host animal. Research complications. Aerosolized virus is infective. Personal protective equipment, including gloves, face mask, and eye shields, must be worn by individuals handling animals that are manifesting neurological disease signs. v. Transmissible Spongiform Encephalopathies i. Bovine spongiform encephalopathy (mad cow disease). Bovine spongiform encephalopathy, a transmissible spongiform encephalopathy (TSE), is not known to occur in the United States, where since 1989 it has been listed as a reportable disease. The profound impact of this disease on the cattle industry in Great Britain during the past two decades is well known. The disease may be caused by a scrapielike (prion) agent. It is believed that the source of infection for cattle was feedstuff derived from sheep meat and bonemeal that had been inadequately treated during processing. The incubation period of years, the lack of detectable host immune response, the debilitating and progressive neurological illness, and the pathology localized to the central nervous system are characteristics of the disease, and are is comparable to the characteristics of other TSE diseases such as scrapie, which affects sheep and goats. In addition, the infectious agent is extremely resistant to dessication and disinfectants. Confirmation of disease is by histological examination of brain tissue collected at necropsy; the vacuolation that occurs during the disease will be symmetrical and in the gray matter of the brain stem. Molecular biology techniques, such as Western blots and immunohistochemistry, may also be used to identify the presence of the prion protein. Differentials include many infectious or toxic agents that affect the bovine nervous and musculoskeletal systems, such as rabies, listeriosis, and lead poisoning. Metabolic disorders such as ketosis, milk fever, and grass tetany are also differentials. There is no vaccine or treatment. Prevention focuses on import regulations and not feeding ruminant protein to ruminants; recent USDA regulations prohibit feeding any mammalian proteins to ruminants. ii. Scrapie Etiology. Scrapie is a sporadic, slow, neurodegenerative disease caused by a prion. Scrapie is a reportable disease. It is much more common in sheep than in goats. The disease is similar to transmissible mink encephalopathy, kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy (mad cow disease). Prions are nonantigenic, replicating protein agents. Clinical signs and diagnosis. During early clinical stages, animals are excitable and hard to control. Tremors of head and neck muscles, as well as uncoordinated movements and unusual "bunny-hopping" gaits are observed. In advanced stages of the disease, animals experience severe pruritus and will self-mutilate while rubbing on fences, trees, and other objects. Blindness and abortion may also be seen. Morbidity may reach 50% within a flock. Most animals invariably die within 4–6 weeks; some animals may survive 6 months. In goats, the disease is also fatal. Pruritus is generally less severe but may be localized. A wide range of clinical signs have also been noted in goats, including listlessness, stiffness or restlessness, or behavioral changes such as irritability, hunched posture, twitching, and erect tail and ears. As with sheep, the disease gradually progresses to anorexia and debilitation. Diagnosis can be made by clinical signs and histopathological lesions. A newer diagnostic test in live animals is based on sampling from the third eyelid. Tests for genetic resistance or susceptibility require a tube of EDTA blood and are reasonably priced. Epizootiology and transmission. The Suffolk breed of sheep tends to be especially susceptible. Scrapie has also been reported in several other breeds, including Cheviot, Dorset, Hampshire, Corriedale, Shropshire, Merino, and Rambouillet. It is believed that there is hereditary susceptibility in these breeds. Targhees tend to be resistant. Genomic research indicates there are two chromosomsal sites governing this trait; these sites are referred to codons 171 (Q, R, or H genes can be present) and 136 (A or V genes can be present). Of the five genes, R genes appear to confer immunity to clinical scrapie in Suffolks in the United States. Affected Suffolks in the United States that have been tested have been AA QQ. The disease is also enzootic is many other countries. The disease tends to affect newborns and young animals; however, because the incubation period tends to range from 2 to 5 years, adult animals display signs of the disease. Scrapie is transmitted horizontally by direct or indirect contact; nasal secretions or placentas serve as sources of the infectious agent. Vertical transmission is questioned, and transplacental transmission is considered unlikely. Necropsy findings. At necropsy, no gross lesion is observed. Histopathologically, neuronal vacuolization, astrogliosis, and spongiform degeneration are visualized in the brain stem, the spinal cord, and especially the thalamus. Inflammatory lesions are not seen. Pathogenesis. Replication of the prions probably occurs first in lymphoid tissues throughout the host's body and then progresses to neural tissue. Differential diagnosis. In sheep and goats, depending on the speed of onset, differentials for the pruritus include ectoparasites, pseudorabies, and photosensitization. Prevention and control. If the disease diagnosed in a flock, quarantine and slaughter, followed by strict sanitation, are usually required. The U.S. Department of Agriculture has approved the use of 2% sodium hydroxide as the only disinfectant for sanitation of scrapie-infected premises. Prions are highly resistant to physicochemical means of disinfection. Artificial insemination or embryo transfer has been shown to decrease the spread of scrapie ( Linnabary et al., 1991 ). Treatment. No vaccine or treatment is available. Research complications. As noted, this is a reportable disease. Stringent regulations exist in the United States regarding importation of small ruminants from scrapie-infected countries. w. Vesicular Stomatitis Virus Etiology. Vesicular stomatitis (VS) is caused by the vesicular stomatitis virus (VSV), a member of the Rhabdoviridae. Three serotypes are recognized: New Jersey, Indiana, and Isfahan. The New Jersey and Indiana strains cause sporadic disease in cattle in the United States. The disease is rare in sheep. Clinical signs and diagnosis. Adult cattle are most likely to develop VS. Fever and development of vesicles on the oral mucous membranes are the initial clinical signs. Lesions on the teats and interdigital spaces also develop. The vesicles progress quickly to ulcers and erosions. The animal's tongue may be severely involved. Anorexia and salivation are common. Weight loss and decreased milk production are noticeable. Morbidity will be high in an outbreak, but mortality will be low to nonexistent. Diagnostic work should be initiated as soon as possible to distinguish this from foot-and-mouth disease. Diagnosis is based on analysis of fluid, serum, or membranes associated with the vesicles. Virus isolation, enzyme-linked immunosorbent assay (ELISA), competitive ELISA (CELISA), complement fixation, and serum neutralization are used for diagnosis. Epizootiology and transmission. This disease occurs in several other mammalian species, including swine, horses, and wild ruminants. VSV is an enveloped virus and survives well in different environmental conditions, including in soil, extremes of pH, and low temperatures. Outbreaks of VS occur sporadically in the United States, but it is not understood how or in what species the virus survives between these outbreaks. Incidence of disease decreases during colder seasons. Equipment, such as milking machines, contaminated by secretions is a mechanical vector, as are human hands. Transmission may also be from contaminated water and feed. Transmission is also believed to occur by insects (blackflies, sand flies, and Culicoides) that may simply be mechanical vectors. It is believed that carrier animals do not occur in this disease. Necropsy. It is rare for animals to be necropsied as the result of this disease. Typical vesicular lesion histology is seen, with ballooning degeneration and edema. There is no inclusion body formation. Pathogenesis. Lesions often begin within 24 hr after exposure. The virus invades oral epithelium. Injuries or trauma in any area typically affected, such as mouth, teats, or interdigital areas, will increase the likelihood of lesions developing there. Animals will develop a long-term immunity; this immunity can be overwhelmed, however, by a large dose of the virus. Differential diagnosis. Foot-and-mouth disease lesions are identical to VS lesions. Other differentials in cattle include bovine viral diarrhea, malignant catarrhal fever, contagious ecthyma, photosensitization, trauma, and caustic agents. Prevention and control. Quarantine and restrictions on shipping infected animals or animals from the premises housing affected animals are required in an outbreak. Vaccines are available for use in outbreaks and have decreased the severity of lesions. Phenolics, quaternaries, and halogens are effective for inactivating and disinfecting equipment and facilities. Treatment. Affected animals should be segregated from the rest of the herd and provided with separate water and softened feed. These animals should be cared for after unaffected animals. Any feed or water contaminated by these animals should not be used for other animals; contaminated equipment should be disinfected. Topical or systemic antibiotics control secondary bacterial infections. Cases of mastitis secondary to teat lesions must be treated as necessary. Any abrasive materials that could cause further trauma to the animals should be removed. Research complications. Animals developing vesicular lesions must be reported promptly to eliminate the possibility of an outbreak of foot-and-mouth disease. Personal protective equipment, especially gloves, should be worn when handling any animals with vesicular lesions. VSV causes a flulike illness in humans. x. Viral Diarrhea Diseases i. Ovine. Rotavirus, of the family Reoviridae, induces an acute, transient diarrhea in lambs within the first few weeks of life. Four antigenic groups (A-D) have been identified by differences in capsid antigens VP3 and VP7. Primarily group A, but also groups B and C, have been isolated from sheep. The disease is characterized by yellow, semifluid to watery diarrhea occurring 1–4 days after infection. The disease can progress to dehydration, anorexia and weight loss, acidosis, depression, and occasionally death. The virus is ingested with contaminated feed and water and selectively infects and destroys the enterocytes at the tips of the small intestinal villi. The villi are replaced with immature cells that lack sufficient digestive enzymes; osmotic diarrhea results. Virus may remain in the environment for several months. The disease is diagnosed by virus isolation, electron microscopy of feces, fecal fluorescent antibody, fecal ELISA tests (marketed tests generally detect group A rotavirus), and fecal latex agglutination tests. Rotavirus diarrhea is treated by supportive therapy, including maintaining hydration, electrolyte, and acid-base balance. A rotavirus vaccine is available for cattle; because of cross-species immunity, oral administration of high-quality bovine colostrum from vaccinated cows to infected sheep may be helpful ( "Current Veterinary Therapy," 1993 ). Coronavirus, of the family Coronaviridae, produces a more severe, long-lasting disease when compared with rotavirus. Clinical signs are similar to above, although the incubation period tends to be shorter (20–36 hr), and animals exhibit less anorexia than those with rotavirus. Additionally, mild respiratory disease may be noted ( Janke, 1989 ). Like rotavirus, coronavirus also destroys enterocytes of the villus tips. The virus can be visualized with electron microscopy. Treatment is supportive; close consideration of hydration and acid-base status is essential. Bovine vaccines are available. ii. Caprine. Rotavirus, coronavirus, and adenoviruses affect neonatal goats; however, little has been documented on the pathology and significance of these agents in this age group. It appears that bacteria play a more important role in neonatal kid diarrheal diseases then in neonatal calf diarrheas. iii. Bovine. Rotaviruses, coronaviruses, parvoviruses, and bovine viral diarrhea virus (BVDV) are associated with diarrheal disease in calves. Each pathogen multiplies within and destroys the intestinal epithelial cells, resulting in villous atrophy and clinical signs of diarrhea (soft to watery feces), dehydration, and abdominal pain. These viral infections may be complicated by parasitic infections (e.g., Cryptosporidium, Eimeria) or bacterial infections (e.g., Escherichia coli, Salmonella, Campylobacter). Treatment is aimed at correcting dehydration, electrolyte imbalances, and acidosis; cessation of milk replacers and administration of fluid therapy intravenously and by stomach tube may be necessary, depending on the presence of suckle reflex and the condition of the animals. Diagnosis is by immunoassays available for some viruses, viral culture, exclusion or identification of presence of other pathogens (by culture or fecal exams), and microscopic examination of necropsy specimens. Prevention focuses on calves suckling good-quality colostrum; other recommendations for calf care are in Section II,B,5. Combination vaccine products are available for immunizing dams against rotavirus, coronavirus, and enterotoxigenic E. coli. Additional supportive care for calves includes providing calves with sufficient energy and vitamins until milk intake can resume. Rotaviruses of serogroup A are the most common type in neonatal calves; 4- to 14-day old calves are typically affected, but younger and older animals may also be affected. The small intestine is the site of infection. Antirotavirus antibody is present in colostrum, and onset of rotavirus diarrhea coincides with the decline of this local protection. Transmission is likely from other affected calves and asymptomatic adult carriers. The diarrhea is typically a distinctive yellow. Colitis with tenesmus, mucus, and blood may be seen. This virus may be zoonotic. Coronaviruses are commonly associated with disease in calves during the first month of life, and they infect small- and large-intestinal epithelial cells. The virus infection may extend to mild pneumonia. Transmission is by infected calves and also by asymptomatic adult cattle, including dams excreting virus at the time of parturition. Calves that appear to have recovered continue to shed virus for several weeks. Parvovirus infections are usually associated with neonatal calves. BVDV infections also are seen in neonates and also affect many systems and produce other clinical signs and syndromes that are described in Section III,A,2,e. iv. Winter Dysentery. Winter dysentery is an acute, winter-seasonal, epizootic diarrheal disease of adult cattle, although it has been reported in 4-month-old calves. The etiology has not yet been defined, but a viral pathogen is suspected. Coronavirus-like viral particles have been isolated from cattle feces, either the same as or similar to the coronavirus of calf diarrhea. Outbreaks typically last a few weeks, and first-lactation or younger cattle are affected first, with waves of illness moving through a herd. Individual cows are ill for only a few days. The incubation period is estimated at 2–8 days. The outbreaks of disease are often seen in herds throughout the local area. Clinical signs include explosive diarrhea, anorexia, depression, and decreased production. The diarrhea has a distinctive musty, sweet odor and is light brown and bubbly, but some blood streaks or clots may be mixed in with the feces. Animals will become dehydrated quickly but are thirsty. Respiratory symptoms such as nasolacrimal discharges and coughing may develop. Recovery is generally spontaneous. Mortalities are rare. Diagnosis is based on characteristic patterns of clinical signs, and elimination of diarrheas caused by parasites such as coccidia, bacterial organisms such as Salmonella or Mycobacterium paratuberculosis, and viruses such as BVDV. Pathology is present in the colonic mucosa, and necrosis is present in the crypts. a. Adenovirus Infections Etiology. The ruminant adenoviruses are DNA viruses that cause respiratory and reproductive tract diseases. Nine antigenic types of the bovine adenovirus have been identified, with type 3 associated with respiratory disease. Two of the ovine and two of the caprine antigenic types have been identified. Clinical Signs. Signs of infection range from subclinical to severe, including pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, weak calf syndrome, and abortion. Respiratory tract and intestinal tract diseases may be concurrent. Infections caused by this virus are often found associated with other viral and bacterial infections. Epizootiology and transmission. The virus is believed to be widespread, but prevalence and characteristics of infection have not been characterized. Transmission of adenoviruses in other species (e.g., canine) is by aerosols or fecal-oral routes. Necropsy findings. Lesions found after experimental infections include atelectasis, edema, and consolidation of the lungs. Etiology. The ruminant adenoviruses are DNA viruses that cause respiratory and reproductive tract diseases. Nine antigenic types of the bovine adenovirus have been identified, with type 3 associated with respiratory disease. Two of the ovine and two of the caprine antigenic types have been identified. Clinical Signs. Signs of infection range from subclinical to severe, including pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, weak calf syndrome, and abortion. Respiratory tract and intestinal tract diseases may be concurrent. Infections caused by this virus are often found associated with other viral and bacterial infections. Epizootiology and transmission. The virus is believed to be widespread, but prevalence and characteristics of infection have not been characterized. Transmission of adenoviruses in other species (e.g., canine) is by aerosols or fecal-oral routes. Necropsy findings. Lesions found after experimental infections include atelectasis, edema, and consolidation of the lungs. Etiology. The ruminant adenoviruses are DNA viruses that cause respiratory and reproductive tract diseases. Nine antigenic types of the bovine adenovirus have been identified, with type 3 associated with respiratory disease. Two of the ovine and two of the caprine antigenic types have been identified. Clinical Signs. Signs of infection range from subclinical to severe, including pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, weak calf syndrome, and abortion. Respiratory tract and intestinal tract diseases may be concurrent. Infections caused by this virus are often found associated with other viral and bacterial infections. Epizootiology and transmission. The virus is believed to be widespread, but prevalence and characteristics of infection have not been characterized. Transmission of adenoviruses in other species (e.g., canine) is by aerosols or fecal-oral routes. Necropsy findings. Lesions found after experimental infections include atelectasis, edema, and consolidation of the lungs. b. Bluetongue Virus Infection (Reoviridae) Etiology. The bluetongue virus is an RNA virus in the Orbivirus genus and Reoviridae family. Five serotypes (2, 10, 11, 13, and 17) have been identified in the United States, where it is seen mostly in western states. Bluetongue is an acute arthropod-borne viral disease of ruminants, characterized by stomatitis, depression, coronary band lesions, and congenital abnormalities ( Bulgin, 1986 ). Clinical signs and diagnosis. Sheep are the most likely to show clinical signs. Clinical disease is less common in goats and cattle. Early in the infection, animals will spike a fever and will develop hyperemia and congestion of tissues of the mouth, lips, and ears. The virus name, bluetongue, is associated with the typical cyanotic membranes. The fever may subside, but tissue lesions erode, causing ulcers. Increased salivary discharges and anorexia are often related to ulcers of the dental pad, lips, gums, and tongue, although salivation and lacrimation may precede apparent ulceration. Chorioretinitis and conjunctivitis are also common signs in cattle and sheep. Lameness may be observed associated with coronitis and is evident in the rear legs. Skin lesions such as drying and cracking of the nose, alopecia, and mammary glands are also observed. Secondary bacterial pneumonia may also occur. Animals may also develop severe diarrhea and become recumbent. Sudden deaths due to cardiomyopathy may occur at any time during the disease. Hematologically, animals will be leukopenic. The course of the disease is about 2 weeks, and mortality may reach 80%. If animals are pregnant, the virus crosses the placenta and causes central nervous system lesions. Abortions may occur at any stage of gestation in cattle. Prolonged gestation may result from cerebellar hypoplasia and lack of normal sequence to induce parturition. Cerebellar hypoplasia will also be present in young born of the infected dams, as well as hydrocephalus, cataracts, gingival hyperplasia, or arthrogryposis. Diagnosis is suspected with the characteristic clinical signs and exposure to viral vectors. Virus isolation is the best diagnostic approach if blood is collected during the febrile stage of the disease or brains from aborted fetuses. Fluorescent antibody tests, ELISA, virus neutralization tests, PCR, and agar gel immunodiffusion (AGID) tests are also used to confirm the diagnosis. Epizootiology and transmission. Severe outbreaks have occurred in other countries during this century. Screening for this disease has limited the strains present in the United States. The disease is most common in outdoor-housed animals primarily in the western United States. The virus is primarily transmitted by biting midges, Culicoides. Culicoides variipennis is the most common vector in the United States. A combination of factors associated with viral strain, available and susceptible hosts, environmental conditions (such as damp areas where flies breed), and vector presence are factors in the severity of outbreaks. The disease is rarely transmitted by animal-to-animal contact or by infected animal products. Virus-contaminated semen, transplacental transfer, and carriage on transferred embyros are other possible means of transmission. Necropsy findings. At necropsy, erosive lesions may be observed around the mouth, tongue, palate, esophagus, and pillars of the rumen. Ulceration or hyperemia of the coronary bands may also be seen. Many of the internal organs will contain petechial and ecchymotic hemorrhages of the surfaces, and hemorrhage may be seen at the base of the pulmonary artery. Pathogenesis. The virus multiplies in the hemocoel and salivary glands of the fly and is excreted in transmissible form in the insect's saliva. After entering the host, the virus causes prolonged viremia. The incubation period is 6–14 days. The virus migrates to and attacks the vascular endothelium. The resulting vasculitis accounts for the lesions of the skin, mouth, tongue, esophagus, and rumen and the edema often found in many tissues. Ballooning degeneration of affected tissues, followed by necrosis and ulceration, occurs. The effects on fetuses appear to be due to generalized infections of developing organs. Differential diagnosis. Differentials include other infectious vesicular diseases such as foot-and-mouth disease, contagious ecthyma, bovine viral diarrhea virus-mucosal disease, infectious bovine rhinotracheitis, bovine papular stomatitis, and malignant catarrhal fever. Rinderpest is a differential in countries where it is endemic. Photosensitization should be considered. Foot rot is a differential for the lameness and coronitis. Differentials for the manifestations such as arthrogryposis include border disease virus and genetic predispositions of some breeds such as Charolais cattle and Merino sheep. Prevention and control. Cellular and humoral immunity are necessary for protection from infection. The bluetongue virus is insidious because the genome is capable of reassortment, and some vaccines will not have the antigenic components represented in the local infection. In addition, there is little to no cross protection between strains. Modified live vaccines are available in some parts of the United States but should not be used in pregnant animals. Vaccinating lambs and rams in an outbreak is worthwhile, for example, but vaccinating late-gestation ewes may cause birth defects or abortions. Congenital defects are more common from vaccine use than from naturally occurring infection. Minimizing exposure to the vector in endemic areas will decrease the incidence of the disease. Treatment. Supportive care and nursing care are helpful, including gruels or softer feeds, easily accessed water, and shaded resting places. Nonsteroidal anti-inflammatory drugs are often administered. For the cases of secondary bacterial pneumonia and some cases of bluetongue conjunctivitis, antibiotics may be administered. Research complications. This is a reportable disease because clinical signs resemble foot-and-mouth disease and other exotic vesicular diseases. Etiology. The bluetongue virus is an RNA virus in the Orbivirus genus and Reoviridae family. Five serotypes (2, 10, 11, 13, and 17) have been identified in the United States, where it is seen mostly in western states. Bluetongue is an acute arthropod-borne viral disease of ruminants, characterized by stomatitis, depression, coronary band lesions, and congenital abnormalities ( Bulgin, 1986 ). Clinical signs and diagnosis. Sheep are the most likely to show clinical signs. Clinical disease is less common in goats and cattle. Early in the infection, animals will spike a fever and will develop hyperemia and congestion of tissues of the mouth, lips, and ears. The virus name, bluetongue, is associated with the typical cyanotic membranes. The fever may subside, but tissue lesions erode, causing ulcers. Increased salivary discharges and anorexia are often related to ulcers of the dental pad, lips, gums, and tongue, although salivation and lacrimation may precede apparent ulceration. Chorioretinitis and conjunctivitis are also common signs in cattle and sheep. Lameness may be observed associated with coronitis and is evident in the rear legs. Skin lesions such as drying and cracking of the nose, alopecia, and mammary glands are also observed. Secondary bacterial pneumonia may also occur. Animals may also develop severe diarrhea and become recumbent. Sudden deaths due to cardiomyopathy may occur at any time during the disease. Hematologically, animals will be leukopenic. The course of the disease is about 2 weeks, and mortality may reach 80%. If animals are pregnant, the virus crosses the placenta and causes central nervous system lesions. Abortions may occur at any stage of gestation in cattle. Prolonged gestation may result from cerebellar hypoplasia and lack of normal sequence to induce parturition. Cerebellar hypoplasia will also be present in young born of the infected dams, as well as hydrocephalus, cataracts, gingival hyperplasia, or arthrogryposis. Diagnosis is suspected with the characteristic clinical signs and exposure to viral vectors. Virus isolation is the best diagnostic approach if blood is collected during the febrile stage of the disease or brains from aborted fetuses. Fluorescent antibody tests, ELISA, virus neutralization tests, PCR, and agar gel immunodiffusion (AGID) tests are also used to confirm the diagnosis. Epizootiology and transmission. Severe outbreaks have occurred in other countries during this century. Screening for this disease has limited the strains present in the United States. The disease is most common in outdoor-housed animals primarily in the western United States. The virus is primarily transmitted by biting midges, Culicoides. Culicoides variipennis is the most common vector in the United States. A combination of factors associated with viral strain, available and susceptible hosts, environmental conditions (such as damp areas where flies breed), and vector presence are factors in the severity of outbreaks. The disease is rarely transmitted by animal-to-animal contact or by infected animal products. Virus-contaminated semen, transplacental transfer, and carriage on transferred embyros are other possible means of transmission. Necropsy findings. At necropsy, erosive lesions may be observed around the mouth, tongue, palate, esophagus, and pillars of the rumen. Ulceration or hyperemia of the coronary bands may also be seen. Many of the internal organs will contain petechial and ecchymotic hemorrhages of the surfaces, and hemorrhage may be seen at the base of the pulmonary artery. Pathogenesis. The virus multiplies in the hemocoel and salivary glands of the fly and is excreted in transmissible form in the insect's saliva. After entering the host, the virus causes prolonged viremia. The incubation period is 6–14 days. The virus migrates to and attacks the vascular endothelium. The resulting vasculitis accounts for the lesions of the skin, mouth, tongue, esophagus, and rumen and the edema often found in many tissues. Ballooning degeneration of affected tissues, followed by necrosis and ulceration, occurs. The effects on fetuses appear to be due to generalized infections of developing organs. Differential diagnosis. Differentials include other infectious vesicular diseases such as foot-and-mouth disease, contagious ecthyma, bovine viral diarrhea virus-mucosal disease, infectious bovine rhinotracheitis, bovine papular stomatitis, and malignant catarrhal fever. Rinderpest is a differential in countries where it is endemic. Photosensitization should be considered. Foot rot is a differential for the lameness and coronitis. Differentials for the manifestations such as arthrogryposis include border disease virus and genetic predispositions of some breeds such as Charolais cattle and Merino sheep. Prevention and control. Cellular and humoral immunity are necessary for protection from infection. The bluetongue virus is insidious because the genome is capable of reassortment, and some vaccines will not have the antigenic components represented in the local infection. In addition, there is little to no cross protection between strains. Modified live vaccines are available in some parts of the United States but should not be used in pregnant animals. Vaccinating lambs and rams in an outbreak is worthwhile, for example, but vaccinating late-gestation ewes may cause birth defects or abortions. Congenital defects are more common from vaccine use than from naturally occurring infection. Minimizing exposure to the vector in endemic areas will decrease the incidence of the disease. Treatment. Supportive care and nursing care are helpful, including gruels or softer feeds, easily accessed water, and shaded resting places. Nonsteroidal anti-inflammatory drugs are often administered. For the cases of secondary bacterial pneumonia and some cases of bluetongue conjunctivitis, antibiotics may be administered. Research complications. This is a reportable disease because clinical signs resemble foot-and-mouth disease and other exotic vesicular diseases. Etiology. The bluetongue virus is an RNA virus in the Orbivirus genus and Reoviridae family. Five serotypes (2, 10, 11, 13, and 17) have been identified in the United States, where it is seen mostly in western states. Bluetongue is an acute arthropod-borne viral disease of ruminants, characterized by stomatitis, depression, coronary band lesions, and congenital abnormalities ( Bulgin, 1986 ). Clinical signs and diagnosis. Sheep are the most likely to show clinical signs. Clinical disease is less common in goats and cattle. Early in the infection, animals will spike a fever and will develop hyperemia and congestion of tissues of the mouth, lips, and ears. The virus name, bluetongue, is associated with the typical cyanotic membranes. The fever may subside, but tissue lesions erode, causing ulcers. Increased salivary discharges and anorexia are often related to ulcers of the dental pad, lips, gums, and tongue, although salivation and lacrimation may precede apparent ulceration. Chorioretinitis and conjunctivitis are also common signs in cattle and sheep. Lameness may be observed associated with coronitis and is evident in the rear legs. Skin lesions such as drying and cracking of the nose, alopecia, and mammary glands are also observed. Secondary bacterial pneumonia may also occur. Animals may also develop severe diarrhea and become recumbent. Sudden deaths due to cardiomyopathy may occur at any time during the disease. Hematologically, animals will be leukopenic. The course of the disease is about 2 weeks, and mortality may reach 80%. If animals are pregnant, the virus crosses the placenta and causes central nervous system lesions. Abortions may occur at any stage of gestation in cattle. Prolonged gestation may result from cerebellar hypoplasia and lack of normal sequence to induce parturition. Cerebellar hypoplasia will also be present in young born of the infected dams, as well as hydrocephalus, cataracts, gingival hyperplasia, or arthrogryposis. Diagnosis is suspected with the characteristic clinical signs and exposure to viral vectors. Virus isolation is the best diagnostic approach if blood is collected during the febrile stage of the disease or brains from aborted fetuses. Fluorescent antibody tests, ELISA, virus neutralization tests, PCR, and agar gel immunodiffusion (AGID) tests are also used to confirm the diagnosis. Epizootiology and transmission. Severe outbreaks have occurred in other countries during this century. Screening for this disease has limited the strains present in the United States. The disease is most common in outdoor-housed animals primarily in the western United States. The virus is primarily transmitted by biting midges, Culicoides. Culicoides variipennis is the most common vector in the United States. A combination of factors associated with viral strain, available and susceptible hosts, environmental conditions (such as damp areas where flies breed), and vector presence are factors in the severity of outbreaks. The disease is rarely transmitted by animal-to-animal contact or by infected animal products. Virus-contaminated semen, transplacental transfer, and carriage on transferred embyros are other possible means of transmission. Necropsy findings. At necropsy, erosive lesions may be observed around the mouth, tongue, palate, esophagus, and pillars of the rumen. Ulceration or hyperemia of the coronary bands may also be seen. Many of the internal organs will contain petechial and ecchymotic hemorrhages of the surfaces, and hemorrhage may be seen at the base of the pulmonary artery. Pathogenesis. The virus multiplies in the hemocoel and salivary glands of the fly and is excreted in transmissible form in the insect's saliva. After entering the host, the virus causes prolonged viremia. The incubation period is 6–14 days. The virus migrates to and attacks the vascular endothelium. The resulting vasculitis accounts for the lesions of the skin, mouth, tongue, esophagus, and rumen and the edema often found in many tissues. Ballooning degeneration of affected tissues, followed by necrosis and ulceration, occurs. The effects on fetuses appear to be due to generalized infections of developing organs. Differential diagnosis. Differentials include other infectious vesicular diseases such as foot-and-mouth disease, contagious ecthyma, bovine viral diarrhea virus-mucosal disease, infectious bovine rhinotracheitis, bovine papular stomatitis, and malignant catarrhal fever. Rinderpest is a differential in countries where it is endemic. Photosensitization should be considered. Foot rot is a differential for the lameness and coronitis. Differentials for the manifestations such as arthrogryposis include border disease virus and genetic predispositions of some breeds such as Charolais cattle and Merino sheep. Prevention and control. Cellular and humoral immunity are necessary for protection from infection. The bluetongue virus is insidious because the genome is capable of reassortment, and some vaccines will not have the antigenic components represented in the local infection. In addition, there is little to no cross protection between strains. Modified live vaccines are available in some parts of the United States but should not be used in pregnant animals. Vaccinating lambs and rams in an outbreak is worthwhile, for example, but vaccinating late-gestation ewes may cause birth defects or abortions. Congenital defects are more common from vaccine use than from naturally occurring infection. Minimizing exposure to the vector in endemic areas will decrease the incidence of the disease. Treatment. Supportive care and nursing care are helpful, including gruels or softer feeds, easily accessed water, and shaded resting places. Nonsteroidal anti-inflammatory drugs are often administered. For the cases of secondary bacterial pneumonia and some cases of bluetongue conjunctivitis, antibiotics may be administered. Research complications. This is a reportable disease because clinical signs resemble foot-and-mouth disease and other exotic vesicular diseases. c. Bovine Lymphosarcoma (Bovine Leukemia Virus Infection, Bovine Leukosis) Etiology. Bovine lymphosarcoma refers to lymphoproliferative diseases in young cattle that are not associated with bovine leukemia virus (BLV) infection, and those in older cattle that are associated with BLV. BLV is a B lymphocyte-associated retrovirus ( Johnson and Kaneene, 1993 a,b,c). Clinical signs. Forms of bovine lymphosarcoma that are not associated with BLV infection are calf, or juvenile; thymic, or adolescent (animals 6 months to 2 years old); and cutaneous (any age). The calf form is rare and characterized by generalized lymphadenopathy. Onset may be sudden, and the disease is usually fatal within a few weeks. Signs include lymphadenopathy, anemia, weight loss, and weakness. Some animals may be paralyzed because of spinal cord compression from subperiosteal infiltration of neoplastic cells. The adolescent form is also rare, the course rapid, and the prognosis poor. The disease is seen most often in beef breeds such as Hereford cattle and is characterized by space-occupying masses in the neck or thorax. These masses are also often present in the brisket. Secondary effects of the masses are loss of condition, dysphagia, rumen tympany, and fatal bloat. The cutaneous presentation has a longer course and may wax and wane. The masses are found at the anus, vulva, escutcheon, shoulder, and flank; they are painful when palpated, raised, and often ulcerated. The animals are anemic, and neoplastic involvement may affect cardiac function. Generalized or limited lymphadenopathy may be apparent. Only the adult, or enzootic, form of bovine lymphosarcoma is associated with BLV infection. Many animals do not develop any malignancies or clinical signs of infection and simply remain permanently infected. Some cows manifest disease only during the periparturient period. Malignant lymphoma is the more common, whereas leukosis, due to B-lymphocyte proliferation, is rare. Clinical signs are loss of condition and a drop in production of dairy cattle, anorexia, diarrhea, ataxia, paresis, and other signs dependent on the location of the neoplastic tissue. Tumors are associated with lymphoid tissues. Common sites also include the abomasum, spinal canal, and uterus. Cardiac tumors develop at the right atrial or left ventricular myocardium, and associated beat and rate abnormalities may be auscultated. The common ocular manifestation of the disease is exophthalmos due to retrobulbar masses. Many internal organs may be involved, and tumors may be palpable per rectum. Secondary infections will be due to immunosuppression and the weakened state of the animal. Sheep have acquired BLV infection naturally and have been used as experimental models; in both situations, this species is susceptible to tumor and leukemia development. Goats seroconvert but do not develop the clinical syndromes. Diagnosis is based on the animal's age, clinical signs, serology, hematology findings according to the form, aspirates or biopsies of masses, and necropsy findings. Kits are available for running AGID, for which the BLV antigens gp-51 and gp-24 are used; antibodies may be detected within weeks after exposure and may also help in predicting disease in clinically normal cattle. ELISA and PCR diagnostic aids will also be helpful. Epizootiology and transmission. This disease is present worldwide. It is estimated that at least 50% of the cattle in the United States are infected with BLV. As few as 1% of these animals develop lymphosarcoma, but the adult form of the disease described here is the most common bovine neoplastic disease in the United States. Larger herds tend to have higher rates. Genetic predisposition may be involved; in addition to the presence of BLV, the type of bovine lymphocyte antigen (BoLA) may be correlated to resistance or susceptibility and to the course of the disease. Transmission is believed to be by inhalation of BLV in secretions; in colostrum; horizontally by contaminated equipment not sanitized between cattle; and by rectum (e.g., mucosal irritation during per-rectum exams or procedures). Natural-service bulls may transmit the infection to cows. Cows infected with BLV may transmit the infection to their calves in utero. Tabanid and other flies also serve as vectors, but these represent a minor means of transmission. Necropsy findings. Neoplastic infiltration of many organs and tissues are found in the calf form and the cutaneous forms. Tumors may be local or widely distributed in the enzootic form. Definitive diagnosis of neoplastic tissue specimens is by histology. Pathogenesis. As with other retroviruses, the BLV integrates viral DNA into host target cell DNA by means of the reverse transcriptase enzyme, creating a provirus. Prevention and control. There is no vaccine for this disease. Development and maintenance of a BLV-free herd, or controlling infection within a herd, requires financial and programmatic commitments: BLV-positive and BLV-negative animals maintained separately; serologic testing (such as at least every 6 months) and separating positive animals; and washing and then disinfecting instruments, needles (or using sterile single-use products), and equipment for ear tagging and dehorning and other such equipment between animals. A fresh rectal exam sleeve and lubricant should be used for each animal examined. Otherwise serologically positive cows may have undetectable antibodies during the periparturient period. Embryo transfer recipients should be negative, and the virus will not be transferred by the embryonic stage. Calves should be fed colostrum from serologically negative cows. Treatment. Treatment regimens of corticosteroids and cancer chemotherapeutic agents provide only short-term improvement. In cases where ova, embryos, or semen need to be collected, supportive care for the affected animals is essential. Research complications. The United States and several countries, some in Europe, have official programs for eradication of enzootic bovine leukosis. Etiology. Bovine lymphosarcoma refers to lymphoproliferative diseases in young cattle that are not associated with bovine leukemia virus (BLV) infection, and those in older cattle that are associated with BLV. BLV is a B lymphocyte-associated retrovirus ( Johnson and Kaneene, 1993 a,b,c). Clinical signs. Forms of bovine lymphosarcoma that are not associated with BLV infection are calf, or juvenile; thymic, or adolescent (animals 6 months to 2 years old); and cutaneous (any age). The calf form is rare and characterized by generalized lymphadenopathy. Onset may be sudden, and the disease is usually fatal within a few weeks. Signs include lymphadenopathy, anemia, weight loss, and weakness. Some animals may be paralyzed because of spinal cord compression from subperiosteal infiltration of neoplastic cells. The adolescent form is also rare, the course rapid, and the prognosis poor. The disease is seen most often in beef breeds such as Hereford cattle and is characterized by space-occupying masses in the neck or thorax. These masses are also often present in the brisket. Secondary effects of the masses are loss of condition, dysphagia, rumen tympany, and fatal bloat. The cutaneous presentation has a longer course and may wax and wane. The masses are found at the anus, vulva, escutcheon, shoulder, and flank; they are painful when palpated, raised, and often ulcerated. The animals are anemic, and neoplastic involvement may affect cardiac function. Generalized or limited lymphadenopathy may be apparent. Only the adult, or enzootic, form of bovine lymphosarcoma is associated with BLV infection. Many animals do not develop any malignancies or clinical signs of infection and simply remain permanently infected. Some cows manifest disease only during the periparturient period. Malignant lymphoma is the more common, whereas leukosis, due to B-lymphocyte proliferation, is rare. Clinical signs are loss of condition and a drop in production of dairy cattle, anorexia, diarrhea, ataxia, paresis, and other signs dependent on the location of the neoplastic tissue. Tumors are associated with lymphoid tissues. Common sites also include the abomasum, spinal canal, and uterus. Cardiac tumors develop at the right atrial or left ventricular myocardium, and associated beat and rate abnormalities may be auscultated. The common ocular manifestation of the disease is exophthalmos due to retrobulbar masses. Many internal organs may be involved, and tumors may be palpable per rectum. Secondary infections will be due to immunosuppression and the weakened state of the animal. Sheep have acquired BLV infection naturally and have been used as experimental models; in both situations, this species is susceptible to tumor and leukemia development. Goats seroconvert but do not develop the clinical syndromes. Diagnosis is based on the animal's age, clinical signs, serology, hematology findings according to the form, aspirates or biopsies of masses, and necropsy findings. Kits are available for running AGID, for which the BLV antigens gp-51 and gp-24 are used; antibodies may be detected within weeks after exposure and may also help in predicting disease in clinically normal cattle. ELISA and PCR diagnostic aids will also be helpful. Epizootiology and transmission. This disease is present worldwide. It is estimated that at least 50% of the cattle in the United States are infected with BLV. As few as 1% of these animals develop lymphosarcoma, but the adult form of the disease described here is the most common bovine neoplastic disease in the United States. Larger herds tend to have higher rates. Genetic predisposition may be involved; in addition to the presence of BLV, the type of bovine lymphocyte antigen (BoLA) may be correlated to resistance or susceptibility and to the course of the disease. Transmission is believed to be by inhalation of BLV in secretions; in colostrum; horizontally by contaminated equipment not sanitized between cattle; and by rectum (e.g., mucosal irritation during per-rectum exams or procedures). Natural-service bulls may transmit the infection to cows. Cows infected with BLV may transmit the infection to their calves in utero. Tabanid and other flies also serve as vectors, but these represent a minor means of transmission. Necropsy findings. Neoplastic infiltration of many organs and tissues are found in the calf form and the cutaneous forms. Tumors may be local or widely distributed in the enzootic form. Definitive diagnosis of neoplastic tissue specimens is by histology. Pathogenesis. As with other retroviruses, the BLV integrates viral DNA into host target cell DNA by means of the reverse transcriptase enzyme, creating a provirus. Prevention and control. There is no vaccine for this disease. Development and maintenance of a BLV-free herd, or controlling infection within a herd, requires financial and programmatic commitments: BLV-positive and BLV-negative animals maintained separately; serologic testing (such as at least every 6 months) and separating positive animals; and washing and then disinfecting instruments, needles (or using sterile single-use products), and equipment for ear tagging and dehorning and other such equipment between animals. A fresh rectal exam sleeve and lubricant should be used for each animal examined. Otherwise serologically positive cows may have undetectable antibodies during the periparturient period. Embryo transfer recipients should be negative, and the virus will not be transferred by the embryonic stage. Calves should be fed colostrum from serologically negative cows. Treatment. Treatment regimens of corticosteroids and cancer chemotherapeutic agents provide only short-term improvement. In cases where ova, embryos, or semen need to be collected, supportive care for the affected animals is essential. Research complications. The United States and several countries, some in Europe, have official programs for eradication of enzootic bovine leukosis. Etiology. Bovine lymphosarcoma refers to lymphoproliferative diseases in young cattle that are not associated with bovine leukemia virus (BLV) infection, and those in older cattle that are associated with BLV. BLV is a B lymphocyte-associated retrovirus ( Johnson and Kaneene, 1993 a,b,c). Clinical signs. Forms of bovine lymphosarcoma that are not associated with BLV infection are calf, or juvenile; thymic, or adolescent (animals 6 months to 2 years old); and cutaneous (any age). The calf form is rare and characterized by generalized lymphadenopathy. Onset may be sudden, and the disease is usually fatal within a few weeks. Signs include lymphadenopathy, anemia, weight loss, and weakness. Some animals may be paralyzed because of spinal cord compression from subperiosteal infiltration of neoplastic cells. The adolescent form is also rare, the course rapid, and the prognosis poor. The disease is seen most often in beef breeds such as Hereford cattle and is characterized by space-occupying masses in the neck or thorax. These masses are also often present in the brisket. Secondary effects of the masses are loss of condition, dysphagia, rumen tympany, and fatal bloat. The cutaneous presentation has a longer course and may wax and wane. The masses are found at the anus, vulva, escutcheon, shoulder, and flank; they are painful when palpated, raised, and often ulcerated. The animals are anemic, and neoplastic involvement may affect cardiac function. Generalized or limited lymphadenopathy may be apparent. Only the adult, or enzootic, form of bovine lymphosarcoma is associated with BLV infection. Many animals do not develop any malignancies or clinical signs of infection and simply remain permanently infected. Some cows manifest disease only during the periparturient period. Malignant lymphoma is the more common, whereas leukosis, due to B-lymphocyte proliferation, is rare. Clinical signs are loss of condition and a drop in production of dairy cattle, anorexia, diarrhea, ataxia, paresis, and other signs dependent on the location of the neoplastic tissue. Tumors are associated with lymphoid tissues. Common sites also include the abomasum, spinal canal, and uterus. Cardiac tumors develop at the right atrial or left ventricular myocardium, and associated beat and rate abnormalities may be auscultated. The common ocular manifestation of the disease is exophthalmos due to retrobulbar masses. Many internal organs may be involved, and tumors may be palpable per rectum. Secondary infections will be due to immunosuppression and the weakened state of the animal. Sheep have acquired BLV infection naturally and have been used as experimental models; in both situations, this species is susceptible to tumor and leukemia development. Goats seroconvert but do not develop the clinical syndromes. Diagnosis is based on the animal's age, clinical signs, serology, hematology findings according to the form, aspirates or biopsies of masses, and necropsy findings. Kits are available for running AGID, for which the BLV antigens gp-51 and gp-24 are used; antibodies may be detected within weeks after exposure and may also help in predicting disease in clinically normal cattle. ELISA and PCR diagnostic aids will also be helpful. Epizootiology and transmission. This disease is present worldwide. It is estimated that at least 50% of the cattle in the United States are infected with BLV. As few as 1% of these animals develop lymphosarcoma, but the adult form of the disease described here is the most common bovine neoplastic disease in the United States. Larger herds tend to have higher rates. Genetic predisposition may be involved; in addition to the presence of BLV, the type of bovine lymphocyte antigen (BoLA) may be correlated to resistance or susceptibility and to the course of the disease. Transmission is believed to be by inhalation of BLV in secretions; in colostrum; horizontally by contaminated equipment not sanitized between cattle; and by rectum (e.g., mucosal irritation during per-rectum exams or procedures). Natural-service bulls may transmit the infection to cows. Cows infected with BLV may transmit the infection to their calves in utero. Tabanid and other flies also serve as vectors, but these represent a minor means of transmission. Necropsy findings. Neoplastic infiltration of many organs and tissues are found in the calf form and the cutaneous forms. Tumors may be local or widely distributed in the enzootic form. Definitive diagnosis of neoplastic tissue specimens is by histology. Pathogenesis. As with other retroviruses, the BLV integrates viral DNA into host target cell DNA by means of the reverse transcriptase enzyme, creating a provirus. Prevention and control. There is no vaccine for this disease. Development and maintenance of a BLV-free herd, or controlling infection within a herd, requires financial and programmatic commitments: BLV-positive and BLV-negative animals maintained separately; serologic testing (such as at least every 6 months) and separating positive animals; and washing and then disinfecting instruments, needles (or using sterile single-use products), and equipment for ear tagging and dehorning and other such equipment between animals. A fresh rectal exam sleeve and lubricant should be used for each animal examined. Otherwise serologically positive cows may have undetectable antibodies during the periparturient period. Embryo transfer recipients should be negative, and the virus will not be transferred by the embryonic stage. Calves should be fed colostrum from serologically negative cows. Treatment. Treatment regimens of corticosteroids and cancer chemotherapeutic agents provide only short-term improvement. In cases where ova, embryos, or semen need to be collected, supportive care for the affected animals is essential. Research complications. The United States and several countries, some in Europe, have official programs for eradication of enzootic bovine leukosis. d. Bovine Herpes Mammillitis (Bovine Herpesvirus 2 Bovine Ulcerative Mammillitis) Etiology. Bovine herpesvirus 2 causes bovine herpes mammillitis, a widespread disease characterized by teat and udder lesions, as well as oral and skin lesions. Clinical signs and diagnosis. Lesions begin suddenly with teat swelling; the tissue will be edematous and tender when touched. The udder lesions may extend to the perineum. The lesions progress to vesicles, then to ulcers; these may take 10 weeks to heal. Lesions rarely may also develop focally around the mouth and generally on the skin of the udder. Secondary mastitis may occur, because of bacteria associated with the scabs. Diagnosis is by clinical signs and serologically. Epizootiology and transmission. The virus is reported to be widespread. Occurrence is often seasonal, and biting insects may be vectors. Transmission with successful infection requires deep penetration of the skin. Transmission may be by contaminated milkers' hands, contaminated equipment, and other fomites. Differential diagnosis. Differential diagnoses include other diseases that cause lesions on teats such as pseudocowpox, papillomatosis, and vesicular stomatitis. Other vesicular diseases may be considered, but other more severe clinical signs might be associated with those. Prevention and control. Established milking hygiene practices are important control measures: having milkers wash their hands with germicidal solutions or wear gloves, cleaning equipment between animals, and separating affected animals. Treatment. There is no treatment, and affected animals should be separated from the herd and milked last. Lesions can be cleaned and treated with topical antibacterials. Etiology. Bovine herpesvirus 2 causes bovine herpes mammillitis, a widespread disease characterized by teat and udder lesions, as well as oral and skin lesions. Clinical signs and diagnosis. Lesions begin suddenly with teat swelling; the tissue will be edematous and tender when touched. The udder lesions may extend to the perineum. The lesions progress to vesicles, then to ulcers; these may take 10 weeks to heal. Lesions rarely may also develop focally around the mouth and generally on the skin of the udder. Secondary mastitis may occur, because of bacteria associated with the scabs. Diagnosis is by clinical signs and serologically. Epizootiology and transmission. The virus is reported to be widespread. Occurrence is often seasonal, and biting insects may be vectors. Transmission with successful infection requires deep penetration of the skin. Transmission may be by contaminated milkers' hands, contaminated equipment, and other fomites. Differential diagnosis. Differential diagnoses include other diseases that cause lesions on teats such as pseudocowpox, papillomatosis, and vesicular stomatitis. Other vesicular diseases may be considered, but other more severe clinical signs might be associated with those. Prevention and control. Established milking hygiene practices are important control measures: having milkers wash their hands with germicidal solutions or wear gloves, cleaning equipment between animals, and separating affected animals. Treatment. There is no treatment, and affected animals should be separated from the herd and milked last. Lesions can be cleaned and treated with topical antibacterials. Etiology. Bovine herpesvirus 2 causes bovine herpes mammillitis, a widespread disease characterized by teat and udder lesions, as well as oral and skin lesions. Clinical signs and diagnosis. Lesions begin suddenly with teat swelling; the tissue will be edematous and tender when touched. The udder lesions may extend to the perineum. The lesions progress to vesicles, then to ulcers; these may take 10 weeks to heal. Lesions rarely may also develop focally around the mouth and generally on the skin of the udder. Secondary mastitis may occur, because of bacteria associated with the scabs. Diagnosis is by clinical signs and serologically. Epizootiology and transmission. The virus is reported to be widespread. Occurrence is often seasonal, and biting insects may be vectors. Transmission with successful infection requires deep penetration of the skin. Transmission may be by contaminated milkers' hands, contaminated equipment, and other fomites. Differential diagnosis. Differential diagnoses include other diseases that cause lesions on teats such as pseudocowpox, papillomatosis, and vesicular stomatitis. Other vesicular diseases may be considered, but other more severe clinical signs might be associated with those. Prevention and control. Established milking hygiene practices are important control measures: having milkers wash their hands with germicidal solutions or wear gloves, cleaning equipment between animals, and separating affected animals. Treatment. There is no treatment, and affected animals should be separated from the herd and milked last. Lesions can be cleaned and treated with topical antibacterials. e. Bovine Viral Diarrhea Virus Etiology. The bovine viral diarrhea virus (BVDV) is a pestivirus of the Flaviviridae family. The Flaviviridae include hog cholera virus and border disease virus of sheep. The virus contains a single strand of positive-sense RNA. A broad range of disease and immune effects is produced by BVDV only in cattle. In addition, this virus is important in the etiology of bovine pneumonias. Bovine viral diarrhea/mucosal disease (BVD/MD) is one of the most important viral diseases and one of the most complex diseases of cattle. Strains of BVDV are characterized as cytopathic (CP) and noncytopathic (NCP), based on cell-culture growth characteristics. The virus has also been categorized as type 1 and type 2 isolates. Heterologous strains exist that may confound even sound vaccination programs. Clinical signs and diagnosis. Signs of BVDV infections may be subclinical but also include abortions, congenital abnormalities, reduced fertility, persistent infection (PI) with gradual debilitation, and acute and fatal disease. The presence of antibodies, whether from passive transfer or immunizations, does not necessarily guarantee protection from the various forms of the disease. An acute form of the disease, caused by type 2 BVDV, occurs in cattle without sufficient immunity. After an incubation period of 5–7 days, clinical signs include fever, anorexia, oculonasal discharge, oral erosions (including on the hard palate), diarrhea, and decreased milk production. The disease course may be shorter with hemorrhagic syndrome and death within 2 days. Clinical signs of BVDV in calves also include severe enteritis and pneumonia. When susceptible cows are infected in utero from gestational days 50–100, or gestational cows are vaccinated with a modified live vaccine, abortion or stillbirth result. Congenital defects caused by BVDV during gestational days 90–170 include impaired immunity (thymic atrophy), cerebellar hypoplasia, ocular defects, alopecia or hypotrichosis, dysmyelinogenesis, hydranencephaly, hydrocephalus, and intrauterine growth retardation. Typical signs of cerebellar dysfunction will be evident in calves, such as wide-based stance, weakness, opisthotonus, hyperflexion, hypermetria, nystagmus, or strabismus. Some severely affected calves will not be able to stand. Ophthalmic effects include retinal degeneration and microphthalmia. Fetuses can also be infected in utero, normal at birth, immunotolerant to the virus, and persistently infected (PI). The term mucosal disease is commonly associated with this form of the infection. Many PI animals do not survive to maturity, however, and many have weakened immune systems. The PI animals are important because they shed virus and will probably show the clinical signs of mucosal disease (MD) caused by a CP BVDV strain derived from an NCP BVDV strain. These MD clinical signs include fever, anorexia, and profuse diarrhea that may include blood and fibrin casts, and oral and pharyngeal erosions, as well as erosion at the interdigital spaces and on the teats and vulva. Many other associated clinical signs include anemia, bloat, lameness, or corneal opacities and discharges. Secondary effects of hemorrhage and dehydration also contribute to the morbidity and mortality. Animals that do not succumb to the disease will be chronically unthrifty, debilitated, and infection-prone. Diagnosis in affected calves is based on herd health history, clinical signs, and antibodies to BVDV in precolostral serum. Viral culturing from blood may be useful. In older animals, oral lesions, serology, detection of viral antigen, and virus isolation contribute to the diagnosis. Leukopenia, and especially lymphopenia, are seen. Serology must be interpreted with the awareness of the possibility of PI immunotolerant animals. Vaccination against the disease carries its own set of side effects and potential problems, especially when using modified live vaccines, whether against CP or NCP strains. The condition of the animals is also a variable. Epizootiology and transmission. BVDV is present throughout the world. Transmission occurs easily by direct contact between cattle, from feed contaminated with secretions or feces, and by aborted fetuses and placentas. PI females transmit the virus to their fetuses. Semen also is a source of virus. Necropsy findings. In affected calves, histopathologic findings include necrosis of external germinal cells, focal hemorrhages, and folial edema. Later in the disease, large cavities develop in the cerebellum, and atrophy of the cerebellar folia and thin neuropil are evident. Older calves may have areas of intestinal necrosis. In cases where oral erosions occur, erosions will be found extending throughout the gastrointestinal tract to the cecum. The respiratory tract lesions will often be complicated by secondary bacterial pneumonia. When the hemorrhagic syndrome develops, petechiation and mucosal bleeding will be present. Pathogenesis. The CP and NCP strains are thought to be related mutations of the BVDV; the CP short-lived isolates are believed to arise from the NCP strains. The NCP strains are those present in the PI animals, and the strains are maintained in cattle populations. CP and NCP isolates vary in virulence, and classification of these types is based on viral surface proteins. Considerable antigenic variation also exists between strains and types. Other viral infections, such as bovine respiratory syncytial virus and infectious bovine rhinotracheitis, may also be present in the same animals. The pathology caused by BVDV is due to its ability to infect epithelial cells and impair the functioning of immune cell populations through out the bovine system. In type 2 BVDV hemorrhagic syndrome, death results from viral-induced thrombocytopenia. In fetuses, the virus infects developing germinal cells of the cerebellum. The Purkinje's cells in the granular layer are killed, and necrosis and inflammation follow. The immune effects are the result of the virus's interfering with neutrophil and macrophage functions and of lymphocyte blastogenesis. All of these predispose the affected animals to bacterial infections with Pasteurella haemolytica. BVDV damages dividing cells in fetal organ systems, resulting in abortions and congenital effects. Differential diagnosis. Many differentials must be considered for the clinical manifestations of BVDV infections. Differentials for enteritis of calves include viral infections, Cryptosporidia, Escherichia coli, Salmonella, and Coccidia. Salmonella, winter dysentery, Johne's disease, intestinal parasites, malignant catarrhal fever (MCF), and copper deficiency are differentials for the diarrhea seen in the disease in adult animals. Respiratory tract pathogens such as bovine respiratory syncytial virus, Pasteurella, Haemophilus, and Mycoplasma must be considered for the respiratory tract manifestations. Oral lesions are also produced by MCF, vesicular stomatitis, bluetongue, and papular stomatitis. Infectious bovine herpesvirus 1, leptospirosis, brucellosis, trichomoniasis, and mycosis should be considered in cases of abortion. Prevention and control. Combined with sound management in a typical cattle herd, vaccination is the best way to prevent BVDV and should be integrated into the herd health program, timed appropriately preceding breeding, gestation, or stressful events. Vaccine preparations for BVDV are modified live virus (MLV) or killed virus. Each has advantages and disadvantages. The former induces rapid immunity (within 1 week) after a single dose, provides longer duration of immunity against several strains, and induces serum neutralizing antibodies. MLV vaccines are not recommended for use in pregnant cattle, may induce mucosal disease, and may be immunosuppressive at the time of vaccination. The immunosuppression is detrimental if cattle are concurrently exposed to field-strain virus because it will facilitate infection and possible clinical disease. The MLV strains may cross the placenta, resulting in fetal infections. The killed vaccines are safer in pregnant animals but require booster doses after the initial immunization, may need to be given 2–3 times per year, and do not induce cell-mediated immunity. Passive immunity may protect most calves for up to 6–8 months of age. Subsequent vaccination with MLV may provide lifelong immunity, but this is not guaranteed. Annual boosters are recommended to protect against vaccine breaks. The virus persists in the environment for 2 weeks and is susceptible to the disfectants chlorhexidine, hypochlorite, iodophors, and aldehydes. Maintenance of a closed herd to prevent any possibility of the introduction of the virus is difficult. Isolation of new animals, avoidance of the purchase of pregnant cows, scrutiny of records from source farms, use of semen tested bulls, minimization of stress, testing of embryo-recipient cows, and maintainenance of populations of ruminants (smaller or wild species) separately on the premises will minimize viral exposure. Other management strategies may require a program for testing and culling PI cattle. This can be expensive but may be a worthwhile investment to remove the virus shedders from a herd. Treatment. No specific treatment is available. Supportive care and treatment with antibiotics to prevent secondary infection are recommended. Animals that survive the infection should be evaluated a month after recovery to determine their status as PI or virus-free. Etiology. The bovine viral diarrhea virus (BVDV) is a pestivirus of the Flaviviridae family. The Flaviviridae include hog cholera virus and border disease virus of sheep. The virus contains a single strand of positive-sense RNA. A broad range of disease and immune effects is produced by BVDV only in cattle. In addition, this virus is important in the etiology of bovine pneumonias. Bovine viral diarrhea/mucosal disease (BVD/MD) is one of the most important viral diseases and one of the most complex diseases of cattle. Strains of BVDV are characterized as cytopathic (CP) and noncytopathic (NCP), based on cell-culture growth characteristics. The virus has also been categorized as type 1 and type 2 isolates. Heterologous strains exist that may confound even sound vaccination programs. Clinical signs and diagnosis. Signs of BVDV infections may be subclinical but also include abortions, congenital abnormalities, reduced fertility, persistent infection (PI) with gradual debilitation, and acute and fatal disease. The presence of antibodies, whether from passive transfer or immunizations, does not necessarily guarantee protection from the various forms of the disease. An acute form of the disease, caused by type 2 BVDV, occurs in cattle without sufficient immunity. After an incubation period of 5–7 days, clinical signs include fever, anorexia, oculonasal discharge, oral erosions (including on the hard palate), diarrhea, and decreased milk production. The disease course may be shorter with hemorrhagic syndrome and death within 2 days. Clinical signs of BVDV in calves also include severe enteritis and pneumonia. When susceptible cows are infected in utero from gestational days 50–100, or gestational cows are vaccinated with a modified live vaccine, abortion or stillbirth result. Congenital defects caused by BVDV during gestational days 90–170 include impaired immunity (thymic atrophy), cerebellar hypoplasia, ocular defects, alopecia or hypotrichosis, dysmyelinogenesis, hydranencephaly, hydrocephalus, and intrauterine growth retardation. Typical signs of cerebellar dysfunction will be evident in calves, such as wide-based stance, weakness, opisthotonus, hyperflexion, hypermetria, nystagmus, or strabismus. Some severely affected calves will not be able to stand. Ophthalmic effects include retinal degeneration and microphthalmia. Fetuses can also be infected in utero, normal at birth, immunotolerant to the virus, and persistently infected (PI). The term mucosal disease is commonly associated with this form of the infection. Many PI animals do not survive to maturity, however, and many have weakened immune systems. The PI animals are important because they shed virus and will probably show the clinical signs of mucosal disease (MD) caused by a CP BVDV strain derived from an NCP BVDV strain. These MD clinical signs include fever, anorexia, and profuse diarrhea that may include blood and fibrin casts, and oral and pharyngeal erosions, as well as erosion at the interdigital spaces and on the teats and vulva. Many other associated clinical signs include anemia, bloat, lameness, or corneal opacities and discharges. Secondary effects of hemorrhage and dehydration also contribute to the morbidity and mortality. Animals that do not succumb to the disease will be chronically unthrifty, debilitated, and infection-prone. Diagnosis in affected calves is based on herd health history, clinical signs, and antibodies to BVDV in precolostral serum. Viral culturing from blood may be useful. In older animals, oral lesions, serology, detection of viral antigen, and virus isolation contribute to the diagnosis. Leukopenia, and especially lymphopenia, are seen. Serology must be interpreted with the awareness of the possibility of PI immunotolerant animals. Vaccination against the disease carries its own set of side effects and potential problems, especially when using modified live vaccines, whether against CP or NCP strains. The condition of the animals is also a variable. Epizootiology and transmission. BVDV is present throughout the world. Transmission occurs easily by direct contact between cattle, from feed contaminated with secretions or feces, and by aborted fetuses and placentas. PI females transmit the virus to their fetuses. Semen also is a source of virus. Necropsy findings. In affected calves, histopathologic findings include necrosis of external germinal cells, focal hemorrhages, and folial edema. Later in the disease, large cavities develop in the cerebellum, and atrophy of the cerebellar folia and thin neuropil are evident. Older calves may have areas of intestinal necrosis. In cases where oral erosions occur, erosions will be found extending throughout the gastrointestinal tract to the cecum. The respiratory tract lesions will often be complicated by secondary bacterial pneumonia. When the hemorrhagic syndrome develops, petechiation and mucosal bleeding will be present. Pathogenesis. The CP and NCP strains are thought to be related mutations of the BVDV; the CP short-lived isolates are believed to arise from the NCP strains. The NCP strains are those present in the PI animals, and the strains are maintained in cattle populations. CP and NCP isolates vary in virulence, and classification of these types is based on viral surface proteins. Considerable antigenic variation also exists between strains and types. Other viral infections, such as bovine respiratory syncytial virus and infectious bovine rhinotracheitis, may also be present in the same animals. The pathology caused by BVDV is due to its ability to infect epithelial cells and impair the functioning of immune cell populations through out the bovine system. In type 2 BVDV hemorrhagic syndrome, death results from viral-induced thrombocytopenia. In fetuses, the virus infects developing germinal cells of the cerebellum. The Purkinje's cells in the granular layer are killed, and necrosis and inflammation follow. The immune effects are the result of the virus's interfering with neutrophil and macrophage functions and of lymphocyte blastogenesis. All of these predispose the affected animals to bacterial infections with Pasteurella haemolytica. BVDV damages dividing cells in fetal organ systems, resulting in abortions and congenital effects. Differential diagnosis. Many differentials must be considered for the clinical manifestations of BVDV infections. Differentials for enteritis of calves include viral infections, Cryptosporidia, Escherichia coli, Salmonella, and Coccidia. Salmonella, winter dysentery, Johne's disease, intestinal parasites, malignant catarrhal fever (MCF), and copper deficiency are differentials for the diarrhea seen in the disease in adult animals. Respiratory tract pathogens such as bovine respiratory syncytial virus, Pasteurella, Haemophilus, and Mycoplasma must be considered for the respiratory tract manifestations. Oral lesions are also produced by MCF, vesicular stomatitis, bluetongue, and papular stomatitis. Infectious bovine herpesvirus 1, leptospirosis, brucellosis, trichomoniasis, and mycosis should be considered in cases of abortion. Prevention and control. Combined with sound management in a typical cattle herd, vaccination is the best way to prevent BVDV and should be integrated into the herd health program, timed appropriately preceding breeding, gestation, or stressful events. Vaccine preparations for BVDV are modified live virus (MLV) or killed virus. Each has advantages and disadvantages. The former induces rapid immunity (within 1 week) after a single dose, provides longer duration of immunity against several strains, and induces serum neutralizing antibodies. MLV vaccines are not recommended for use in pregnant cattle, may induce mucosal disease, and may be immunosuppressive at the time of vaccination. The immunosuppression is detrimental if cattle are concurrently exposed to field-strain virus because it will facilitate infection and possible clinical disease. The MLV strains may cross the placenta, resulting in fetal infections. The killed vaccines are safer in pregnant animals but require booster doses after the initial immunization, may need to be given 2–3 times per year, and do not induce cell-mediated immunity. Passive immunity may protect most calves for up to 6–8 months of age. Subsequent vaccination with MLV may provide lifelong immunity, but this is not guaranteed. Annual boosters are recommended to protect against vaccine breaks. The virus persists in the environment for 2 weeks and is susceptible to the disfectants chlorhexidine, hypochlorite, iodophors, and aldehydes. Maintenance of a closed herd to prevent any possibility of the introduction of the virus is difficult. Isolation of new animals, avoidance of the purchase of pregnant cows, scrutiny of records from source farms, use of semen tested bulls, minimization of stress, testing of embryo-recipient cows, and maintainenance of populations of ruminants (smaller or wild species) separately on the premises will minimize viral exposure. Other management strategies may require a program for testing and culling PI cattle. This can be expensive but may be a worthwhile investment to remove the virus shedders from a herd. Treatment. No specific treatment is available. Supportive care and treatment with antibiotics to prevent secondary infection are recommended. Animals that survive the infection should be evaluated a month after recovery to determine their status as PI or virus-free. Etiology. The bovine viral diarrhea virus (BVDV) is a pestivirus of the Flaviviridae family. The Flaviviridae include hog cholera virus and border disease virus of sheep. The virus contains a single strand of positive-sense RNA. A broad range of disease and immune effects is produced by BVDV only in cattle. In addition, this virus is important in the etiology of bovine pneumonias. Bovine viral diarrhea/mucosal disease (BVD/MD) is one of the most important viral diseases and one of the most complex diseases of cattle. Strains of BVDV are characterized as cytopathic (CP) and noncytopathic (NCP), based on cell-culture growth characteristics. The virus has also been categorized as type 1 and type 2 isolates. Heterologous strains exist that may confound even sound vaccination programs. Clinical signs and diagnosis. Signs of BVDV infections may be subclinical but also include abortions, congenital abnormalities, reduced fertility, persistent infection (PI) with gradual debilitation, and acute and fatal disease. The presence of antibodies, whether from passive transfer or immunizations, does not necessarily guarantee protection from the various forms of the disease. An acute form of the disease, caused by type 2 BVDV, occurs in cattle without sufficient immunity. After an incubation period of 5–7 days, clinical signs include fever, anorexia, oculonasal discharge, oral erosions (including on the hard palate), diarrhea, and decreased milk production. The disease course may be shorter with hemorrhagic syndrome and death within 2 days. Clinical signs of BVDV in calves also include severe enteritis and pneumonia. When susceptible cows are infected in utero from gestational days 50–100, or gestational cows are vaccinated with a modified live vaccine, abortion or stillbirth result. Congenital defects caused by BVDV during gestational days 90–170 include impaired immunity (thymic atrophy), cerebellar hypoplasia, ocular defects, alopecia or hypotrichosis, dysmyelinogenesis, hydranencephaly, hydrocephalus, and intrauterine growth retardation. Typical signs of cerebellar dysfunction will be evident in calves, such as wide-based stance, weakness, opisthotonus, hyperflexion, hypermetria, nystagmus, or strabismus. Some severely affected calves will not be able to stand. Ophthalmic effects include retinal degeneration and microphthalmia. Fetuses can also be infected in utero, normal at birth, immunotolerant to the virus, and persistently infected (PI). The term mucosal disease is commonly associated with this form of the infection. Many PI animals do not survive to maturity, however, and many have weakened immune systems. The PI animals are important because they shed virus and will probably show the clinical signs of mucosal disease (MD) caused by a CP BVDV strain derived from an NCP BVDV strain. These MD clinical signs include fever, anorexia, and profuse diarrhea that may include blood and fibrin casts, and oral and pharyngeal erosions, as well as erosion at the interdigital spaces and on the teats and vulva. Many other associated clinical signs include anemia, bloat, lameness, or corneal opacities and discharges. Secondary effects of hemorrhage and dehydration also contribute to the morbidity and mortality. Animals that do not succumb to the disease will be chronically unthrifty, debilitated, and infection-prone. Diagnosis in affected calves is based on herd health history, clinical signs, and antibodies to BVDV in precolostral serum. Viral culturing from blood may be useful. In older animals, oral lesions, serology, detection of viral antigen, and virus isolation contribute to the diagnosis. Leukopenia, and especially lymphopenia, are seen. Serology must be interpreted with the awareness of the possibility of PI immunotolerant animals. Vaccination against the disease carries its own set of side effects and potential problems, especially when using modified live vaccines, whether against CP or NCP strains. The condition of the animals is also a variable. Epizootiology and transmission. BVDV is present throughout the world. Transmission occurs easily by direct contact between cattle, from feed contaminated with secretions or feces, and by aborted fetuses and placentas. PI females transmit the virus to their fetuses. Semen also is a source of virus. Necropsy findings. In affected calves, histopathologic findings include necrosis of external germinal cells, focal hemorrhages, and folial edema. Later in the disease, large cavities develop in the cerebellum, and atrophy of the cerebellar folia and thin neuropil are evident. Older calves may have areas of intestinal necrosis. In cases where oral erosions occur, erosions will be found extending throughout the gastrointestinal tract to the cecum. The respiratory tract lesions will often be complicated by secondary bacterial pneumonia. When the hemorrhagic syndrome develops, petechiation and mucosal bleeding will be present. Pathogenesis. The CP and NCP strains are thought to be related mutations of the BVDV; the CP short-lived isolates are believed to arise from the NCP strains. The NCP strains are those present in the PI animals, and the strains are maintained in cattle populations. CP and NCP isolates vary in virulence, and classification of these types is based on viral surface proteins. Considerable antigenic variation also exists between strains and types. Other viral infections, such as bovine respiratory syncytial virus and infectious bovine rhinotracheitis, may also be present in the same animals. The pathology caused by BVDV is due to its ability to infect epithelial cells and impair the functioning of immune cell populations through out the bovine system. In type 2 BVDV hemorrhagic syndrome, death results from viral-induced thrombocytopenia. In fetuses, the virus infects developing germinal cells of the cerebellum. The Purkinje's cells in the granular layer are killed, and necrosis and inflammation follow. The immune effects are the result of the virus's interfering with neutrophil and macrophage functions and of lymphocyte blastogenesis. All of these predispose the affected animals to bacterial infections with Pasteurella haemolytica. BVDV damages dividing cells in fetal organ systems, resulting in abortions and congenital effects. Differential diagnosis. Many differentials must be considered for the clinical manifestations of BVDV infections. Differentials for enteritis of calves include viral infections, Cryptosporidia, Escherichia coli, Salmonella, and Coccidia. Salmonella, winter dysentery, Johne's disease, intestinal parasites, malignant catarrhal fever (MCF), and copper deficiency are differentials for the diarrhea seen in the disease in adult animals. Respiratory tract pathogens such as bovine respiratory syncytial virus, Pasteurella, Haemophilus, and Mycoplasma must be considered for the respiratory tract manifestations. Oral lesions are also produced by MCF, vesicular stomatitis, bluetongue, and papular stomatitis. Infectious bovine herpesvirus 1, leptospirosis, brucellosis, trichomoniasis, and mycosis should be considered in cases of abortion. Prevention and control. Combined with sound management in a typical cattle herd, vaccination is the best way to prevent BVDV and should be integrated into the herd health program, timed appropriately preceding breeding, gestation, or stressful events. Vaccine preparations for BVDV are modified live virus (MLV) or killed virus. Each has advantages and disadvantages. The former induces rapid immunity (within 1 week) after a single dose, provides longer duration of immunity against several strains, and induces serum neutralizing antibodies. MLV vaccines are not recommended for use in pregnant cattle, may induce mucosal disease, and may be immunosuppressive at the time of vaccination. The immunosuppression is detrimental if cattle are concurrently exposed to field-strain virus because it will facilitate infection and possible clinical disease. The MLV strains may cross the placenta, resulting in fetal infections. The killed vaccines are safer in pregnant animals but require booster doses after the initial immunization, may need to be given 2–3 times per year, and do not induce cell-mediated immunity. Passive immunity may protect most calves for up to 6–8 months of age. Subsequent vaccination with MLV may provide lifelong immunity, but this is not guaranteed. Annual boosters are recommended to protect against vaccine breaks. The virus persists in the environment for 2 weeks and is susceptible to the disfectants chlorhexidine, hypochlorite, iodophors, and aldehydes. Maintenance of a closed herd to prevent any possibility of the introduction of the virus is difficult. Isolation of new animals, avoidance of the purchase of pregnant cows, scrutiny of records from source farms, use of semen tested bulls, minimization of stress, testing of embryo-recipient cows, and maintainenance of populations of ruminants (smaller or wild species) separately on the premises will minimize viral exposure. Other management strategies may require a program for testing and culling PI cattle. This can be expensive but may be a worthwhile investment to remove the virus shedders from a herd. Treatment. No specific treatment is available. Supportive care and treatment with antibiotics to prevent secondary infection are recommended. Animals that survive the infection should be evaluated a month after recovery to determine their status as PI or virus-free. f. Cache Valley Virus Etiology. Cache Valley virus (CVV), of the arbovirus genus of the Bunyaviridae family, is a cause of congenital defects in lambs. Clinical signs and diagnosis. Teratogenic effects of in utero CVV infection in fetal and newborn lambs include arthrogryposis, microencephaly, hydranencephaly, porencephaly, cerebellar hypoplasia, and micromyelia. Stillbirths and mummified fetuses are seen. Lambs will be born weak and will act abnormally. Diagnosis is by evidence of seroconversion in precolostral blood samples or fetal fluids, as the result of in utero infection. Epizootiology and transmission. The virus is present in the western United States, although it has been isolated in a few Midwestern states. Although considered a disease of sheep, virus has been isolated from cattle and from wild ruminants and antibodies found in white-tailed deer. Transmission is by arthropods during the first trimester of pregnancy. Etiology. Cache Valley virus (CVV), of the arbovirus genus of the Bunyaviridae family, is a cause of congenital defects in lambs. Clinical signs and diagnosis. Teratogenic effects of in utero CVV infection in fetal and newborn lambs include arthrogryposis, microencephaly, hydranencephaly, porencephaly, cerebellar hypoplasia, and micromyelia. Stillbirths and mummified fetuses are seen. Lambs will be born weak and will act abnormally. Diagnosis is by evidence of seroconversion in precolostral blood samples or fetal fluids, as the result of in utero infection. Epizootiology and transmission. The virus is present in the western United States, although it has been isolated in a few Midwestern states. Although considered a disease of sheep, virus has been isolated from cattle and from wild ruminants and antibodies found in white-tailed deer. Transmission is by arthropods during the first trimester of pregnancy. Etiology. Cache Valley virus (CVV), of the arbovirus genus of the Bunyaviridae family, is a cause of congenital defects in lambs. Clinical signs and diagnosis. Teratogenic effects of in utero CVV infection in fetal and newborn lambs include arthrogryposis, microencephaly, hydranencephaly, porencephaly, cerebellar hypoplasia, and micromyelia. Stillbirths and mummified fetuses are seen. Lambs will be born weak and will act abnormally. Diagnosis is by evidence of seroconversion in precolostral blood samples or fetal fluids, as the result of in utero infection. Epizootiology and transmission. The virus is present in the western United States, although it has been isolated in a few Midwestern states. Although considered a disease of sheep, virus has been isolated from cattle and from wild ruminants and antibodies found in white-tailed deer. Transmission is by arthropods during the first trimester of pregnancy. g. Caprine Arthritis Encephalitis Virus Etiology. Caprine arthritis encephalitis virus (CAEV) occurs worldwide, with a high prevalence in the United States. Caprine arthritis encephalitis (CAE) is considered the most important viral disease of goats. The CAEV is in the Lentivirus genus of the Retroviridae family. It causes chronic arthritis in adults and encephalitis in young. CAEV is in the same viral genus as the ovine progressive pneumonia virus (OPPV). Clinical signs and diagnosis. The most common presentation in goats is an insidious, progressive arthritis in animals 6 months of age and older. Animals become stiff, have difficulty getting up, and may be clinically lame in one or both forelimbs. Carpal joints are so swollen and painful that the animal prefers to eat, drink, and walk on its "knees." In dairy goats, milk production decreases, and udders may become firmer. This retrovirus also causes neurological clinical signs in young kids 2–6 months old. Kids may be bright and alert, afebrile, and able to eat normally even when recumbent. Some kids may initially show unilateral weakness in a rear limb, which progresses to hemiplegia or tetraplegia. Mild to severe lower motor neuron deficits may be noted, but spinal reflexes are intact. Clinical signs may also include head tilt, blindness, ataxia, and facial nerve paralysis. Older animals in the group may experience interstitial pneumonia or chronic arthritis. The pneumonia is similar to the pneumonia in sheep caused by OPPV; the course is gradual but progressive, and animals will eventually lose weight and have respiratory distress. Some animals in a herd may not develop any clinical signs. Diagnosis is based on clinical signs, postmortem lesions, and positive serology for viral antibodies to CAEV. An agar gel immunodiffusion (AGID) test identifies antibodies to the virus and is used for diagnosis. Kids acquire an anti-CAEV antibody in colostrum, and this passive immunity may be interpreted as indicative of infection with the virus. The antibody does not prevent viral transmission. Epizootiology and transmission. The virus is prevalent in most industrialized countries. The common means of transmission, from adults to kids, is in the colostrum and milk in spite of the presence of anti-CAEV antibody in the colostrum. Transmission may occur among adult goats by contact. Intrauterine transmission is believed to be rare. Transmission to sheep has occurred only experimentally; there is no documented case of natural transmission. Necropsy findings. Necropsy and histopathology reveal a striking synovial hyperplasia of the joints with infiltrates of lymphocytes, macrophages, and plasma cells. Other histologic lesions include demyelination in the brain and spinal cord, with multifocal invasion of lymphocytes, macrophages, and plasma cells. In severe cases of mastitis, the udder may appear to be composed of lymphoid tissue. Pathogenesis. The virus infects cells of the mononuclear system, resulting in the formation of non-neutralizing antibody to viral core proteins and envelope proteins. Immune complex formation in synovial, mammary gland, and neurological tissue is thought to result in the clinical changes observed. Most commonly, the carpal joint is affected, followed by the stifle, hock, and hip. The infection is lifelong. Differential diagnosis. The differential diagnosis for the neurologic form of CAEV should include copper deficiency, enzootic pneumonia, white muscle disease, listeriosis, and spinal cord disease or injury. The differential diagnosis for CAEV arthritis should include chlamydia and mycoplasma. Prevention and control. Herds can be screened for CAE by testing serologically, using an AGID or an enzyme-linked immunosorbent assay (ELISA) test. The ELISA is purported to be more sensitive, whereas the AGID is more specific. Individual animals show great variation in development of antibody. Because CAE is highly prevalent in the United States, and because seronegative animals can shed organisms in the milk, retesting herds at least annually may be necessary. Recently, an immuno-precipitation test for CAE has been developed that has high sensitivity and specificity. Control measures include management practices such as test and cull, prevention of milk transmission, and isolation of affected animals. Parturition must be monitored, and kids must be removed immediately and fed heat-treated colostrum (56° C for 1 hr). CAEV-negative goats should be separated from CAEV-positive goats. Treatment. There is no treatment for CAEV. Etiology. Caprine arthritis encephalitis virus (CAEV) occurs worldwide, with a high prevalence in the United States. Caprine arthritis encephalitis (CAE) is considered the most important viral disease of goats. The CAEV is in the Lentivirus genus of the Retroviridae family. It causes chronic arthritis in adults and encephalitis in young. CAEV is in the same viral genus as the ovine progressive pneumonia virus (OPPV). Clinical signs and diagnosis. The most common presentation in goats is an insidious, progressive arthritis in animals 6 months of age and older. Animals become stiff, have difficulty getting up, and may be clinically lame in one or both forelimbs. Carpal joints are so swollen and painful that the animal prefers to eat, drink, and walk on its "knees." In dairy goats, milk production decreases, and udders may become firmer. This retrovirus also causes neurological clinical signs in young kids 2–6 months old. Kids may be bright and alert, afebrile, and able to eat normally even when recumbent. Some kids may initially show unilateral weakness in a rear limb, which progresses to hemiplegia or tetraplegia. Mild to severe lower motor neuron deficits may be noted, but spinal reflexes are intact. Clinical signs may also include head tilt, blindness, ataxia, and facial nerve paralysis. Older animals in the group may experience interstitial pneumonia or chronic arthritis. The pneumonia is similar to the pneumonia in sheep caused by OPPV; the course is gradual but progressive, and animals will eventually lose weight and have respiratory distress. Some animals in a herd may not develop any clinical signs. Diagnosis is based on clinical signs, postmortem lesions, and positive serology for viral antibodies to CAEV. An agar gel immunodiffusion (AGID) test identifies antibodies to the virus and is used for diagnosis. Kids acquire an anti-CAEV antibody in colostrum, and this passive immunity may be interpreted as indicative of infection with the virus. The antibody does not prevent viral transmission. Epizootiology and transmission. The virus is prevalent in most industrialized countries. The common means of transmission, from adults to kids, is in the colostrum and milk in spite of the presence of anti-CAEV antibody in the colostrum. Transmission may occur among adult goats by contact. Intrauterine transmission is believed to be rare. Transmission to sheep has occurred only experimentally; there is no documented case of natural transmission. Necropsy findings. Necropsy and histopathology reveal a striking synovial hyperplasia of the joints with infiltrates of lymphocytes, macrophages, and plasma cells. Other histologic lesions include demyelination in the brain and spinal cord, with multifocal invasion of lymphocytes, macrophages, and plasma cells. In severe cases of mastitis, the udder may appear to be composed of lymphoid tissue. Pathogenesis. The virus infects cells of the mononuclear system, resulting in the formation of non-neutralizing antibody to viral core proteins and envelope proteins. Immune complex formation in synovial, mammary gland, and neurological tissue is thought to result in the clinical changes observed. Most commonly, the carpal joint is affected, followed by the stifle, hock, and hip. The infection is lifelong. Differential diagnosis. The differential diagnosis for the neurologic form of CAEV should include copper deficiency, enzootic pneumonia, white muscle disease, listeriosis, and spinal cord disease or injury. The differential diagnosis for CAEV arthritis should include chlamydia and mycoplasma. Prevention and control. Herds can be screened for CAE by testing serologically, using an AGID or an enzyme-linked immunosorbent assay (ELISA) test. The ELISA is purported to be more sensitive, whereas the AGID is more specific. Individual animals show great variation in development of antibody. Because CAE is highly prevalent in the United States, and because seronegative animals can shed organisms in the milk, retesting herds at least annually may be necessary. Recently, an immuno-precipitation test for CAE has been developed that has high sensitivity and specificity. Control measures include management practices such as test and cull, prevention of milk transmission, and isolation of affected animals. Parturition must be monitored, and kids must be removed immediately and fed heat-treated colostrum (56° C for 1 hr). CAEV-negative goats should be separated from CAEV-positive goats. Treatment. There is no treatment for CAEV. Etiology. Caprine arthritis encephalitis virus (CAEV) occurs worldwide, with a high prevalence in the United States. Caprine arthritis encephalitis (CAE) is considered the most important viral disease of goats. The CAEV is in the Lentivirus genus of the Retroviridae family. It causes chronic arthritis in adults and encephalitis in young. CAEV is in the same viral genus as the ovine progressive pneumonia virus (OPPV). Clinical signs and diagnosis. The most common presentation in goats is an insidious, progressive arthritis in animals 6 months of age and older. Animals become stiff, have difficulty getting up, and may be clinically lame in one or both forelimbs. Carpal joints are so swollen and painful that the animal prefers to eat, drink, and walk on its "knees." In dairy goats, milk production decreases, and udders may become firmer. This retrovirus also causes neurological clinical signs in young kids 2–6 months old. Kids may be bright and alert, afebrile, and able to eat normally even when recumbent. Some kids may initially show unilateral weakness in a rear limb, which progresses to hemiplegia or tetraplegia. Mild to severe lower motor neuron deficits may be noted, but spinal reflexes are intact. Clinical signs may also include head tilt, blindness, ataxia, and facial nerve paralysis. Older animals in the group may experience interstitial pneumonia or chronic arthritis. The pneumonia is similar to the pneumonia in sheep caused by OPPV; the course is gradual but progressive, and animals will eventually lose weight and have respiratory distress. Some animals in a herd may not develop any clinical signs. Diagnosis is based on clinical signs, postmortem lesions, and positive serology for viral antibodies to CAEV. An agar gel immunodiffusion (AGID) test identifies antibodies to the virus and is used for diagnosis. Kids acquire an anti-CAEV antibody in colostrum, and this passive immunity may be interpreted as indicative of infection with the virus. The antibody does not prevent viral transmission. Epizootiology and transmission. The virus is prevalent in most industrialized countries. The common means of transmission, from adults to kids, is in the colostrum and milk in spite of the presence of anti-CAEV antibody in the colostrum. Transmission may occur among adult goats by contact. Intrauterine transmission is believed to be rare. Transmission to sheep has occurred only experimentally; there is no documented case of natural transmission. Necropsy findings. Necropsy and histopathology reveal a striking synovial hyperplasia of the joints with infiltrates of lymphocytes, macrophages, and plasma cells. Other histologic lesions include demyelination in the brain and spinal cord, with multifocal invasion of lymphocytes, macrophages, and plasma cells. In severe cases of mastitis, the udder may appear to be composed of lymphoid tissue. Pathogenesis. The virus infects cells of the mononuclear system, resulting in the formation of non-neutralizing antibody to viral core proteins and envelope proteins. Immune complex formation in synovial, mammary gland, and neurological tissue is thought to result in the clinical changes observed. Most commonly, the carpal joint is affected, followed by the stifle, hock, and hip. The infection is lifelong. Differential diagnosis. The differential diagnosis for the neurologic form of CAEV should include copper deficiency, enzootic pneumonia, white muscle disease, listeriosis, and spinal cord disease or injury. The differential diagnosis for CAEV arthritis should include chlamydia and mycoplasma. Prevention and control. Herds can be screened for CAE by testing serologically, using an AGID or an enzyme-linked immunosorbent assay (ELISA) test. The ELISA is purported to be more sensitive, whereas the AGID is more specific. Individual animals show great variation in development of antibody. Because CAE is highly prevalent in the United States, and because seronegative animals can shed organisms in the milk, retesting herds at least annually may be necessary. Recently, an immuno-precipitation test for CAE has been developed that has high sensitivity and specificity. Control measures include management practices such as test and cull, prevention of milk transmission, and isolation of affected animals. Parturition must be monitored, and kids must be removed immediately and fed heat-treated colostrum (56° C for 1 hr). CAEV-negative goats should be separated from CAEV-positive goats. Treatment. There is no treatment for CAEV. h. Infectious Bovine Rhinotracheitis Virus (Infectious Bovine Rhinotracheitis-Infectious Pustular Vulvovaginitis) Etiology. The infectious bovine rhinotracheitis virus (IBRV) is also referred to as bovine herpesvirus 1 (BHV-1) and is an alphaherpesvirus. IBRV causes or contributes to several bovine syndromes, including respiratory and reproductive tract diseases. It is one of the primary pathogens in the bovine respiratory disease complex. Strains include BHV-1.1 (associated with respiratory disease), BHV 1.2 (associated with respiratory and genital diseases), and BHV 1.4 (associated with neurological diseases), which has been reclassified as bovine herpesvirus 5. Clinical signs and diagnosis. Diseases caused by the virus include conjunctivitis, rhinotracheitis, pustular vulvovaginitis, balanoposthitis, abortion, encephalomyelitis, and mastitis. The respiratory form is known as infectious bovine rhinotracheitis, and clinical signs may range from mild to severe, the latter particularly when there are additional respiratory viral infections or secondary bacterial infections. The mortality rate in more mature cattle is low, however, unless there is secondary bacterial pneumonia. Fever, anorexia, restlessness, hyperemia of the muzzle, gray pustules on the muzzle (that later form plaques), nasal discharge (that may progress from serous to mucopurulent), hyperpnea, coughing, salivation, conjunctivitis with excessive epiphora, and decreased production in dairy animals are typical signs. Open-mouth breathing may be seen if the larynx or nasopharygneal areas are blocked by mucopurulent discharges. Neonatal calves may develop respiratory as well as general systemic disease. In these cases, in addition to the symptoms already noted, the soft palate may become necrotic, and gastrointestinal tract ulceration occurs. Young calves are most susceptible to the encephalitic form; signs include dull attitude, head pressing, vocalizations, nystagmus, head tilt, blindness, convulsions, and coma, as well as some signs, such as discharges, seen with respiratory tract presentations. This form is usually fatal within 5 days. Abortion may occur simultaneously with the conjunctival or respiratory tract diseases, when the respiratory infection appears to be mild, or may be delayed by as much as 3 months after the respiratory tract disease signs. Infectious pustular vulvovaginitis is most commonly seen in dairy cows, and clinical signs may be mild and not noticed. Otherwise, signs are fever, depression, anorexia, swelling of the vulvar labia, vulvar discharge, and vestibular mucosa reddened by pustules. The cow will often carry her tail elevated away from these lesions. These soon coalesce, and a fibrous membrane covers the ulcerated area. If uncomplicated, the infection lasts about 4–5 days, and lesions heal in 2 weeks. Younger infected bulls may develop balanoposthitis with edema, swelling, and pain such that the animals will not service cows. Epizootiology and transmission. IBRV is widely distributed throughout the world, and adult animals are the reservoirs of infection. The disease is more common in intensive calf-rearing situations and in grouped or stressed cattle. Transmission is primarily by secretions, such as nasal, during and after clinical signs of disease. Modified live vaccines are capable of causing latent infections. Necropsy findings. Fibrinonecrotic rhinotracheitis is considered pathognomic for IBRV respiratory tract infections. There will be adherent necrotic lesions in the respiratory, ocular, and reproductive mucosa. When there are secondary bacterial infections, such as Pasteurella bronchopneumonia, findings will include congested tracheal mucosa and petechial and ecchymotic hemorrhages in that tissue. Lesions from the encephalitic form include lymphocytic meningoencephalitis and will be found throughout the gray matter (neuronal degeneration, perivascular cuffing) and white matter (myelitis, demyelination). Intranuclear inclusion bodies are not a common finding with this herpesvirus. Pathogenesis. In the encephalitic form, the virus first grows in nasal mucosa and produces plaques. These resolve within 11 days, and the encephalitis develops after the virus spreads centripetally to the brain stem by the trigeminal nerve dendrites. Latent infections are also established in neural tissue. Differential diagnosis. The severe oral erosions seen with BVDV infections are rare with infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV). The conjunctivitis of IBR may initially be mistaken for that of a Moraxella bovis (pinkeye) infection; the IBR will be peripheral, and there will not be corneal ulceration. Bovine viral diarrhea virus and IBRV are the most common viral causes of bovine abortion. Differentials for balanoposthitis include trauma from service. Prevention and control. Vaccination options include inactivated, attenuated, modified live, and genetically altered preparations. Some are in combination with parainfluenza 3 (PI-3) virus. The MLV preparations are administered intranasally; these are advantageous in calves for inducing mucosal immunity even when serologic passive immunity is already present and adequate. Some newer vaccines, with gene deletion, allow for serologic differentiation between antibody responses from infection or immunization. Bulls with the venereal form of the infection will transmit the virus in semen; intranasal vaccine may be used to provide some immunity. Treatment. Uncomplicated mild infections will resolve over a few weeks; palliative treatments, such as cleaning ocular discharges and supplying softened food, are helpful in recovery. Antibiotics are usually administered because of the high likelihood of secondary bacterial pneumonia. The encephalitic animals may need to be treated with anticonvulsants. Etiology. The infectious bovine rhinotracheitis virus (IBRV) is also referred to as bovine herpesvirus 1 (BHV-1) and is an alphaherpesvirus. IBRV causes or contributes to several bovine syndromes, including respiratory and reproductive tract diseases. It is one of the primary pathogens in the bovine respiratory disease complex. Strains include BHV-1.1 (associated with respiratory disease), BHV 1.2 (associated with respiratory and genital diseases), and BHV 1.4 (associated with neurological diseases), which has been reclassified as bovine herpesvirus 5. Clinical signs and diagnosis. Diseases caused by the virus include conjunctivitis, rhinotracheitis, pustular vulvovaginitis, balanoposthitis, abortion, encephalomyelitis, and mastitis. The respiratory form is known as infectious bovine rhinotracheitis, and clinical signs may range from mild to severe, the latter particularly when there are additional respiratory viral infections or secondary bacterial infections. The mortality rate in more mature cattle is low, however, unless there is secondary bacterial pneumonia. Fever, anorexia, restlessness, hyperemia of the muzzle, gray pustules on the muzzle (that later form plaques), nasal discharge (that may progress from serous to mucopurulent), hyperpnea, coughing, salivation, conjunctivitis with excessive epiphora, and decreased production in dairy animals are typical signs. Open-mouth breathing may be seen if the larynx or nasopharygneal areas are blocked by mucopurulent discharges. Neonatal calves may develop respiratory as well as general systemic disease. In these cases, in addition to the symptoms already noted, the soft palate may become necrotic, and gastrointestinal tract ulceration occurs. Young calves are most susceptible to the encephalitic form; signs include dull attitude, head pressing, vocalizations, nystagmus, head tilt, blindness, convulsions, and coma, as well as some signs, such as discharges, seen with respiratory tract presentations. This form is usually fatal within 5 days. Abortion may occur simultaneously with the conjunctival or respiratory tract diseases, when the respiratory infection appears to be mild, or may be delayed by as much as 3 months after the respiratory tract disease signs. Infectious pustular vulvovaginitis is most commonly seen in dairy cows, and clinical signs may be mild and not noticed. Otherwise, signs are fever, depression, anorexia, swelling of the vulvar labia, vulvar discharge, and vestibular mucosa reddened by pustules. The cow will often carry her tail elevated away from these lesions. These soon coalesce, and a fibrous membrane covers the ulcerated area. If uncomplicated, the infection lasts about 4–5 days, and lesions heal in 2 weeks. Younger infected bulls may develop balanoposthitis with edema, swelling, and pain such that the animals will not service cows. Epizootiology and transmission. IBRV is widely distributed throughout the world, and adult animals are the reservoirs of infection. The disease is more common in intensive calf-rearing situations and in grouped or stressed cattle. Transmission is primarily by secretions, such as nasal, during and after clinical signs of disease. Modified live vaccines are capable of causing latent infections. Necropsy findings. Fibrinonecrotic rhinotracheitis is considered pathognomic for IBRV respiratory tract infections. There will be adherent necrotic lesions in the respiratory, ocular, and reproductive mucosa. When there are secondary bacterial infections, such as Pasteurella bronchopneumonia, findings will include congested tracheal mucosa and petechial and ecchymotic hemorrhages in that tissue. Lesions from the encephalitic form include lymphocytic meningoencephalitis and will be found throughout the gray matter (neuronal degeneration, perivascular cuffing) and white matter (myelitis, demyelination). Intranuclear inclusion bodies are not a common finding with this herpesvirus. Pathogenesis. In the encephalitic form, the virus first grows in nasal mucosa and produces plaques. These resolve within 11 days, and the encephalitis develops after the virus spreads centripetally to the brain stem by the trigeminal nerve dendrites. Latent infections are also established in neural tissue. Differential diagnosis. The severe oral erosions seen with BVDV infections are rare with infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV). The conjunctivitis of IBR may initially be mistaken for that of a Moraxella bovis (pinkeye) infection; the IBR will be peripheral, and there will not be corneal ulceration. Bovine viral diarrhea virus and IBRV are the most common viral causes of bovine abortion. Differentials for balanoposthitis include trauma from service. Prevention and control. Vaccination options include inactivated, attenuated, modified live, and genetically altered preparations. Some are in combination with parainfluenza 3 (PI-3) virus. The MLV preparations are administered intranasally; these are advantageous in calves for inducing mucosal immunity even when serologic passive immunity is already present and adequate. Some newer vaccines, with gene deletion, allow for serologic differentiation between antibody responses from infection or immunization. Bulls with the venereal form of the infection will transmit the virus in semen; intranasal vaccine may be used to provide some immunity. Treatment. Uncomplicated mild infections will resolve over a few weeks; palliative treatments, such as cleaning ocular discharges and supplying softened food, are helpful in recovery. Antibiotics are usually administered because of the high likelihood of secondary bacterial pneumonia. The encephalitic animals may need to be treated with anticonvulsants. Etiology. The infectious bovine rhinotracheitis virus (IBRV) is also referred to as bovine herpesvirus 1 (BHV-1) and is an alphaherpesvirus. IBRV causes or contributes to several bovine syndromes, including respiratory and reproductive tract diseases. It is one of the primary pathogens in the bovine respiratory disease complex. Strains include BHV-1.1 (associated with respiratory disease), BHV 1.2 (associated with respiratory and genital diseases), and BHV 1.4 (associated with neurological diseases), which has been reclassified as bovine herpesvirus 5. Clinical signs and diagnosis. Diseases caused by the virus include conjunctivitis, rhinotracheitis, pustular vulvovaginitis, balanoposthitis, abortion, encephalomyelitis, and mastitis. The respiratory form is known as infectious bovine rhinotracheitis, and clinical signs may range from mild to severe, the latter particularly when there are additional respiratory viral infections or secondary bacterial infections. The mortality rate in more mature cattle is low, however, unless there is secondary bacterial pneumonia. Fever, anorexia, restlessness, hyperemia of the muzzle, gray pustules on the muzzle (that later form plaques), nasal discharge (that may progress from serous to mucopurulent), hyperpnea, coughing, salivation, conjunctivitis with excessive epiphora, and decreased production in dairy animals are typical signs. Open-mouth breathing may be seen if the larynx or nasopharygneal areas are blocked by mucopurulent discharges. Neonatal calves may develop respiratory as well as general systemic disease. In these cases, in addition to the symptoms already noted, the soft palate may become necrotic, and gastrointestinal tract ulceration occurs. Young calves are most susceptible to the encephalitic form; signs include dull attitude, head pressing, vocalizations, nystagmus, head tilt, blindness, convulsions, and coma, as well as some signs, such as discharges, seen with respiratory tract presentations. This form is usually fatal within 5 days. Abortion may occur simultaneously with the conjunctival or respiratory tract diseases, when the respiratory infection appears to be mild, or may be delayed by as much as 3 months after the respiratory tract disease signs. Infectious pustular vulvovaginitis is most commonly seen in dairy cows, and clinical signs may be mild and not noticed. Otherwise, signs are fever, depression, anorexia, swelling of the vulvar labia, vulvar discharge, and vestibular mucosa reddened by pustules. The cow will often carry her tail elevated away from these lesions. These soon coalesce, and a fibrous membrane covers the ulcerated area. If uncomplicated, the infection lasts about 4–5 days, and lesions heal in 2 weeks. Younger infected bulls may develop balanoposthitis with edema, swelling, and pain such that the animals will not service cows. Epizootiology and transmission. IBRV is widely distributed throughout the world, and adult animals are the reservoirs of infection. The disease is more common in intensive calf-rearing situations and in grouped or stressed cattle. Transmission is primarily by secretions, such as nasal, during and after clinical signs of disease. Modified live vaccines are capable of causing latent infections. Necropsy findings. Fibrinonecrotic rhinotracheitis is considered pathognomic for IBRV respiratory tract infections. There will be adherent necrotic lesions in the respiratory, ocular, and reproductive mucosa. When there are secondary bacterial infections, such as Pasteurella bronchopneumonia, findings will include congested tracheal mucosa and petechial and ecchymotic hemorrhages in that tissue. Lesions from the encephalitic form include lymphocytic meningoencephalitis and will be found throughout the gray matter (neuronal degeneration, perivascular cuffing) and white matter (myelitis, demyelination). Intranuclear inclusion bodies are not a common finding with this herpesvirus. Pathogenesis. In the encephalitic form, the virus first grows in nasal mucosa and produces plaques. These resolve within 11 days, and the encephalitis develops after the virus spreads centripetally to the brain stem by the trigeminal nerve dendrites. Latent infections are also established in neural tissue. Differential diagnosis. The severe oral erosions seen with BVDV infections are rare with infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV). The conjunctivitis of IBR may initially be mistaken for that of a Moraxella bovis (pinkeye) infection; the IBR will be peripheral, and there will not be corneal ulceration. Bovine viral diarrhea virus and IBRV are the most common viral causes of bovine abortion. Differentials for balanoposthitis include trauma from service. Prevention and control. Vaccination options include inactivated, attenuated, modified live, and genetically altered preparations. Some are in combination with parainfluenza 3 (PI-3) virus. The MLV preparations are administered intranasally; these are advantageous in calves for inducing mucosal immunity even when serologic passive immunity is already present and adequate. Some newer vaccines, with gene deletion, allow for serologic differentiation between antibody responses from infection or immunization. Bulls with the venereal form of the infection will transmit the virus in semen; intranasal vaccine may be used to provide some immunity. Treatment. Uncomplicated mild infections will resolve over a few weeks; palliative treatments, such as cleaning ocular discharges and supplying softened food, are helpful in recovery. Antibiotics are usually administered because of the high likelihood of secondary bacterial pneumonia. The encephalitic animals may need to be treated with anticonvulsants. i. Parainfluenza 3 (PI-3) Etiology. Parainfluenza 3, an RNA virus of the family Paramyxoviridae, causes mild respiratory disease of ruminants when it is the sole pathogen. The viral infection often predisposes the respiratory system to severe disease associated with concurrent viral or bacterial pathogens. Viral strains are reported to vary in virulence. Serotypes seen in the smaller ruminants are distinct from those isolated from cattle. Clinical signs and diagnosis. Infections ranging from asymptomatic to mild signs of upper respiratory tract disease are associated with this virus by itself; infections are almost never fatal. Clinical signs include ocular and nasal discharges, cough, fever, and increased respiratory rate and breath sounds. In pregnant animals, exposure to PI-3 can result in abortions. Clinical signs become apparent or more severe when additional viral pathogens are present, such as bovine viral diarrhea virus, or a secondary bacterial infection, such as Pasteurella haemolytica infection, is involved. Greater morbidity and mortality will be sequelae of the bacterial infections. Viral isolation or direct immunofluorescence antibody (IFA) from nasal swabs can be used for definitive diagnosis. Epizootiology and transmission. The virus is considered ubiquitous in cattle and is a common infection in sheep. Presently it is assumed that the virus is widespread in goats, but firm evidence is lacking. Necropsy findings. For an infection of PI-3 only, findings will be negligible. Some congestion of respiratory mucosa, swelling of respiratory tract-associated lymph nodes, and mild pneumonitis may be noted grossly and histologically. Intranuclear and intracytoplasmic inclusion bodies may be present in the mucosal epithelial cells. Findings will be similar but not as severe as those caused by bovine respiratory syncytial virus. Immunohistochemistry may also be used. Pathogenesis. PI-3 infects the epithelial mucosa of the respiratory tract; however, the disease is often asymptomatic when uncomplicated. Differential diagnosis. Differentials, particularly in cattle, include infections with other respiratory tract viruses of ruminants: IBRV, BVDV, bovine respiratory syncytial virus, and type 3 bovine adenovirus. Prevention and control. Immunization, management, and nutrition are important for this respiratory pathogen, as for others. In cattle, modified live vaccines for intramuscular (IM), subcutaneous (SC), or intranasal (IN) administration are available. The IM and SC routes provide immune protection within 1 week after administration but will not provide protection in the presence of passively acquired antibodies. It is contraindicated for pregnant animals because it will cause abortion. The IN route immunizes in the presence of passively acquired antibodies, provides immunity within 3 days of administration, and stimulates the production of interferon. Other vaccine formulations, about which less information is reported, include inactivated or chemically altered live-virus preparations; both are administered IM, and followup immunizations are needed within 4 weeks. Booster vaccinations are recommended for all preparations within 2–6 months after the initial immunization. All presently marketed vaccine products come in combination with other bovine respiratory viruses as multivaccine products. The humoral immunity protects against PI-3 abortions. There is no approved PI-3 vaccine for sheep and goats. The use of the cattle formulation in these smaller ruminants is not recommended. Sound management of housing, sanitation, nutrition, and preventive medicine programs are all equally important components in prevention and control. Treatment. Uncomplicated disease is not treated. Etiology. Parainfluenza 3, an RNA virus of the family Paramyxoviridae, causes mild respiratory disease of ruminants when it is the sole pathogen. The viral infection often predisposes the respiratory system to severe disease associated with concurrent viral or bacterial pathogens. Viral strains are reported to vary in virulence. Serotypes seen in the smaller ruminants are distinct from those isolated from cattle. Clinical signs and diagnosis. Infections ranging from asymptomatic to mild signs of upper respiratory tract disease are associated with this virus by itself; infections are almost never fatal. Clinical signs include ocular and nasal discharges, cough, fever, and increased respiratory rate and breath sounds. In pregnant animals, exposure to PI-3 can result in abortions. Clinical signs become apparent or more severe when additional viral pathogens are present, such as bovine viral diarrhea virus, or a secondary bacterial infection, such as Pasteurella haemolytica infection, is involved. Greater morbidity and mortality will be sequelae of the bacterial infections. Viral isolation or direct immunofluorescence antibody (IFA) from nasal swabs can be used for definitive diagnosis. Epizootiology and transmission. The virus is considered ubiquitous in cattle and is a common infection in sheep. Presently it is assumed that the virus is widespread in goats, but firm evidence is lacking. Necropsy findings. For an infection of PI-3 only, findings will be negligible. Some congestion of respiratory mucosa, swelling of respiratory tract-associated lymph nodes, and mild pneumonitis may be noted grossly and histologically. Intranuclear and intracytoplasmic inclusion bodies may be present in the mucosal epithelial cells. Findings will be similar but not as severe as those caused by bovine respiratory syncytial virus. Immunohistochemistry may also be used. Pathogenesis. PI-3 infects the epithelial mucosa of the respiratory tract; however, the disease is often asymptomatic when uncomplicated. Differential diagnosis. Differentials, particularly in cattle, include infections with other respiratory tract viruses of ruminants: IBRV, BVDV, bovine respiratory syncytial virus, and type 3 bovine adenovirus. Prevention and control. Immunization, management, and nutrition are important for this respiratory pathogen, as for others. In cattle, modified live vaccines for intramuscular (IM), subcutaneous (SC), or intranasal (IN) administration are available. The IM and SC routes provide immune protection within 1 week after administration but will not provide protection in the presence of passively acquired antibodies. It is contraindicated for pregnant animals because it will cause abortion. The IN route immunizes in the presence of passively acquired antibodies, provides immunity within 3 days of administration, and stimulates the production of interferon. Other vaccine formulations, about which less information is reported, include inactivated or chemically altered live-virus preparations; both are administered IM, and followup immunizations are needed within 4 weeks. Booster vaccinations are recommended for all preparations within 2–6 months after the initial immunization. All presently marketed vaccine products come in combination with other bovine respiratory viruses as multivaccine products. The humoral immunity protects against PI-3 abortions. There is no approved PI-3 vaccine for sheep and goats. The use of the cattle formulation in these smaller ruminants is not recommended. Sound management of housing, sanitation, nutrition, and preventive medicine programs are all equally important components in prevention and control. Treatment. Uncomplicated disease is not treated. Etiology. Parainfluenza 3, an RNA virus of the family Paramyxoviridae, causes mild respiratory disease of ruminants when it is the sole pathogen. The viral infection often predisposes the respiratory system to severe disease associated with concurrent viral or bacterial pathogens. Viral strains are reported to vary in virulence. Serotypes seen in the smaller ruminants are distinct from those isolated from cattle. Clinical signs and diagnosis. Infections ranging from asymptomatic to mild signs of upper respiratory tract disease are associated with this virus by itself; infections are almost never fatal. Clinical signs include ocular and nasal discharges, cough, fever, and increased respiratory rate and breath sounds. In pregnant animals, exposure to PI-3 can result in abortions. Clinical signs become apparent or more severe when additional viral pathogens are present, such as bovine viral diarrhea virus, or a secondary bacterial infection, such as Pasteurella haemolytica infection, is involved. Greater morbidity and mortality will be sequelae of the bacterial infections. Viral isolation or direct immunofluorescence antibody (IFA) from nasal swabs can be used for definitive diagnosis. Epizootiology and transmission. The virus is considered ubiquitous in cattle and is a common infection in sheep. Presently it is assumed that the virus is widespread in goats, but firm evidence is lacking. Necropsy findings. For an infection of PI-3 only, findings will be negligible. Some congestion of respiratory mucosa, swelling of respiratory tract-associated lymph nodes, and mild pneumonitis may be noted grossly and histologically. Intranuclear and intracytoplasmic inclusion bodies may be present in the mucosal epithelial cells. Findings will be similar but not as severe as those caused by bovine respiratory syncytial virus. Immunohistochemistry may also be used. Pathogenesis. PI-3 infects the epithelial mucosa of the respiratory tract; however, the disease is often asymptomatic when uncomplicated. Differential diagnosis. Differentials, particularly in cattle, include infections with other respiratory tract viruses of ruminants: IBRV, BVDV, bovine respiratory syncytial virus, and type 3 bovine adenovirus. Prevention and control. Immunization, management, and nutrition are important for this respiratory pathogen, as for others. In cattle, modified live vaccines for intramuscular (IM), subcutaneous (SC), or intranasal (IN) administration are available. The IM and SC routes provide immune protection within 1 week after administration but will not provide protection in the presence of passively acquired antibodies. It is contraindicated for pregnant animals because it will cause abortion. The IN route immunizes in the presence of passively acquired antibodies, provides immunity within 3 days of administration, and stimulates the production of interferon. Other vaccine formulations, about which less information is reported, include inactivated or chemically altered live-virus preparations; both are administered IM, and followup immunizations are needed within 4 weeks. Booster vaccinations are recommended for all preparations within 2–6 months after the initial immunization. All presently marketed vaccine products come in combination with other bovine respiratory viruses as multivaccine products. The humoral immunity protects against PI-3 abortions. There is no approved PI-3 vaccine for sheep and goats. The use of the cattle formulation in these smaller ruminants is not recommended. Sound management of housing, sanitation, nutrition, and preventive medicine programs are all equally important components in prevention and control. Treatment. Uncomplicated disease is not treated. j. Respiratory Syncytial Viruses of Ruminants Etiology. The respiratory syncytial viruses are pneumoviruses of the Paramyxoviridae family and are common causes of severe disease in ruminants, especially calves and yearling cattle. Two serotypes of the bovine respiratory syncytial virus (BRSV) have been described for cattle; these may be similar or identical to the virus seen in sheep and goats. Clinical findings and diagnosis. Infections may be subclinical or develop into severe illness. Clinical signs include fever, hyperpnea, spontaneous or easily induced cough, nasal discharge, and conjunctivitis. Interstitial pneumonia usually develops, and harsh respiratory sounds are evident on auscultation. Development of emphysema indicates a poor prognosis, and death may occur in the severe cases of the viral infection. Secondary bacterial pneumonia, especially with Pasteurella haemolytica, with morbidity and mortality, is also a common sequela. Abortions have been assciated with BRSV outbreaks. Diagnosis is based on virus isolation and serology (acute and convalescent). Nasal swabs for virus isolation should be taken when animals have fever and before onset of respiratory disease. Epizootiology and transmission. These viruses are considered ubiquitous in domestic cattle and are transmitted by aerosols. Necropsy findings. Gross lesions include consolidation of anteroventral lung lobes. Edema and emphysema are present. As the name indicates, syncytia, which may have inclusions, form in areas of the lungs infected with the virus. Necrotizing bronchiolitis, bronchiolitis obliterans, and hyaline membrane formation will be evident microscopically. Pathogenesis. The severe form of the disease, which often follows a mild preliminary infection, is thought to be caused by immune-mediated factors during the process of infection in the lung. Virulence may vary greatly among viral strains. Differential diagnosis. Differentials should include other ruminant respiratory tract viruses. Prevention and control. Vaccination should be part of the standard health program, and all animals should be vaccinated regularly. Vaccinations should be administered within 1–2 months of stressful events, such as weaning, shipping, and introduction to new surroundings. Currently available vaccines include an inactivated preparation and a modified live virus preparation administered intramuscularly or subcutaneously; immunity develops well in yearling animals, and colostral antibodies develop when cows are vaccinated during late gestation. Passive immunity from colostrum provides at least partial protection to calves in herds where disease is prevalent. But this immunity suppresses the mucosal IgA response and serum antibody responses. The basis for successful immune protection is the mucosal memory IgA, but this is difficult to achieve with present vaccine formulations. The virus is easily inactivated in the environment. Preventive measures in preweaning animals should include preconditioning to minimize weaning stress. Treatment. Recovery can be spontaneous; however, antibiotics and supportive therapy are useful to prevent or control secondary bacterial pneumonia. In severe cases, antihistamines and corticosteriods may also be necessary. Use of vaccine during natural infection is not productive and may result in severe disease. Etiology. The respiratory syncytial viruses are pneumoviruses of the Paramyxoviridae family and are common causes of severe disease in ruminants, especially calves and yearling cattle. Two serotypes of the bovine respiratory syncytial virus (BRSV) have been described for cattle; these may be similar or identical to the virus seen in sheep and goats. Clinical findings and diagnosis. Infections may be subclinical or develop into severe illness. Clinical signs include fever, hyperpnea, spontaneous or easily induced cough, nasal discharge, and conjunctivitis. Interstitial pneumonia usually develops, and harsh respiratory sounds are evident on auscultation. Development of emphysema indicates a poor prognosis, and death may occur in the severe cases of the viral infection. Secondary bacterial pneumonia, especially with Pasteurella haemolytica, with morbidity and mortality, is also a common sequela. Abortions have been assciated with BRSV outbreaks. Diagnosis is based on virus isolation and serology (acute and convalescent). Nasal swabs for virus isolation should be taken when animals have fever and before onset of respiratory disease. Epizootiology and transmission. These viruses are considered ubiquitous in domestic cattle and are transmitted by aerosols. Necropsy findings. Gross lesions include consolidation of anteroventral lung lobes. Edema and emphysema are present. As the name indicates, syncytia, which may have inclusions, form in areas of the lungs infected with the virus. Necrotizing bronchiolitis, bronchiolitis obliterans, and hyaline membrane formation will be evident microscopically. Pathogenesis. The severe form of the disease, which often follows a mild preliminary infection, is thought to be caused by immune-mediated factors during the process of infection in the lung. Virulence may vary greatly among viral strains. Differential diagnosis. Differentials should include other ruminant respiratory tract viruses. Prevention and control. Vaccination should be part of the standard health program, and all animals should be vaccinated regularly. Vaccinations should be administered within 1–2 months of stressful events, such as weaning, shipping, and introduction to new surroundings. Currently available vaccines include an inactivated preparation and a modified live virus preparation administered intramuscularly or subcutaneously; immunity develops well in yearling animals, and colostral antibodies develop when cows are vaccinated during late gestation. Passive immunity from colostrum provides at least partial protection to calves in herds where disease is prevalent. But this immunity suppresses the mucosal IgA response and serum antibody responses. The basis for successful immune protection is the mucosal memory IgA, but this is difficult to achieve with present vaccine formulations. The virus is easily inactivated in the environment. Preventive measures in preweaning animals should include preconditioning to minimize weaning stress. Treatment. Recovery can be spontaneous; however, antibiotics and supportive therapy are useful to prevent or control secondary bacterial pneumonia. In severe cases, antihistamines and corticosteriods may also be necessary. Use of vaccine during natural infection is not productive and may result in severe disease. Etiology. The respiratory syncytial viruses are pneumoviruses of the Paramyxoviridae family and are common causes of severe disease in ruminants, especially calves and yearling cattle. Two serotypes of the bovine respiratory syncytial virus (BRSV) have been described for cattle; these may be similar or identical to the virus seen in sheep and goats. Clinical findings and diagnosis. Infections may be subclinical or develop into severe illness. Clinical signs include fever, hyperpnea, spontaneous or easily induced cough, nasal discharge, and conjunctivitis. Interstitial pneumonia usually develops, and harsh respiratory sounds are evident on auscultation. Development of emphysema indicates a poor prognosis, and death may occur in the severe cases of the viral infection. Secondary bacterial pneumonia, especially with Pasteurella haemolytica, with morbidity and mortality, is also a common sequela. Abortions have been assciated with BRSV outbreaks. Diagnosis is based on virus isolation and serology (acute and convalescent). Nasal swabs for virus isolation should be taken when animals have fever and before onset of respiratory disease. Epizootiology and transmission. These viruses are considered ubiquitous in domestic cattle and are transmitted by aerosols. Necropsy findings. Gross lesions include consolidation of anteroventral lung lobes. Edema and emphysema are present. As the name indicates, syncytia, which may have inclusions, form in areas of the lungs infected with the virus. Necrotizing bronchiolitis, bronchiolitis obliterans, and hyaline membrane formation will be evident microscopically. Pathogenesis. The severe form of the disease, which often follows a mild preliminary infection, is thought to be caused by immune-mediated factors during the process of infection in the lung. Virulence may vary greatly among viral strains. Differential diagnosis. Differentials should include other ruminant respiratory tract viruses. Prevention and control. Vaccination should be part of the standard health program, and all animals should be vaccinated regularly. Vaccinations should be administered within 1–2 months of stressful events, such as weaning, shipping, and introduction to new surroundings. Currently available vaccines include an inactivated preparation and a modified live virus preparation administered intramuscularly or subcutaneously; immunity develops well in yearling animals, and colostral antibodies develop when cows are vaccinated during late gestation. Passive immunity from colostrum provides at least partial protection to calves in herds where disease is prevalent. But this immunity suppresses the mucosal IgA response and serum antibody responses. The basis for successful immune protection is the mucosal memory IgA, but this is difficult to achieve with present vaccine formulations. The virus is easily inactivated in the environment. Preventive measures in preweaning animals should include preconditioning to minimize weaning stress. Treatment. Recovery can be spontaneous; however, antibiotics and supportive therapy are useful to prevent or control secondary bacterial pneumonia. In severe cases, antihistamines and corticosteriods may also be necessary. Use of vaccine during natural infection is not productive and may result in severe disease. k. Ulcerative Dermatosis (Ovine Venereal Disease, Balanoposthitis) Etiology. Ulcerative dermatosis is a contagious disease of sheep only. It is caused by a poxvirus similar to but distinct from the causative agent of contagious ecthyma ( "Current Veterinary Therapy," 1993 ). Clinical signs and diagnosis. Lesions include ulcers and crusts associated with the skin and mucous membranes of the genitalia, face, and feet ( Bulgin, 1986 ). Genital lesions are much more common than the facial or coronal lesions. Discomfort may be associated with the lesions. Paraphimosis occasionally occurs. These lesions are painful; during breeding season, animals will avoid coitus. Morbidity is low to moderate, and mortality negligible if the flock is otherwise healthy. Diagnosis is based on clinical signs. Epizootiology and transmission. Endemic to the western United States, ulcerative dermatosis is transmitted through direct contact with abraded skin of the prepuce, vulva, face, and feet. Necropsy findings. Necropsy would rarely be necessary to diagnose an outbreak in a healthy flock. Findings will be similar to those described for contagious ecthyma. Pathogenesis. Following an incubation period of 2–5 days, the virus replicates in the epidermal cells and leads to necrosis and pustule formation. Pustules rapidly break, forming weeping ulcers. The ulcers scab over and eventually form a fibrotic scar. The disease usually resolves in 2–6 weeks. Rarely, the disease will persist for many months to more than a year. Differential diagnosis. The main differential is contagious ecthyma, which is grossly and histopathologically associated with epithelial hyperplasia. This is also a feature of ulcerative dermatosis. Prevention and control. No vaccine is available. Affected animals, especially males, should not be used for breeding. Treatment. Affected animals should be separated from the rest of the flock. Treatment is supportive, including antiseptic ointments and astringents. Research complications. Breeding and maintenance of the flocks' condition, because of the pain associated with eating, will be compromised during an outbreak. Etiology. Ulcerative dermatosis is a contagious disease of sheep only. It is caused by a poxvirus similar to but distinct from the causative agent of contagious ecthyma ( "Current Veterinary Therapy," 1993 ). Clinical signs and diagnosis. Lesions include ulcers and crusts associated with the skin and mucous membranes of the genitalia, face, and feet ( Bulgin, 1986 ). Genital lesions are much more common than the facial or coronal lesions. Discomfort may be associated with the lesions. Paraphimosis occasionally occurs. These lesions are painful; during breeding season, animals will avoid coitus. Morbidity is low to moderate, and mortality negligible if the flock is otherwise healthy. Diagnosis is based on clinical signs. Epizootiology and transmission. Endemic to the western United States, ulcerative dermatosis is transmitted through direct contact with abraded skin of the prepuce, vulva, face, and feet. Necropsy findings. Necropsy would rarely be necessary to diagnose an outbreak in a healthy flock. Findings will be similar to those described for contagious ecthyma. Pathogenesis. Following an incubation period of 2–5 days, the virus replicates in the epidermal cells and leads to necrosis and pustule formation. Pustules rapidly break, forming weeping ulcers. The ulcers scab over and eventually form a fibrotic scar. The disease usually resolves in 2–6 weeks. Rarely, the disease will persist for many months to more than a year. Differential diagnosis. The main differential is contagious ecthyma, which is grossly and histopathologically associated with epithelial hyperplasia. This is also a feature of ulcerative dermatosis. Prevention and control. No vaccine is available. Affected animals, especially males, should not be used for breeding. Treatment. Affected animals should be separated from the rest of the flock. Treatment is supportive, including antiseptic ointments and astringents. Research complications. Breeding and maintenance of the flocks' condition, because of the pain associated with eating, will be compromised during an outbreak. Etiology. Ulcerative dermatosis is a contagious disease of sheep only. It is caused by a poxvirus similar to but distinct from the causative agent of contagious ecthyma ( "Current Veterinary Therapy," 1993 ). Clinical signs and diagnosis. Lesions include ulcers and crusts associated with the skin and mucous membranes of the genitalia, face, and feet ( Bulgin, 1986 ). Genital lesions are much more common than the facial or coronal lesions. Discomfort may be associated with the lesions. Paraphimosis occasionally occurs. These lesions are painful; during breeding season, animals will avoid coitus. Morbidity is low to moderate, and mortality negligible if the flock is otherwise healthy. Diagnosis is based on clinical signs. Epizootiology and transmission. Endemic to the western United States, ulcerative dermatosis is transmitted through direct contact with abraded skin of the prepuce, vulva, face, and feet. Necropsy findings. Necropsy would rarely be necessary to diagnose an outbreak in a healthy flock. Findings will be similar to those described for contagious ecthyma. Pathogenesis. Following an incubation period of 2–5 days, the virus replicates in the epidermal cells and leads to necrosis and pustule formation. Pustules rapidly break, forming weeping ulcers. The ulcers scab over and eventually form a fibrotic scar. The disease usually resolves in 2–6 weeks. Rarely, the disease will persist for many months to more than a year. Differential diagnosis. The main differential is contagious ecthyma, which is grossly and histopathologically associated with epithelial hyperplasia. This is also a feature of ulcerative dermatosis. Prevention and control. No vaccine is available. Affected animals, especially males, should not be used for breeding. Treatment. Affected animals should be separated from the rest of the flock. Treatment is supportive, including antiseptic ointments and astringents. Research complications. Breeding and maintenance of the flocks' condition, because of the pain associated with eating, will be compromised during an outbreak. l. Border Disease Etiology. Border disease, also known as hairy shaker disease (or "fuzzies" in the southwestern United States), is a disease of sheep caused by a virus closely related to the bovine viral diarrhea virus (BVDV), a pestivirus of the Togaviridae family. Goats are also affected. The virus causes few pathogenic effects in cattle. Clinical signs and diagnosis. Border disease in ewes causes early embryonic death, abortion of macerated or mummified fetuses, or birth of lambs with developmental abnormalities. Lambs infected in utero that survive until parturition may be born weak and often exhibit a number of congenital defects such as tremor, hirsutism (sometimes darkly pigmented over the shoulders and head), hypothyroidism, central nervous system defects, and joint abnormalities, including arthrogryposis. Later, survivors may be more susceptible to diseases and may develop persistent, sometimes fatal, diarrhea. The virus infection produces similar clinical manifestations in goats, except that the hair changes are not seen. Diagnosis includes the typical signs described above, as well as serological evidence of viral infection. Virus isolation confirms the diagnosis. Epizootiology and transmission. The virus is present worldwide, and reports of disease are sporadic. Disease has occurred when no contact with cattle has occurred. Persistently infected animals, such as lambs, are shedding reservoirs of the virus in urine, feces, and saliva throughout their lives. Necropsy findings. Lesions include placentitis, and characteristic joint and hair-coat changes in the fetus. Histologically, axonal swelling, neuronal vacuolation, dysmyelination, and focal microgliosis are observed in central nervous system structures. Pathogenesis. The virus entering the ewe via the gastrointestinal or respiratory tracts penetrates the mucous membranes and causes maternal and fetal viremia. Infection during the first 45 days of gestation causes embryonic death. In lambs infected between 45 and 80 days, the virus activates follicular development, diminishes the myelination of neurons, and causes dysfunction of the thyroid gland. Infection after 80 days of gestation results in lambs that are born persistently infected. Infected lambs have high perinatal mortality; survivors have diminished signs over time but, as noted, continue to shed the virus. Prevention and control. Border disease can be prevented by vaccinating breeding ewes with killed-BVDV vaccine. Congenitally affected lambs should be maintained separately and disposed of as soon as humanely possible. New animals to the flock should be screened serologically. If cattle are housed nearby, vaccination programs for BVDV should be maintained. Treatment. There is no treatment other than supportive care for affected animals. Etiology. Border disease, also known as hairy shaker disease (or "fuzzies" in the southwestern United States), is a disease of sheep caused by a virus closely related to the bovine viral diarrhea virus (BVDV), a pestivirus of the Togaviridae family. Goats are also affected. The virus causes few pathogenic effects in cattle. Clinical signs and diagnosis. Border disease in ewes causes early embryonic death, abortion of macerated or mummified fetuses, or birth of lambs with developmental abnormalities. Lambs infected in utero that survive until parturition may be born weak and often exhibit a number of congenital defects such as tremor, hirsutism (sometimes darkly pigmented over the shoulders and head), hypothyroidism, central nervous system defects, and joint abnormalities, including arthrogryposis. Later, survivors may be more susceptible to diseases and may develop persistent, sometimes fatal, diarrhea. The virus infection produces similar clinical manifestations in goats, except that the hair changes are not seen. Diagnosis includes the typical signs described above, as well as serological evidence of viral infection. Virus isolation confirms the diagnosis. Epizootiology and transmission. The virus is present worldwide, and reports of disease are sporadic. Disease has occurred when no contact with cattle has occurred. Persistently infected animals, such as lambs, are shedding reservoirs of the virus in urine, feces, and saliva throughout their lives. Necropsy findings. Lesions include placentitis, and characteristic joint and hair-coat changes in the fetus. Histologically, axonal swelling, neuronal vacuolation, dysmyelination, and focal microgliosis are observed in central nervous system structures. Pathogenesis. The virus entering the ewe via the gastrointestinal or respiratory tracts penetrates the mucous membranes and causes maternal and fetal viremia. Infection during the first 45 days of gestation causes embryonic death. In lambs infected between 45 and 80 days, the virus activates follicular development, diminishes the myelination of neurons, and causes dysfunction of the thyroid gland. Infection after 80 days of gestation results in lambs that are born persistently infected. Infected lambs have high perinatal mortality; survivors have diminished signs over time but, as noted, continue to shed the virus. Prevention and control. Border disease can be prevented by vaccinating breeding ewes with killed-BVDV vaccine. Congenitally affected lambs should be maintained separately and disposed of as soon as humanely possible. New animals to the flock should be screened serologically. If cattle are housed nearby, vaccination programs for BVDV should be maintained. Treatment. There is no treatment other than supportive care for affected animals. Etiology. Border disease, also known as hairy shaker disease (or "fuzzies" in the southwestern United States), is a disease of sheep caused by a virus closely related to the bovine viral diarrhea virus (BVDV), a pestivirus of the Togaviridae family. Goats are also affected. The virus causes few pathogenic effects in cattle. Clinical signs and diagnosis. Border disease in ewes causes early embryonic death, abortion of macerated or mummified fetuses, or birth of lambs with developmental abnormalities. Lambs infected in utero that survive until parturition may be born weak and often exhibit a number of congenital defects such as tremor, hirsutism (sometimes darkly pigmented over the shoulders and head), hypothyroidism, central nervous system defects, and joint abnormalities, including arthrogryposis. Later, survivors may be more susceptible to diseases and may develop persistent, sometimes fatal, diarrhea. The virus infection produces similar clinical manifestations in goats, except that the hair changes are not seen. Diagnosis includes the typical signs described above, as well as serological evidence of viral infection. Virus isolation confirms the diagnosis. Epizootiology and transmission. The virus is present worldwide, and reports of disease are sporadic. Disease has occurred when no contact with cattle has occurred. Persistently infected animals, such as lambs, are shedding reservoirs of the virus in urine, feces, and saliva throughout their lives. Necropsy findings. Lesions include placentitis, and characteristic joint and hair-coat changes in the fetus. Histologically, axonal swelling, neuronal vacuolation, dysmyelination, and focal microgliosis are observed in central nervous system structures. Pathogenesis. The virus entering the ewe via the gastrointestinal or respiratory tracts penetrates the mucous membranes and causes maternal and fetal viremia. Infection during the first 45 days of gestation causes embryonic death. In lambs infected between 45 and 80 days, the virus activates follicular development, diminishes the myelination of neurons, and causes dysfunction of the thyroid gland. Infection after 80 days of gestation results in lambs that are born persistently infected. Infected lambs have high perinatal mortality; survivors have diminished signs over time but, as noted, continue to shed the virus. Prevention and control. Border disease can be prevented by vaccinating breeding ewes with killed-BVDV vaccine. Congenitally affected lambs should be maintained separately and disposed of as soon as humanely possible. New animals to the flock should be screened serologically. If cattle are housed nearby, vaccination programs for BVDV should be maintained. Treatment. There is no treatment other than supportive care for affected animals. m. Contagious Ecthyma (Contagious Pustular Dermatitis, Sore Mouth, Orf) Etiology. Contagious ecthyma, also known as contagious pustular dermatitis, sore mouth, or orf, is an acute dermatitis of sheep and goats caused by a parapoxvirus. This disease occurs worldwide and is zoonotic. Naturally occurring disease has also been reported in other species such as musk ox and reindeer. Other parapoxviruses infect the mucous membranes and skin of cattle, causing the diseases bovine pustular dermatitis and pseudocowpox. Clinical signs and diagnosis. The disease is characterized by the presence of papules, vesicles, or pustules and subsequently scabs of the skin of the face, genitals of both sexes, and coronary bands of the feet. Lesions develop most frequently at mucocutaneous junctions and are found most commonly at the commissures of the mouth. Orf is usually found in young animals less than 1 year of age. Younger lambs and kids will have difficulty nursing and become weak. Lesions may also develop on udders of nursing dams, which may resist suckling by offspring to nurse, leading to secondary mastitis. The scabs may appear nodular and raised above the surface of the surrounding skin. Morbidity in a susceptible group of animals may exceed 90%. Mortality is low, but the course of the disease may last up to 6 weeks. Diagnosis is based on characteristic lesions. Biopsies may reveal eosinophilic cytoplasmic inclusions and proliferative lesions under the skin. Electron microscopy will reveal the virus itself. Disease is confirmed by virus isolation. Epizootiology and transmission. All ages of sheep and goats are susceptible. Seasonal occurrences immediately after lambing and after entry into a feedlot are common; stress likely plays a role in susceptibility to this viral disease. Older animals develop immunity that usually prevents reinfection for at least 1 or more years. Resistant animals may be present in some flocks or herds. The virus is very resistant to environmental conditions and may contaminate small-ruminant facilities, pens, feedlots, and the like for many years as the result of scabs that have been shed from infected animals. Transmission occurs through superficial lesions such as punctures from grass awns, scrapes, shearing, and other common injuries. Necropsy findings. Necropsy findings include ballooning degeneration of epidermal and dermal layers, edema, granulomatous inflammation, vesiculation, and cellular hyperplasia. Secondary bacterial infection may also be evident. Pathogenesis. The virus is typical of the Poxviridae, resembling sheep poxvirus (not found in the United States) and vaccinia virus and replicating in the cytoplasm of epithelial cells. Following an incubation period of 2–14 days, papules and vesicles develop around the margins of the lips, nostrils, eyelids, gums, tongue, or teats; skin of the genitalia; or coronary band of the feet. The vesicles form pustules that rupture and finally scab over. Differential diagnosis. Ulcerative dermatosis and bluetongue virus should be considered in both sheep and goats. An important differential in goats is staphylococcal dermatitis. Prevention and control. Individuals handling infected animals should be advised of precautions beforehand, should wear gloves, and should separate work clothing and other personal protective equipment. Clippers, ear tagging devices, and other similar equipment should always be cleaned and disinfected after each use. Colostral antibodies may not be protective. Vaccinating lambs and kids with commercial vaccine best prevents the disease. Dried scabs from previous outbreaks may also be used by rubbing the material into scarified skin on the inner thigh or axilla. Animals newly introduced to infected premises should be vaccinated upon arrival. Precautions must be taken when vaccinating animals, because the vaccine may induce orf in the animal handlers; it is not recommended to vaccinate animals in flocks already free of the disease. Affected dairy goats should be milked last, using disposable towels for cleaning teat ends. Treatment. Affected animals should be isolated and provided supportive care, especially tube feeding for young animals whose mouths are too sore to nurse. Treatment should also address secondary bacterial infections of the orf lesions, including systemic antibiotics for more severe infections. Treatment for myiasis may also be necessary. The viral infection is self-limiting, with recovery in about 4 weeks. Research complications. Carrier animals may be a factor in flock or herd outbreaks. Contagious ecthyma is a zoonotic disease, and human-to-human transmission can also occur. The virus typically enters through abrasions on the hands and results in a large (several centimeters) nodule that is described as being extremely painful and lasting for as many as 6 weeks. Lesions heal without scarring. Etiology. Contagious ecthyma, also known as contagious pustular dermatitis, sore mouth, or orf, is an acute dermatitis of sheep and goats caused by a parapoxvirus. This disease occurs worldwide and is zoonotic. Naturally occurring disease has also been reported in other species such as musk ox and reindeer. Other parapoxviruses infect the mucous membranes and skin of cattle, causing the diseases bovine pustular dermatitis and pseudocowpox. Clinical signs and diagnosis. The disease is characterized by the presence of papules, vesicles, or pustules and subsequently scabs of the skin of the face, genitals of both sexes, and coronary bands of the feet. Lesions develop most frequently at mucocutaneous junctions and are found most commonly at the commissures of the mouth. Orf is usually found in young animals less than 1 year of age. Younger lambs and kids will have difficulty nursing and become weak. Lesions may also develop on udders of nursing dams, which may resist suckling by offspring to nurse, leading to secondary mastitis. The scabs may appear nodular and raised above the surface of the surrounding skin. Morbidity in a susceptible group of animals may exceed 90%. Mortality is low, but the course of the disease may last up to 6 weeks. Diagnosis is based on characteristic lesions. Biopsies may reveal eosinophilic cytoplasmic inclusions and proliferative lesions under the skin. Electron microscopy will reveal the virus itself. Disease is confirmed by virus isolation. Epizootiology and transmission. All ages of sheep and goats are susceptible. Seasonal occurrences immediately after lambing and after entry into a feedlot are common; stress likely plays a role in susceptibility to this viral disease. Older animals develop immunity that usually prevents reinfection for at least 1 or more years. Resistant animals may be present in some flocks or herds. The virus is very resistant to environmental conditions and may contaminate small-ruminant facilities, pens, feedlots, and the like for many years as the result of scabs that have been shed from infected animals. Transmission occurs through superficial lesions such as punctures from grass awns, scrapes, shearing, and other common injuries. Necropsy findings. Necropsy findings include ballooning degeneration of epidermal and dermal layers, edema, granulomatous inflammation, vesiculation, and cellular hyperplasia. Secondary bacterial infection may also be evident. Pathogenesis. The virus is typical of the Poxviridae, resembling sheep poxvirus (not found in the United States) and vaccinia virus and replicating in the cytoplasm of epithelial cells. Following an incubation period of 2–14 days, papules and vesicles develop around the margins of the lips, nostrils, eyelids, gums, tongue, or teats; skin of the genitalia; or coronary band of the feet. The vesicles form pustules that rupture and finally scab over. Differential diagnosis. Ulcerative dermatosis and bluetongue virus should be considered in both sheep and goats. An important differential in goats is staphylococcal dermatitis. Prevention and control. Individuals handling infected animals should be advised of precautions beforehand, should wear gloves, and should separate work clothing and other personal protective equipment. Clippers, ear tagging devices, and other similar equipment should always be cleaned and disinfected after each use. Colostral antibodies may not be protective. Vaccinating lambs and kids with commercial vaccine best prevents the disease. Dried scabs from previous outbreaks may also be used by rubbing the material into scarified skin on the inner thigh or axilla. Animals newly introduced to infected premises should be vaccinated upon arrival. Precautions must be taken when vaccinating animals, because the vaccine may induce orf in the animal handlers; it is not recommended to vaccinate animals in flocks already free of the disease. Affected dairy goats should be milked last, using disposable towels for cleaning teat ends. Treatment. Affected animals should be isolated and provided supportive care, especially tube feeding for young animals whose mouths are too sore to nurse. Treatment should also address secondary bacterial infections of the orf lesions, including systemic antibiotics for more severe infections. Treatment for myiasis may also be necessary. The viral infection is self-limiting, with recovery in about 4 weeks. Research complications. Carrier animals may be a factor in flock or herd outbreaks. Contagious ecthyma is a zoonotic disease, and human-to-human transmission can also occur. The virus typically enters through abrasions on the hands and results in a large (several centimeters) nodule that is described as being extremely painful and lasting for as many as 6 weeks. Lesions heal without scarring. Etiology. Contagious ecthyma, also known as contagious pustular dermatitis, sore mouth, or orf, is an acute dermatitis of sheep and goats caused by a parapoxvirus. This disease occurs worldwide and is zoonotic. Naturally occurring disease has also been reported in other species such as musk ox and reindeer. Other parapoxviruses infect the mucous membranes and skin of cattle, causing the diseases bovine pustular dermatitis and pseudocowpox. Clinical signs and diagnosis. The disease is characterized by the presence of papules, vesicles, or pustules and subsequently scabs of the skin of the face, genitals of both sexes, and coronary bands of the feet. Lesions develop most frequently at mucocutaneous junctions and are found most commonly at the commissures of the mouth. Orf is usually found in young animals less than 1 year of age. Younger lambs and kids will have difficulty nursing and become weak. Lesions may also develop on udders of nursing dams, which may resist suckling by offspring to nurse, leading to secondary mastitis. The scabs may appear nodular and raised above the surface of the surrounding skin. Morbidity in a susceptible group of animals may exceed 90%. Mortality is low, but the course of the disease may last up to 6 weeks. Diagnosis is based on characteristic lesions. Biopsies may reveal eosinophilic cytoplasmic inclusions and proliferative lesions under the skin. Electron microscopy will reveal the virus itself. Disease is confirmed by virus isolation. Epizootiology and transmission. All ages of sheep and goats are susceptible. Seasonal occurrences immediately after lambing and after entry into a feedlot are common; stress likely plays a role in susceptibility to this viral disease. Older animals develop immunity that usually prevents reinfection for at least 1 or more years. Resistant animals may be present in some flocks or herds. The virus is very resistant to environmental conditions and may contaminate small-ruminant facilities, pens, feedlots, and the like for many years as the result of scabs that have been shed from infected animals. Transmission occurs through superficial lesions such as punctures from grass awns, scrapes, shearing, and other common injuries. Necropsy findings. Necropsy findings include ballooning degeneration of epidermal and dermal layers, edema, granulomatous inflammation, vesiculation, and cellular hyperplasia. Secondary bacterial infection may also be evident. Pathogenesis. The virus is typical of the Poxviridae, resembling sheep poxvirus (not found in the United States) and vaccinia virus and replicating in the cytoplasm of epithelial cells. Following an incubation period of 2–14 days, papules and vesicles develop around the margins of the lips, nostrils, eyelids, gums, tongue, or teats; skin of the genitalia; or coronary band of the feet. The vesicles form pustules that rupture and finally scab over. Differential diagnosis. Ulcerative dermatosis and bluetongue virus should be considered in both sheep and goats. An important differential in goats is staphylococcal dermatitis. Prevention and control. Individuals handling infected animals should be advised of precautions beforehand, should wear gloves, and should separate work clothing and other personal protective equipment. Clippers, ear tagging devices, and other similar equipment should always be cleaned and disinfected after each use. Colostral antibodies may not be protective. Vaccinating lambs and kids with commercial vaccine best prevents the disease. Dried scabs from previous outbreaks may also be used by rubbing the material into scarified skin on the inner thigh or axilla. Animals newly introduced to infected premises should be vaccinated upon arrival. Precautions must be taken when vaccinating animals, because the vaccine may induce orf in the animal handlers; it is not recommended to vaccinate animals in flocks already free of the disease. Affected dairy goats should be milked last, using disposable towels for cleaning teat ends. Treatment. Affected animals should be isolated and provided supportive care, especially tube feeding for young animals whose mouths are too sore to nurse. Treatment should also address secondary bacterial infections of the orf lesions, including systemic antibiotics for more severe infections. Treatment for myiasis may also be necessary. The viral infection is self-limiting, with recovery in about 4 weeks. Research complications. Carrier animals may be a factor in flock or herd outbreaks. Contagious ecthyma is a zoonotic disease, and human-to-human transmission can also occur. The virus typically enters through abrasions on the hands and results in a large (several centimeters) nodule that is described as being extremely painful and lasting for as many as 6 weeks. Lesions heal without scarring. n. Foot-and-Mouth Disease Etiology. Foot-and-mouth disease (FMD) is caused by the foot-and-mouth disease virus, a Picornavirus in the Aphthovirus genus. The disease is also referred to as aftosa or aphthous fever. Seven immunologically distinct types of the virus have been identified, with 60 subtypes within those 7. Epidemics of the disease have occurred worldwide. North and Central America have been free of the virus since the mid-1950s. This is a reportable disease in the United States; clinical signs are very similar to other vesicular diseases. Cattle (and swine) are primarily affected, but disease can occur in sheep and is usually subclinical in goats. Clinical signs and diagnosis. In addition to vesicle formation around and in the mouth, hooves, and teats, fever, anorexia, weakness, and salivation occur. Vesicles may be as large as 10 cm, rupture after 2 days, and subsequently erode. Secondary bacterial infections often occur at the erosions. Anorexia is likely due to the pain associated with the oral lesions. High morbidity and low mortality, except for the high mortality in young cattle, are typical. Diagnosis must be based on ELISA, virus neutralization, fluorescent antibody tests, and complement fixation. Epizootiology and transmission. Domestic and wild ruminants and several other species, such as swine, rats, bears, and llamas are hosts. Asymptomatic goats can serve as virus reservoirs for more susceptible cohoused species such as cattle. Greater mortality occurs in younger animals. The United States, Great Britain, Canada, Japan, New Zealand, and Australia are FMD-free, whereas the disease is endemic in most of South America, parts of Europe, and throughout Asia and Africa. The virus is very contagious and is spread primarily by the inhalation of aerosols, which can be carried over long distances. Transmission may also occur by fomites, such as shoes, clothing, and equipment. Human hands, soiled bedding, and animal products such as frozen or partially cooked meat and meat products, hides, semen, and pasteurized milk also serve as sources of virus. Necropsy findings. Vesicles, erosions, and ulcers are present in the oral cavity as well as on the rumen pillars and mammary alveolar epithelium. Myocardial and skeletal muscle degeneration (Zenker's) is most common (and accounts for the greater mortality) in younger animals. Histological findings include lack of inclusion bodies. Vesicular lesions include intracellular and extracellular edema, cellular degeneration, and separation of the basal epithelium. Pathogenesis. The incubation period is 2–8 days. The virus replicates in the pharynx and digestive tract in the cells of the stratum spinosum, and viremia and spread of virus to many tissues occur before clinical signs develop. Virus shedding begins about 24 hr before clinical signs are apparent. Vesicles result from the separation of the superficial epithelium from the basal epithelium. Fluid fills the basal epithelium, and erosions develop when the epithelium sloughs. Persistent infection also occurs, and virus can be found for months or years in the pharnyx; the mechanisms for the persistence are not known. Differential diagnosis. Vesicular stomatitis is the principal differential. Other differentials include contagious ecthyma (orf), rinderpest, bluetongue, malignant catarrhal fever, bovine papular stomatitis, bovine herpes mammillitis, and infectious bovine rhinotracheitis virus infection. Prevention and control. Movement of animals and animal products from endemic areas is regulated. Quarantine and slaughter are practiced in outbreaks in endemic areas. Quarantine and vaccination are also used in endemic areas, but vaccines must be type-specific and repeated 2 or 3 times per year to be effective and will provide only partial protection. Autogenous vaccines are best in an outbreak. Passive immunity protects calves for up to 5 months after birth. The virus is inactivated by extremes of pH, sunlight, high temperatures, sodium hydroxide, sodium carbonate, and acetic acid. Treatment. Nursing care and antibiotic therapy to minimize secondary reactions help with recovery. Humoral immunity is considered the more important immune mechanism, with cell-mediated immunity of less importance. Research complications. Rare cases in humans have been reported. Importation into the United States of animal products from endemic areas is prohibited. Etiology. Foot-and-mouth disease (FMD) is caused by the foot-and-mouth disease virus, a Picornavirus in the Aphthovirus genus. The disease is also referred to as aftosa or aphthous fever. Seven immunologically distinct types of the virus have been identified, with 60 subtypes within those 7. Epidemics of the disease have occurred worldwide. North and Central America have been free of the virus since the mid-1950s. This is a reportable disease in the United States; clinical signs are very similar to other vesicular diseases. Cattle (and swine) are primarily affected, but disease can occur in sheep and is usually subclinical in goats. Clinical signs and diagnosis. In addition to vesicle formation around and in the mouth, hooves, and teats, fever, anorexia, weakness, and salivation occur. Vesicles may be as large as 10 cm, rupture after 2 days, and subsequently erode. Secondary bacterial infections often occur at the erosions. Anorexia is likely due to the pain associated with the oral lesions. High morbidity and low mortality, except for the high mortality in young cattle, are typical. Diagnosis must be based on ELISA, virus neutralization, fluorescent antibody tests, and complement fixation. Epizootiology and transmission. Domestic and wild ruminants and several other species, such as swine, rats, bears, and llamas are hosts. Asymptomatic goats can serve as virus reservoirs for more susceptible cohoused species such as cattle. Greater mortality occurs in younger animals. The United States, Great Britain, Canada, Japan, New Zealand, and Australia are FMD-free, whereas the disease is endemic in most of South America, parts of Europe, and throughout Asia and Africa. The virus is very contagious and is spread primarily by the inhalation of aerosols, which can be carried over long distances. Transmission may also occur by fomites, such as shoes, clothing, and equipment. Human hands, soiled bedding, and animal products such as frozen or partially cooked meat and meat products, hides, semen, and pasteurized milk also serve as sources of virus. Necropsy findings. Vesicles, erosions, and ulcers are present in the oral cavity as well as on the rumen pillars and mammary alveolar epithelium. Myocardial and skeletal muscle degeneration (Zenker's) is most common (and accounts for the greater mortality) in younger animals. Histological findings include lack of inclusion bodies. Vesicular lesions include intracellular and extracellular edema, cellular degeneration, and separation of the basal epithelium. Pathogenesis. The incubation period is 2–8 days. The virus replicates in the pharynx and digestive tract in the cells of the stratum spinosum, and viremia and spread of virus to many tissues occur before clinical signs develop. Virus shedding begins about 24 hr before clinical signs are apparent. Vesicles result from the separation of the superficial epithelium from the basal epithelium. Fluid fills the basal epithelium, and erosions develop when the epithelium sloughs. Persistent infection also occurs, and virus can be found for months or years in the pharnyx; the mechanisms for the persistence are not known. Differential diagnosis. Vesicular stomatitis is the principal differential. Other differentials include contagious ecthyma (orf), rinderpest, bluetongue, malignant catarrhal fever, bovine papular stomatitis, bovine herpes mammillitis, and infectious bovine rhinotracheitis virus infection. Prevention and control. Movement of animals and animal products from endemic areas is regulated. Quarantine and slaughter are practiced in outbreaks in endemic areas. Quarantine and vaccination are also used in endemic areas, but vaccines must be type-specific and repeated 2 or 3 times per year to be effective and will provide only partial protection. Autogenous vaccines are best in an outbreak. Passive immunity protects calves for up to 5 months after birth. The virus is inactivated by extremes of pH, sunlight, high temperatures, sodium hydroxide, sodium carbonate, and acetic acid. Treatment. Nursing care and antibiotic therapy to minimize secondary reactions help with recovery. Humoral immunity is considered the more important immune mechanism, with cell-mediated immunity of less importance. Research complications. Rare cases in humans have been reported. Importation into the United States of animal products from endemic areas is prohibited. Etiology. Foot-and-mouth disease (FMD) is caused by the foot-and-mouth disease virus, a Picornavirus in the Aphthovirus genus. The disease is also referred to as aftosa or aphthous fever. Seven immunologically distinct types of the virus have been identified, with 60 subtypes within those 7. Epidemics of the disease have occurred worldwide. North and Central America have been free of the virus since the mid-1950s. This is a reportable disease in the United States; clinical signs are very similar to other vesicular diseases. Cattle (and swine) are primarily affected, but disease can occur in sheep and is usually subclinical in goats. Clinical signs and diagnosis. In addition to vesicle formation around and in the mouth, hooves, and teats, fever, anorexia, weakness, and salivation occur. Vesicles may be as large as 10 cm, rupture after 2 days, and subsequently erode. Secondary bacterial infections often occur at the erosions. Anorexia is likely due to the pain associated with the oral lesions. High morbidity and low mortality, except for the high mortality in young cattle, are typical. Diagnosis must be based on ELISA, virus neutralization, fluorescent antibody tests, and complement fixation. Epizootiology and transmission. Domestic and wild ruminants and several other species, such as swine, rats, bears, and llamas are hosts. Asymptomatic goats can serve as virus reservoirs for more susceptible cohoused species such as cattle. Greater mortality occurs in younger animals. The United States, Great Britain, Canada, Japan, New Zealand, and Australia are FMD-free, whereas the disease is endemic in most of South America, parts of Europe, and throughout Asia and Africa. The virus is very contagious and is spread primarily by the inhalation of aerosols, which can be carried over long distances. Transmission may also occur by fomites, such as shoes, clothing, and equipment. Human hands, soiled bedding, and animal products such as frozen or partially cooked meat and meat products, hides, semen, and pasteurized milk also serve as sources of virus. Necropsy findings. Vesicles, erosions, and ulcers are present in the oral cavity as well as on the rumen pillars and mammary alveolar epithelium. Myocardial and skeletal muscle degeneration (Zenker's) is most common (and accounts for the greater mortality) in younger animals. Histological findings include lack of inclusion bodies. Vesicular lesions include intracellular and extracellular edema, cellular degeneration, and separation of the basal epithelium. Pathogenesis. The incubation period is 2–8 days. The virus replicates in the pharynx and digestive tract in the cells of the stratum spinosum, and viremia and spread of virus to many tissues occur before clinical signs develop. Virus shedding begins about 24 hr before clinical signs are apparent. Vesicles result from the separation of the superficial epithelium from the basal epithelium. Fluid fills the basal epithelium, and erosions develop when the epithelium sloughs. Persistent infection also occurs, and virus can be found for months or years in the pharnyx; the mechanisms for the persistence are not known. Differential diagnosis. Vesicular stomatitis is the principal differential. Other differentials include contagious ecthyma (orf), rinderpest, bluetongue, malignant catarrhal fever, bovine papular stomatitis, bovine herpes mammillitis, and infectious bovine rhinotracheitis virus infection. Prevention and control. Movement of animals and animal products from endemic areas is regulated. Quarantine and slaughter are practiced in outbreaks in endemic areas. Quarantine and vaccination are also used in endemic areas, but vaccines must be type-specific and repeated 2 or 3 times per year to be effective and will provide only partial protection. Autogenous vaccines are best in an outbreak. Passive immunity protects calves for up to 5 months after birth. The virus is inactivated by extremes of pH, sunlight, high temperatures, sodium hydroxide, sodium carbonate, and acetic acid. Treatment. Nursing care and antibiotic therapy to minimize secondary reactions help with recovery. Humoral immunity is considered the more important immune mechanism, with cell-mediated immunity of less importance. Research complications. Rare cases in humans have been reported. Importation into the United States of animal products from endemic areas is prohibited. o. Malignant Catarrhal Fever Etiology. Malignant catarrhal fever (MCF) is a severe disease primarily of cattle. The agents of MCF are viruses of the Gammaherpesvirinae subfamily. Alcelaphine herpesvirus 1 and 2 and ovine herpesvirus 2 are known strains. The alcelaphine strains are seen in Africa. The ovine strain is seen in North America. The alcelaphine and ovine strains differ in incubation times and duration of illness. Disease may occur sporadically or as outbreaks. Clinical signs and diagnosis. Signs range from subclinical to recrudescing latent infections to the lethal disease seen in susceptible species, such as cattle. Sudden death may also occur in cattle. Presentations of the disease may be categorized as alimentary, encephalitis, or skin forms; all three may occur in an animal. Corneal edema starting at the limbus and progressing centripetally is a nearly pathognomonic sign; photophobia, severe keratoconjunctivitis, and ocular involvement may follow. Other signs include prolonged fever, oral mucosal erosions, salivation, lacrimation, purulent nasal discharge, encephalitis, and pronounced lymphadenopathy. As the disease progresses, cattle may shed horns and hooves. In North America, cattle will also have severe diarrhea. The course of the disease may extend to 1 week. Recovery is usually prolonged, and some permanent debilitation may occur. The disease is fatal in severely affected individuals. History of exposure, as well as the clinical signs and lesions, contributes to the diagnosis. Serology, PCR-based assays, viral isolation, and cell-culture assays, such as cytopathic effects on thyroid cell cultures, are also used. Because of the susceptibility of rabbits, inoculation of this species may be used. In less severe outbreaks or individual animal disease, definitive diagnosis may never be made. Epizootiology and transmission. Most ruminant species are susceptible to MCF. Sheep are sources of infection for cattle, which are dead-end hosts. Other ruminants, including goats, may harbor the virus. Both the African and North American strains are transmissible to rabbits; these animals develop a fatal lymphoproliferative disease. The virus is shed from the nasopharynx. Infection of lambs is horizontal from direct contact. Other sources of the virus include water troughs, placental tissues, contaminated fomites, aerosols, birds, and caretakers. Necropsy. Gross findings at necropsy include necrotic and ulcerated nasal and oral mucosa; thickened, edematous, ulcerated, and hemorrhagic areas of the intestinal tract; swollen, friable, and hemorrhagic lymph nodes and other lymphatic tissues; and erosion of affected mucosal surfaces. Lymph nodes should be submitted for histological examination. Histological findings include nonsuppurative vasculitis and encephalitis; large numbers of lymphocytes and lymphoblasts will be present without evidence of virus. Pathogenesis. The incubation period may be up to 3 months. Vascular endothelium and all epithelial surfaces will be affected. The virus is believed to cause proliferation of cytotoxic T lymphocytes with natural killer cell activities, and the resulting lesions are due to an autoimmune type of phenomenon. Differential diagnoses. The differentials for this disease are bovine viral diarrhea/mucosal disease, bovine respiratory disease complex, infectious bovine rhinotracheitis, bluetongue, vesicular stomatitis, and foot-and-mouth disease. Causes of encephalitis, such as bovine spongiform encephalopathy and rabies, should be considered. In Africa, rinderpest is also a differential. Other differentials are arsenic toxicity and chlorinated naphthalene toxicity. Prevention and control. No vaccine is available at this time. In North America, sheep, as well as cattle that have been either exposed or that have survived the disease, are reservoirs for outbreaks in other cattle. If there is concern regarding presence of the virus, animals should be screened serologically; once an animal has been infected, it remains infected indefinitely. Lambs can be free of the infection if removed from the flock at weaning. The virus is very fragile outside of host's cells and will not survive in the environment for more than a few hours. Treatment. Affected and any exposed animals should be isolated from healthy animals. There is no specific treatment for MCF; supportive treatment may improve recovery rates. Corticosteroids may be useful. Etiology. Malignant catarrhal fever (MCF) is a severe disease primarily of cattle. The agents of MCF are viruses of the Gammaherpesvirinae subfamily. Alcelaphine herpesvirus 1 and 2 and ovine herpesvirus 2 are known strains. The alcelaphine strains are seen in Africa. The ovine strain is seen in North America. The alcelaphine and ovine strains differ in incubation times and duration of illness. Disease may occur sporadically or as outbreaks. Clinical signs and diagnosis. Signs range from subclinical to recrudescing latent infections to the lethal disease seen in susceptible species, such as cattle. Sudden death may also occur in cattle. Presentations of the disease may be categorized as alimentary, encephalitis, or skin forms; all three may occur in an animal. Corneal edema starting at the limbus and progressing centripetally is a nearly pathognomonic sign; photophobia, severe keratoconjunctivitis, and ocular involvement may follow. Other signs include prolonged fever, oral mucosal erosions, salivation, lacrimation, purulent nasal discharge, encephalitis, and pronounced lymphadenopathy. As the disease progresses, cattle may shed horns and hooves. In North America, cattle will also have severe diarrhea. The course of the disease may extend to 1 week. Recovery is usually prolonged, and some permanent debilitation may occur. The disease is fatal in severely affected individuals. History of exposure, as well as the clinical signs and lesions, contributes to the diagnosis. Serology, PCR-based assays, viral isolation, and cell-culture assays, such as cytopathic effects on thyroid cell cultures, are also used. Because of the susceptibility of rabbits, inoculation of this species may be used. In less severe outbreaks or individual animal disease, definitive diagnosis may never be made. Epizootiology and transmission. Most ruminant species are susceptible to MCF. Sheep are sources of infection for cattle, which are dead-end hosts. Other ruminants, including goats, may harbor the virus. Both the African and North American strains are transmissible to rabbits; these animals develop a fatal lymphoproliferative disease. The virus is shed from the nasopharynx. Infection of lambs is horizontal from direct contact. Other sources of the virus include water troughs, placental tissues, contaminated fomites, aerosols, birds, and caretakers. Necropsy. Gross findings at necropsy include necrotic and ulcerated nasal and oral mucosa; thickened, edematous, ulcerated, and hemorrhagic areas of the intestinal tract; swollen, friable, and hemorrhagic lymph nodes and other lymphatic tissues; and erosion of affected mucosal surfaces. Lymph nodes should be submitted for histological examination. Histological findings include nonsuppurative vasculitis and encephalitis; large numbers of lymphocytes and lymphoblasts will be present without evidence of virus. Pathogenesis. The incubation period may be up to 3 months. Vascular endothelium and all epithelial surfaces will be affected. The virus is believed to cause proliferation of cytotoxic T lymphocytes with natural killer cell activities, and the resulting lesions are due to an autoimmune type of phenomenon. Differential diagnoses. The differentials for this disease are bovine viral diarrhea/mucosal disease, bovine respiratory disease complex, infectious bovine rhinotracheitis, bluetongue, vesicular stomatitis, and foot-and-mouth disease. Causes of encephalitis, such as bovine spongiform encephalopathy and rabies, should be considered. In Africa, rinderpest is also a differential. Other differentials are arsenic toxicity and chlorinated naphthalene toxicity. Prevention and control. No vaccine is available at this time. In North America, sheep, as well as cattle that have been either exposed or that have survived the disease, are reservoirs for outbreaks in other cattle. If there is concern regarding presence of the virus, animals should be screened serologically; once an animal has been infected, it remains infected indefinitely. Lambs can be free of the infection if removed from the flock at weaning. The virus is very fragile outside of host's cells and will not survive in the environment for more than a few hours. Treatment. Affected and any exposed animals should be isolated from healthy animals. There is no specific treatment for MCF; supportive treatment may improve recovery rates. Corticosteroids may be useful. Etiology. Malignant catarrhal fever (MCF) is a severe disease primarily of cattle. The agents of MCF are viruses of the Gammaherpesvirinae subfamily. Alcelaphine herpesvirus 1 and 2 and ovine herpesvirus 2 are known strains. The alcelaphine strains are seen in Africa. The ovine strain is seen in North America. The alcelaphine and ovine strains differ in incubation times and duration of illness. Disease may occur sporadically or as outbreaks. Clinical signs and diagnosis. Signs range from subclinical to recrudescing latent infections to the lethal disease seen in susceptible species, such as cattle. Sudden death may also occur in cattle. Presentations of the disease may be categorized as alimentary, encephalitis, or skin forms; all three may occur in an animal. Corneal edema starting at the limbus and progressing centripetally is a nearly pathognomonic sign; photophobia, severe keratoconjunctivitis, and ocular involvement may follow. Other signs include prolonged fever, oral mucosal erosions, salivation, lacrimation, purulent nasal discharge, encephalitis, and pronounced lymphadenopathy. As the disease progresses, cattle may shed horns and hooves. In North America, cattle will also have severe diarrhea. The course of the disease may extend to 1 week. Recovery is usually prolonged, and some permanent debilitation may occur. The disease is fatal in severely affected individuals. History of exposure, as well as the clinical signs and lesions, contributes to the diagnosis. Serology, PCR-based assays, viral isolation, and cell-culture assays, such as cytopathic effects on thyroid cell cultures, are also used. Because of the susceptibility of rabbits, inoculation of this species may be used. In less severe outbreaks or individual animal disease, definitive diagnosis may never be made. Epizootiology and transmission. Most ruminant species are susceptible to MCF. Sheep are sources of infection for cattle, which are dead-end hosts. Other ruminants, including goats, may harbor the virus. Both the African and North American strains are transmissible to rabbits; these animals develop a fatal lymphoproliferative disease. The virus is shed from the nasopharynx. Infection of lambs is horizontal from direct contact. Other sources of the virus include water troughs, placental tissues, contaminated fomites, aerosols, birds, and caretakers. Necropsy. Gross findings at necropsy include necrotic and ulcerated nasal and oral mucosa; thickened, edematous, ulcerated, and hemorrhagic areas of the intestinal tract; swollen, friable, and hemorrhagic lymph nodes and other lymphatic tissues; and erosion of affected mucosal surfaces. Lymph nodes should be submitted for histological examination. Histological findings include nonsuppurative vasculitis and encephalitis; large numbers of lymphocytes and lymphoblasts will be present without evidence of virus. Pathogenesis. The incubation period may be up to 3 months. Vascular endothelium and all epithelial surfaces will be affected. The virus is believed to cause proliferation of cytotoxic T lymphocytes with natural killer cell activities, and the resulting lesions are due to an autoimmune type of phenomenon. Differential diagnoses. The differentials for this disease are bovine viral diarrhea/mucosal disease, bovine respiratory disease complex, infectious bovine rhinotracheitis, bluetongue, vesicular stomatitis, and foot-and-mouth disease. Causes of encephalitis, such as bovine spongiform encephalopathy and rabies, should be considered. In Africa, rinderpest is also a differential. Other differentials are arsenic toxicity and chlorinated naphthalene toxicity. Prevention and control. No vaccine is available at this time. In North America, sheep, as well as cattle that have been either exposed or that have survived the disease, are reservoirs for outbreaks in other cattle. If there is concern regarding presence of the virus, animals should be screened serologically; once an animal has been infected, it remains infected indefinitely. Lambs can be free of the infection if removed from the flock at weaning. The virus is very fragile outside of host's cells and will not survive in the environment for more than a few hours. Treatment. Affected and any exposed animals should be isolated from healthy animals. There is no specific treatment for MCF; supportive treatment may improve recovery rates. Corticosteroids may be useful. p. Ovine Progressive Pneumonia (Maedi/Visna) Etiology. An RNA virus in the lentivirus group of the Retroviridae family causes ovine progressive pneumonia (OPP), or maedi/visna. Maedi refers to the progressive pneumonia presentation of the disease; visna refers to the central nervous system disease, which is reported predominantly in Iceland. Visna has been reported in goats but may have been due to caprine arthritis encephalitis infection. Clinical signs and diagnosis. OPP is a viral disease of adult sheep characterized by weakness, unthriftiness, weight loss, and pneumonia ( Pepin et al., 1998 ; de la Concha Bermejillo, 1997 ). Clinically, animals exhibit signs of progressive pulmonary disease after an extremely long incubation period of up to 2 years. Respiratory rate and dyspnea gradually increase as the disease progresses. The animal continues to eat throughout the disease; however, animals progressively lose weight and become weak. Additionally, mastitis is a common clinical feature. Thoracic auscultation reveals consolidation of ventral lung lobes; and hematological findings indicate anemia and leukocytosis. The rare neurological signs include flexion of fetlock and pastern joints, tremors of facial muscles, progressive paresis and paralysis, depression, and prostration. Death occurs in weeks to months. The disease can be serologically diagnosed with agar gel immunodiffusion (AGID) tests, virus isolation, serum neutralization, complement fixation, and enzyme-linked immunosorbent assay (ELISA) tests. Epizootiology and transmission. Sixty-eight percent of sheep in some states have been infected with the virus ( Radostits et al., 1994 ). It is transmitted horizontally via inhalation of aerosolized virus particles and vertically between the infected dam and fetus. In addition, transmission through the milk or colostrum is considered common ( Knowles, 1997 ). Necropsy findings. Lesions are observed in lungs, mammary glands, joints, and the brain. Pulmonary adhesions, ventral lung lobe consolidation, bronchial lymph node enlargement, mastitis, and degenerative arthritis are visualized grossly. Meningeal edema, thickening of the choroid plexus, and foci of leukoencephalomalacia are seen in the central nervous system (CNS). Histologically, interalveolar septal thickening, lymphoid hyperplasia, histiocyte and fibrocyte proliferation, and squamous epithelial changes are seen in the lungs. Meningitis, lymphoid hyperplasia, demyelination, and glial fibrosis are seen in the CNS. Pathogenesis. The virus has a predilection for the lungs, mediastinal lymph nodes, udder, spleen, joints, and rarely the brain. After initial infection, the virus integrates into the DNA of mature monocytes and persists as a provirus. Later in the animal's life, infected monocytes mature as lung (and other tissue) macrophages and establish active infection. The virus induces lymphoproliferative disease, histiocyte and fibrocyte proliferation in the alveolar septa, and squamous metaplasia. Pulmonary alveolar and vascular changes impinge on oxygen and carbon dioxide exchange and lead to serious hypoxia and pulmonary hypertension. Secondary bacterial pneumonia may contribute to the animal's death. Differential diagnosis. Pulmonary adenomatosis is the differential diagnosis. Prevention and control. Isolating or removing infected animals can prevent the disease. Facilities and equipment should also be disinfected. Treatment. Treatment is unsuccessful. Etiology. An RNA virus in the lentivirus group of the Retroviridae family causes ovine progressive pneumonia (OPP), or maedi/visna. Maedi refers to the progressive pneumonia presentation of the disease; visna refers to the central nervous system disease, which is reported predominantly in Iceland. Visna has been reported in goats but may have been due to caprine arthritis encephalitis infection. Clinical signs and diagnosis. OPP is a viral disease of adult sheep characterized by weakness, unthriftiness, weight loss, and pneumonia ( Pepin et al., 1998 ; de la Concha Bermejillo, 1997 ). Clinically, animals exhibit signs of progressive pulmonary disease after an extremely long incubation period of up to 2 years. Respiratory rate and dyspnea gradually increase as the disease progresses. The animal continues to eat throughout the disease; however, animals progressively lose weight and become weak. Additionally, mastitis is a common clinical feature. Thoracic auscultation reveals consolidation of ventral lung lobes; and hematological findings indicate anemia and leukocytosis. The rare neurological signs include flexion of fetlock and pastern joints, tremors of facial muscles, progressive paresis and paralysis, depression, and prostration. Death occurs in weeks to months. The disease can be serologically diagnosed with agar gel immunodiffusion (AGID) tests, virus isolation, serum neutralization, complement fixation, and enzyme-linked immunosorbent assay (ELISA) tests. Epizootiology and transmission. Sixty-eight percent of sheep in some states have been infected with the virus ( Radostits et al., 1994 ). It is transmitted horizontally via inhalation of aerosolized virus particles and vertically between the infected dam and fetus. In addition, transmission through the milk or colostrum is considered common ( Knowles, 1997 ). Necropsy findings. Lesions are observed in lungs, mammary glands, joints, and the brain. Pulmonary adhesions, ventral lung lobe consolidation, bronchial lymph node enlargement, mastitis, and degenerative arthritis are visualized grossly. Meningeal edema, thickening of the choroid plexus, and foci of leukoencephalomalacia are seen in the central nervous system (CNS). Histologically, interalveolar septal thickening, lymphoid hyperplasia, histiocyte and fibrocyte proliferation, and squamous epithelial changes are seen in the lungs. Meningitis, lymphoid hyperplasia, demyelination, and glial fibrosis are seen in the CNS. Pathogenesis. The virus has a predilection for the lungs, mediastinal lymph nodes, udder, spleen, joints, and rarely the brain. After initial infection, the virus integrates into the DNA of mature monocytes and persists as a provirus. Later in the animal's life, infected monocytes mature as lung (and other tissue) macrophages and establish active infection. The virus induces lymphoproliferative disease, histiocyte and fibrocyte proliferation in the alveolar septa, and squamous metaplasia. Pulmonary alveolar and vascular changes impinge on oxygen and carbon dioxide exchange and lead to serious hypoxia and pulmonary hypertension. Secondary bacterial pneumonia may contribute to the animal's death. Differential diagnosis. Pulmonary adenomatosis is the differential diagnosis. Prevention and control. Isolating or removing infected animals can prevent the disease. Facilities and equipment should also be disinfected. Treatment. Treatment is unsuccessful. Etiology. An RNA virus in the lentivirus group of the Retroviridae family causes ovine progressive pneumonia (OPP), or maedi/visna. Maedi refers to the progressive pneumonia presentation of the disease; visna refers to the central nervous system disease, which is reported predominantly in Iceland. Visna has been reported in goats but may have been due to caprine arthritis encephalitis infection. Clinical signs and diagnosis. OPP is a viral disease of adult sheep characterized by weakness, unthriftiness, weight loss, and pneumonia ( Pepin et al., 1998 ; de la Concha Bermejillo, 1997 ). Clinically, animals exhibit signs of progressive pulmonary disease after an extremely long incubation period of up to 2 years. Respiratory rate and dyspnea gradually increase as the disease progresses. The animal continues to eat throughout the disease; however, animals progressively lose weight and become weak. Additionally, mastitis is a common clinical feature. Thoracic auscultation reveals consolidation of ventral lung lobes; and hematological findings indicate anemia and leukocytosis. The rare neurological signs include flexion of fetlock and pastern joints, tremors of facial muscles, progressive paresis and paralysis, depression, and prostration. Death occurs in weeks to months. The disease can be serologically diagnosed with agar gel immunodiffusion (AGID) tests, virus isolation, serum neutralization, complement fixation, and enzyme-linked immunosorbent assay (ELISA) tests. Epizootiology and transmission. Sixty-eight percent of sheep in some states have been infected with the virus ( Radostits et al., 1994 ). It is transmitted horizontally via inhalation of aerosolized virus particles and vertically between the infected dam and fetus. In addition, transmission through the milk or colostrum is considered common ( Knowles, 1997 ). Necropsy findings. Lesions are observed in lungs, mammary glands, joints, and the brain. Pulmonary adhesions, ventral lung lobe consolidation, bronchial lymph node enlargement, mastitis, and degenerative arthritis are visualized grossly. Meningeal edema, thickening of the choroid plexus, and foci of leukoencephalomalacia are seen in the central nervous system (CNS). Histologically, interalveolar septal thickening, lymphoid hyperplasia, histiocyte and fibrocyte proliferation, and squamous epithelial changes are seen in the lungs. Meningitis, lymphoid hyperplasia, demyelination, and glial fibrosis are seen in the CNS. Pathogenesis. The virus has a predilection for the lungs, mediastinal lymph nodes, udder, spleen, joints, and rarely the brain. After initial infection, the virus integrates into the DNA of mature monocytes and persists as a provirus. Later in the animal's life, infected monocytes mature as lung (and other tissue) macrophages and establish active infection. The virus induces lymphoproliferative disease, histiocyte and fibrocyte proliferation in the alveolar septa, and squamous metaplasia. Pulmonary alveolar and vascular changes impinge on oxygen and carbon dioxide exchange and lead to serious hypoxia and pulmonary hypertension. Secondary bacterial pneumonia may contribute to the animal's death. Differential diagnosis. Pulmonary adenomatosis is the differential diagnosis. Prevention and control. Isolating or removing infected animals can prevent the disease. Facilities and equipment should also be disinfected. Treatment. Treatment is unsuccessful. q. Poxviruses of Ruminants i. Ovine viral dermatosis. Ovine viral dermatosis is a venereal disease of sheep caused by a parapoxvirus distinct from contagious ecthyma. The disease resolves within 2 weeks in healthy animals, but lesions are painful and resemble those of Corynebacterium renale posthitis/vulvovaginitis. Symptomatic treatment may be necessary in some cases. There is no vaccine. Animals should not be used for breeding while clinical signs are present. ii. Proliferative stomatitis (bovine papular stomatitis) Etiology. A parapoxvirus is the causative agent of bovine papular stomatitis. This virus is considered to be closely related to the parapoxvirus that causes contagious ecthyma and pseudocowpox. It is also a zoonotic disease. The disease is not considered of major consequence, but high morbidity and mortality may be seen in severe outbreaks. In addition, lesions are comparable in appearance to those seen with vesicular stomatitis, bovine viral diarrhea virus, and foot-and-mouth disease. The disease occurs worldwide. Clinical signs and diagnosis. Raised red papules or erosions or shallow ulcers on the muzzle, nose, oral mucosa (including the hard palate), esophagus, and rumen of younger cattle are the most common findings. In some outbreaks, the papules will be associated with ulcerative esophagitis, salivation, diarrhea, and subsequent weight loss. Lesions persist or may come and go over a span of several months. Morbidity among herds may be 100%. Mortalities are rare. Bovine papular stomatitis is associated with "rat tail" in feedlot cattle. Animals continue to eat and usually do not show a fever. No lesion is seen on the feet. The infection may also be asymptomatic. Diagnosis is based on clinical signs, histological findings, and viral isolation. Epizootiology and transmission. Cattle less than 1 year of age are most commonly affected, and disease is rare in older cattle. Transmission is by animal-to-animal contact. Necropsy findings. Raised papules may be found around the muzzle and mouth and involve the mucosa of the esophagus and rumen. Histologically, epithelial cells will show hydropic degeneration and hyperplasia of the lamina propria. Eosinophilic inclusions will be in the cytoplasm of infected epithelial cells. Pathogenesis. Following exposure to the virus, erythematous macules most commonly appear on the nares, followed by the mouth. These become raised papules within a day, regressing after days to weeks; the lesions that remain will be persistent yellow, red, or brown spots. Some infections may recur or persist, with animals showing lesions intermittently or continuously over several months. Differential diagnosis. Pseudocowpox, vesicular stomatitis, foot-and-mouth disease, and bovine viral diarrhea virus infection are the differentials for this disease. The differential for the "rat tail" clinical sign is Sarcocystis infection. Prevention and control. There is no vaccine available for bovine papular stomatitis. Because of the similarity of this virus to the parapoxvirus of contagious ecthyma, it is important to be aware of the persistence in the environment and susceptibility of younger cattle. Vaccination using the local strain, and the skin scarification technique for orf, have been protective. Handlers should wear gloves and protective clothing. Treatment. Cattle usually will not require extensive nursing care, but lesions with secondary bacterial infections should be treated with antibiotics. Research complications. Handlers may develop lesions on their hands at sites of contact with lesions of cattle. iii. Pseudocowpox Etiology. Pseudocowpox is a worldwide cattle disease caused by a parapoxvirus related to the causative agents of contagious ecthyma and bovine papular stomatitis (see Sections III,A,2,m and III,A,2,q,ii). Lesions are confined to the teats. This is also a zoonotic disease. Clinical signs and diagnosis. Minor lesions are usually confined to the teats. These are distinctive because of the ring- or horseshoe-shaped scab that develops after 10 days. Additional lesions sometimes develop on the udder, the medial aspect of the thighs, and the scrotum. The teat lesions may predispose to mastitis. Pathogenesis. The virus is spread by contaminated hands, equipment, and fomites. Differential diagnosis. Differentials include bovine herpes mammillitis and papillomatosis. Prevention and control. Milking hygiene is helpful in control. Treatment. Lesions should be treated symptomatically, and affected animals milked last. Research complications. Like other related poxviruses, this virus causes nodular lesions on humans. i. Ovine viral dermatosis. Ovine viral dermatosis is a venereal disease of sheep caused by a parapoxvirus distinct from contagious ecthyma. The disease resolves within 2 weeks in healthy animals, but lesions are painful and resemble those of Corynebacterium renale posthitis/vulvovaginitis. Symptomatic treatment may be necessary in some cases. There is no vaccine. Animals should not be used for breeding while clinical signs are present. ii. Proliferative stomatitis (bovine papular stomatitis) Etiology. A parapoxvirus is the causative agent of bovine papular stomatitis. This virus is considered to be closely related to the parapoxvirus that causes contagious ecthyma and pseudocowpox. It is also a zoonotic disease. The disease is not considered of major consequence, but high morbidity and mortality may be seen in severe outbreaks. In addition, lesions are comparable in appearance to those seen with vesicular stomatitis, bovine viral diarrhea virus, and foot-and-mouth disease. The disease occurs worldwide. Clinical signs and diagnosis. Raised red papules or erosions or shallow ulcers on the muzzle, nose, oral mucosa (including the hard palate), esophagus, and rumen of younger cattle are the most common findings. In some outbreaks, the papules will be associated with ulcerative esophagitis, salivation, diarrhea, and subsequent weight loss. Lesions persist or may come and go over a span of several months. Morbidity among herds may be 100%. Mortalities are rare. Bovine papular stomatitis is associated with "rat tail" in feedlot cattle. Animals continue to eat and usually do not show a fever. No lesion is seen on the feet. The infection may also be asymptomatic. Diagnosis is based on clinical signs, histological findings, and viral isolation. Epizootiology and transmission. Cattle less than 1 year of age are most commonly affected, and disease is rare in older cattle. Transmission is by animal-to-animal contact. Necropsy findings. Raised papules may be found around the muzzle and mouth and involve the mucosa of the esophagus and rumen. Histologically, epithelial cells will show hydropic degeneration and hyperplasia of the lamina propria. Eosinophilic inclusions will be in the cytoplasm of infected epithelial cells. Pathogenesis. Following exposure to the virus, erythematous macules most commonly appear on the nares, followed by the mouth. These become raised papules within a day, regressing after days to weeks; the lesions that remain will be persistent yellow, red, or brown spots. Some infections may recur or persist, with animals showing lesions intermittently or continuously over several months. Differential diagnosis. Pseudocowpox, vesicular stomatitis, foot-and-mouth disease, and bovine viral diarrhea virus infection are the differentials for this disease. The differential for the "rat tail" clinical sign is Sarcocystis infection. Prevention and control. There is no vaccine available for bovine papular stomatitis. Because of the similarity of this virus to the parapoxvirus of contagious ecthyma, it is important to be aware of the persistence in the environment and susceptibility of younger cattle. Vaccination using the local strain, and the skin scarification technique for orf, have been protective. Handlers should wear gloves and protective clothing. Treatment. Cattle usually will not require extensive nursing care, but lesions with secondary bacterial infections should be treated with antibiotics. Research complications. Handlers may develop lesions on their hands at sites of contact with lesions of cattle. Etiology. A parapoxvirus is the causative agent of bovine papular stomatitis. This virus is considered to be closely related to the parapoxvirus that causes contagious ecthyma and pseudocowpox. It is also a zoonotic disease. The disease is not considered of major consequence, but high morbidity and mortality may be seen in severe outbreaks. In addition, lesions are comparable in appearance to those seen with vesicular stomatitis, bovine viral diarrhea virus, and foot-and-mouth disease. The disease occurs worldwide. Clinical signs and diagnosis. Raised red papules or erosions or shallow ulcers on the muzzle, nose, oral mucosa (including the hard palate), esophagus, and rumen of younger cattle are the most common findings. In some outbreaks, the papules will be associated with ulcerative esophagitis, salivation, diarrhea, and subsequent weight loss. Lesions persist or may come and go over a span of several months. Morbidity among herds may be 100%. Mortalities are rare. Bovine papular stomatitis is associated with "rat tail" in feedlot cattle. Animals continue to eat and usually do not show a fever. No lesion is seen on the feet. The infection may also be asymptomatic. Diagnosis is based on clinical signs, histological findings, and viral isolation. Epizootiology and transmission. Cattle less than 1 year of age are most commonly affected, and disease is rare in older cattle. Transmission is by animal-to-animal contact. Necropsy findings. Raised papules may be found around the muzzle and mouth and involve the mucosa of the esophagus and rumen. Histologically, epithelial cells will show hydropic degeneration and hyperplasia of the lamina propria. Eosinophilic inclusions will be in the cytoplasm of infected epithelial cells. Pathogenesis. Following exposure to the virus, erythematous macules most commonly appear on the nares, followed by the mouth. These become raised papules within a day, regressing after days to weeks; the lesions that remain will be persistent yellow, red, or brown spots. Some infections may recur or persist, with animals showing lesions intermittently or continuously over several months. Differential diagnosis. Pseudocowpox, vesicular stomatitis, foot-and-mouth disease, and bovine viral diarrhea virus infection are the differentials for this disease. The differential for the "rat tail" clinical sign is Sarcocystis infection. Prevention and control. There is no vaccine available for bovine papular stomatitis. Because of the similarity of this virus to the parapoxvirus of contagious ecthyma, it is important to be aware of the persistence in the environment and susceptibility of younger cattle. Vaccination using the local strain, and the skin scarification technique for orf, have been protective. Handlers should wear gloves and protective clothing. Treatment. Cattle usually will not require extensive nursing care, but lesions with secondary bacterial infections should be treated with antibiotics. Research complications. Handlers may develop lesions on their hands at sites of contact with lesions of cattle. iii. Pseudocowpox Etiology. Pseudocowpox is a worldwide cattle disease caused by a parapoxvirus related to the causative agents of contagious ecthyma and bovine papular stomatitis (see Sections III,A,2,m and III,A,2,q,ii). Lesions are confined to the teats. This is also a zoonotic disease. Clinical signs and diagnosis. Minor lesions are usually confined to the teats. These are distinctive because of the ring- or horseshoe-shaped scab that develops after 10 days. Additional lesions sometimes develop on the udder, the medial aspect of the thighs, and the scrotum. The teat lesions may predispose to mastitis. Pathogenesis. The virus is spread by contaminated hands, equipment, and fomites. Differential diagnosis. Differentials include bovine herpes mammillitis and papillomatosis. Prevention and control. Milking hygiene is helpful in control. Treatment. Lesions should be treated symptomatically, and affected animals milked last. Research complications. Like other related poxviruses, this virus causes nodular lesions on humans. Etiology. Pseudocowpox is a worldwide cattle disease caused by a parapoxvirus related to the causative agents of contagious ecthyma and bovine papular stomatitis (see Sections III,A,2,m and III,A,2,q,ii). Lesions are confined to the teats. This is also a zoonotic disease. Clinical signs and diagnosis. Minor lesions are usually confined to the teats. These are distinctive because of the ring- or horseshoe-shaped scab that develops after 10 days. Additional lesions sometimes develop on the udder, the medial aspect of the thighs, and the scrotum. The teat lesions may predispose to mastitis. Pathogenesis. The virus is spread by contaminated hands, equipment, and fomites. Differential diagnosis. Differentials include bovine herpes mammillitis and papillomatosis. Prevention and control. Milking hygiene is helpful in control. Treatment. Lesions should be treated symptomatically, and affected animals milked last. Research complications. Like other related poxviruses, this virus causes nodular lesions on humans. r. Pulmonary Adenomatosis (Jaagsiekte) Etiology. Pulmonary adenomatosis is a rare but progressive wasting disease of sheep, with worldwide distribution. Pulmonary adenomatosis is caused by a type D retrovirus antigenically related to the Mason-Pfizer monkey virus. Jaagsiekte was the designation when the disease was described originally in South Africa. Clinical signs and diagnosis. Typical clinical signs include progressive respiratory signs such as dyspnea, rapid respiration, and wasting. The disease is diagnosed by these chronic clinical signs and histology. Epizootiology and transmission. The disease is transmitted by aerosols. Body fluids of viremic animals, such as milk, blood, saliva, tears, semen, and bronchial secretions, will contain the virus or cells carrying the virus. Necropsy. The adenomas and adenocarcinomas will be small firm lesions distributed throughout the lungs. The adenocarcinomas metastasize to regional lymph nodes. Pathogenesis. As with ovine progressive pneumonia (OPP), the incubation period is up to 2 years long. Adenocarcinomatous lesions arising from type II alveolar epithelial cells may be discrete or confluent and involve all lung lobes. Differential diagnosis. This disease occurs coincidentally with or is a differential diagnosis for OPP. Treatment. No treatment is effective. Etiology. Pulmonary adenomatosis is a rare but progressive wasting disease of sheep, with worldwide distribution. Pulmonary adenomatosis is caused by a type D retrovirus antigenically related to the Mason-Pfizer monkey virus. Jaagsiekte was the designation when the disease was described originally in South Africa. Clinical signs and diagnosis. Typical clinical signs include progressive respiratory signs such as dyspnea, rapid respiration, and wasting. The disease is diagnosed by these chronic clinical signs and histology. Epizootiology and transmission. The disease is transmitted by aerosols. Body fluids of viremic animals, such as milk, blood, saliva, tears, semen, and bronchial secretions, will contain the virus or cells carrying the virus. Necropsy. The adenomas and adenocarcinomas will be small firm lesions distributed throughout the lungs. The adenocarcinomas metastasize to regional lymph nodes. Pathogenesis. As with ovine progressive pneumonia (OPP), the incubation period is up to 2 years long. Adenocarcinomatous lesions arising from type II alveolar epithelial cells may be discrete or confluent and involve all lung lobes. Differential diagnosis. This disease occurs coincidentally with or is a differential diagnosis for OPP. Treatment. No treatment is effective. Etiology. Pulmonary adenomatosis is a rare but progressive wasting disease of sheep, with worldwide distribution. Pulmonary adenomatosis is caused by a type D retrovirus antigenically related to the Mason-Pfizer monkey virus. Jaagsiekte was the designation when the disease was described originally in South Africa. Clinical signs and diagnosis. Typical clinical signs include progressive respiratory signs such as dyspnea, rapid respiration, and wasting. The disease is diagnosed by these chronic clinical signs and histology. Epizootiology and transmission. The disease is transmitted by aerosols. Body fluids of viremic animals, such as milk, blood, saliva, tears, semen, and bronchial secretions, will contain the virus or cells carrying the virus. Necropsy. The adenomas and adenocarcinomas will be small firm lesions distributed throughout the lungs. The adenocarcinomas metastasize to regional lymph nodes. Pathogenesis. As with ovine progressive pneumonia (OPP), the incubation period is up to 2 years long. Adenocarcinomatous lesions arising from type II alveolar epithelial cells may be discrete or confluent and involve all lung lobes. Differential diagnosis. This disease occurs coincidentally with or is a differential diagnosis for OPP. Treatment. No treatment is effective. s. Papillomatosis (Warts, Verrucae) Etiology. Cutaneous papillomatosis is a very common disease in cattle and is much less common among sheep and goats. The disease is a viral-induced proliferation of the epithelium of the neck, face, back, and legs. These tumors are caused by a papillomavirus (DNA virus) of the Papovaviridae family, and the viruses are host-specific and often body site-specific. Most are benign, although some forms in cattle and one form in goats can become malignant. In cattle, the site specificity of the papillomavirus strains are particularly well recognized. Designations of the currently recognized bovine papillomavirus (BPV) types are BPV-1 through BPV-5. Clinical signs and diagnosis. The papillomas may last up to 12 months and are seen more frequently in younger animals. Lesions have typical wart appearances and may be single or multiple, small (1 mm) or very large (500 mm). The infections will generally be benign, but pain will be evident when warts develop on occlusal surfaces or within the gastrointestinal tract. In addition, when infections are severe, weight loss may occur. When warts occur on teats, secondary mastitis may develop. In cattle, BPV-1 and BPV-2 cause fibropapillomas on teats and penises or on head, neck, and dewlap, respectively. BPV-3 causes flat warts that occur in all body locations, BPV-4 causes warts in the gastrointestinal tract, and BPV-5 causes small white warts (called rice-grain warts) on teats. Warts caused by BPV-3 and BPV-5 do not regress spontaneously. Prognosis in cattle is poor only when papillomatosis involves more than 20% of the body surface. In sheep, warts are the verrucous type. The disease is of little consequence unless the warts develop in an area that causes discomfort or incapacitation such as between the digits, on the lips, or over the joints. In adult sheep, warts may transform to squamous cell carcinoma. In goats, the disease is rare, and the warts are also of the verrucous type and occasionally may develop into squamous cell carcinoma. Warts on goat udders tend to be persistent. Diagnosis is made by observing the typical proliferative lesions. Epizootiology and transmission. Older animals are less sensitive to papillomatosis than young animals, although immunosu-pressed animals of any age may develop warts as the result of harbored latent infections. The virus is transmitted by direct and indirect (fomite) contact, entering through surface wounds and sites such as tattoos. Pathogenesis. The incubation period ranges from 1 to 6 months. The virus induces epidermal and fibrous tissue proliferation, often described as cauliflower-like skin tumors. The disease is generally self-limiting. Differential diagnosis. In sheep and goats, differentials include contagious ecthyma, ulcerative dermatosis, strawberry foot rot, and sheep and goat pox. Prevention and control. Commercial vaccines (available only for cattle) or autogenous vaccines must be used with a recognition that papovavirus strains are host-specific and that immunity from infection or vaccination is viral-type-specific. Autogenous vaccines are generally considered more effective. Some vaccine preparations are effective at prevention but not treatment of outbreaks. Viricidal products are recommended for disinfection of contaminated environments. Minimizing cutaneous injuries and sanitizing equipment (tattoo devices, dehorners, ear taggers, etc.) in a virucidal solution between uses are also recommended preventive and control measures. Halters, brushes, and other items may also be sources of virus. Treatment. Warts will often spontaneously resolve as immunity develops. In severe cases or with flockwide or herdwide problems, affected animals should be isolated from nonaffected animals, and premises disinfected. Warts can be surgically excised and autogenous vaccines can be made and administered to help prevent disease spread. Cryosurgery with liquid nitrogen or dry ice has also proven to be successful for wart removal. Topical agents such as podophyllin (various formulations) and dimethyl sulfoxide may be applied to individual lesions once daily until regression. Etiology. Cutaneous papillomatosis is a very common disease in cattle and is much less common among sheep and goats. The disease is a viral-induced proliferation of the epithelium of the neck, face, back, and legs. These tumors are caused by a papillomavirus (DNA virus) of the Papovaviridae family, and the viruses are host-specific and often body site-specific. Most are benign, although some forms in cattle and one form in goats can become malignant. In cattle, the site specificity of the papillomavirus strains are particularly well recognized. Designations of the currently recognized bovine papillomavirus (BPV) types are BPV-1 through BPV-5. Clinical signs and diagnosis. The papillomas may last up to 12 months and are seen more frequently in younger animals. Lesions have typical wart appearances and may be single or multiple, small (1 mm) or very large (500 mm). The infections will generally be benign, but pain will be evident when warts develop on occlusal surfaces or within the gastrointestinal tract. In addition, when infections are severe, weight loss may occur. When warts occur on teats, secondary mastitis may develop. In cattle, BPV-1 and BPV-2 cause fibropapillomas on teats and penises or on head, neck, and dewlap, respectively. BPV-3 causes flat warts that occur in all body locations, BPV-4 causes warts in the gastrointestinal tract, and BPV-5 causes small white warts (called rice-grain warts) on teats. Warts caused by BPV-3 and BPV-5 do not regress spontaneously. Prognosis in cattle is poor only when papillomatosis involves more than 20% of the body surface. In sheep, warts are the verrucous type. The disease is of little consequence unless the warts develop in an area that causes discomfort or incapacitation such as between the digits, on the lips, or over the joints. In adult sheep, warts may transform to squamous cell carcinoma. In goats, the disease is rare, and the warts are also of the verrucous type and occasionally may develop into squamous cell carcinoma. Warts on goat udders tend to be persistent. Diagnosis is made by observing the typical proliferative lesions. Epizootiology and transmission. Older animals are less sensitive to papillomatosis than young animals, although immunosu-pressed animals of any age may develop warts as the result of harbored latent infections. The virus is transmitted by direct and indirect (fomite) contact, entering through surface wounds and sites such as tattoos. Pathogenesis. The incubation period ranges from 1 to 6 months. The virus induces epidermal and fibrous tissue proliferation, often described as cauliflower-like skin tumors. The disease is generally self-limiting. Differential diagnosis. In sheep and goats, differentials include contagious ecthyma, ulcerative dermatosis, strawberry foot rot, and sheep and goat pox. Prevention and control. Commercial vaccines (available only for cattle) or autogenous vaccines must be used with a recognition that papovavirus strains are host-specific and that immunity from infection or vaccination is viral-type-specific. Autogenous vaccines are generally considered more effective. Some vaccine preparations are effective at prevention but not treatment of outbreaks. Viricidal products are recommended for disinfection of contaminated environments. Minimizing cutaneous injuries and sanitizing equipment (tattoo devices, dehorners, ear taggers, etc.) in a virucidal solution between uses are also recommended preventive and control measures. Halters, brushes, and other items may also be sources of virus. Treatment. Warts will often spontaneously resolve as immunity develops. In severe cases or with flockwide or herdwide problems, affected animals should be isolated from nonaffected animals, and premises disinfected. Warts can be surgically excised and autogenous vaccines can be made and administered to help prevent disease spread. Cryosurgery with liquid nitrogen or dry ice has also proven to be successful for wart removal. Topical agents such as podophyllin (various formulations) and dimethyl sulfoxide may be applied to individual lesions once daily until regression. Etiology. Cutaneous papillomatosis is a very common disease in cattle and is much less common among sheep and goats. The disease is a viral-induced proliferation of the epithelium of the neck, face, back, and legs. These tumors are caused by a papillomavirus (DNA virus) of the Papovaviridae family, and the viruses are host-specific and often body site-specific. Most are benign, although some forms in cattle and one form in goats can become malignant. In cattle, the site specificity of the papillomavirus strains are particularly well recognized. Designations of the currently recognized bovine papillomavirus (BPV) types are BPV-1 through BPV-5. Clinical signs and diagnosis. The papillomas may last up to 12 months and are seen more frequently in younger animals. Lesions have typical wart appearances and may be single or multiple, small (1 mm) or very large (500 mm). The infections will generally be benign, but pain will be evident when warts develop on occlusal surfaces or within the gastrointestinal tract. In addition, when infections are severe, weight loss may occur. When warts occur on teats, secondary mastitis may develop. In cattle, BPV-1 and BPV-2 cause fibropapillomas on teats and penises or on head, neck, and dewlap, respectively. BPV-3 causes flat warts that occur in all body locations, BPV-4 causes warts in the gastrointestinal tract, and BPV-5 causes small white warts (called rice-grain warts) on teats. Warts caused by BPV-3 and BPV-5 do not regress spontaneously. Prognosis in cattle is poor only when papillomatosis involves more than 20% of the body surface. In sheep, warts are the verrucous type. The disease is of little consequence unless the warts develop in an area that causes discomfort or incapacitation such as between the digits, on the lips, or over the joints. In adult sheep, warts may transform to squamous cell carcinoma. In goats, the disease is rare, and the warts are also of the verrucous type and occasionally may develop into squamous cell carcinoma. Warts on goat udders tend to be persistent. Diagnosis is made by observing the typical proliferative lesions. Epizootiology and transmission. Older animals are less sensitive to papillomatosis than young animals, although immunosu-pressed animals of any age may develop warts as the result of harbored latent infections. The virus is transmitted by direct and indirect (fomite) contact, entering through surface wounds and sites such as tattoos. Pathogenesis. The incubation period ranges from 1 to 6 months. The virus induces epidermal and fibrous tissue proliferation, often described as cauliflower-like skin tumors. The disease is generally self-limiting. Differential diagnosis. In sheep and goats, differentials include contagious ecthyma, ulcerative dermatosis, strawberry foot rot, and sheep and goat pox. Prevention and control. Commercial vaccines (available only for cattle) or autogenous vaccines must be used with a recognition that papovavirus strains are host-specific and that immunity from infection or vaccination is viral-type-specific. Autogenous vaccines are generally considered more effective. Some vaccine preparations are effective at prevention but not treatment of outbreaks. Viricidal products are recommended for disinfection of contaminated environments. Minimizing cutaneous injuries and sanitizing equipment (tattoo devices, dehorners, ear taggers, etc.) in a virucidal solution between uses are also recommended preventive and control measures. Halters, brushes, and other items may also be sources of virus. Treatment. Warts will often spontaneously resolve as immunity develops. In severe cases or with flockwide or herdwide problems, affected animals should be isolated from nonaffected animals, and premises disinfected. Warts can be surgically excised and autogenous vaccines can be made and administered to help prevent disease spread. Cryosurgery with liquid nitrogen or dry ice has also proven to be successful for wart removal. Topical agents such as podophyllin (various formulations) and dimethyl sulfoxide may be applied to individual lesions once daily until regression. t. Pseudorabies (Mad Itch, Aujeszky's Disease) Etiology. Pseudorabies is an acute encephalitic disease caused by a neurotropic alphaherpesvirus, the porcine herpesvirus 1. One serotype is recognized, but strain differences exist. The disease has worldwide distribution. It is a primarily a clinical disease of cattle, with less frequent reports (but no less severe clinical manifestations) in sheep and goats. Clinical signs and diagnosis. A range of clinical signs is seen during the rapid course of this usually fatal disease. At the site of virus inoculation or in other locations, abrasions, swelling, intense pruritus, and alopecia are seen. Pruritus will not be asymmetric. Animals will also become hyperthermic and will vocalize frantically. Other neurological signs range from hoof stamping, kicking at the pruritic area, salivation, tongue chewing, head pressing and circling, to paresthesia or hyperesthesia, ataxia, and conscious proprioceptive deficits. Nystagmus and strabismus are also seen. Animals will be fearful or depressed, and aggression is sometimes seen. Recumbency and coma precede death. Diagnostic evidence includes clinical findings; virus isolation from nasal or pharyngeal secretions or postmortem tissues; and histological findings at necropsy. Serology of affected animals is not productive, because of the rapid course. If swine are housed nearby, or if swine were transported in the same vehicles as affected animals, serological evaluations are worthwhile from those animals. Epizootiology and transmission. Swine are the primary hosts for pseudorabies virus, but they are usually asymptomatic and serve as reservoirs for the virus. The infection can remain latent in the trigeminal ganglion of pigs and recrudesce during stressful conditions. Other animals are dead-end hosts. The unprotected virus will survive only a few weeks in the environment but may remain viable in meat (including carcasses) or saliva and will survive outside the host, in favorable conditions, in the summer for several weeks and the winter for several months. Transmission is by oral, intranasal, intradermal, or subcutaneous introduction of the virus. When the virus is inhaled, the clinical signs of pruritus are less likely to be seen. Transmission can also be by inadvertent exposure (e.g., contaminated syringes) of ruminants to the modified live vaccines developed for use in swine. Spread between infected ruminants is a less likely means of transmission, because of the relatively short period of virus shedding. Transport vehicles used for swine may also be sources of the virus. Raccoons are believed to be vectors of the virus. Horses are resistant to infection. Necropsy findings. There is no pathognomonic gross lesion. Definitive histologic findings include severe, focal, nonsuppurative encephalitis and myelitis. Eosinophilic intranuclear inclusion bodies (Cowdry type A) may be present in some affected neurons. Methods such as immunofluorescence and immunoperoxidase staining can be used to show presence of the porcine herpesvirus 1. Pathogenesis. The incubation period is 90–156 hr and duration of the illness is 8–72 hr. The longest duration is seen in animals with pruritus around the head. Differential diagnoses. Differentials for the neurologic signs of pseudorabies infection include rabies, polioencephalomalacia, salt poisoning, meningitis, lead poisoning, hypomagnesemia, and enterotoxemia. Those for the intense pruritus include psoroptic mange and scrapie in sheep, sarcoptic mange, and pediculosis. Prevention and control. Pseudorabies is a reportable disease in the United States, where a nationwide eradication program exists; states are rated regarding status. Effective disinfectants include sodium hypochlorite (10% solution), formalin, peracetic acid, tamed iodines, and quaternary ammonium compounds. Five minutes of contact time is required, and then surfaces must be rinsed. Other disinfectant methods for viral killing include 6 hr of formaldehyde fumigation, or 360 min of ultraviolet light. Transport vehicles should be cleaned and disinfected between species. Serological screening for pseudorabies of swine housed near ruminants is essential. Treatment. There is no treatment, and most affected animals die. Research complications. Swine housed close to research ruminants should be serologically screened prior to purchase, and all transport vehicles should be cleaned and disinfected between loads of large animals. Humans have been reported to seroconvert. The porcine herpesvirus 1 shares antigens with the infectious bovine rhinotracheitis virus. Etiology. Pseudorabies is an acute encephalitic disease caused by a neurotropic alphaherpesvirus, the porcine herpesvirus 1. One serotype is recognized, but strain differences exist. The disease has worldwide distribution. It is a primarily a clinical disease of cattle, with less frequent reports (but no less severe clinical manifestations) in sheep and goats. Clinical signs and diagnosis. A range of clinical signs is seen during the rapid course of this usually fatal disease. At the site of virus inoculation or in other locations, abrasions, swelling, intense pruritus, and alopecia are seen. Pruritus will not be asymmetric. Animals will also become hyperthermic and will vocalize frantically. Other neurological signs range from hoof stamping, kicking at the pruritic area, salivation, tongue chewing, head pressing and circling, to paresthesia or hyperesthesia, ataxia, and conscious proprioceptive deficits. Nystagmus and strabismus are also seen. Animals will be fearful or depressed, and aggression is sometimes seen. Recumbency and coma precede death. Diagnostic evidence includes clinical findings; virus isolation from nasal or pharyngeal secretions or postmortem tissues; and histological findings at necropsy. Serology of affected animals is not productive, because of the rapid course. If swine are housed nearby, or if swine were transported in the same vehicles as affected animals, serological evaluations are worthwhile from those animals. Epizootiology and transmission. Swine are the primary hosts for pseudorabies virus, but they are usually asymptomatic and serve as reservoirs for the virus. The infection can remain latent in the trigeminal ganglion of pigs and recrudesce during stressful conditions. Other animals are dead-end hosts. The unprotected virus will survive only a few weeks in the environment but may remain viable in meat (including carcasses) or saliva and will survive outside the host, in favorable conditions, in the summer for several weeks and the winter for several months. Transmission is by oral, intranasal, intradermal, or subcutaneous introduction of the virus. When the virus is inhaled, the clinical signs of pruritus are less likely to be seen. Transmission can also be by inadvertent exposure (e.g., contaminated syringes) of ruminants to the modified live vaccines developed for use in swine. Spread between infected ruminants is a less likely means of transmission, because of the relatively short period of virus shedding. Transport vehicles used for swine may also be sources of the virus. Raccoons are believed to be vectors of the virus. Horses are resistant to infection. Necropsy findings. There is no pathognomonic gross lesion. Definitive histologic findings include severe, focal, nonsuppurative encephalitis and myelitis. Eosinophilic intranuclear inclusion bodies (Cowdry type A) may be present in some affected neurons. Methods such as immunofluorescence and immunoperoxidase staining can be used to show presence of the porcine herpesvirus 1. Pathogenesis. The incubation period is 90–156 hr and duration of the illness is 8–72 hr. The longest duration is seen in animals with pruritus around the head. Differential diagnoses. Differentials for the neurologic signs of pseudorabies infection include rabies, polioencephalomalacia, salt poisoning, meningitis, lead poisoning, hypomagnesemia, and enterotoxemia. Those for the intense pruritus include psoroptic mange and scrapie in sheep, sarcoptic mange, and pediculosis. Prevention and control. Pseudorabies is a reportable disease in the United States, where a nationwide eradication program exists; states are rated regarding status. Effective disinfectants include sodium hypochlorite (10% solution), formalin, peracetic acid, tamed iodines, and quaternary ammonium compounds. Five minutes of contact time is required, and then surfaces must be rinsed. Other disinfectant methods for viral killing include 6 hr of formaldehyde fumigation, or 360 min of ultraviolet light. Transport vehicles should be cleaned and disinfected between species. Serological screening for pseudorabies of swine housed near ruminants is essential. Treatment. There is no treatment, and most affected animals die. Research complications. Swine housed close to research ruminants should be serologically screened prior to purchase, and all transport vehicles should be cleaned and disinfected between loads of large animals. Humans have been reported to seroconvert. The porcine herpesvirus 1 shares antigens with the infectious bovine rhinotracheitis virus. Etiology. Pseudorabies is an acute encephalitic disease caused by a neurotropic alphaherpesvirus, the porcine herpesvirus 1. One serotype is recognized, but strain differences exist. The disease has worldwide distribution. It is a primarily a clinical disease of cattle, with less frequent reports (but no less severe clinical manifestations) in sheep and goats. Clinical signs and diagnosis. A range of clinical signs is seen during the rapid course of this usually fatal disease. At the site of virus inoculation or in other locations, abrasions, swelling, intense pruritus, and alopecia are seen. Pruritus will not be asymmetric. Animals will also become hyperthermic and will vocalize frantically. Other neurological signs range from hoof stamping, kicking at the pruritic area, salivation, tongue chewing, head pressing and circling, to paresthesia or hyperesthesia, ataxia, and conscious proprioceptive deficits. Nystagmus and strabismus are also seen. Animals will be fearful or depressed, and aggression is sometimes seen. Recumbency and coma precede death. Diagnostic evidence includes clinical findings; virus isolation from nasal or pharyngeal secretions or postmortem tissues; and histological findings at necropsy. Serology of affected animals is not productive, because of the rapid course. If swine are housed nearby, or if swine were transported in the same vehicles as affected animals, serological evaluations are worthwhile from those animals. Epizootiology and transmission. Swine are the primary hosts for pseudorabies virus, but they are usually asymptomatic and serve as reservoirs for the virus. The infection can remain latent in the trigeminal ganglion of pigs and recrudesce during stressful conditions. Other animals are dead-end hosts. The unprotected virus will survive only a few weeks in the environment but may remain viable in meat (including carcasses) or saliva and will survive outside the host, in favorable conditions, in the summer for several weeks and the winter for several months. Transmission is by oral, intranasal, intradermal, or subcutaneous introduction of the virus. When the virus is inhaled, the clinical signs of pruritus are less likely to be seen. Transmission can also be by inadvertent exposure (e.g., contaminated syringes) of ruminants to the modified live vaccines developed for use in swine. Spread between infected ruminants is a less likely means of transmission, because of the relatively short period of virus shedding. Transport vehicles used for swine may also be sources of the virus. Raccoons are believed to be vectors of the virus. Horses are resistant to infection. Necropsy findings. There is no pathognomonic gross lesion. Definitive histologic findings include severe, focal, nonsuppurative encephalitis and myelitis. Eosinophilic intranuclear inclusion bodies (Cowdry type A) may be present in some affected neurons. Methods such as immunofluorescence and immunoperoxidase staining can be used to show presence of the porcine herpesvirus 1. Pathogenesis. The incubation period is 90–156 hr and duration of the illness is 8–72 hr. The longest duration is seen in animals with pruritus around the head. Differential diagnoses. Differentials for the neurologic signs of pseudorabies infection include rabies, polioencephalomalacia, salt poisoning, meningitis, lead poisoning, hypomagnesemia, and enterotoxemia. Those for the intense pruritus include psoroptic mange and scrapie in sheep, sarcoptic mange, and pediculosis. Prevention and control. Pseudorabies is a reportable disease in the United States, where a nationwide eradication program exists; states are rated regarding status. Effective disinfectants include sodium hypochlorite (10% solution), formalin, peracetic acid, tamed iodines, and quaternary ammonium compounds. Five minutes of contact time is required, and then surfaces must be rinsed. Other disinfectant methods for viral killing include 6 hr of formaldehyde fumigation, or 360 min of ultraviolet light. Transport vehicles should be cleaned and disinfected between species. Serological screening for pseudorabies of swine housed near ruminants is essential. Treatment. There is no treatment, and most affected animals die. Research complications. Swine housed close to research ruminants should be serologically screened prior to purchase, and all transport vehicles should be cleaned and disinfected between loads of large animals. Humans have been reported to seroconvert. The porcine herpesvirus 1 shares antigens with the infectious bovine rhinotracheitis virus. u. Rabies (Hydrophobia) Etiology. Rabies is a sporadic but fatal, acute viral disease affecting the central nervous system. The rabies virus is a neurotropic RNA virus of the Lyssavirus genus and the Rhabdoviridae family. Sheep, goats, and cattle are susceptible. The zoonotic potential of this virus must be kept in mind at all times when handling moribund animals with neurological signs characteristic of the disease. Rabies is endemic in many areas of the world and within areas of the Unites States. This is a reportable disease in North America. Clinical findings and diagnosis. Animals generally progress through three phases: prodromal, excitatory, and paralytic. Many signs in the different species during these stages are nonspecific, and forms of the disease are also referred to as dumb or furious. During the short prodromal phase, animals are hyperthermic and apprehensive. Animals progress to the excitatory phase, during which they refuse to eat or drink and are active and aggressive. Repeated vocalizations, tenesmus, sexual excitement, and salivation occur during this phase. The final paralytic stage, with recumbency and death, occurs over several hours to days. This paralytic stage is common in cattle, and animals may simply be found dead. The clinical course is usually 1–4 days. Diagnosis is based on clinical signs, with a progressive and fatal course. Confirmation presently is made with the fluorescent antibody technique on brain tissue. Epizootiology and transmission. The rabies virus is transmitted via a bite wound inflicted by a rabid animal. Cats, dogs, raccoons, skunks, foxes, wild canids, and bats are the common disease vectors in North America. Virus is also transmitted in milk and aerosols. Necropsy findings. Few lesions are seen at necropsy. Many secondary lesions from manic behaviors during the course of disease may be evident. Histological findings will include nonsuppurative encephalitis. Negri bodies in the cytoplasm of neurons of the hippocampus and in Purkinje's cells are pathognomonic histologic findings. Pathogenesis. After exposure, the incubation period is variable, from 2 weeks to several months, depending on the distance that the virus has to travel to reach the central nervous system. The rabies virus proliferates locally, gains access to neurons by attaching to acetylcholine receptors, via a viral surface glycoprotein, migrates along sensory nerves to the spinal cord and brain, and then descends via cranial nerves (trigeminal, facial, olfactory, glossopharyngeal) to oral and nasal cavity structures (i.e., salivary glands). The fatal outcome is currently believed to be multifactorial, related to anorexia, respiratory paralysis, and effects on the pituitary. Differential diagnosis. Rabies should be included on the differential list when clinical signs of neurologic disease are evident. Other differentials for ruminants include herpesvirus encephalitis, thromboemobolic meningoencephalitis, nervous ketosis, grass tetany, and nervous cocciodiosis. Prevention and control. Vaccines approved for use cattle and sheep are commercially available and contain inactivated virus; there is not one available in the United States for goats. Ruminants in endemic areas, such as the East Coast of the United States, should be routinely vaccinated. Any animals housed outside that may be exposed to rabid animals should be vaccinated. Vaccination programs generally begin at 3 months of age, with a booster at 1 year of age and then annual or triennial boosters. Awareness of the current rabies case reports for the region and wildlife reservoirs, however, is important. Monitoring for and exclusion of wildlife from large-animal facilities are worthwhile preventive measures. The virus is fragile and unstable outside of a host animal. Research complications. Aerosolized virus is infective. Personal protective equipment, including gloves, face mask, and eye shields, must be worn by individuals handling animals that are manifesting neurological disease signs. Etiology. Rabies is a sporadic but fatal, acute viral disease affecting the central nervous system. The rabies virus is a neurotropic RNA virus of the Lyssavirus genus and the Rhabdoviridae family. Sheep, goats, and cattle are susceptible. The zoonotic potential of this virus must be kept in mind at all times when handling moribund animals with neurological signs characteristic of the disease. Rabies is endemic in many areas of the world and within areas of the Unites States. This is a reportable disease in North America. Clinical findings and diagnosis. Animals generally progress through three phases: prodromal, excitatory, and paralytic. Many signs in the different species during these stages are nonspecific, and forms of the disease are also referred to as dumb or furious. During the short prodromal phase, animals are hyperthermic and apprehensive. Animals progress to the excitatory phase, during which they refuse to eat or drink and are active and aggressive. Repeated vocalizations, tenesmus, sexual excitement, and salivation occur during this phase. The final paralytic stage, with recumbency and death, occurs over several hours to days. This paralytic stage is common in cattle, and animals may simply be found dead. The clinical course is usually 1–4 days. Diagnosis is based on clinical signs, with a progressive and fatal course. Confirmation presently is made with the fluorescent antibody technique on brain tissue. Epizootiology and transmission. The rabies virus is transmitted via a bite wound inflicted by a rabid animal. Cats, dogs, raccoons, skunks, foxes, wild canids, and bats are the common disease vectors in North America. Virus is also transmitted in milk and aerosols. Necropsy findings. Few lesions are seen at necropsy. Many secondary lesions from manic behaviors during the course of disease may be evident. Histological findings will include nonsuppurative encephalitis. Negri bodies in the cytoplasm of neurons of the hippocampus and in Purkinje's cells are pathognomonic histologic findings. Pathogenesis. After exposure, the incubation period is variable, from 2 weeks to several months, depending on the distance that the virus has to travel to reach the central nervous system. The rabies virus proliferates locally, gains access to neurons by attaching to acetylcholine receptors, via a viral surface glycoprotein, migrates along sensory nerves to the spinal cord and brain, and then descends via cranial nerves (trigeminal, facial, olfactory, glossopharyngeal) to oral and nasal cavity structures (i.e., salivary glands). The fatal outcome is currently believed to be multifactorial, related to anorexia, respiratory paralysis, and effects on the pituitary. Differential diagnosis. Rabies should be included on the differential list when clinical signs of neurologic disease are evident. Other differentials for ruminants include herpesvirus encephalitis, thromboemobolic meningoencephalitis, nervous ketosis, grass tetany, and nervous cocciodiosis. Prevention and control. Vaccines approved for use cattle and sheep are commercially available and contain inactivated virus; there is not one available in the United States for goats. Ruminants in endemic areas, such as the East Coast of the United States, should be routinely vaccinated. Any animals housed outside that may be exposed to rabid animals should be vaccinated. Vaccination programs generally begin at 3 months of age, with a booster at 1 year of age and then annual or triennial boosters. Awareness of the current rabies case reports for the region and wildlife reservoirs, however, is important. Monitoring for and exclusion of wildlife from large-animal facilities are worthwhile preventive measures. The virus is fragile and unstable outside of a host animal. Research complications. Aerosolized virus is infective. Personal protective equipment, including gloves, face mask, and eye shields, must be worn by individuals handling animals that are manifesting neurological disease signs. Etiology. Rabies is a sporadic but fatal, acute viral disease affecting the central nervous system. The rabies virus is a neurotropic RNA virus of the Lyssavirus genus and the Rhabdoviridae family. Sheep, goats, and cattle are susceptible. The zoonotic potential of this virus must be kept in mind at all times when handling moribund animals with neurological signs characteristic of the disease. Rabies is endemic in many areas of the world and within areas of the Unites States. This is a reportable disease in North America. Clinical findings and diagnosis. Animals generally progress through three phases: prodromal, excitatory, and paralytic. Many signs in the different species during these stages are nonspecific, and forms of the disease are also referred to as dumb or furious. During the short prodromal phase, animals are hyperthermic and apprehensive. Animals progress to the excitatory phase, during which they refuse to eat or drink and are active and aggressive. Repeated vocalizations, tenesmus, sexual excitement, and salivation occur during this phase. The final paralytic stage, with recumbency and death, occurs over several hours to days. This paralytic stage is common in cattle, and animals may simply be found dead. The clinical course is usually 1–4 days. Diagnosis is based on clinical signs, with a progressive and fatal course. Confirmation presently is made with the fluorescent antibody technique on brain tissue. Epizootiology and transmission. The rabies virus is transmitted via a bite wound inflicted by a rabid animal. Cats, dogs, raccoons, skunks, foxes, wild canids, and bats are the common disease vectors in North America. Virus is also transmitted in milk and aerosols. Necropsy findings. Few lesions are seen at necropsy. Many secondary lesions from manic behaviors during the course of disease may be evident. Histological findings will include nonsuppurative encephalitis. Negri bodies in the cytoplasm of neurons of the hippocampus and in Purkinje's cells are pathognomonic histologic findings. Pathogenesis. After exposure, the incubation period is variable, from 2 weeks to several months, depending on the distance that the virus has to travel to reach the central nervous system. The rabies virus proliferates locally, gains access to neurons by attaching to acetylcholine receptors, via a viral surface glycoprotein, migrates along sensory nerves to the spinal cord and brain, and then descends via cranial nerves (trigeminal, facial, olfactory, glossopharyngeal) to oral and nasal cavity structures (i.e., salivary glands). The fatal outcome is currently believed to be multifactorial, related to anorexia, respiratory paralysis, and effects on the pituitary. Differential diagnosis. Rabies should be included on the differential list when clinical signs of neurologic disease are evident. Other differentials for ruminants include herpesvirus encephalitis, thromboemobolic meningoencephalitis, nervous ketosis, grass tetany, and nervous cocciodiosis. Prevention and control. Vaccines approved for use cattle and sheep are commercially available and contain inactivated virus; there is not one available in the United States for goats. Ruminants in endemic areas, such as the East Coast of the United States, should be routinely vaccinated. Any animals housed outside that may be exposed to rabid animals should be vaccinated. Vaccination programs generally begin at 3 months of age, with a booster at 1 year of age and then annual or triennial boosters. Awareness of the current rabies case reports for the region and wildlife reservoirs, however, is important. Monitoring for and exclusion of wildlife from large-animal facilities are worthwhile preventive measures. The virus is fragile and unstable outside of a host animal. Research complications. Aerosolized virus is infective. Personal protective equipment, including gloves, face mask, and eye shields, must be worn by individuals handling animals that are manifesting neurological disease signs. v. Transmissible Spongiform Encephalopathies i. Bovine spongiform encephalopathy (mad cow disease). Bovine spongiform encephalopathy, a transmissible spongiform encephalopathy (TSE), is not known to occur in the United States, where since 1989 it has been listed as a reportable disease. The profound impact of this disease on the cattle industry in Great Britain during the past two decades is well known. The disease may be caused by a scrapielike (prion) agent. It is believed that the source of infection for cattle was feedstuff derived from sheep meat and bonemeal that had been inadequately treated during processing. The incubation period of years, the lack of detectable host immune response, the debilitating and progressive neurological illness, and the pathology localized to the central nervous system are characteristics of the disease, and are is comparable to the characteristics of other TSE diseases such as scrapie, which affects sheep and goats. In addition, the infectious agent is extremely resistant to dessication and disinfectants. Confirmation of disease is by histological examination of brain tissue collected at necropsy; the vacuolation that occurs during the disease will be symmetrical and in the gray matter of the brain stem. Molecular biology techniques, such as Western blots and immunohistochemistry, may also be used to identify the presence of the prion protein. Differentials include many infectious or toxic agents that affect the bovine nervous and musculoskeletal systems, such as rabies, listeriosis, and lead poisoning. Metabolic disorders such as ketosis, milk fever, and grass tetany are also differentials. There is no vaccine or treatment. Prevention focuses on import regulations and not feeding ruminant protein to ruminants; recent USDA regulations prohibit feeding any mammalian proteins to ruminants. ii. Scrapie Etiology. Scrapie is a sporadic, slow, neurodegenerative disease caused by a prion. Scrapie is a reportable disease. It is much more common in sheep than in goats. The disease is similar to transmissible mink encephalopathy, kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy (mad cow disease). Prions are nonantigenic, replicating protein agents. Clinical signs and diagnosis. During early clinical stages, animals are excitable and hard to control. Tremors of head and neck muscles, as well as uncoordinated movements and unusual "bunny-hopping" gaits are observed. In advanced stages of the disease, animals experience severe pruritus and will self-mutilate while rubbing on fences, trees, and other objects. Blindness and abortion may also be seen. Morbidity may reach 50% within a flock. Most animals invariably die within 4–6 weeks; some animals may survive 6 months. In goats, the disease is also fatal. Pruritus is generally less severe but may be localized. A wide range of clinical signs have also been noted in goats, including listlessness, stiffness or restlessness, or behavioral changes such as irritability, hunched posture, twitching, and erect tail and ears. As with sheep, the disease gradually progresses to anorexia and debilitation. Diagnosis can be made by clinical signs and histopathological lesions. A newer diagnostic test in live animals is based on sampling from the third eyelid. Tests for genetic resistance or susceptibility require a tube of EDTA blood and are reasonably priced. Epizootiology and transmission. The Suffolk breed of sheep tends to be especially susceptible. Scrapie has also been reported in several other breeds, including Cheviot, Dorset, Hampshire, Corriedale, Shropshire, Merino, and Rambouillet. It is believed that there is hereditary susceptibility in these breeds. Targhees tend to be resistant. Genomic research indicates there are two chromosomsal sites governing this trait; these sites are referred to codons 171 (Q, R, or H genes can be present) and 136 (A or V genes can be present). Of the five genes, R genes appear to confer immunity to clinical scrapie in Suffolks in the United States. Affected Suffolks in the United States that have been tested have been AA QQ. The disease is also enzootic is many other countries. The disease tends to affect newborns and young animals; however, because the incubation period tends to range from 2 to 5 years, adult animals display signs of the disease. Scrapie is transmitted horizontally by direct or indirect contact; nasal secretions or placentas serve as sources of the infectious agent. Vertical transmission is questioned, and transplacental transmission is considered unlikely. Necropsy findings. At necropsy, no gross lesion is observed. Histopathologically, neuronal vacuolization, astrogliosis, and spongiform degeneration are visualized in the brain stem, the spinal cord, and especially the thalamus. Inflammatory lesions are not seen. Pathogenesis. Replication of the prions probably occurs first in lymphoid tissues throughout the host's body and then progresses to neural tissue. Differential diagnosis. In sheep and goats, depending on the speed of onset, differentials for the pruritus include ectoparasites, pseudorabies, and photosensitization. Prevention and control. If the disease diagnosed in a flock, quarantine and slaughter, followed by strict sanitation, are usually required. The U.S. Department of Agriculture has approved the use of 2% sodium hydroxide as the only disinfectant for sanitation of scrapie-infected premises. Prions are highly resistant to physicochemical means of disinfection. Artificial insemination or embryo transfer has been shown to decrease the spread of scrapie ( Linnabary et al., 1991 ). Treatment. No vaccine or treatment is available. Research complications. As noted, this is a reportable disease. Stringent regulations exist in the United States regarding importation of small ruminants from scrapie-infected countries. i. Bovine spongiform encephalopathy (mad cow disease). Bovine spongiform encephalopathy, a transmissible spongiform encephalopathy (TSE), is not known to occur in the United States, where since 1989 it has been listed as a reportable disease. The profound impact of this disease on the cattle industry in Great Britain during the past two decades is well known. The disease may be caused by a scrapielike (prion) agent. It is believed that the source of infection for cattle was feedstuff derived from sheep meat and bonemeal that had been inadequately treated during processing. The incubation period of years, the lack of detectable host immune response, the debilitating and progressive neurological illness, and the pathology localized to the central nervous system are characteristics of the disease, and are is comparable to the characteristics of other TSE diseases such as scrapie, which affects sheep and goats. In addition, the infectious agent is extremely resistant to dessication and disinfectants. Confirmation of disease is by histological examination of brain tissue collected at necropsy; the vacuolation that occurs during the disease will be symmetrical and in the gray matter of the brain stem. Molecular biology techniques, such as Western blots and immunohistochemistry, may also be used to identify the presence of the prion protein. Differentials include many infectious or toxic agents that affect the bovine nervous and musculoskeletal systems, such as rabies, listeriosis, and lead poisoning. Metabolic disorders such as ketosis, milk fever, and grass tetany are also differentials. There is no vaccine or treatment. Prevention focuses on import regulations and not feeding ruminant protein to ruminants; recent USDA regulations prohibit feeding any mammalian proteins to ruminants. ii. Scrapie Etiology. Scrapie is a sporadic, slow, neurodegenerative disease caused by a prion. Scrapie is a reportable disease. It is much more common in sheep than in goats. The disease is similar to transmissible mink encephalopathy, kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy (mad cow disease). Prions are nonantigenic, replicating protein agents. Clinical signs and diagnosis. During early clinical stages, animals are excitable and hard to control. Tremors of head and neck muscles, as well as uncoordinated movements and unusual "bunny-hopping" gaits are observed. In advanced stages of the disease, animals experience severe pruritus and will self-mutilate while rubbing on fences, trees, and other objects. Blindness and abortion may also be seen. Morbidity may reach 50% within a flock. Most animals invariably die within 4–6 weeks; some animals may survive 6 months. In goats, the disease is also fatal. Pruritus is generally less severe but may be localized. A wide range of clinical signs have also been noted in goats, including listlessness, stiffness or restlessness, or behavioral changes such as irritability, hunched posture, twitching, and erect tail and ears. As with sheep, the disease gradually progresses to anorexia and debilitation. Diagnosis can be made by clinical signs and histopathological lesions. A newer diagnostic test in live animals is based on sampling from the third eyelid. Tests for genetic resistance or susceptibility require a tube of EDTA blood and are reasonably priced. Epizootiology and transmission. The Suffolk breed of sheep tends to be especially susceptible. Scrapie has also been reported in several other breeds, including Cheviot, Dorset, Hampshire, Corriedale, Shropshire, Merino, and Rambouillet. It is believed that there is hereditary susceptibility in these breeds. Targhees tend to be resistant. Genomic research indicates there are two chromosomsal sites governing this trait; these sites are referred to codons 171 (Q, R, or H genes can be present) and 136 (A or V genes can be present). Of the five genes, R genes appear to confer immunity to clinical scrapie in Suffolks in the United States. Affected Suffolks in the United States that have been tested have been AA QQ. The disease is also enzootic is many other countries. The disease tends to affect newborns and young animals; however, because the incubation period tends to range from 2 to 5 years, adult animals display signs of the disease. Scrapie is transmitted horizontally by direct or indirect contact; nasal secretions or placentas serve as sources of the infectious agent. Vertical transmission is questioned, and transplacental transmission is considered unlikely. Necropsy findings. At necropsy, no gross lesion is observed. Histopathologically, neuronal vacuolization, astrogliosis, and spongiform degeneration are visualized in the brain stem, the spinal cord, and especially the thalamus. Inflammatory lesions are not seen. Pathogenesis. Replication of the prions probably occurs first in lymphoid tissues throughout the host's body and then progresses to neural tissue. Differential diagnosis. In sheep and goats, depending on the speed of onset, differentials for the pruritus include ectoparasites, pseudorabies, and photosensitization. Prevention and control. If the disease diagnosed in a flock, quarantine and slaughter, followed by strict sanitation, are usually required. The U.S. Department of Agriculture has approved the use of 2% sodium hydroxide as the only disinfectant for sanitation of scrapie-infected premises. Prions are highly resistant to physicochemical means of disinfection. Artificial insemination or embryo transfer has been shown to decrease the spread of scrapie ( Linnabary et al., 1991 ). Treatment. No vaccine or treatment is available. Research complications. As noted, this is a reportable disease. Stringent regulations exist in the United States regarding importation of small ruminants from scrapie-infected countries. Etiology. Scrapie is a sporadic, slow, neurodegenerative disease caused by a prion. Scrapie is a reportable disease. It is much more common in sheep than in goats. The disease is similar to transmissible mink encephalopathy, kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy (mad cow disease). Prions are nonantigenic, replicating protein agents. Clinical signs and diagnosis. During early clinical stages, animals are excitable and hard to control. Tremors of head and neck muscles, as well as uncoordinated movements and unusual "bunny-hopping" gaits are observed. In advanced stages of the disease, animals experience severe pruritus and will self-mutilate while rubbing on fences, trees, and other objects. Blindness and abortion may also be seen. Morbidity may reach 50% within a flock. Most animals invariably die within 4–6 weeks; some animals may survive 6 months. In goats, the disease is also fatal. Pruritus is generally less severe but may be localized. A wide range of clinical signs have also been noted in goats, including listlessness, stiffness or restlessness, or behavioral changes such as irritability, hunched posture, twitching, and erect tail and ears. As with sheep, the disease gradually progresses to anorexia and debilitation. Diagnosis can be made by clinical signs and histopathological lesions. A newer diagnostic test in live animals is based on sampling from the third eyelid. Tests for genetic resistance or susceptibility require a tube of EDTA blood and are reasonably priced. Epizootiology and transmission. The Suffolk breed of sheep tends to be especially susceptible. Scrapie has also been reported in several other breeds, including Cheviot, Dorset, Hampshire, Corriedale, Shropshire, Merino, and Rambouillet. It is believed that there is hereditary susceptibility in these breeds. Targhees tend to be resistant. Genomic research indicates there are two chromosomsal sites governing this trait; these sites are referred to codons 171 (Q, R, or H genes can be present) and 136 (A or V genes can be present). Of the five genes, R genes appear to confer immunity to clinical scrapie in Suffolks in the United States. Affected Suffolks in the United States that have been tested have been AA QQ. The disease is also enzootic is many other countries. The disease tends to affect newborns and young animals; however, because the incubation period tends to range from 2 to 5 years, adult animals display signs of the disease. Scrapie is transmitted horizontally by direct or indirect contact; nasal secretions or placentas serve as sources of the infectious agent. Vertical transmission is questioned, and transplacental transmission is considered unlikely. Necropsy findings. At necropsy, no gross lesion is observed. Histopathologically, neuronal vacuolization, astrogliosis, and spongiform degeneration are visualized in the brain stem, the spinal cord, and especially the thalamus. Inflammatory lesions are not seen. Pathogenesis. Replication of the prions probably occurs first in lymphoid tissues throughout the host's body and then progresses to neural tissue. Differential diagnosis. In sheep and goats, depending on the speed of onset, differentials for the pruritus include ectoparasites, pseudorabies, and photosensitization. Prevention and control. If the disease diagnosed in a flock, quarantine and slaughter, followed by strict sanitation, are usually required. The U.S. Department of Agriculture has approved the use of 2% sodium hydroxide as the only disinfectant for sanitation of scrapie-infected premises. Prions are highly resistant to physicochemical means of disinfection. Artificial insemination or embryo transfer has been shown to decrease the spread of scrapie ( Linnabary et al., 1991 ). Treatment. No vaccine or treatment is available. Research complications. As noted, this is a reportable disease. Stringent regulations exist in the United States regarding importation of small ruminants from scrapie-infected countries. w. Vesicular Stomatitis Virus Etiology. Vesicular stomatitis (VS) is caused by the vesicular stomatitis virus (VSV), a member of the Rhabdoviridae. Three serotypes are recognized: New Jersey, Indiana, and Isfahan. The New Jersey and Indiana strains cause sporadic disease in cattle in the United States. The disease is rare in sheep. Clinical signs and diagnosis. Adult cattle are most likely to develop VS. Fever and development of vesicles on the oral mucous membranes are the initial clinical signs. Lesions on the teats and interdigital spaces also develop. The vesicles progress quickly to ulcers and erosions. The animal's tongue may be severely involved. Anorexia and salivation are common. Weight loss and decreased milk production are noticeable. Morbidity will be high in an outbreak, but mortality will be low to nonexistent. Diagnostic work should be initiated as soon as possible to distinguish this from foot-and-mouth disease. Diagnosis is based on analysis of fluid, serum, or membranes associated with the vesicles. Virus isolation, enzyme-linked immunosorbent assay (ELISA), competitive ELISA (CELISA), complement fixation, and serum neutralization are used for diagnosis. Epizootiology and transmission. This disease occurs in several other mammalian species, including swine, horses, and wild ruminants. VSV is an enveloped virus and survives well in different environmental conditions, including in soil, extremes of pH, and low temperatures. Outbreaks of VS occur sporadically in the United States, but it is not understood how or in what species the virus survives between these outbreaks. Incidence of disease decreases during colder seasons. Equipment, such as milking machines, contaminated by secretions is a mechanical vector, as are human hands. Transmission may also be from contaminated water and feed. Transmission is also believed to occur by insects (blackflies, sand flies, and Culicoides) that may simply be mechanical vectors. It is believed that carrier animals do not occur in this disease. Necropsy. It is rare for animals to be necropsied as the result of this disease. Typical vesicular lesion histology is seen, with ballooning degeneration and edema. There is no inclusion body formation. Pathogenesis. Lesions often begin within 24 hr after exposure. The virus invades oral epithelium. Injuries or trauma in any area typically affected, such as mouth, teats, or interdigital areas, will increase the likelihood of lesions developing there. Animals will develop a long-term immunity; this immunity can be overwhelmed, however, by a large dose of the virus. Differential diagnosis. Foot-and-mouth disease lesions are identical to VS lesions. Other differentials in cattle include bovine viral diarrhea, malignant catarrhal fever, contagious ecthyma, photosensitization, trauma, and caustic agents. Prevention and control. Quarantine and restrictions on shipping infected animals or animals from the premises housing affected animals are required in an outbreak. Vaccines are available for use in outbreaks and have decreased the severity of lesions. Phenolics, quaternaries, and halogens are effective for inactivating and disinfecting equipment and facilities. Treatment. Affected animals should be segregated from the rest of the herd and provided with separate water and softened feed. These animals should be cared for after unaffected animals. Any feed or water contaminated by these animals should not be used for other animals; contaminated equipment should be disinfected. Topical or systemic antibiotics control secondary bacterial infections. Cases of mastitis secondary to teat lesions must be treated as necessary. Any abrasive materials that could cause further trauma to the animals should be removed. Research complications. Animals developing vesicular lesions must be reported promptly to eliminate the possibility of an outbreak of foot-and-mouth disease. Personal protective equipment, especially gloves, should be worn when handling any animals with vesicular lesions. VSV causes a flulike illness in humans. Etiology. Vesicular stomatitis (VS) is caused by the vesicular stomatitis virus (VSV), a member of the Rhabdoviridae. Three serotypes are recognized: New Jersey, Indiana, and Isfahan. The New Jersey and Indiana strains cause sporadic disease in cattle in the United States. The disease is rare in sheep. Clinical signs and diagnosis. Adult cattle are most likely to develop VS. Fever and development of vesicles on the oral mucous membranes are the initial clinical signs. Lesions on the teats and interdigital spaces also develop. The vesicles progress quickly to ulcers and erosions. The animal's tongue may be severely involved. Anorexia and salivation are common. Weight loss and decreased milk production are noticeable. Morbidity will be high in an outbreak, but mortality will be low to nonexistent. Diagnostic work should be initiated as soon as possible to distinguish this from foot-and-mouth disease. Diagnosis is based on analysis of fluid, serum, or membranes associated with the vesicles. Virus isolation, enzyme-linked immunosorbent assay (ELISA), competitive ELISA (CELISA), complement fixation, and serum neutralization are used for diagnosis. Epizootiology and transmission. This disease occurs in several other mammalian species, including swine, horses, and wild ruminants. VSV is an enveloped virus and survives well in different environmental conditions, including in soil, extremes of pH, and low temperatures. Outbreaks of VS occur sporadically in the United States, but it is not understood how or in what species the virus survives between these outbreaks. Incidence of disease decreases during colder seasons. Equipment, such as milking machines, contaminated by secretions is a mechanical vector, as are human hands. Transmission may also be from contaminated water and feed. Transmission is also believed to occur by insects (blackflies, sand flies, and Culicoides) that may simply be mechanical vectors. It is believed that carrier animals do not occur in this disease. Necropsy. It is rare for animals to be necropsied as the result of this disease. Typical vesicular lesion histology is seen, with ballooning degeneration and edema. There is no inclusion body formation. Pathogenesis. Lesions often begin within 24 hr after exposure. The virus invades oral epithelium. Injuries or trauma in any area typically affected, such as mouth, teats, or interdigital areas, will increase the likelihood of lesions developing there. Animals will develop a long-term immunity; this immunity can be overwhelmed, however, by a large dose of the virus. Differential diagnosis. Foot-and-mouth disease lesions are identical to VS lesions. Other differentials in cattle include bovine viral diarrhea, malignant catarrhal fever, contagious ecthyma, photosensitization, trauma, and caustic agents. Prevention and control. Quarantine and restrictions on shipping infected animals or animals from the premises housing affected animals are required in an outbreak. Vaccines are available for use in outbreaks and have decreased the severity of lesions. Phenolics, quaternaries, and halogens are effective for inactivating and disinfecting equipment and facilities. Treatment. Affected animals should be segregated from the rest of the herd and provided with separate water and softened feed. These animals should be cared for after unaffected animals. Any feed or water contaminated by these animals should not be used for other animals; contaminated equipment should be disinfected. Topical or systemic antibiotics control secondary bacterial infections. Cases of mastitis secondary to teat lesions must be treated as necessary. Any abrasive materials that could cause further trauma to the animals should be removed. Research complications. Animals developing vesicular lesions must be reported promptly to eliminate the possibility of an outbreak of foot-and-mouth disease. Personal protective equipment, especially gloves, should be worn when handling any animals with vesicular lesions. VSV causes a flulike illness in humans. Etiology. Vesicular stomatitis (VS) is caused by the vesicular stomatitis virus (VSV), a member of the Rhabdoviridae. Three serotypes are recognized: New Jersey, Indiana, and Isfahan. The New Jersey and Indiana strains cause sporadic disease in cattle in the United States. The disease is rare in sheep. Clinical signs and diagnosis. Adult cattle are most likely to develop VS. Fever and development of vesicles on the oral mucous membranes are the initial clinical signs. Lesions on the teats and interdigital spaces also develop. The vesicles progress quickly to ulcers and erosions. The animal's tongue may be severely involved. Anorexia and salivation are common. Weight loss and decreased milk production are noticeable. Morbidity will be high in an outbreak, but mortality will be low to nonexistent. Diagnostic work should be initiated as soon as possible to distinguish this from foot-and-mouth disease. Diagnosis is based on analysis of fluid, serum, or membranes associated with the vesicles. Virus isolation, enzyme-linked immunosorbent assay (ELISA), competitive ELISA (CELISA), complement fixation, and serum neutralization are used for diagnosis. Epizootiology and transmission. This disease occurs in several other mammalian species, including swine, horses, and wild ruminants. VSV is an enveloped virus and survives well in different environmental conditions, including in soil, extremes of pH, and low temperatures. Outbreaks of VS occur sporadically in the United States, but it is not understood how or in what species the virus survives between these outbreaks. Incidence of disease decreases during colder seasons. Equipment, such as milking machines, contaminated by secretions is a mechanical vector, as are human hands. Transmission may also be from contaminated water and feed. Transmission is also believed to occur by insects (blackflies, sand flies, and Culicoides) that may simply be mechanical vectors. It is believed that carrier animals do not occur in this disease. Necropsy. It is rare for animals to be necropsied as the result of this disease. Typical vesicular lesion histology is seen, with ballooning degeneration and edema. There is no inclusion body formation. Pathogenesis. Lesions often begin within 24 hr after exposure. The virus invades oral epithelium. Injuries or trauma in any area typically affected, such as mouth, teats, or interdigital areas, will increase the likelihood of lesions developing there. Animals will develop a long-term immunity; this immunity can be overwhelmed, however, by a large dose of the virus. Differential diagnosis. Foot-and-mouth disease lesions are identical to VS lesions. Other differentials in cattle include bovine viral diarrhea, malignant catarrhal fever, contagious ecthyma, photosensitization, trauma, and caustic agents. Prevention and control. Quarantine and restrictions on shipping infected animals or animals from the premises housing affected animals are required in an outbreak. Vaccines are available for use in outbreaks and have decreased the severity of lesions. Phenolics, quaternaries, and halogens are effective for inactivating and disinfecting equipment and facilities. Treatment. Affected animals should be segregated from the rest of the herd and provided with separate water and softened feed. These animals should be cared for after unaffected animals. Any feed or water contaminated by these animals should not be used for other animals; contaminated equipment should be disinfected. Topical or systemic antibiotics control secondary bacterial infections. Cases of mastitis secondary to teat lesions must be treated as necessary. Any abrasive materials that could cause further trauma to the animals should be removed. Research complications. Animals developing vesicular lesions must be reported promptly to eliminate the possibility of an outbreak of foot-and-mouth disease. Personal protective equipment, especially gloves, should be worn when handling any animals with vesicular lesions. VSV causes a flulike illness in humans. x. Viral Diarrhea Diseases i. Ovine. Rotavirus, of the family Reoviridae, induces an acute, transient diarrhea in lambs within the first few weeks of life. Four antigenic groups (A-D) have been identified by differences in capsid antigens VP3 and VP7. Primarily group A, but also groups B and C, have been isolated from sheep. The disease is characterized by yellow, semifluid to watery diarrhea occurring 1–4 days after infection. The disease can progress to dehydration, anorexia and weight loss, acidosis, depression, and occasionally death. The virus is ingested with contaminated feed and water and selectively infects and destroys the enterocytes at the tips of the small intestinal villi. The villi are replaced with immature cells that lack sufficient digestive enzymes; osmotic diarrhea results. Virus may remain in the environment for several months. The disease is diagnosed by virus isolation, electron microscopy of feces, fecal fluorescent antibody, fecal ELISA tests (marketed tests generally detect group A rotavirus), and fecal latex agglutination tests. Rotavirus diarrhea is treated by supportive therapy, including maintaining hydration, electrolyte, and acid-base balance. A rotavirus vaccine is available for cattle; because of cross-species immunity, oral administration of high-quality bovine colostrum from vaccinated cows to infected sheep may be helpful ( "Current Veterinary Therapy," 1993 ). Coronavirus, of the family Coronaviridae, produces a more severe, long-lasting disease when compared with rotavirus. Clinical signs are similar to above, although the incubation period tends to be shorter (20–36 hr), and animals exhibit less anorexia than those with rotavirus. Additionally, mild respiratory disease may be noted ( Janke, 1989 ). Like rotavirus, coronavirus also destroys enterocytes of the villus tips. The virus can be visualized with electron microscopy. Treatment is supportive; close consideration of hydration and acid-base status is essential. Bovine vaccines are available. ii. Caprine. Rotavirus, coronavirus, and adenoviruses affect neonatal goats; however, little has been documented on the pathology and significance of these agents in this age group. It appears that bacteria play a more important role in neonatal kid diarrheal diseases then in neonatal calf diarrheas. iii. Bovine. Rotaviruses, coronaviruses, parvoviruses, and bovine viral diarrhea virus (BVDV) are associated with diarrheal disease in calves. Each pathogen multiplies within and destroys the intestinal epithelial cells, resulting in villous atrophy and clinical signs of diarrhea (soft to watery feces), dehydration, and abdominal pain. These viral infections may be complicated by parasitic infections (e.g., Cryptosporidium, Eimeria) or bacterial infections (e.g., Escherichia coli, Salmonella, Campylobacter). Treatment is aimed at correcting dehydration, electrolyte imbalances, and acidosis; cessation of milk replacers and administration of fluid therapy intravenously and by stomach tube may be necessary, depending on the presence of suckle reflex and the condition of the animals. Diagnosis is by immunoassays available for some viruses, viral culture, exclusion or identification of presence of other pathogens (by culture or fecal exams), and microscopic examination of necropsy specimens. Prevention focuses on calves suckling good-quality colostrum; other recommendations for calf care are in Section II,B,5. Combination vaccine products are available for immunizing dams against rotavirus, coronavirus, and enterotoxigenic E. coli. Additional supportive care for calves includes providing calves with sufficient energy and vitamins until milk intake can resume. Rotaviruses of serogroup A are the most common type in neonatal calves; 4- to 14-day old calves are typically affected, but younger and older animals may also be affected. The small intestine is the site of infection. Antirotavirus antibody is present in colostrum, and onset of rotavirus diarrhea coincides with the decline of this local protection. Transmission is likely from other affected calves and asymptomatic adult carriers. The diarrhea is typically a distinctive yellow. Colitis with tenesmus, mucus, and blood may be seen. This virus may be zoonotic. Coronaviruses are commonly associated with disease in calves during the first month of life, and they infect small- and large-intestinal epithelial cells. The virus infection may extend to mild pneumonia. Transmission is by infected calves and also by asymptomatic adult cattle, including dams excreting virus at the time of parturition. Calves that appear to have recovered continue to shed virus for several weeks. Parvovirus infections are usually associated with neonatal calves. BVDV infections also are seen in neonates and also affect many systems and produce other clinical signs and syndromes that are described in Section III,A,2,e. iv. Winter Dysentery. Winter dysentery is an acute, winter-seasonal, epizootic diarrheal disease of adult cattle, although it has been reported in 4-month-old calves. The etiology has not yet been defined, but a viral pathogen is suspected. Coronavirus-like viral particles have been isolated from cattle feces, either the same as or similar to the coronavirus of calf diarrhea. Outbreaks typically last a few weeks, and first-lactation or younger cattle are affected first, with waves of illness moving through a herd. Individual cows are ill for only a few days. The incubation period is estimated at 2–8 days. The outbreaks of disease are often seen in herds throughout the local area. Clinical signs include explosive diarrhea, anorexia, depression, and decreased production. The diarrhea has a distinctive musty, sweet odor and is light brown and bubbly, but some blood streaks or clots may be mixed in with the feces. Animals will become dehydrated quickly but are thirsty. Respiratory symptoms such as nasolacrimal discharges and coughing may develop. Recovery is generally spontaneous. Mortalities are rare. Diagnosis is based on characteristic patterns of clinical signs, and elimination of diarrheas caused by parasites such as coccidia, bacterial organisms such as Salmonella or Mycobacterium paratuberculosis, and viruses such as BVDV. Pathology is present in the colonic mucosa, and necrosis is present in the crypts. i. Ovine. Rotavirus, of the family Reoviridae, induces an acute, transient diarrhea in lambs within the first few weeks of life. Four antigenic groups (A-D) have been identified by differences in capsid antigens VP3 and VP7. Primarily group A, but also groups B and C, have been isolated from sheep. The disease is characterized by yellow, semifluid to watery diarrhea occurring 1–4 days after infection. The disease can progress to dehydration, anorexia and weight loss, acidosis, depression, and occasionally death. The virus is ingested with contaminated feed and water and selectively infects and destroys the enterocytes at the tips of the small intestinal villi. The villi are replaced with immature cells that lack sufficient digestive enzymes; osmotic diarrhea results. Virus may remain in the environment for several months. The disease is diagnosed by virus isolation, electron microscopy of feces, fecal fluorescent antibody, fecal ELISA tests (marketed tests generally detect group A rotavirus), and fecal latex agglutination tests. Rotavirus diarrhea is treated by supportive therapy, including maintaining hydration, electrolyte, and acid-base balance. A rotavirus vaccine is available for cattle; because of cross-species immunity, oral administration of high-quality bovine colostrum from vaccinated cows to infected sheep may be helpful ( "Current Veterinary Therapy," 1993 ). Coronavirus, of the family Coronaviridae, produces a more severe, long-lasting disease when compared with rotavirus. Clinical signs are similar to above, although the incubation period tends to be shorter (20–36 hr), and animals exhibit less anorexia than those with rotavirus. Additionally, mild respiratory disease may be noted ( Janke, 1989 ). Like rotavirus, coronavirus also destroys enterocytes of the villus tips. The virus can be visualized with electron microscopy. Treatment is supportive; close consideration of hydration and acid-base status is essential. Bovine vaccines are available. ii. Caprine. Rotavirus, coronavirus, and adenoviruses affect neonatal goats; however, little has been documented on the pathology and significance of these agents in this age group. It appears that bacteria play a more important role in neonatal kid diarrheal diseases then in neonatal calf diarrheas. iii. Bovine. Rotaviruses, coronaviruses, parvoviruses, and bovine viral diarrhea virus (BVDV) are associated with diarrheal disease in calves. Each pathogen multiplies within and destroys the intestinal epithelial cells, resulting in villous atrophy and clinical signs of diarrhea (soft to watery feces), dehydration, and abdominal pain. These viral infections may be complicated by parasitic infections (e.g., Cryptosporidium, Eimeria) or bacterial infections (e.g., Escherichia coli, Salmonella, Campylobacter). Treatment is aimed at correcting dehydration, electrolyte imbalances, and acidosis; cessation of milk replacers and administration of fluid therapy intravenously and by stomach tube may be necessary, depending on the presence of suckle reflex and the condition of the animals. Diagnosis is by immunoassays available for some viruses, viral culture, exclusion or identification of presence of other pathogens (by culture or fecal exams), and microscopic examination of necropsy specimens. Prevention focuses on calves suckling good-quality colostrum; other recommendations for calf care are in Section II,B,5. Combination vaccine products are available for immunizing dams against rotavirus, coronavirus, and enterotoxigenic E. coli. Additional supportive care for calves includes providing calves with sufficient energy and vitamins until milk intake can resume. Rotaviruses of serogroup A are the most common type in neonatal calves; 4- to 14-day old calves are typically affected, but younger and older animals may also be affected. The small intestine is the site of infection. Antirotavirus antibody is present in colostrum, and onset of rotavirus diarrhea coincides with the decline of this local protection. Transmission is likely from other affected calves and asymptomatic adult carriers. The diarrhea is typically a distinctive yellow. Colitis with tenesmus, mucus, and blood may be seen. This virus may be zoonotic. Coronaviruses are commonly associated with disease in calves during the first month of life, and they infect small- and large-intestinal epithelial cells. The virus infection may extend to mild pneumonia. Transmission is by infected calves and also by asymptomatic adult cattle, including dams excreting virus at the time of parturition. Calves that appear to have recovered continue to shed virus for several weeks. Parvovirus infections are usually associated with neonatal calves. BVDV infections also are seen in neonates and also affect many systems and produce other clinical signs and syndromes that are described in Section III,A,2,e. iv. Winter Dysentery. Winter dysentery is an acute, winter-seasonal, epizootic diarrheal disease of adult cattle, although it has been reported in 4-month-old calves. The etiology has not yet been defined, but a viral pathogen is suspected. Coronavirus-like viral particles have been isolated from cattle feces, either the same as or similar to the coronavirus of calf diarrhea. Outbreaks typically last a few weeks, and first-lactation or younger cattle are affected first, with waves of illness moving through a herd. Individual cows are ill for only a few days. The incubation period is estimated at 2–8 days. The outbreaks of disease are often seen in herds throughout the local area. Clinical signs include explosive diarrhea, anorexia, depression, and decreased production. The diarrhea has a distinctive musty, sweet odor and is light brown and bubbly, but some blood streaks or clots may be mixed in with the feces. Animals will become dehydrated quickly but are thirsty. Respiratory symptoms such as nasolacrimal discharges and coughing may develop. Recovery is generally spontaneous. Mortalities are rare. Diagnosis is based on characteristic patterns of clinical signs, and elimination of diarrheas caused by parasites such as coccidia, bacterial organisms such as Salmonella or Mycobacterium paratuberculosis, and viruses such as BVDV. Pathology is present in the colonic mucosa, and necrosis is present in the crypts. i. Ovine. Rotavirus, of the family Reoviridae, induces an acute, transient diarrhea in lambs within the first few weeks of life. Four antigenic groups (A-D) have been identified by differences in capsid antigens VP3 and VP7. Primarily group A, but also groups B and C, have been isolated from sheep. The disease is characterized by yellow, semifluid to watery diarrhea occurring 1–4 days after infection. The disease can progress to dehydration, anorexia and weight loss, acidosis, depression, and occasionally death. The virus is ingested with contaminated feed and water and selectively infects and destroys the enterocytes at the tips of the small intestinal villi. The villi are replaced with immature cells that lack sufficient digestive enzymes; osmotic diarrhea results. Virus may remain in the environment for several months. The disease is diagnosed by virus isolation, electron microscopy of feces, fecal fluorescent antibody, fecal ELISA tests (marketed tests generally detect group A rotavirus), and fecal latex agglutination tests. Rotavirus diarrhea is treated by supportive therapy, including maintaining hydration, electrolyte, and acid-base balance. A rotavirus vaccine is available for cattle; because of cross-species immunity, oral administration of high-quality bovine colostrum from vaccinated cows to infected sheep may be helpful ( "Current Veterinary Therapy," 1993 ). Coronavirus, of the family Coronaviridae, produces a more severe, long-lasting disease when compared with rotavirus. Clinical signs are similar to above, although the incubation period tends to be shorter (20–36 hr), and animals exhibit less anorexia than those with rotavirus. Additionally, mild respiratory disease may be noted ( Janke, 1989 ). Like rotavirus, coronavirus also destroys enterocytes of the villus tips. The virus can be visualized with electron microscopy. Treatment is supportive; close consideration of hydration and acid-base status is essential. Bovine vaccines are available. ii. Caprine. Rotavirus, coronavirus, and adenoviruses affect neonatal goats; however, little has been documented on the pathology and significance of these agents in this age group. It appears that bacteria play a more important role in neonatal kid diarrheal diseases then in neonatal calf diarrheas. iii. Bovine. Rotaviruses, coronaviruses, parvoviruses, and bovine viral diarrhea virus (BVDV) are associated with diarrheal disease in calves. Each pathogen multiplies within and destroys the intestinal epithelial cells, resulting in villous atrophy and clinical signs of diarrhea (soft to watery feces), dehydration, and abdominal pain. These viral infections may be complicated by parasitic infections (e.g., Cryptosporidium, Eimeria) or bacterial infections (e.g., Escherichia coli, Salmonella, Campylobacter). Treatment is aimed at correcting dehydration, electrolyte imbalances, and acidosis; cessation of milk replacers and administration of fluid therapy intravenously and by stomach tube may be necessary, depending on the presence of suckle reflex and the condition of the animals. Diagnosis is by immunoassays available for some viruses, viral culture, exclusion or identification of presence of other pathogens (by culture or fecal exams), and microscopic examination of necropsy specimens. Prevention focuses on calves suckling good-quality colostrum; other recommendations for calf care are in Section II,B,5. Combination vaccine products are available for immunizing dams against rotavirus, coronavirus, and enterotoxigenic E. coli. Additional supportive care for calves includes providing calves with sufficient energy and vitamins until milk intake can resume. Rotaviruses of serogroup A are the most common type in neonatal calves; 4- to 14-day old calves are typically affected, but younger and older animals may also be affected. The small intestine is the site of infection. Antirotavirus antibody is present in colostrum, and onset of rotavirus diarrhea coincides with the decline of this local protection. Transmission is likely from other affected calves and asymptomatic adult carriers. The diarrhea is typically a distinctive yellow. Colitis with tenesmus, mucus, and blood may be seen. This virus may be zoonotic. Coronaviruses are commonly associated with disease in calves during the first month of life, and they infect small- and large-intestinal epithelial cells. The virus infection may extend to mild pneumonia. Transmission is by infected calves and also by asymptomatic adult cattle, including dams excreting virus at the time of parturition. Calves that appear to have recovered continue to shed virus for several weeks. Parvovirus infections are usually associated with neonatal calves. BVDV infections also are seen in neonates and also affect many systems and produce other clinical signs and syndromes that are described in Section III,A,2,e. iv. Winter Dysentery. Winter dysentery is an acute, winter-seasonal, epizootic diarrheal disease of adult cattle, although it has been reported in 4-month-old calves. The etiology has not yet been defined, but a viral pathogen is suspected. Coronavirus-like viral particles have been isolated from cattle feces, either the same as or similar to the coronavirus of calf diarrhea. Outbreaks typically last a few weeks, and first-lactation or younger cattle are affected first, with waves of illness moving through a herd. Individual cows are ill for only a few days. The incubation period is estimated at 2–8 days. The outbreaks of disease are often seen in herds throughout the local area. Clinical signs include explosive diarrhea, anorexia, depression, and decreased production. The diarrhea has a distinctive musty, sweet odor and is light brown and bubbly, but some blood streaks or clots may be mixed in with the feces. Animals will become dehydrated quickly but are thirsty. Respiratory symptoms such as nasolacrimal discharges and coughing may develop. Recovery is generally spontaneous. Mortalities are rare. Diagnosis is based on characteristic patterns of clinical signs, and elimination of diarrheas caused by parasites such as coccidia, bacterial organisms such as Salmonella or Mycobacterium paratuberculosis, and viruses such as BVDV. Pathology is present in the colonic mucosa, and necrosis is present in the crypts. 3. Chlamydial Diseases a. Enzootic Abortion of Ewes (Chlamydial Abortion) Etiology. Chlamydia psittaci is a nonmotile, obligate, intracytoplasmic, gram-negative bacterium. Clinical signs. Enzootic abortion in sheep and goats is a contagious disease characterized by hyperthermia and late abortion or by birth of stillborn or weak lambs or kids ( Rodolakis et al., 1998 ). The only presenting clinical sign may be serosanguineous vulvar discharges. Other animals may present with arthritis or pneumonia. Infection of animals prior to about 120 days of gestation results in abortion, stillbirths, or birth of weak lambs. Infection after 120 days results in potentially normal births, but the dams or offspring may be latently infected. Latently infected animals that were infected during their dry period may abort during the next pregnancy. Ewes or does generally only abort once, and thus recovered animals will be immune to future infections. Epizootiology and transmission. Chlamydia possess group and specific antigens associated with the cell surface. The group antigen is common among all Chlamydia; the specific antigen is common to related subgroups. Two subgroups are recognized, one that causes EAE and one that causes polyarthritis and conjunctivitis. The disease is transmitted by direct contact with infectious secretions such as placental, fetal, and uterine fluids or by indirect contact with contaminated feed and water. Necropsy. Placental lesions include intercotyledonary plaques and necrosis and cotyledonary hemorrhages. Histopathological evidence of leukocytic infiltration, edema, and necrosis is found throughout the placentome. Fetal lesions include giant-cell accumulation in mesenteric lymph nodes and lymphohistiocytic proliferations around the blood vessels within the liver. Diagnosis is based on clinical signs and laboratory (serological or histopathological) identification of the organism. Impression smears in placental tissues stained with Giemsa, Gimenez, or modified Ziehl-Neelsen can provide preliminary indications of the causative agent. Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) methods also aid in diagnosis. Differential diagnosis. Q fever will be the major differential for late-term abortion and necrotizing placentitis. Campylobacter and Toxoplasma should also be considered for late-term abortion. Treatment. Animals may respond to treatment with oxytetra cycline. Abortions are prevented through administration of a commercial vaccine, but the vaccine will not eliminate infections. This is a sheep vaccine and should be administered before breeding and annually to at least the young females entering the breeding herd or flock. Research complications. In addition to losses or compromise of research animals, pregnant women should not handle aborted tissues. b. Chlamydial Polyarthritis of Sheep Etiology. Chlamydia psittaci is a nonmotile, obligate intracellular, gram-negative bacterium. Chlamydial polyarthritis is an acute, contagious disease characterized by fever, lameness ( Bulgin, 1986 ), and conjunctivitis (see Section III,A,3,c) in growing and nursing lambs. Clinical signs. Clinically, animals will appear lame on one or all legs and in major joints, including the scapulohumeral, humeroradioulnar, coxofemoral, femorotibial, and tibiotarsal joints. Lambs may be anorexic and febrile. Animals frequently also exhibit concurrent conjunctivitis. The disease usually resolves in approximately 4 weeks. Joint inflammation usually resolves without causing chronic articular changes. Epizootiology and transmission. The disease is transmitted to susceptible animals by direct contact as well as by contaminated feed and water. The organism penetrates the gastrointestinal tract and migrates to joints and synovial membranes as well as to the conjunctiva. The organism causes acute inflammation and associated fibrinopurulent exudates. Necropsy findings. Lesions are found in joints, tendon sheaths, conjunctiva, and lungs. Pathological sites will be edematous and hyperemic, with fibrinous exudates but without articular changes. Lesions will be infiltrated with mononuclear cells. Lung lesions include atelectasis and alveolar inspissation. Diagnosis is based on clinical signs. Synovial taps and subsequent smears may allow the identification of chlamydial inclusion bodies. Treatment. Animals respond to treatment with parenteral oxytetracycline. c. Chlamydial Conjunctivitis (Infectious Keratoconjunctivitis, Pinkeye) Etiology. Chlamydia psittaci, a nonmotile, obligate intracellular, gram-negative bacterium, is the most common cause of infectious keratoconjunctivitis in sheep. Chlamydia and Mycoplasma are considered to be the most common causes of this disease in goats. Chlamydial conjunctivitis is not a disease of cattle. Clinical signs. Infectious keratoconjunctivitis is an acute, contagious disease characterized in earlier stages by conjunctival hyperemia, epiphora, and edema and in later stages by, corneal edema, ulceration, and opacity. Perforation may result from the ulceration. Animals will be photophobic. In less severe cases, corneal healing associated with fibrosis and neovascularization occurs in 3–4 days. Lymphoid tissues associated with the conjunctiva and nictitating membrane may enlarge and prolapse the eyelids. Morbidity may reach 80–90%. Bilateral and symmetrical infections characterize most outbreaks. Relapses may occur. Other concurrent systemic infections may be seen, such as polyarthritis or abortion in sheep and polyarthritis, mastitis, and uterine infections in goats. Epizootiology and transmission. Direct contact, and mechanical vectors such as flies easily spread the organism. Necropsy. If the chlamydial or mycoplasmal agents are suspected, diagnostic laboratories should be contacted for recommendations regarding sampling. Conjunctival smears are also useful. Pathogenesis. The pathogen penetrates the conjunctival epithelium and replicates in the cytoplasm by forming initial and elementary bodies. The infection moves from cell to cell and causes an acute inflammation and resultant purulent exudate. The chlamydial organism may penetrate the bloodstream and migrate to the opposite eye or joints, leading to arthritis. Diagnosis is suggested by the clinical signs. Cytoplasmic inclusions observed on conjunctival scrapings and immunofluorescent techniques help confirm the diagnosis. Differential diagnosis. Nonchlamydial keratoconjunctivitis also occurs in sheep and goats. The primary agents involved include Mycoplasma conjunctiva, M. agalactiae in goats, and Branhamella (Neisseria) ovis. A less common differential for sheep and cattle is Listeria monocytogenes. Other differentials include eye worms, trauma, and foreign bodies such as windblown materials (pollen, dust) and poor-quality hay; these latter irritants and stress may predispose the animals' eyes to the infectious agents. Prevention and control. Source of mechanical irritation should be minimized whenever possible. Quarantine of new animals and treatment, if necessary, before introduction into the flock or herd are important measures. Shade should be provided for all animals. Treatment. The infections can be self-limiting in 2–3 weeks without treatment. Treatment consists of topical application of tetracycline ophthalmic ointments. Systemic or oral oxytetracycline treatments have been used with the topical treatment. Atropine may be added to the treatment regimen when uveitis is present. Shade should be provided. a. Enzootic Abortion of Ewes (Chlamydial Abortion) Etiology. Chlamydia psittaci is a nonmotile, obligate, intracytoplasmic, gram-negative bacterium. Clinical signs. Enzootic abortion in sheep and goats is a contagious disease characterized by hyperthermia and late abortion or by birth of stillborn or weak lambs or kids ( Rodolakis et al., 1998 ). The only presenting clinical sign may be serosanguineous vulvar discharges. Other animals may present with arthritis or pneumonia. Infection of animals prior to about 120 days of gestation results in abortion, stillbirths, or birth of weak lambs. Infection after 120 days results in potentially normal births, but the dams or offspring may be latently infected. Latently infected animals that were infected during their dry period may abort during the next pregnancy. Ewes or does generally only abort once, and thus recovered animals will be immune to future infections. Epizootiology and transmission. Chlamydia possess group and specific antigens associated with the cell surface. The group antigen is common among all Chlamydia; the specific antigen is common to related subgroups. Two subgroups are recognized, one that causes EAE and one that causes polyarthritis and conjunctivitis. The disease is transmitted by direct contact with infectious secretions such as placental, fetal, and uterine fluids or by indirect contact with contaminated feed and water. Necropsy. Placental lesions include intercotyledonary plaques and necrosis and cotyledonary hemorrhages. Histopathological evidence of leukocytic infiltration, edema, and necrosis is found throughout the placentome. Fetal lesions include giant-cell accumulation in mesenteric lymph nodes and lymphohistiocytic proliferations around the blood vessels within the liver. Diagnosis is based on clinical signs and laboratory (serological or histopathological) identification of the organism. Impression smears in placental tissues stained with Giemsa, Gimenez, or modified Ziehl-Neelsen can provide preliminary indications of the causative agent. Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) methods also aid in diagnosis. Differential diagnosis. Q fever will be the major differential for late-term abortion and necrotizing placentitis. Campylobacter and Toxoplasma should also be considered for late-term abortion. Treatment. Animals may respond to treatment with oxytetra cycline. Abortions are prevented through administration of a commercial vaccine, but the vaccine will not eliminate infections. This is a sheep vaccine and should be administered before breeding and annually to at least the young females entering the breeding herd or flock. Research complications. In addition to losses or compromise of research animals, pregnant women should not handle aborted tissues. Etiology. Chlamydia psittaci is a nonmotile, obligate, intracytoplasmic, gram-negative bacterium. Clinical signs. Enzootic abortion in sheep and goats is a contagious disease characterized by hyperthermia and late abortion or by birth of stillborn or weak lambs or kids ( Rodolakis et al., 1998 ). The only presenting clinical sign may be serosanguineous vulvar discharges. Other animals may present with arthritis or pneumonia. Infection of animals prior to about 120 days of gestation results in abortion, stillbirths, or birth of weak lambs. Infection after 120 days results in potentially normal births, but the dams or offspring may be latently infected. Latently infected animals that were infected during their dry period may abort during the next pregnancy. Ewes or does generally only abort once, and thus recovered animals will be immune to future infections. Epizootiology and transmission. Chlamydia possess group and specific antigens associated with the cell surface. The group antigen is common among all Chlamydia; the specific antigen is common to related subgroups. Two subgroups are recognized, one that causes EAE and one that causes polyarthritis and conjunctivitis. The disease is transmitted by direct contact with infectious secretions such as placental, fetal, and uterine fluids or by indirect contact with contaminated feed and water. Necropsy. Placental lesions include intercotyledonary plaques and necrosis and cotyledonary hemorrhages. Histopathological evidence of leukocytic infiltration, edema, and necrosis is found throughout the placentome. Fetal lesions include giant-cell accumulation in mesenteric lymph nodes and lymphohistiocytic proliferations around the blood vessels within the liver. Diagnosis is based on clinical signs and laboratory (serological or histopathological) identification of the organism. Impression smears in placental tissues stained with Giemsa, Gimenez, or modified Ziehl-Neelsen can provide preliminary indications of the causative agent. Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) methods also aid in diagnosis. Differential diagnosis. Q fever will be the major differential for late-term abortion and necrotizing placentitis. Campylobacter and Toxoplasma should also be considered for late-term abortion. Treatment. Animals may respond to treatment with oxytetra cycline. Abortions are prevented through administration of a commercial vaccine, but the vaccine will not eliminate infections. This is a sheep vaccine and should be administered before breeding and annually to at least the young females entering the breeding herd or flock. Research complications. In addition to losses or compromise of research animals, pregnant women should not handle aborted tissues. Etiology. Chlamydia psittaci is a nonmotile, obligate, intracytoplasmic, gram-negative bacterium. Clinical signs. Enzootic abortion in sheep and goats is a contagious disease characterized by hyperthermia and late abortion or by birth of stillborn or weak lambs or kids ( Rodolakis et al., 1998 ). The only presenting clinical sign may be serosanguineous vulvar discharges. Other animals may present with arthritis or pneumonia. Infection of animals prior to about 120 days of gestation results in abortion, stillbirths, or birth of weak lambs. Infection after 120 days results in potentially normal births, but the dams or offspring may be latently infected. Latently infected animals that were infected during their dry period may abort during the next pregnancy. Ewes or does generally only abort once, and thus recovered animals will be immune to future infections. Epizootiology and transmission. Chlamydia possess group and specific antigens associated with the cell surface. The group antigen is common among all Chlamydia; the specific antigen is common to related subgroups. Two subgroups are recognized, one that causes EAE and one that causes polyarthritis and conjunctivitis. The disease is transmitted by direct contact with infectious secretions such as placental, fetal, and uterine fluids or by indirect contact with contaminated feed and water. Necropsy. Placental lesions include intercotyledonary plaques and necrosis and cotyledonary hemorrhages. Histopathological evidence of leukocytic infiltration, edema, and necrosis is found throughout the placentome. Fetal lesions include giant-cell accumulation in mesenteric lymph nodes and lymphohistiocytic proliferations around the blood vessels within the liver. Diagnosis is based on clinical signs and laboratory (serological or histopathological) identification of the organism. Impression smears in placental tissues stained with Giemsa, Gimenez, or modified Ziehl-Neelsen can provide preliminary indications of the causative agent. Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) methods also aid in diagnosis. Differential diagnosis. Q fever will be the major differential for late-term abortion and necrotizing placentitis. Campylobacter and Toxoplasma should also be considered for late-term abortion. Treatment. Animals may respond to treatment with oxytetra cycline. Abortions are prevented through administration of a commercial vaccine, but the vaccine will not eliminate infections. This is a sheep vaccine and should be administered before breeding and annually to at least the young females entering the breeding herd or flock. Research complications. In addition to losses or compromise of research animals, pregnant women should not handle aborted tissues. b. Chlamydial Polyarthritis of Sheep Etiology. Chlamydia psittaci is a nonmotile, obligate intracellular, gram-negative bacterium. Chlamydial polyarthritis is an acute, contagious disease characterized by fever, lameness ( Bulgin, 1986 ), and conjunctivitis (see Section III,A,3,c) in growing and nursing lambs. Clinical signs. Clinically, animals will appear lame on one or all legs and in major joints, including the scapulohumeral, humeroradioulnar, coxofemoral, femorotibial, and tibiotarsal joints. Lambs may be anorexic and febrile. Animals frequently also exhibit concurrent conjunctivitis. The disease usually resolves in approximately 4 weeks. Joint inflammation usually resolves without causing chronic articular changes. Epizootiology and transmission. The disease is transmitted to susceptible animals by direct contact as well as by contaminated feed and water. The organism penetrates the gastrointestinal tract and migrates to joints and synovial membranes as well as to the conjunctiva. The organism causes acute inflammation and associated fibrinopurulent exudates. Necropsy findings. Lesions are found in joints, tendon sheaths, conjunctiva, and lungs. Pathological sites will be edematous and hyperemic, with fibrinous exudates but without articular changes. Lesions will be infiltrated with mononuclear cells. Lung lesions include atelectasis and alveolar inspissation. Diagnosis is based on clinical signs. Synovial taps and subsequent smears may allow the identification of chlamydial inclusion bodies. Treatment. Animals respond to treatment with parenteral oxytetracycline. Etiology. Chlamydia psittaci is a nonmotile, obligate intracellular, gram-negative bacterium. Chlamydial polyarthritis is an acute, contagious disease characterized by fever, lameness ( Bulgin, 1986 ), and conjunctivitis (see Section III,A,3,c) in growing and nursing lambs. Clinical signs. Clinically, animals will appear lame on one or all legs and in major joints, including the scapulohumeral, humeroradioulnar, coxofemoral, femorotibial, and tibiotarsal joints. Lambs may be anorexic and febrile. Animals frequently also exhibit concurrent conjunctivitis. The disease usually resolves in approximately 4 weeks. Joint inflammation usually resolves without causing chronic articular changes. Epizootiology and transmission. The disease is transmitted to susceptible animals by direct contact as well as by contaminated feed and water. The organism penetrates the gastrointestinal tract and migrates to joints and synovial membranes as well as to the conjunctiva. The organism causes acute inflammation and associated fibrinopurulent exudates. Necropsy findings. Lesions are found in joints, tendon sheaths, conjunctiva, and lungs. Pathological sites will be edematous and hyperemic, with fibrinous exudates but without articular changes. Lesions will be infiltrated with mononuclear cells. Lung lesions include atelectasis and alveolar inspissation. Diagnosis is based on clinical signs. Synovial taps and subsequent smears may allow the identification of chlamydial inclusion bodies. Treatment. Animals respond to treatment with parenteral oxytetracycline. Etiology. Chlamydia psittaci is a nonmotile, obligate intracellular, gram-negative bacterium. Chlamydial polyarthritis is an acute, contagious disease characterized by fever, lameness ( Bulgin, 1986 ), and conjunctivitis (see Section III,A,3,c) in growing and nursing lambs. Clinical signs. Clinically, animals will appear lame on one or all legs and in major joints, including the scapulohumeral, humeroradioulnar, coxofemoral, femorotibial, and tibiotarsal joints. Lambs may be anorexic and febrile. Animals frequently also exhibit concurrent conjunctivitis. The disease usually resolves in approximately 4 weeks. Joint inflammation usually resolves without causing chronic articular changes. Epizootiology and transmission. The disease is transmitted to susceptible animals by direct contact as well as by contaminated feed and water. The organism penetrates the gastrointestinal tract and migrates to joints and synovial membranes as well as to the conjunctiva. The organism causes acute inflammation and associated fibrinopurulent exudates. Necropsy findings. Lesions are found in joints, tendon sheaths, conjunctiva, and lungs. Pathological sites will be edematous and hyperemic, with fibrinous exudates but without articular changes. Lesions will be infiltrated with mononuclear cells. Lung lesions include atelectasis and alveolar inspissation. Diagnosis is based on clinical signs. Synovial taps and subsequent smears may allow the identification of chlamydial inclusion bodies. Treatment. Animals respond to treatment with parenteral oxytetracycline. c. Chlamydial Conjunctivitis (Infectious Keratoconjunctivitis, Pinkeye) Etiology. Chlamydia psittaci, a nonmotile, obligate intracellular, gram-negative bacterium, is the most common cause of infectious keratoconjunctivitis in sheep. Chlamydia and Mycoplasma are considered to be the most common causes of this disease in goats. Chlamydial conjunctivitis is not a disease of cattle. Clinical signs. Infectious keratoconjunctivitis is an acute, contagious disease characterized in earlier stages by conjunctival hyperemia, epiphora, and edema and in later stages by, corneal edema, ulceration, and opacity. Perforation may result from the ulceration. Animals will be photophobic. In less severe cases, corneal healing associated with fibrosis and neovascularization occurs in 3–4 days. Lymphoid tissues associated with the conjunctiva and nictitating membrane may enlarge and prolapse the eyelids. Morbidity may reach 80–90%. Bilateral and symmetrical infections characterize most outbreaks. Relapses may occur. Other concurrent systemic infections may be seen, such as polyarthritis or abortion in sheep and polyarthritis, mastitis, and uterine infections in goats. Epizootiology and transmission. Direct contact, and mechanical vectors such as flies easily spread the organism. Necropsy. If the chlamydial or mycoplasmal agents are suspected, diagnostic laboratories should be contacted for recommendations regarding sampling. Conjunctival smears are also useful. Pathogenesis. The pathogen penetrates the conjunctival epithelium and replicates in the cytoplasm by forming initial and elementary bodies. The infection moves from cell to cell and causes an acute inflammation and resultant purulent exudate. The chlamydial organism may penetrate the bloodstream and migrate to the opposite eye or joints, leading to arthritis. Diagnosis is suggested by the clinical signs. Cytoplasmic inclusions observed on conjunctival scrapings and immunofluorescent techniques help confirm the diagnosis. Differential diagnosis. Nonchlamydial keratoconjunctivitis also occurs in sheep and goats. The primary agents involved include Mycoplasma conjunctiva, M. agalactiae in goats, and Branhamella (Neisseria) ovis. A less common differential for sheep and cattle is Listeria monocytogenes. Other differentials include eye worms, trauma, and foreign bodies such as windblown materials (pollen, dust) and poor-quality hay; these latter irritants and stress may predispose the animals' eyes to the infectious agents. Prevention and control. Source of mechanical irritation should be minimized whenever possible. Quarantine of new animals and treatment, if necessary, before introduction into the flock or herd are important measures. Shade should be provided for all animals. Treatment. The infections can be self-limiting in 2–3 weeks without treatment. Treatment consists of topical application of tetracycline ophthalmic ointments. Systemic or oral oxytetracycline treatments have been used with the topical treatment. Atropine may be added to the treatment regimen when uveitis is present. Shade should be provided. Etiology. Chlamydia psittaci, a nonmotile, obligate intracellular, gram-negative bacterium, is the most common cause of infectious keratoconjunctivitis in sheep. Chlamydia and Mycoplasma are considered to be the most common causes of this disease in goats. Chlamydial conjunctivitis is not a disease of cattle. Clinical signs. Infectious keratoconjunctivitis is an acute, contagious disease characterized in earlier stages by conjunctival hyperemia, epiphora, and edema and in later stages by, corneal edema, ulceration, and opacity. Perforation may result from the ulceration. Animals will be photophobic. In less severe cases, corneal healing associated with fibrosis and neovascularization occurs in 3–4 days. Lymphoid tissues associated with the conjunctiva and nictitating membrane may enlarge and prolapse the eyelids. Morbidity may reach 80–90%. Bilateral and symmetrical infections characterize most outbreaks. Relapses may occur. Other concurrent systemic infections may be seen, such as polyarthritis or abortion in sheep and polyarthritis, mastitis, and uterine infections in goats. Epizootiology and transmission. Direct contact, and mechanical vectors such as flies easily spread the organism. Necropsy. If the chlamydial or mycoplasmal agents are suspected, diagnostic laboratories should be contacted for recommendations regarding sampling. Conjunctival smears are also useful. Pathogenesis. The pathogen penetrates the conjunctival epithelium and replicates in the cytoplasm by forming initial and elementary bodies. The infection moves from cell to cell and causes an acute inflammation and resultant purulent exudate. The chlamydial organism may penetrate the bloodstream and migrate to the opposite eye or joints, leading to arthritis. Diagnosis is suggested by the clinical signs. Cytoplasmic inclusions observed on conjunctival scrapings and immunofluorescent techniques help confirm the diagnosis. Differential diagnosis. Nonchlamydial keratoconjunctivitis also occurs in sheep and goats. The primary agents involved include Mycoplasma conjunctiva, M. agalactiae in goats, and Branhamella (Neisseria) ovis. A less common differential for sheep and cattle is Listeria monocytogenes. Other differentials include eye worms, trauma, and foreign bodies such as windblown materials (pollen, dust) and poor-quality hay; these latter irritants and stress may predispose the animals' eyes to the infectious agents. Prevention and control. Source of mechanical irritation should be minimized whenever possible. Quarantine of new animals and treatment, if necessary, before introduction into the flock or herd are important measures. Shade should be provided for all animals. Treatment. The infections can be self-limiting in 2–3 weeks without treatment. Treatment consists of topical application of tetracycline ophthalmic ointments. Systemic or oral oxytetracycline treatments have been used with the topical treatment. Atropine may be added to the treatment regimen when uveitis is present. Shade should be provided. Etiology. Chlamydia psittaci, a nonmotile, obligate intracellular, gram-negative bacterium, is the most common cause of infectious keratoconjunctivitis in sheep. Chlamydia and Mycoplasma are considered to be the most common causes of this disease in goats. Chlamydial conjunctivitis is not a disease of cattle. Clinical signs. Infectious keratoconjunctivitis is an acute, contagious disease characterized in earlier stages by conjunctival hyperemia, epiphora, and edema and in later stages by, corneal edema, ulceration, and opacity. Perforation may result from the ulceration. Animals will be photophobic. In less severe cases, corneal healing associated with fibrosis and neovascularization occurs in 3–4 days. Lymphoid tissues associated with the conjunctiva and nictitating membrane may enlarge and prolapse the eyelids. Morbidity may reach 80–90%. Bilateral and symmetrical infections characterize most outbreaks. Relapses may occur. Other concurrent systemic infections may be seen, such as polyarthritis or abortion in sheep and polyarthritis, mastitis, and uterine infections in goats. Epizootiology and transmission. Direct contact, and mechanical vectors such as flies easily spread the organism. Necropsy. If the chlamydial or mycoplasmal agents are suspected, diagnostic laboratories should be contacted for recommendations regarding sampling. Conjunctival smears are also useful. Pathogenesis. The pathogen penetrates the conjunctival epithelium and replicates in the cytoplasm by forming initial and elementary bodies. The infection moves from cell to cell and causes an acute inflammation and resultant purulent exudate. The chlamydial organism may penetrate the bloodstream and migrate to the opposite eye or joints, leading to arthritis. Diagnosis is suggested by the clinical signs. Cytoplasmic inclusions observed on conjunctival scrapings and immunofluorescent techniques help confirm the diagnosis. Differential diagnosis. Nonchlamydial keratoconjunctivitis also occurs in sheep and goats. The primary agents involved include Mycoplasma conjunctiva, M. agalactiae in goats, and Branhamella (Neisseria) ovis. A less common differential for sheep and cattle is Listeria monocytogenes. Other differentials include eye worms, trauma, and foreign bodies such as windblown materials (pollen, dust) and poor-quality hay; these latter irritants and stress may predispose the animals' eyes to the infectious agents. Prevention and control. Source of mechanical irritation should be minimized whenever possible. Quarantine of new animals and treatment, if necessary, before introduction into the flock or herd are important measures. Shade should be provided for all animals. Treatment. The infections can be self-limiting in 2–3 weeks without treatment. Treatment consists of topical application of tetracycline ophthalmic ointments. Systemic or oral oxytetracycline treatments have been used with the topical treatment. Atropine may be added to the treatment regimen when uveitis is present. Shade should be provided. 4. Parasitic Diseases a. Protozoa i. Anaplasmosis Etiology. Anaplasmosis is an infectious, hemolytic, noncontagious, transmissible disease of cattle caused by the protozoan Anaplasma marginale. Anaplasma is a member of the Anaplas-matacae family within the order Rickettsiales. In sheep and goats, the disease is caused by A. ovis and is an uncommon cause of hemolytic disease. Anaplasmosis has not been reported in goats in the United States. Some controversy exists regarding the classification. Most recently it is classified as a protozoal disease because of similarities to babesiosis. It has also been classified as a rickettsial pathogen. This summary addresses the disease in cattle with limited reference to A. ovis infections, but there are many similarities to the disease in cattle. Clinical signs and diagnosis. Acute anemia is the predominant sign in anaplasmosis, and fever coincides with parasitemia. Weakness, pallor, lethargy, dehydration, and anorexia are the result of the anemia. Four disease stages—incubation, developmental, convalescent, and carrier—are recognized. The incubation stage may be long, 3–8 weeks, and is characterized by a rise in body temperature as the infection moves to the next stage. Most clinical signs occur during the 4- to 9-day developmental stage, with hemolytic anemia being common. Death is most likely to occur at this stage or at the beginning of the convalescent stage. Death may also occur from anoxia, because of the animal's inability to handle any exertion or stress, especially if treatment is initiated when severe anemia exists. Reticulocytosis characterizes the convalescent stage, which may continue for many weeks. Morbidity is high, and mortality is low. The carrier stage is defined as the time in the convalescent stage when the animal host becomes a reservoir of the disease, and Anaplasma organisms and any parasitemia are not discernible. Common serologic tests are the complement fixation test and the rapid card test. These become positive after the incubation phase and do not distinguish between the later three stages of disease. Definitive diagnosis is made by clinical and necropsy findings. Staining of thin blood smears with Wright's or Giemsa stain allows detection of basophilic, spherical A. marginale bodies near the red blood cell peripheries. Evidence will most likely be found before a hemolytic episode. A negative finding should not eliminate the pathogen from consideration. Epizootiology and transmission. The disease is common in cattle in the southern and western United States. Anaplasma organisms are spread biologically or mechanically. Mechanical transmission occurs when infected red blood cells are passed from one host to another on the mouthparts of seasonal biting flies. Sometimes mosquitoes or instruments such as dehorners or hypodermic needles may facilitate transfer of infected red cells from one animal to another. Biological transmission occurs when the tick stage of the organism is passed by Dermacentor andersoni and D. occidentalis ticks. The carrier stage covers the time when discernible Anaplasma organisms can be found on host blood smears. Recovered animals serve as immune carriers and disease reservoirs. Necropsy. Pale tissues and watery, thin blood are typical findings. Splenomegaly, hepatomegaly, and gallbladder distension are common findings. Pathogenesis. The parasites infect the host's red blood cells, and acute hemolysis occurs during the parasites' developmental stage. The four stages of the parasite's life cycle are described above because these are closely linked to the clinical stages. Differential diagnosis. The clinical disease closely resembles the protozoal disease babesiosis. Prevention and control. Offspring of immune carriers resist infection up to 6 months of age because of passive immunity. Vector control and attention to hygiene are essential, such as between-animal rinsing in disinfectant of mechanical vectors such as dehorners. There is no entirely effective means, however, to prevent and control the disease. Vaccination (killed whole organism) programs are not entirely effective, and vaccine should not be administered to pregnant cows. Neonatal isoerythrolysis may occur because of the antierythrocyte antibodies stimulated by one vaccine product. Vaccinated animals can still become infected and become carriers. The cattle vaccine has shown no efficacy in smaller ruminants, and there is no A. ovis vaccine. Identifying carriers serologically and treating with tetracycline during and/or after vector seasons may be an option. Removing carriers to a separate herd is also an approach. Interstate movement of infected animals is regulated. Treatment. Oxytetracycline, administered once, helps reduce the severity of the infection during the developmental stage. Other tetracycline treatment programs have been described to help control carriers. ii. Babesiosis (red water, Texas cattle fever, cattle tick fever) Etiology. Babesia bovis and Ba. bigemina are protozoa that cause subclinical infections or disease in cattle. These are intraerythrocytic parasites. Babesia bovis is regarded as the more virulent of the two organisms. This disease is not seen in the smaller ruminants in the United States. Clinical signs and diagnosis. The more common presentation is liver and kidney failure due to hemolysis with icterus, hemoglobinuria, and fever. Hemoglobinuria indicates a poor prognosis. Acute encephalitis is a less common presentation and begins acutely with fever, ataxia, depression, deficits in conscious proprioception, mania, convulsions, and coma. The encephalitic form generally also has a poor prognosis. Sudden death may occur. Thin blood smears stained with Giemsa will show Babesia trophozoites at some stages of the disease, but lack of these cannot be interpreted as a negative. The trophozoites occur in a variety of shapes, such as piriform, round, or rod. Complement fixation, immunofluorescent antibody, and enzyme immunoassay are the most favored of the available serologic tests. Epizootiology and transmission. Babesiosis is present on several continents, including the Americas. In addition to domestic cattle, some wild ruminants, such as white-tailed deer and American buffalo, are also susceptible. Bos indicus breeds have resistance to the disease and the tick vectors. Innate resistance factors have been found in all calves. If infected, these animals will not show many signs of disease during the first year of life and will become carriers. Stress can cause disease development. Necropsy findings. Signs of acute hemolytic crisis are the most common findings, including hepatomegaly, splenomegaly, dark and distended gallbladder, pale tissues, thin blood, scattered hemorrhages, and petechiation. Animals dying after a longer course of disease will be emaciated and icteric, with thin blood, pale kidneys, and enlarged liver. Pathogenesis. The protozoon is transmitted by the cattle fever ticks Boophilus annulatus, B. microplus, and B. decoloratus; these one-host ticks acquire the protozoon from infected animals. It is passed transovarially, and both nymph and adult ticks may transmit to other cattle. Only B. ovis is transmitted by the larval stage. Clinical signs develop about 2 weeks after tick infestations or mechanical transmission but may develop sooner with the mechanical transmission. Hemolysis is due to intracellular reproduction of the parasites and occurs intra- and extravascularly. In addition to the release of merozoites, proteolytic enzymes are also released, and these contribute to the clinical metabolic acidosis and anoxia. The development of the encephalitis form is believed to be the result of direct invasion of the central nervous system, disseminated intravascular coagulation, capillary thrombosis by the parasites and infarction, and/or tissue anoxia. Differential diagnosis. In addition to anaplasmosis, other differentials for the hemolytic form of the disease are leptospirosis, chronic copper toxicity, and bacillary hemoglobinuria. Several differentials in the United States for the encephalitic presentation include rabies, nervous system coccidiosis, polioencephalomalacia, lead poisoning, infectious bovine rhinotracheitis, salt poisoning, and chlorinated hydrocarbon toxicity. Prevention and control. Control or eradication of ticks and cleaning of equipment to prevent mechanical transmission, as noted in Section III,A,3,a,i, are important preventive measures. Some vaccination approaches have been effective, but a commercial product is not available. Treatment. Supportive care is indicated, including blood transfusions, fluids, and antibiotics. Medications such as diminazene diaceturate, phenamidine diisethionate, imidocarb diprionate, or amicarbalide diisethionate are most commonly used. Treatment outcomes will be either elimination of the parasite or development of a chronic carrier state immune to further disease. Research complications. This is a reportable disease in the United States. iii. Coccidiosis Etiology. Coccidiosis is an important acute and chronic protozoal disease of ruminants. In young ruminants, it is characterized primarily by hemorrhagic diarrhea. Adult ruminants may carry and shed the protozoa, but they rarely display clinical signs. Intensive rearing and housing conditions and stress increase the severity of the disease in all age groups. Coccidia are protozoal organisms of the phylum Apicomplexa, members of which are obligatory intracellular parasites. There are at least 11 reported species of coccidia in sheep, of which several are considered pathogenic: Eimeria ashata, E. crandallis, and E. ovinoidalis ( Schillhorn van Veen, 1986 ). At least 9 species of Eimeria have been recognized in the goat ( Foreyt, 1990 ). Eimeria ninakohlyakimovae, E. arloingi, and E. christenseni are regarded as the most pathogenic. Eimeria bovis and E. zuernii (highly pathogenic), and E. auburnensis and E. alabamensis (moderately pathogenic), are among the 13 species known to infect cattle. Eimeria zuernii is more commonly seen in older cattle and is the agent of "winter coccidiosis." Clinical signs and diagnosis. Hemorrhagic diarrhea develops 10 days to 3 weeks after infection. Fecal staining of the tail and perineum will be present. Animals will frequently display tenesmus; rectal prolapses may also develop. Anorexia, weight loss, dehydration, anemia, fever (infrequently), depression, and weakness may also be seen in all ruminants. The diarrhea is watery and malodorous and will contain variable amounts of blood and fibrinous, necrotic tissues. The intestinal hemorrhage may subsequently lead to anemia and hypoproteinemia. Depending on the predilection of the coccidial species for small and/or large intestines, malabsorption of nutrients or water may occur, and electrolyte imbalances may be severe. Concurrent disease with other enteropathogens may also be part of the clinical picture. In sheep, secondary bacterial infection with organisms such as Fusobacterium necrophorum may ensue. Young goats may die peracutely or suffer severe anemia from blood loss into the bowel. Older goats may lose the pelleted form of feces. Cattle may have explosive diarrhea and develop anal paralysis. The disease is usually diagnosed by history and clinical signs. Numerous oocysts will frequently be observed in fresh fecal flotation (salt or sugar solution) samples as the diarrhea begins. Laboratory results are usually reported as number of oocysts per gram of feces. Coccidia seen on routine fecal evaluations reflect shedding, possibly of nonpathogenic species, without necessarily being indicative of impending or resolving mild disease. Epizootiology and transmission. As noted, coccidiosis is a common disease in young ruminants. In goats, young animals aged 3 weeks to 5 months are primarily affected, but isolated outbreaks in adults may occur after stressful conditions such as transportation or diet changes. Coccidia are host-specific and also host cell-specific. The disease is transmitted via ingestion of sporulated oocysts. Coccidial oocysts remain viable for long periods of time when in moist, shady conditions. Necropsy. Necropsies provide information on specific locations and severity of lesions that correlate with the species involved. Ileitis, typhlitis, and colitis with associated necrosis and hemorrhage will be observed. Mucosal scrapings will frequently yield oocysts. Various coccidial stages associated with schizogony or gametogony may be observed in histopathological sections of the intestines. Fibrin and cellular infiltrates will be found in the lamina propria. Pathogenesis. This parasite has a complex life cycle in which sexual and asexual reproduction occurs in gastrointestinal enterocytes ( Speer, 1996 ). The severity of the disease is correlated primarily with the number of ingested oocysts. Specifics of life cycles vary with the species, and those characteristics contribute to the pathogenicity. In most cases, the disease is well established by the time clinical signs are seen. Oocysts must undergo sporulation over a 3- to 10-day period in the environment. After ingestion of the sporulated oocysts, sporozoites are released and penetrate the intestinal mucosa and form schizonts. Schizonts initially undergo replication by fission to form merozoites and eventually undergo sexual reproduction, forming new oocysts. The organisms cause edema and hyperemia; penetration into the lamina propria may lead to necrosis of capillaries and hemorrhage. Differential diagnosis. Differential diagnoses include the many enteropathogens associated with acute diarrhea in young ruminants: cryptosporidia, colibacilli, salmonella, enterotoxins, Yersinia, viruses, and other intestinal parasites such as helminths. In cattle, for example, bovine viral diarrhea virus and helminthiasis caused by Ostergia must be considered. Management factors, such as dietary-induced diarrheas, are also differentials. In older animals, differentials in addition to stress are malnutrition, grain engorgement, and other intestinal parasitisms. Prevention and control. Good management practices will help prevent the disease. Oocysts are resistant to disinfectants but are susceptible to dry or freezing conditions. Proper sanitation of animal housing and minimizing overcrowding are essential. Coccidiostats added to the feed and water are helpful in preventing the disease in areas of high exposure. Treatment. Affected animals should be isolated. On an individual basis, treatment should also include provision of a dry, warm environment, fluids, electrolytes (orally or intravenously), antibiotics (to prevent bacterial invasion and septicemia), and administration of coccidiostats. Coccidiostats are preferred to coccidiocidals because the former allow immunity to develop. Although many coccidial infections tend to be self-limiting, sulfonamides and amprolium may be used to aid in the treatment of disease. Other anticoccidial drugs include decoquinate, lasalocid, and monensin; labels should be checked for specific approval in a species or specific indications. Animals treated with amprolium should be monitored for development of secondary polioencephalomalacia. Pen mates of affected animals should be considered exposed and should be treated to control early stages of infection. Mechanisms of immunity have not been well defined but appear to be correlated with the particular coccidial species and their characteristics (for example, the extent of intracellular penetration). Immunity may result when low numbers are ingested and there is only mild disease. Immunity also may develop after more severe infections. iv. Cryptosporidiosis Etiology. Cryptosporidium organisms are a very common cause of diarrhea in young ruminants. Four Cryptosporidium species have been described in vertebrates: C. baileyi and C. meleagridis in birds and C. parvum and C. muris in mammals. Cryptosporidium parvum is the species affecting sheep ( Rings and Rings, 1996 ). Debate continues regarding whether there are definite host-specific variants. Clinical signs and diagnosis. Cryptosporidiosis is characterized by protracted, watery diarrhea and debilitation. The diarrhea may last only 6–10 days or may be persistent and fatal. The diarrhea is watery and yellow, and blood, mucus, bile, and undigested milk may also be present. Infected animals will display tenesmus, anorexia and weight loss, dehydration, and depression. In relapsing cases, animals become cachectic. Overall, morbidity will be high, and mortality variable. Mucosal scrapings or fixed stained tissue sections may be useful in diagnosis. The disease is also diagnosed by detecting the oocysts in iodine-stained feces or in tissues stained with periodic acid-Schiff stain or methenamine silver. Cryptosporidium also stains red on acid-fast stains such as Kinyoun or Ziehl-Neelsen. Fecal flotations should be performed without sugar solutions or with sugar solutions at specific gravity of 1.27 (Foryet, 1990). Fecal immunofluorescent antibody (IFA) techniques have also been described. Epizootiology and transmission. Younger ruminants are commonly affected: lambs, kids (especially kids between the ages of 5 and 10 days old), and calves less than 30 days old. Like other coccidians, Cryptosporidium is transmitted via the fecal-oral route. In addition to local contamination, water supplies have also been sources of the infecting oocysts. The oocysts are extremely resistant to desiccation in the environment and may survive in the soil and manure for many months. Necropsy findings. The lesions caused by Cryptosporidium are nonspecific. Animals will be emaciated. Moderate enteritis and hyperplasia of the crypt epithelial cells with villous atrophy as well as villous fusion, primarily in the lower small intestines, will be present. Cecal and colonic mucosae may sometimes be involved. Gastrointestinal smears may be made at necropsy and stained as described above. Pathogenesis. Although Cryptosporidium infections are clinically similar to Eimeria infections ( Moore, 1989 ), Cryptosporidium, in contrast to Eimeria, invades just under the surface but does not invade the cytoplasm of enterocytes. There is no intermediate host. The oocysts are half the size of Eimeria oocysts and are shed sporulated; they are, therefore, immediately infective. Within 2–7 days of exposure, diarrhea and oocyst shedding occur. The diarrhea is the result of malabsorption and, in younger animals, intraluminal milk fermentation. Autoinfection within the lumen of the intestines may also occur and result in persistent infections. In addition, several other pathogens may be involved, such as concurrent coronavirus and rotavirus infections in calves. Environmental stressors such as cold weather increase mortality. Intensive housing arrangements increase morbidity and mortality. Differential diagnosis. Other causes of diarrhea in younger ruminants include rotavirus, coronavirus, and other enteric viral infections; enterotoxigenic Escherichia coli; Clostridium; other coccidial pathogens; and dietary causes (inappropriate use of milk replacers). In addition, these other agents may also be causing illness in the affected animals and may complicate the diagnosis and the treatment picture. Eimeria is more likely to cause diarrhea in calves and lambs at 3–4 weeks of age. Giardia organisms may be seen in fecal preparations from young ruminants but are not considered to play a significant role in enteric disease. Prevention and control. Precautions should be taken when handling infected animals. Affected animals must be removed and isolated as soon as possible. Animal housing areas should be disinfected with undiluted commercial bleach or 5% ammonia. Formalin (10%) fumigation has proven successful (Foryet, 1990). After being cleaned, areas should be allowed to dry thoroughly and should remain unpopulated for a period of time. Because enteric disease often is multifactorial, other pathogens should also be considered, and management and husbandry should be examined. Treatment. No known drug treatment is available. The disease is generally self-limiting, so symptomatic, supportive therapy aimed at rehydrating, correcting electrolyte and acid-base balance, and providing energy is often effective. Supplementation with vitamin A may be helpful. Age resistance begins to develop when the animals are about 1 month old. Research complications. Cryptosporidiosis is a zoonotic disease. It is easily spread from calves to humans, for example, even as the result of simply handling clothing soiled by calf diarrhea. Adult immunocompetent humans are reported to experience watery diarrhea, cramping, flatulence, and headache. The disease can be life-threatening in immunocompromised individuals. v. Giardiasis Etiology. Giardia lamblia (also called G. intestinalis and G. duodenalis) is a flagellate protozoon. Giardiasis is a worldwide protozoal-induced diarrheal disease of mammals and some birds ( Kirkpatrick, 1989 ), but it not considered to be a significant pathogen in ruminants. Clinical signs and diagnosis. Diarrhea may be continuous or intermittent, is pasty to watery, is yellow, and may contain blood. Animals exhibit fever, dehydration, and depression. Chronic cases may result in a "poor doer" syndrome with weight loss and unthriftiness. Giardia can be diagnosed by identifying the motile piriform trophozoites in fresh fecal mounts. Oval cysts can be floated with zinc sulfate solution (33%). Standard solutions tend to be too hyperosmotic and to distort the cysts. Newer enzyme-linked immunosorbent assay (ELISA) and IFA tests are sensitive and specific. Epizootiology and transmission. Giardia infection may occur at any age, but young animals are predisposed. Chronic oocyst shedding is common. Transmission of the cyst stage is fecal-oral. Wild animals may serve as reservoirs. Necropsy findings. Gross lesions may not be evident. Villous atrophy and cuboidal enterocytes may be evident histologically. Pathogenesis. Following ingestion, each Giardia cyst releases four trophozoites, which attach to the enterocytes of the duodenum and proximal jejunum and subsequently divide by binary fission or encyst. The organism causes little intestinal pathology, and the cause of diarrhea is unknown but is thought to be related to disruption of digestive enzyme function, leading to malabsorption. Disturbances in intestinal motility may also occur ( Rings and Rings, 1996 ). Prevention and control. Intensive housing and warm environments should be minimized. Cysts can survive in the environment for long periods of time but are susceptible to desiccation. Effective disinfectants include quaternary ammonium compounds, bleach-water solution (1:16 or 1:32), steam, or boiling water. After cleaning, areas should be left empty and allowed to dry completely. Treatment. Giardia has been successfully treated with oral metronidazole. Benzimidazole anthelmintics are also effective, but these are not approved for use in animals for this purpose. Research complications. Giardia is zoonotic. Precautions should be taken when handling infected animals. vi. Neosporosis Etiology. Neosporosis is a common, worldwide cause of bovine abortion caused by the protozoal species Neospora caninum. Abortions have also been reported in sheep and goats. Neonatal disease is seen in lambs, kids, and calves. Until 1988, these infections were misdiagnosed as caused by Toxoplasma gondii. Some similarities exist between the life cycles and pathogeneses of both organisms. Clinical signs and diagnosis. Abortion is the only clinical sign seen in adult cattle and occurs sporadically, endemically, or as abortion storms. Bovine abortions occur between the third and seventh month of gestation; fetal age at abortion correlates with the parity of the dam as well as with pattern of abortion in the herd. Although cows that abort tend to be culled after the first or second abortion, repeated N. caninum- caused abortions will occur progressively later in gestation (up to about 6 months) and within a shorter time frame in the same cow ( Thurmond and Hietala, 1997 ). Although infections in adults are asymptomatic other than the abortions, decreased milk production has been noted in congenitally infected cows. Many Neospo ra-infected calves will be born asymptomatic. Weakness will be evident in some infected calves, but this resolves. Rare clinical signs include exophthalmos or asymmetric eyes, weight loss, ataxia, hyperflexion or hyperextension of all limbs, decreased patellar reflexes, and loss of conscious proprioception. Some fetal deaths will occur, and resorption, mummification, autolysis, or stillbirth will follow. Immunohistochemistry and histopathology of fetal tissue are the most efficient and reliable means of establishing a postmortem diagnosis. Serology (IFA and ELISA) is useful, including precolostral levels in weak neonates, but this indicates only exposure. Titers of dams will not be elevated at the time of abortion; fetal serology is influenced by the stage of gestation and course of infection. Earlier and rapid infections are less likely to yield antibodies against Neospora. None of the currently available tests is predictive of disease. Epizootiology and transmission. The parasite is now acknowledged to be widespread in dairy and cattle herds. The life cycle of N. caninum is complex, and many aspects remain to be clarified. The definitive host is the dog ( McAllister et al., 1998 ). Placental or aborted tissues are the most likely sources of infection for the definitive host and play a minor role in transmission to the intermediate hosts. The many intermediate hosts include ruminants, deer, and horses. Transplacental transmission is the major mode of transmission in dairy cattle and is the means by which a herd's infection is perpetuated. A less significant mode of transmission is by ingestion of oocysts, which sporulate in the environment or in the intermediate host's body. Reactivation in a chronically infected animal's body is the result of rupture of tissue cysts in neural tissue. Seropositive immunity does not protect a cow from future abortions. Many seropositive cows and calves will never abort or show clinical signs, respectively. Some immunological cross-reactivity may exist among Neospora, Cryptosporidia, and Coccidium. Necropsy findings. Aborted fetuses will usually be autolysed. In those from which tissue can be recovered, tissue cysts are most commonly found in the brain. Spinal cord is also useful. Histological lesions include mild to moderate gliosis, nonsuppurative encephalitis, and perivascular infiltration by mixed mononuclear cells. Pathogenesis. As with Toxoplasma, cell death is the result of intracellular multiplication of Neospora tachyzoites. Neospora undergoes sexual replication in the dog's intestinal tract, and oocysts are shed in the feces. The intermediate hosts develop nonclinical systemic infections, with tachyzoites in several organs, and parasites then localize and become encysted in particular tissues, especially the brain. Infections of this type are latent and lifelong. Except when immunocompromised, most cattle do not usually develop clinical signs and do not have fetal loss. Fetuses become infected, leading to fetal death, mid-gestation abortions, or live calves with latent infections or congenital brain disease. It usually takes 2–4 weeks for a fetus to die and to be expelled. Many aspects of the role of the maternal immune response and pregnancy-associated immunodeficiency in the patterns of Neospora abortions remain to be elucidated. Differential diagnosis. Even when there is a herd history of confirmed Neospora abortions, leptospirosis, bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), salmonellosis, and campylobacteriosis should be considered. BVDV in particular should be considered for abortion storms. Differentials for weak calves are BVDV, perinatal hypoxia following dystocia (immediate postpartum time), bluetongue virus, Toxoplasma, exposure to teratogens, or congenital defects. Prevention and control. The primary preventive measure is preventing contact with contaminated feces. Oocysts will not survive dry environments or extremes of temperature. Dog populations should be controlled, and dogs and other canids should not have access to placentas or aborted fetuses. Dogs should also be restricted from feed bunks and other feed storage areas. Preventive culling is not economically practical for most producers. A vaccine recently became available. If embryo transfer is practiced, recipients should be screened serologically before use. Treatment. There is no known treatment or immunoprophylaxis. vii. Sarcocystosis Etiology. Sarcocystosis is the disease caused by the cyst-forming sporozoon Sarcocystis. Sarcocystis capricanus, S. ovi-canus, and S. tenella are the species that infect sheep and goats. Sarcocystis cruzi, S. hirsuta, and S. hominis are the species that infect cattle. Definitive hosts are carnivores, and all ruminant species are intermediate hosts. Clinical signs and diagnosis. Clinical signs of sarcocystosis infection are seen in cattle during the stage when the parasite encysts in soft tissues. Often the infections are asymptomatic. Fever, anemia, ataxia, symmetric lameness, tremors, tail-switch hair loss, excessive salivation, diarrhea, and weight loss are clinical signs. Abortions in cattle occur during the second trimester and in smaller ruminants 28 days after ingestion of the sporulated oocysts. Definitive diagnosis is based on finding merozoites and meronts in neural tissue lesions. Clinical hematology results include decreased hematocrit, decreased serum protein, and prolonged prothrombin times. Sarcocystis-specific IgG will increase dramatically by 5–6 weeks after infection. There is no cross-reaction between Sarcocystis and Toxoplasma. Epizootiology and transmission. Infection rates among cattle in the United States are estimated to be very high. Transmission is by ingestion of feed and water contaminated by feces of the definitive hosts. Dogs are the definitive hosts for the species that infect the smaller ruminants. Cats, dogs, and primates (including humans when S. hominis is involved) are the definitive hosts for the species that infect cattle. Necropsy. Aborted fetuses may be autolysed. Lesions in neural tissues, including meningoencephalomyelitis, focal malacia, perivascular cuffing, neuronal degeneration, and gliosis, are most marked in the cerebellum and midbrain. Lesions may be found in other tissues, such as lymphadenopathy, and hemorrhages may be found in muscles and on serous surfaces. Cysts in cardiac and skeletal muscles are common incidental findings during necropsies. Pathogenesis. Ingestion of muscle flesh from an infected ruminant results in Sarcocystis cysts' being broken down in the carnivore's digestive system, release of bradyzoites, infection of intestinal mucosal cells by the bradyzoites, differentiation into sexual stages, fusion of the male and female gametes to form oocysts, and shedding as sporocysts by the definitive hosts. The sporocysts are eaten by the ruminant and penetrate the bowel walls; several stages of development occur in endothelial cells of arteries. Merozoites are the form that enters soft tissues, such as muscle, and subsequently encysts. Prevention and control. Feed supplies of ruminants must be protected from fecal contamination by domestic and wild carnivores. These animals should be controlled and must also not have access to carcasses. In larger production situations, monensin may be fed as a prophylactic measure. Treatment. Monensin fed during incubation is prophylactic, but the efficacy in clinically affected cattle is not known. viii. Toxoplasmosis Etiology. Toxoplasmosis is caused by the obligate intracellular protozoon Toxoplasma gondii, a coccidial parasite of the family Eimeridae. Cats are the only definitive hosts, and several warm-blooded animals, including ruminants, have been shown to be intermediate hosts. The disease is a major cause of abortion in sheep and goats and less common in cattle. Clinical signs and diagnosis. Clinical signs depend on the organ or tissue parasitized. Toxoplasmosis is typically associated with placentitis, abortion, stillbirths, or birth of weak young ( Underwood and Rook, 1992 ; Buxton, 1998 ). It has also been shown to cause pneumonia and nonsuppurative encephalitis. The enteritis at the early stage of infection may be fatal in some hosts. Hydrocephalus does not occur in animals as it does in human fetal Toxoplasma infections. Rare clinical presentations in ruminants include retinitis and chorioretinitis; these are usually asymptomatic. Infection of the ewe during the first trimester usually leads to fetal resorption, during the second trimester leads to abortion, and during the third trimester leads to birth of weak to normal lambs with subsequent high perinatal mortality. Congenitally infected lambs may display encephalitic signs of circling, incoordination, muscular paresis, and prostration. In sheep, weak young will develop normally if they survive the first week after birth. Infected adult sheep show no systemic illness. Infected adult goats, however, may die. Diagnosis may be difficult, and biological, serological, and histological methods are helpful. Serological tests are the most readily available. Complement fixation and the Sabin-Feldman antibody test may assist in diagnosis. Antibodies found in fetuses are indicative of congenital infection and are typically detectable 35 days after infection; fetal thoracic fluid is especially useful in demonstrating serological evidence of exposure. Biological methods, such as tissue culture or inoculation of mice with maternal body fluids, or with postmortem or necropsy tissues, are more time-consuming and expensive. Epizootiology and transmission. This protozoon is considered ubiquitous. Fifty percent (50%) of adult western sheep and 20% of feedlot lambs have positive hemagglutination titers (1:64 or higher) ( Jensen and Swift, 1982 ). Transmission among the definitive host is by ingestion of tissue cysts. Necropsy findings. At necropsy, placental cotyledons contain multiple small white areas that are sites of necrosis, edema, and calcification. Fetal brains may show nonspecific lesions such as coagulative necrosis, nonsuppurative encephalomyelitis, pneumonia, myocarditis, and hepatitis. Histologically, granulomas with Toxoplasma organisms may be seen in the retina, myocardium, liver, kidney, brain, and other tissues. Impression smears of these tissues, stained appropriately (e.g., with Giemsa), provide a rapid means of diagnosis. Identification of the organism in tissue sections (especially of the heart and the brain) also confirms the findings. Toxoplasma gondii is crescent-shaped, with a clearly visible nuclei, and will be found within macrophages. Pathogenesis. The protozoon has three infectious stages: the tachyzoite, the bradyzoite, and the sporozoite within the oocyst. The definitive hosts, felids, become infected by ingesting cyst stages in mammalian tissues, by ingesting oocysts in feces, and by transplacental transfer. Ingested zoites invade epithelial cells and eventually undergo sexual reproduction, resulting in new oocysts, which the cats will shed in the feces. Cats rarely show clinical signs of infection. One cat can shed millions of oocysts in 1 gm of feces, but the asymptomatic shedding takes place for only a few weeks in its life. Oocysts sporulate in cat feces after 1 day. Ruminants are intermediate hosts of toxoplasmosis and become infected by ingesting sporulated oocyst-contaminated water or feed. As in the definitive host, the ingested sporozoite invades epithelial cells within the intestine but also further invades the bloodstream and is transported throughout the host. The organism migrates to tissues such as the brain, liver, muscles, and placenta. Placental infection develops about 14 days after ingestion of the oocysts. The damage caused by an infection is due to multiplication within cells. Toxoplasma does not produce any toxin. Differential diagnosis. Differentials for abortion include Campylobacter, Chlamydia, and Q fever. Prevention and control. Feline populations on source farms should be controlled. Eliminating contamination of feed and water with cat feces is the best preventive measure. Sporulated oocysts can survive in soil and other places for long periods of time and are resistant to desiccation and freezing. Vaccines for abortion prevention in sheep are available in New Zealand and Europe. Treatment. Toxoplasmosis treatment is ineffective, although feeding monensin during pregnancy may be helpful ( Underwood and Rook, 1992 ). (Monensin is not approved for this use in the Unites States.) Weak lambs that survive the first week after birth will mature normally and will not deliver Toxoplasma- infected young. Research complications. Because toxoplasmosis is zoonotic, precautions must be taken when handling tissues from any abortions or neurological cases. Infections in immunocompromised humans have been fatal. ix. Trichomoniasis Etiology. Trichomoniasis is an insidious venereal disease of cattle caused by Tritrichomonas (also referred to as Trichomonas) fetus, a large, pear-shaped, flagellated protozoon. The organism is an obligate parasite of the reproductive tract, and it requires a microaerophilic environment to establish chronic infections. In the United States, it is now primarily a disease seen in western beef herds. There are many similarities between trichomoniasis and campylobacteriosis; both diseases cause herd infertility problems. Clinical signs and diagnosis. Clinical signs include infertility manifested by high nonpregnancy rates as well as periodic pyometras and abortions during the first half of gestation. Often the problem is not recognized until herd pregnancy checks indicate many "open," delayed-estrus, late-bred cows, or cows with postcoital pyometras. The abortion rate varies from 5% to 30%, and placentas will be expelled or retained. Tritrichomonas fetus also causes mild salpingitis but this does not result in permanent damage. Other than these manifestations, infection with T. fetus causes no systemic signs. Diagnosis is based on patterns of infertility and pyometras. For example, pyometras in postcoital heifers or cows are suggestive of this pathogen. Diagnostic methods include identifying or culturing the trichomonads from preputial smegma, cervicovaginal mucus, uterine exudates, placental fluids, or abomasal contents of aborted fetuses. Other nonpathogenic protozoa from fecal contamination may be present in the sample. The trichomonad has three anterior flagellae, one posterior flagella, and an undulating membrane; it travels in fluids with a characteristic jerky movement. Culturing must be done on specific media, such as Diamond's or modified Pastridge. Epizootiology and transmission. All transmission is by venereal exposure from breeding bulls or cows or, in some cases, contaminated breeding equipment. Necropsy findings. Nonspecific lesions, such as pyogranulomatous bronchopneumonia of fetuses and placentitis, may be seen in aborted material; some cases will have no gross lesions. Histologically, trichomonads may be visible in the fetal lung lesions and the placenta; those tissues are also the most useful for culturing. Pathogenesis. Tritrichomonas fetus colonizes the female reproductive tract, and subsequent clinical manifestations may be related to the size of the initial infecting dose. Tritrichomonas fetus does not interfere with conception. Embryonic death occurs within the first 2 months of infection. Affected cows will clear the infection over a span of months and maintain immunity for about 6 months. Infections in younger bulls are transient; apparently organisms are cleared by the bulls' immune systems and are dependent on exposure to infected females. Older bulls become chronic carriers, probably because of the ability of T. fetus to colonize deeper epithelial crypts of the prepuce and penis. Differential diagnosis. Campylobacteriosis is the other primary differential for reduced reproductive efficiency of a herd. Other venereal diseases should be considered when infertility problems are noted in a herd: brucellosis, mycoplasmosis, ureaplasmosis, and infectious pustular vulvovaginitis. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. A bacterin vaccine is available. Heifers, cows, and breeding bulls are vaccinated subcutaneously twice at 2 to 4 week intervals, with the booster dose administered 4 weeks before breeding season starts. Similar timing is recommended for administration of the annual booster; a long, anamnestic response does not occur. Bulls used for artificial insemination (AI) are screened routinely for T. fetus (and Campylobacter). AI reduces but does not eliminate the disease. The use of younger, vaccinated bulls is recommended in all circumstances. New animals should be tested before introduction to the herd. Control measures also include culling affected cows or else removing them from the breeding herd for 3 months to rest and clear the infection. Culling chronically infected bulls is strongly recommended. Treatment. Imidazole compounds have been effective, but the use of these is not permitted in food animals in the United States. Therapeutic immunizations are worthwhile when a positive diagnosis has been made. These will not curtail fetal losses but will shorten the convalescence of the affected cows and improve immunity of breeding bulls. Research complications. Trichomoniasis should be considered whenever natural service is used and fertility problems are encountered. b. Nematodes Nematodes are important ruminant pathogens that cause acute, chronic, subclinical, and clinical disease in adults and adolescents. The major helminths may cause gastroenteritis associated with intestinal hemorrhage and malnutrition. Nematodiasis is associated with grazing exposure to infective larvae; animals procured for research may have had exposure to these helminths. Mixed infections of these parasites are common. Generally, older animals develop resistance to some of the species; thus, animals between about 2 months and 2 years of age are most susceptible to infection. Because of the parasites' effects on the animals' physiology, infection in these younger animals is a major contributor to a cycle of poor nutrition and digestion, compromised immune responses, and impaired growth and development. Diagnosis is primarily based on fecal flotation techniques; however, because many of these nematodes have similar-appearing ova, hatching the ova and identifying the larvae are often required (Baermann technique). A number of anthelmintics can be used to interrupt nematode life cycles. See Zajac and Moore (1993) and Pugh et al. (1998 ) for comprehensive reviews of treatment and control of nematodiasis. i. Haemonchus contortus, H. placei (barber's pole worm, large stomach worm). Haemonchus contortus is the most important internal parasite of sheep and goats, and the brief description here focuses on the disease in the smaller ruminants. Haemonchus contortus and H. placei infections do occur in younger cattle and are similar to the disease in sheep. Haemonchus is extremely pathogenic, and the adults feed by sucking blood from the mucosa of the abomasum. Severe anemia may lead to death. Weight loss, decreased milk production, poor wool growth, and intermandibular and cervical edema due to hypoproteinemia ("bottle jaw") are also common clinical signs. Diarrhea is not seen in all cases but may sometimes be severe or chronic. The life cycle is direct. Under optimal conditions, a complete life cycle, from ingestion of larvae to eggs passed in the feces, occurs in 3 weeks. Embryonated eggs may develop into infective larvae within a week. Hypobiotic (arrested) larvae may exist for several months in animal tissues, serving as a reservoir for future pasture contamination. Periparturient increases in egg shedding by ewes contribute to large numbers of eggs spread on spring pastures ("spring rise"). Resistance to common anthelmintics has developed; currently ivermectin or benzimidazole products are used, with a minimum of 2 dosings given 2–3 weeks apart. Levamisole is also used. In severe cases, animals may benefit from blood transfusions and iron supplementation. Because animals may easily acquire infective larvae from ingestion of contaminated feed and from contaminated pastures, general facility sanitation and pasture management and rotation are important preventive and control measures. Haemonchus contortus is susceptible to destruction by freezing temperatures and dry conditions. ii. Ostertagia (Teladorsagia) circumcincta (medium stomach worm). Ostertagia circumcincta is also highly pathogenic for sheep and goats and, like Haemonchus, attaches to the abomasal mucosa and ingests blood. The life cycle is comparable to that of Haemonchus, including the phenomenon of hypobiosis. Larvae are especially resistant to cool temperatures, however, and will overwinter on pastures. Larvae-induced hyperplasia of abomasal epithelial glands results in a change of gastric pH from about 2.0 to near 7.0, leading to decreased digestive enzyme activity and malnutrition. Clinical syndromes are categorized as type 1 or type 2. The former type is associated with infections acquired in fall or spring and is seen in younger animals. The latter type is associated with emergence of the arrested larvae during spring or fall. Clinical signs include anemia, weight loss, decreased milk production, and unthriftiness. Diarrhea is usually seen in type 1 only; the symptoms of type 2 are comparable to those of Haemonchus infections. Anthelmintic drug therapy is comparable to that for Haemonchus, and drug resistance is also a problem with Ostertagia. iii. Ostertagia ostertagi (cattle stomach worm). Ostertagia ostertagi is the most pathogenic and most costly of the cattle nematodes. Ostertagia leptospicularis and O. bisonis also cause disease. The life cycle is direct, and egg shedding by the cattle may occur within 3–4 weeks of ingestion of infective larvae. Hypobiosis is also a characteristic of O. ostertagi. In the initial steps of infection, the normal processes of the abomasum are profoundly disrupted and cells are destroyed as the larvae develop within and emerge from the glands. Moroccan leather appearance is the term to describe the result of cellular hyperplasia and loss of cell differentiation. Cycles of infection and morbidity depend on geographic location, climate, and production cycles. Type 1 cattle ostertagiasis is associated with ingestion of large numbers of infective larvae, occurs in animals less than 2 years old, and causes diarrhea and anorexia. Type 2 ostertagiasis occurs in cattle 2–4 years old and older adults, is the result of the emergence and development of hypobiotic larvae, and in addition to signs seen with type 1, hypoproteinemia with development of submandibular edema, fever, and anemia is a clinical sign. Treatment options include ivermectin, fenbendazole, and levamisole; all are effective against the arrested larvae. Ostertagia is susceptible to desiccation but is resistant to freezing. iv. Trichostrongylus vitrinus, T. axei, T. colubriformis (hair worms). Trichostrongylus species favor cooler conditions, and some larvae may overwinter. Although the different species may affect different segments of the gastrointestinal tract, the nematode attaches to the mucosa and affects secretion and/or absorption. Trichostrongylus vitrinus and T. colubriformis infect the small intestine of sheep and goats. Trichostrongylus axei infects the abomasum of cattle, sheep, and goats and causes increases in abomasal pH similar to those seen with Ostertagia. Mucosal hyperplasia is not seen. The prepatent period is about 3 weeks. Affected animals display unthriftiness, anorexia, decreased milk production, weight loss, diarrhea, and dehydration. These worms show intermediate resistance to freezing temperatures and dry conditions. v. Nematodirus spathiger, N. battus (thread-necked worms). Nematodirus has lower pathogenicity compared with other gastrointestinal nematodes. The larvae cause small-intestinal necrosis and inflammation. The larvae are especially resistant to desiccation and freezing. Clinical signs include depression, weight loss, anorexia, and diarrhea. vi. Cooperia (small intestinal worms). Cooperia primarily affects younger animals less than 1 year of age. Cooperia curticei infects the small intestine of sheep and goats; C. punctata and C. oncophora infect the small intestines of cattle, sheep, and goats. Cooperia pectinata infects the stomach of cattle. Large numbers lead to clinical infection, and the prepatent period is about 3 weeks. Cooperia and Osteragia infections, like infections of some other nematode species, may act synergistically. Because these nematodes suck blood, clinical signs include anemia, gastrointestinal hemorrhage, and malnutrition. Animals exhibit weight loss, diarrhea, and depression. Cooperia species are intermediate to resistant to the effects of cold temperatures. vii. Strongyloides papillosus. Strongyloides papillosus is a small-intestinal parasite of sheep and cattle. Strongyloides has a different life cycle from that of many nematodes. The eggs, expelled in the feces, are larvated, and when they hatch, they form both free-living males and females or parasitic females only. The parasitic females may enter the gastrointestinal tract through oral ingestion, such as in milk during nursing, or through direct penetration of the skin. Penetrating larvae enter the bloodstream and are transported to the lungs, where they penetrate the alveoli, are coughed up, and then swallowed to ultimately enter the gastrointestinal tract. Adult females may reproduce in the small intestines by parthenogenesis. Clinical signs associated with Strongyloides include weight loss, diarrhea, unthriftiness, and dermatitis in cases where large numbers migrate through the skin. The current broad-spectrum anthelmintics are effective against Strongyloides. viii. Bunostomum trigonocephalum (hookworm). Bunostomum trigonocephalum is a hookworm that occasionally infects sheep in locales in the southwestern United States. Like Strongyloides, Bunostomum infection may involve oral ingestion or direct penetration of the skin (followed by tracheal migration and swallowing). The larvae mature in the small intestines and suck blood. Larvae are susceptible to desiccation and freezing. Heavy infection with Bunostomum may result in anemia, diarrhea, intestinal hemorrhage, edema, and weight loss. ix. Oesophagostomum columbianum, O. venulosum (nodule worms). Oesophagostomum spp. primarily infect the large intestine and occasionally the distal small intestine, causing nodule worm disease, or simply gut. Oesophagostomum columbianum and O. venulosum infect sheep and cattle. These nematodes may affect sheep from 3 months to 2 years of age, and the prepatent period is about 6 weeks. Larvae are highly sensitive to freezing and desiccation and rarely overwinter. Larvae penetrate the large-intestinal mucosa but occasionally move into the deeper areas of the intestinal wall near the serosa. The resultant inflammatory reaction may lead to the formation of a caseous nodule that may mineralize over time. Intestinal lesions may accelerate peristalsis, leading to diarrhea, or may inhibit peristalsis (later stages), resulting in constipation. Clinical signs include weakness, unthriftiness, alternating episodes of diarrhea and constipation, and severe weight loss. Nodular lesions are typical at necropsy. x. Chabertia ovis (large-mouth bowel worm). Chabertia ovis is a minor colon parasite of sheep, goats, and cattle and is seen primarily in sheep. Signs of infection are not usually seen in cattle. Prepatent periods are up to 50 days. Heavy infection, which may result from as few as 100 worms located at the proximal end of the colon, may lead to hemorrhagic mucoid diarrhea, weight loss, weakness, colitis, and mild anemia. xi. Trichuris (whipworms). Trichuris spp. are mildly pathogenic nematodes and are usually attached to the cecal mucosa. Trichuris has a rather long prepatent period, extending from 1 to 3 months. The oval eggs are double-operculated and survive well in pasture environmental extremes. The adult worms also have a characterisitic morphology, with one thicker end appearing as a whip handle. The nematodes cause a minor cecitis and will feed on blood. Clinical infection is rare and results in diarrhea with mucus and blood. Treatment and prevention methods are similar to those for other nematodes. xii. Dictyocaulus (lungworms). Dictyocaulus spp., or lungworms, are nematodes that cause varying clinical signs in ruminants. In sheep, Dictyocaulus filaria, Protostrongylus rufescens, and Muellerius capillaris cause disease; Dictyocaulus is the most pathogenic. Goats are infected by the same species as sheep, but infections are uncommon. Dictyocaulus viviparus is the only lungworm found in cattle, causing "fog fever." Infections with these parasites in the United States tend to be associated with cooler, moister climates. Lungworms induce a severe parasitic bronchitis (known as husk, or verminous pneumonia) in sheep between approximately 2 and 18 months of age. Sheep infected with any of the lungworm species may display coughing, dyspnea, nasal discharge, weight loss, unthriftiness, and occasionally fever. Coughing and dyspnea are symptoms in goats. Diagnosis is suggested by persistent coughing and nasal discharge and is confirmed by identifying larvae in the feces or adults in pathological samples. The Baermann technique, involving prompt examination of room-temperature feces, is usually used; zinc sulfate flotation is also used. Dictyocaulus has a direct life cycle. The adult worms reside in the large bronchi. Dictyocaulus produces embryonated eggs that are coughed up and swallowed; the eggs then hatch in the intestines, and larvae are expelled in the feces. The expelled larvae are infectious in about 7–10 days and, after ingestion, penetrate the intestinal mucosa and move through the lymphatics and blood into the lungs, where they develop into adults in about 5 weeks. Dictyocaulus filaria causes an especially severe bronchitis in sheep. Protostrongylus inhabits smaller bronchioles. Muellerius is of minor pathogenicity. Protostrongylus and Muellerius require the snail or slug as an intermediate host. Infection occurs through ingestion of infected snails; infections are less likely than those caused by the direct ingestion of Dictyocaulus larvae. Immunity wanes over a year. Viral and bacterial respiratory tract infections may be associated with the parasitic infection. Dictyocaulus viviparus causes the obvious signs in cattle. More severe illness is seen after infections with Cooperia and Ostertagia, because of a synergism between the nematodes even if the cattle are not currently infected with those parasites. Hypobiosis (arrested development of immature worms in lung tissue) is associated with Dictyocaulus infections; cattle will be silent carriers, showing no clinical signs and serving as a means for the infection to survive over winter or a dry season. Pastures can be heavily contaminated during the next grazing season. Necropsy lesions include bronchiolitis and bronchitis, atelectasis, and hyperplasia of peribronchiolar lymphoid tissue. Nematodes frequently reside in the bronchi of the diaphragmatic lung lobes and are frequently enmeshed with frothy exudate. Prevention and control of the disease involve appropriate pasture management. Elimination of intermediate hosts is important in sheep and goat pastures. In a laboratory setting, animals may be procured that are already harboring the disease. Infected animals can be treated with anthelmintics such as ivermectin or levamisole. Muellerius tends to be resistant to levamisole. There is no anthelmintic currently approved for goats, but fenbendazole, administered 2 weeks apart, has been effective for all three nematodes. Treating D. viviparus depends on the type and stage of life of the cattle; label directions must be followed. There is no vaccine for D. viviparus in the United States. Even if infections are not severe and do resolve with treatment, permanent lesions may be inflicted on the lung tissue. c. Cestodes (Tapeworms) i. Moniezia expansa and Thysanosoma actinoides infections. Tapeworms are rarely of clinical or economic importance. In younger animals, heavy infections result in potbellies, constipation or mild diarrhea, poor growth, rough coat, and anemia. Moniezia expansa, and less commonly Moniezia benedini, inhabit the small intestines of grazing ruminants. Moniezia expansa has the widest distribution of the tapeworm species in North America. Soil mites (Galumna spp. and Oribatula spp.) contribute to the life cycle as intermediate hosts, a period that lasts up to 16 weeks. Cysticercoids released from the mites are grazed, pass into the small intestines, and mature. No clinical or pathological sign is usually observed with Moniezia infection; diagnosis is made by observing the characteristic triangular-shaped eggs in fecal flotation examinations. Infection is treated with cestocides. Thysanosoma actinoides, or the fringed tapeworm, is a cestode that resides in the duodenum, bile duct, and pancreatic duct of sheep and cattle raised primarily west of the Mississippi River in the United States. Thysanosoma is of the family Anoplocephalidae. The life cycle is indirect, and the intermediate host is the psocid louse. Larval forms, or cysticercoids, are ingested by grazing animals, and the prepatent period is several months. Typically, no clinical signs are observed with Thysanosoma infection; nonetheless, liver damage, resulting in liver condemnation at slaughter, occurs. Necropsy lesions include bile and/or ductal hyperplasia and fibrosis. Thysanosoma is diagnosed premortem by identifying the gravid segments in the feces. ii. Abdominal or visceral cysticercosis. Abdominal or visceral cysticercosis is an occasional finding at slaughter. The so-called bladder worms typically affect the liver or peritoneal cavity and are the larval form of Taenia hydatigena, the common tapeworm of the dog family. Taenia hydatigena resides in the small intestines of canids, and its gravid segments, oncospheres, contaminate feed and water sources. After ingestion, the larvae penetrate the intestinal mucosa, are transported via the bloodstream to the liver, and cause migration tracts throughout the liver parenchyma. The larvae may leave the liver and migrate into the peritoneal cavity, where they attach and develop over the next 1–9 months into small fluid-filled bladders. The life cycle is completed only after these bladders are ingested by a carnivore, thus completing the maturation of the adult tapeworms. Although larval migration may cause nonspecific signs such as anorexia, hyperthermia, and weight loss, affected animals are usually asymptomatic. At necropsy, the bladder worms will be observed attached to the peritoneal or organ surfaces. Migration tracts may result in fibrosis and inflammation. Diagnosis is usually made at necropsy. Because of the migration through the liver, Fasciola hepatica is a differential diagnosis. Minimizing exposure to canine feces-contaminated feeds and water effectively interrupts the life cycle. Research animals may have been exposed prior to purchase. iii. Echinococcosis (hydatidosis, hydatid cyst disease). Echinococcosis, like cysticercosis, is an occasional finding at slaughter or necropsy. The hydatid cyst is the larval intermediate of the adult tapeworm Echinococcus granulosus, which resides in the small intestines of dogs and wild canids. Embryonated ova are expelled in the feces of the primary host and are ingested by herbivores, swine, and potentially humans. The eggs hatch in the gastrointestinal tract, and the oncospheres penetrate the mucosal lining, enter the bloodstream, and are transported to various organs such as the liver and lungs. The cystic structure develops and potentially ruptures, forming new cystic structures. Clinically, echinococcosis presents minimal clinical signs; unthriftiness or pneumonic lesions may be associated with infected organs. Cysts are typically observed at necropsy. Prevention should be aimed at decreasing fecal contamination of feed and water by canids. Additionally, tapeworm-infected dogs can be treated with standard tapeworm therapies. Treatment of infected ruminants is uncommon. iv. Gid. Coenuris cerebralis, the larval form of the canid tapeworm Taenia (Multiceps) multiceps, is the causative agent of the rare condition called gid. The disease occurs in ruminants as well as many other mammalian species. The larval parasite, ingested from fecal-contaminated food and water, invades the brain and spinal cord and develops as a bladder worm that causes pressure necrosis of the nervous tissues. The resultant signs of hyperesthesia, meningitis, paresis, paralysis, ataxia, and convulsions are observed. Diagnosis is usually made at necropsy. Eliminating transfer from the canid hosts prevents the disease. d. Trematodes i. Fascioliasis (liver fluke disease). Liver flukes are an important cause of acute and chronic disease in grazing sheep and cattle. There are three common species of flukes in ruminants of the continental United States: Fasciola hepatica, Fascioloides magna, and Dicrocoelium dendriticum. Fasciola hepática infections are primarily seen in Gulf Coast and western states. Fascioloides magna infections are typically seen in Gulf, Great Lake, and northwestern states, where ruminants share pasture with deer, elk, and moose. Dicrocoelium dendriticum infections occur only in New York State. Liver fluke eggs are passed in the bile and feces and hatch in 2–3 weeks to form the free-swimming miracidia. It is important to note that each fluke egg represents the source of eventually thousands of cercariae or metacercariae. The miracidia penetrate the body of an intermediate host (usually freshwater snails) and develop through sporocyst and redia stages, finally forming cercariae. (Dicrocoelium is unique because it utilizes a land snail that expels slime balls, each containing several hundred cercariae. These are eaten by a second intermediate host, the ant Formica fusca.) The cercariae leave the intermediate host, swim to grassy vegetation, lose their tail, and become a cystlike metacercaria. The metacercariae may remain in a dormant stage on the grass for 6 months or longer until ingested by a ruminant. The ingested metacercariae penetrate the small-intestinal wall and migrate through the abdominal cavity to the liver. There they locate in a bile duct, mature, and remain for up to 4 years. Acute liver fluke disease is related to the damage caused by the migration of immature flukes. Migratory flukes may lead to liver inflammation, hemorrhage, necrosis, and fibrosis. Fascioloides magna infections in sheep and goats can be fatal as the result of just one fluke tunneling through hepatic tissue. In cattle, infections are often asymptomatic because of the host's encapsulation of the parasite. Liver fluke damage may predispose to invasion by anaerobic Clostridium species such as C. novyi that could lead to fatal black disease or bacillary hemoglobinuria. Chronic disease may result from fluke-induced physical damage to the bile ducts and cholangiohepatitis. Blood loss into the bile may lead to anemia and hypoproteinemia. Liver damage also is evidenced by increases in liver enzymes such as γ-glutamyl transpeptidase (GGT). Persistent eosinophilia is also seen with liver fluke disease. Other clinical signs of liver fluke disease include anorexia, weight loss, unthriftiness, edema, and ascites. At necropsy, livers will be pale and friable and may have distinct migration tunnels along the serosal surfaces. Bile ducts will be enlarged, and areas of fibrosis will be evident. Diagnosis can be made from clinical signs and postmortem analyses. Blood chemistries suggestive of liver disease and eosinophilia support the diagnosis. Liver fluke control involves removal of the intermediate hosts. In a laboratory setting, liver fluke infection is unlikely. Nonetheless, incoming animals from pasture environments may be infected. Liver flukes can be treated by using the anthelmintic albendazole. ii. Rumen fluke infections (paramphistomosis). Paramphistomosis is an uncommon disease found in sheep and cattle in southern states. Paramphistomum microbothrioides and P. cervi inhabit the duodenum and rumen of affected sheep. Eggs are passed in the feces and hatch in approximately 1 month, and the miracidia penetrate the intermediate snail hosts. Cercariae develop in the snail over the next month, emerge, and encyst on grasses as metacercariae. When eaten, the metacercariae develop into adult flukes and attach to the mucosal lining. The life cycle is complete in approximately 100 days. The flukes cause localized injury to the mucosa and, by interfering with digestive processes, cause diarrhea and protein loss. Clinically, animals may experience anorexia, dehydration, weight loss, and diarrhea with or without blood. Mortality may reach 25%. Diagnosis is based on clinical findings as well as the identification of flukes or eggs in the feces. Animals can be treated with fluki-cides. Eliminating the intermediate host prevents the disease. e. Mites (Mange) Mites cause a chronic dermatitis. The principal symptom of these infections is intense pruritus. In addition, papules, crusts, alopecia, and secondary dermatitis are seen. Anemia, disruption of reproductive cycles, and increased susceptibility to other diseases may also occur. Mites are rare in ruminants in the United States, but infections of Sarcoptes and Psorergates mange must be reported to animal health officials. Ruminants in poorly managed facilities are generally the most susceptible to infection, and infections are more frequent during winter months. Diagnosis is based on signs, examination of skin scrapings, and response to therapy. No effective treatment for demodectic mange in large animals has been found. The differential for mite infestations is pediculosis. Several genera of mites may affect sheep. These have been eradicated from flocks in the United States or are very rare and include Psoroptes ovis (common scabies), Sarcoptes scabiei (head scabies, barn itch), Psorergates ovis (sheep itch mite), Chorioptes ovis (foot scabies, tail mange), and Demodex ovis (follicular mange). Goats can also be infected by sarcoptic, chorioptic, and psoroptic mange. The scabies mite Sarcoptes rupicaprae invades epidermal tissue and causes focal pruritic areas around the head and neck. The chorioptic mite, either Chorioptes bovis or C. caprae, does not invade epidermal tissue but rather feeds on dead skin tissue. The chorioptic mite prefers distal limbs, the udder, and the scrotum and can be a significant cause of pruritus. The psoroptic mite Psoroptes cuniculi commonly occurs in the ear canal and causes head shaking and scratching. Repeated treatments of lime sulfur, amitraz, or ivermectin may be effective ( Smith and Sherman, 1994 ). Goats are also susceptible to demodectic mange caused by Demodex caprae. Adult mites invade hair follicles and sebaceous glands. Pustules may develop with secondary bacterial infection. Psoroptes bovis continues to be present in cattle in the United States, although it has been eradicated from sheep. Chorioptes bovis typically infects lower hindlimbs, perineum, tail, and scrotum but can become generalized. The sarcoptic mange mite S. scabei can survive off the host, so fomite transmission is a factor. The mange usually begins around the head but then spreads. This parasite can be transmitted to humans. Demodex bovis infects cattle; nodules on the face and neck are typical. Demodex bovis infections may resolve without treatment. Lindane, coumaphos, malathion, and lime sulfur are used to treat Psoroptes and Psorergates. Ivermectin is effective against Sarcoptes and is approved for use in cattle. f. Lice (Pediculosis) Lice that infect ruminants are of the orders Mallophaga, biting or chewing lice, and Anoplura, sucking lice. These are wingless insects. Members of the Mallophaga are colored yellow to red; members of the Anoplura are blue gray. Lice produce a seasonal (winter-to-spring), chronic dermatitis. In sheep, biting lice include Damalinia (Bovicola) ovis (sheep body louse). Sucking lice that infect sheep include Linognathus ovillus (blue body louse) and L. pedalis (sheep foot louse). In goats, biting lice infection are caused by D. caprae (goat biting louse), D. limbatus (Angora goat biting louse), and D. crassipes. Sucking louse infections in goats are caused by L. stenopis and L. africanus. Damalinia bovis is the cattle biting louse. Sucking lice include L. vituli, Solenopotes capillatus, Haematopinus eurysternus, and H. quadripertusus. Pruritus is the most common sign and often results in alopecia and excoriation. The host's rubbing and grooming may not correlate with the extent of infestation. Hairballs can result from overgrooming in cattle. In severe cases, the organisms can lead to anemia, weight loss, and damaged wool in sheep and damaged pelts in other ruminants. Young animals with severe infestations of sucking lice may become anemic or even die. Pregnant animals with heavy infestations may abort. In sheep infected with the foot louse, lameness may result. Lice are generally species-specific. Those infecting ruminants are usually smaller than 5 mm. Goats may serve as a source of infection for sheep by harboring Damalinia ovis. Transmission is primarily by direct contact between animals. Transmission can also occur by attachment to flies or by fomites. Some animals are identified as carriers and seem to be particularly susceptible to infestations. Biting or chewing lice inhabit the host's face, lower legs, and flanks and feed on epidermal debris and sebaceous secretions. Sucking lice inhabit the host's neck, back, and body region and feed on blood. Lice eggs or nits are attached to hairs near the skin. Three nymphal stages, or instars, occur between egg and adult, and the growth cycle takes about 1 month for all species. Lice cannot survive for more than a few days off the host. All ruminant mite infestations are differentials for the clinical signs seen with pediculosis. Animals that are carriers should be culled, because these individuals may perpetuate the infection in the group. Lice are effectively treated with a variety of insecticides, including coumaphos, dichlorvos, crotoxyphos, avermectin, and pyrethroids. Label directions should be read and adhered to, including withdrawal times. Products should not be used on female dairy animals. Treatments must be repeated at least twice at intervals appropriate for nit hatches (about every 16 days) because nits will not be killed. Fall treatments are useful in managing the infections. Systemic treatments in cattle are contraindicated when there may be concurrent larvae of cattle grubs (Hypoderma lineatum and H. bovis). Back rubbers with insecticides, capitalizing on self-treatment, are useful for cattle. Sustained-release insecticide-containing ear tags are approved for use in cattle. g. Ticks Etiology. Ruminants are susceptible to many species of Ixodidae (hard-shell ticks) and Argasidae (softshell ticks). Many diseases, including anaplasmosis, babesiosis, and Q fever are transmitted by ticks. Clinical signs and diagnosis. Tick infestations are associated with decreased productivity, loss of blood and blood proteins, transmission of diseases, debilitation, and even death. Feeding sites on the host vary with the tick species. Ticks are associated with an acute paralytic syndrome called tick paralysis. This disease is characterized by ascending paralysis and may lead to death if the tick is not removed before the paralysis reaches the respiratory muscles. Diagnosis is based on identification of the species. Epizootiology and transmission. Ticks are not as host-specific as lice. Ticks are classified as one-host, two-host, or three-host; this refers to whether they drop off the host between larval and nymphal stages to molt. Pathogenesis of tick infestations. Patterns of feeding on the host differ between Argasidae and Ixodidae. The former feed repeatedly, whereas the latter feed once during each life stage. Pathogenesis of tick paralysis. Following a tick-feeding period of 4–6 days, the tick salivary toxin travels hematogenously to the myoneural junctions and spinal cord and inhibits nerve transmission. Removal of the ticks reverses the syndrome unless paralysis has migrated anteriorly to the respiratory centers of the medulla. In these cases, death due to respiratory failure occurs. Treatment. Ticks can be treated using systemic or topical insecticides. h. Other Parasites i. Nasal bots (nasal myiasis, head grubs). Nasal myiasis causes a chronic rhinitis and sinusitis. The disease is caused by the larval forms of the botfly Oestrus ovis. The botfly deposits eggs around the nostrils of sheep. The ova hatch, and the larvae migrate throughout the nasal cavity and sinuses, feeding on mucus and debris. In 2–10 months, the larvae complete their growing phase, migrate back to the nasal cavity, and are sneezed out. The mature larvae penetrate the soil and pupate for 1–1.5 months and emerge as botflies. Clinically, early in the disease course, animals display unique behaviors such as stamping, snorting, sneezing, and rubbing their noses against each other or objects. Hypersensitivity to the larvae occurs ( Dorchies et al., 1998 ). Later, mucopurulent nasal discharges associated with the larval-induced inflammation of mucosal linings will be observed. At necropsy, larvae will be observed in the nasal cavity or sinuses. Mild inflammatory reactions, mucosal thickening, and exudates will accompany the larvae. The disease is diagnosed by observing the behaviors or identifying organisms at necropsy. Up to 80% of a flock will potentially be infected; treatment should be employed on the rest of the flock. Ivermectins and other insecticides will eliminate the larvae; but treatment should be done in the early fall, when larvae are small. Fly repellents may be helpful at preventing additional infections. ii. Screwworm flies. Cochliomyia hominivorax (Callitroga americana) is the the screwworm that causes occasional disease in the southwestern United States along the Mexico border. Eradication programs have been pursued, and the disease is reportable. Large greenish flies lay large numbers of white eggs as shinglelike layers at the edges of open wounds (including docking and castration sites), soiled skin, or abrasions. Eggs hatch within 24 hr. Larvae are obligate parasites of living tissue, and the cycle is perpetuated because the increasingly large wound continues to be attractive to the next generation of flies. Larvae eventually drop off, pupate best in hot climates, and hatch in 3 weeks. Large cavities in parasitized tissue are formed, and lesions are characterized by malodor, large volumes of brown exudate, and necrosis. Single animals or entire herds may be affected. Treatment is intensive, with dressings and larvicidal applications. If there is no intervention, the host succumbs to secondary infections and fluid loss. Effective current control regimens include subcutaneous injection of ivermectin and programs that release sterile male flies. iii. Sheep keds ("sheep ticks"). In sheep and goats, sheep keds produce a chronic irritation and dermatitis with associated pruritus. The disease is caused by Melophagus ovinus, which is a flat, brown, blood-sucking, wingless fly; the term sheep tick is incorrectly used. The adult fly lives entirely on the skin of sheep. Females mate and produce 10–15 larvae following a gestation of about 10–12 days. The larvae attach to the wool or hair and then pupate for about 3 weeks. The adult female feeds on blood and lives for 4–5 months; the life cycle is completed in about 5–6 weeks. Infection is highest in fall and winter. Pruritus develops around the neck, sides, abdomen, and rump. In severe cases, anemia may occur. Keds can transmit bluetongue virus. Keds are diagnosed by gross or microscopic identification. Ivermectin or other insecticides are useful treatment agents. a. Protozoa i. Anaplasmosis Etiology. Anaplasmosis is an infectious, hemolytic, noncontagious, transmissible disease of cattle caused by the protozoan Anaplasma marginale. Anaplasma is a member of the Anaplas-matacae family within the order Rickettsiales. In sheep and goats, the disease is caused by A. ovis and is an uncommon cause of hemolytic disease. Anaplasmosis has not been reported in goats in the United States. Some controversy exists regarding the classification. Most recently it is classified as a protozoal disease because of similarities to babesiosis. It has also been classified as a rickettsial pathogen. This summary addresses the disease in cattle with limited reference to A. ovis infections, but there are many similarities to the disease in cattle. Clinical signs and diagnosis. Acute anemia is the predominant sign in anaplasmosis, and fever coincides with parasitemia. Weakness, pallor, lethargy, dehydration, and anorexia are the result of the anemia. Four disease stages—incubation, developmental, convalescent, and carrier—are recognized. The incubation stage may be long, 3–8 weeks, and is characterized by a rise in body temperature as the infection moves to the next stage. Most clinical signs occur during the 4- to 9-day developmental stage, with hemolytic anemia being common. Death is most likely to occur at this stage or at the beginning of the convalescent stage. Death may also occur from anoxia, because of the animal's inability to handle any exertion or stress, especially if treatment is initiated when severe anemia exists. Reticulocytosis characterizes the convalescent stage, which may continue for many weeks. Morbidity is high, and mortality is low. The carrier stage is defined as the time in the convalescent stage when the animal host becomes a reservoir of the disease, and Anaplasma organisms and any parasitemia are not discernible. Common serologic tests are the complement fixation test and the rapid card test. These become positive after the incubation phase and do not distinguish between the later three stages of disease. Definitive diagnosis is made by clinical and necropsy findings. Staining of thin blood smears with Wright's or Giemsa stain allows detection of basophilic, spherical A. marginale bodies near the red blood cell peripheries. Evidence will most likely be found before a hemolytic episode. A negative finding should not eliminate the pathogen from consideration. Epizootiology and transmission. The disease is common in cattle in the southern and western United States. Anaplasma organisms are spread biologically or mechanically. Mechanical transmission occurs when infected red blood cells are passed from one host to another on the mouthparts of seasonal biting flies. Sometimes mosquitoes or instruments such as dehorners or hypodermic needles may facilitate transfer of infected red cells from one animal to another. Biological transmission occurs when the tick stage of the organism is passed by Dermacentor andersoni and D. occidentalis ticks. The carrier stage covers the time when discernible Anaplasma organisms can be found on host blood smears. Recovered animals serve as immune carriers and disease reservoirs. Necropsy. Pale tissues and watery, thin blood are typical findings. Splenomegaly, hepatomegaly, and gallbladder distension are common findings. Pathogenesis. The parasites infect the host's red blood cells, and acute hemolysis occurs during the parasites' developmental stage. The four stages of the parasite's life cycle are described above because these are closely linked to the clinical stages. Differential diagnosis. The clinical disease closely resembles the protozoal disease babesiosis. Prevention and control. Offspring of immune carriers resist infection up to 6 months of age because of passive immunity. Vector control and attention to hygiene are essential, such as between-animal rinsing in disinfectant of mechanical vectors such as dehorners. There is no entirely effective means, however, to prevent and control the disease. Vaccination (killed whole organism) programs are not entirely effective, and vaccine should not be administered to pregnant cows. Neonatal isoerythrolysis may occur because of the antierythrocyte antibodies stimulated by one vaccine product. Vaccinated animals can still become infected and become carriers. The cattle vaccine has shown no efficacy in smaller ruminants, and there is no A. ovis vaccine. Identifying carriers serologically and treating with tetracycline during and/or after vector seasons may be an option. Removing carriers to a separate herd is also an approach. Interstate movement of infected animals is regulated. Treatment. Oxytetracycline, administered once, helps reduce the severity of the infection during the developmental stage. Other tetracycline treatment programs have been described to help control carriers. ii. Babesiosis (red water, Texas cattle fever, cattle tick fever) Etiology. Babesia bovis and Ba. bigemina are protozoa that cause subclinical infections or disease in cattle. These are intraerythrocytic parasites. Babesia bovis is regarded as the more virulent of the two organisms. This disease is not seen in the smaller ruminants in the United States. Clinical signs and diagnosis. The more common presentation is liver and kidney failure due to hemolysis with icterus, hemoglobinuria, and fever. Hemoglobinuria indicates a poor prognosis. Acute encephalitis is a less common presentation and begins acutely with fever, ataxia, depression, deficits in conscious proprioception, mania, convulsions, and coma. The encephalitic form generally also has a poor prognosis. Sudden death may occur. Thin blood smears stained with Giemsa will show Babesia trophozoites at some stages of the disease, but lack of these cannot be interpreted as a negative. The trophozoites occur in a variety of shapes, such as piriform, round, or rod. Complement fixation, immunofluorescent antibody, and enzyme immunoassay are the most favored of the available serologic tests. Epizootiology and transmission. Babesiosis is present on several continents, including the Americas. In addition to domestic cattle, some wild ruminants, such as white-tailed deer and American buffalo, are also susceptible. Bos indicus breeds have resistance to the disease and the tick vectors. Innate resistance factors have been found in all calves. If infected, these animals will not show many signs of disease during the first year of life and will become carriers. Stress can cause disease development. Necropsy findings. Signs of acute hemolytic crisis are the most common findings, including hepatomegaly, splenomegaly, dark and distended gallbladder, pale tissues, thin blood, scattered hemorrhages, and petechiation. Animals dying after a longer course of disease will be emaciated and icteric, with thin blood, pale kidneys, and enlarged liver. Pathogenesis. The protozoon is transmitted by the cattle fever ticks Boophilus annulatus, B. microplus, and B. decoloratus; these one-host ticks acquire the protozoon from infected animals. It is passed transovarially, and both nymph and adult ticks may transmit to other cattle. Only B. ovis is transmitted by the larval stage. Clinical signs develop about 2 weeks after tick infestations or mechanical transmission but may develop sooner with the mechanical transmission. Hemolysis is due to intracellular reproduction of the parasites and occurs intra- and extravascularly. In addition to the release of merozoites, proteolytic enzymes are also released, and these contribute to the clinical metabolic acidosis and anoxia. The development of the encephalitis form is believed to be the result of direct invasion of the central nervous system, disseminated intravascular coagulation, capillary thrombosis by the parasites and infarction, and/or tissue anoxia. Differential diagnosis. In addition to anaplasmosis, other differentials for the hemolytic form of the disease are leptospirosis, chronic copper toxicity, and bacillary hemoglobinuria. Several differentials in the United States for the encephalitic presentation include rabies, nervous system coccidiosis, polioencephalomalacia, lead poisoning, infectious bovine rhinotracheitis, salt poisoning, and chlorinated hydrocarbon toxicity. Prevention and control. Control or eradication of ticks and cleaning of equipment to prevent mechanical transmission, as noted in Section III,A,3,a,i, are important preventive measures. Some vaccination approaches have been effective, but a commercial product is not available. Treatment. Supportive care is indicated, including blood transfusions, fluids, and antibiotics. Medications such as diminazene diaceturate, phenamidine diisethionate, imidocarb diprionate, or amicarbalide diisethionate are most commonly used. Treatment outcomes will be either elimination of the parasite or development of a chronic carrier state immune to further disease. Research complications. This is a reportable disease in the United States. iii. Coccidiosis Etiology. Coccidiosis is an important acute and chronic protozoal disease of ruminants. In young ruminants, it is characterized primarily by hemorrhagic diarrhea. Adult ruminants may carry and shed the protozoa, but they rarely display clinical signs. Intensive rearing and housing conditions and stress increase the severity of the disease in all age groups. Coccidia are protozoal organisms of the phylum Apicomplexa, members of which are obligatory intracellular parasites. There are at least 11 reported species of coccidia in sheep, of which several are considered pathogenic: Eimeria ashata, E. crandallis, and E. ovinoidalis ( Schillhorn van Veen, 1986 ). At least 9 species of Eimeria have been recognized in the goat ( Foreyt, 1990 ). Eimeria ninakohlyakimovae, E. arloingi, and E. christenseni are regarded as the most pathogenic. Eimeria bovis and E. zuernii (highly pathogenic), and E. auburnensis and E. alabamensis (moderately pathogenic), are among the 13 species known to infect cattle. Eimeria zuernii is more commonly seen in older cattle and is the agent of "winter coccidiosis." Clinical signs and diagnosis. Hemorrhagic diarrhea develops 10 days to 3 weeks after infection. Fecal staining of the tail and perineum will be present. Animals will frequently display tenesmus; rectal prolapses may also develop. Anorexia, weight loss, dehydration, anemia, fever (infrequently), depression, and weakness may also be seen in all ruminants. The diarrhea is watery and malodorous and will contain variable amounts of blood and fibrinous, necrotic tissues. The intestinal hemorrhage may subsequently lead to anemia and hypoproteinemia. Depending on the predilection of the coccidial species for small and/or large intestines, malabsorption of nutrients or water may occur, and electrolyte imbalances may be severe. Concurrent disease with other enteropathogens may also be part of the clinical picture. In sheep, secondary bacterial infection with organisms such as Fusobacterium necrophorum may ensue. Young goats may die peracutely or suffer severe anemia from blood loss into the bowel. Older goats may lose the pelleted form of feces. Cattle may have explosive diarrhea and develop anal paralysis. The disease is usually diagnosed by history and clinical signs. Numerous oocysts will frequently be observed in fresh fecal flotation (salt or sugar solution) samples as the diarrhea begins. Laboratory results are usually reported as number of oocysts per gram of feces. Coccidia seen on routine fecal evaluations reflect shedding, possibly of nonpathogenic species, without necessarily being indicative of impending or resolving mild disease. Epizootiology and transmission. As noted, coccidiosis is a common disease in young ruminants. In goats, young animals aged 3 weeks to 5 months are primarily affected, but isolated outbreaks in adults may occur after stressful conditions such as transportation or diet changes. Coccidia are host-specific and also host cell-specific. The disease is transmitted via ingestion of sporulated oocysts. Coccidial oocysts remain viable for long periods of time when in moist, shady conditions. Necropsy. Necropsies provide information on specific locations and severity of lesions that correlate with the species involved. Ileitis, typhlitis, and colitis with associated necrosis and hemorrhage will be observed. Mucosal scrapings will frequently yield oocysts. Various coccidial stages associated with schizogony or gametogony may be observed in histopathological sections of the intestines. Fibrin and cellular infiltrates will be found in the lamina propria. Pathogenesis. This parasite has a complex life cycle in which sexual and asexual reproduction occurs in gastrointestinal enterocytes ( Speer, 1996 ). The severity of the disease is correlated primarily with the number of ingested oocysts. Specifics of life cycles vary with the species, and those characteristics contribute to the pathogenicity. In most cases, the disease is well established by the time clinical signs are seen. Oocysts must undergo sporulation over a 3- to 10-day period in the environment. After ingestion of the sporulated oocysts, sporozoites are released and penetrate the intestinal mucosa and form schizonts. Schizonts initially undergo replication by fission to form merozoites and eventually undergo sexual reproduction, forming new oocysts. The organisms cause edema and hyperemia; penetration into the lamina propria may lead to necrosis of capillaries and hemorrhage. Differential diagnosis. Differential diagnoses include the many enteropathogens associated with acute diarrhea in young ruminants: cryptosporidia, colibacilli, salmonella, enterotoxins, Yersinia, viruses, and other intestinal parasites such as helminths. In cattle, for example, bovine viral diarrhea virus and helminthiasis caused by Ostergia must be considered. Management factors, such as dietary-induced diarrheas, are also differentials. In older animals, differentials in addition to stress are malnutrition, grain engorgement, and other intestinal parasitisms. Prevention and control. Good management practices will help prevent the disease. Oocysts are resistant to disinfectants but are susceptible to dry or freezing conditions. Proper sanitation of animal housing and minimizing overcrowding are essential. Coccidiostats added to the feed and water are helpful in preventing the disease in areas of high exposure. Treatment. Affected animals should be isolated. On an individual basis, treatment should also include provision of a dry, warm environment, fluids, electrolytes (orally or intravenously), antibiotics (to prevent bacterial invasion and septicemia), and administration of coccidiostats. Coccidiostats are preferred to coccidiocidals because the former allow immunity to develop. Although many coccidial infections tend to be self-limiting, sulfonamides and amprolium may be used to aid in the treatment of disease. Other anticoccidial drugs include decoquinate, lasalocid, and monensin; labels should be checked for specific approval in a species or specific indications. Animals treated with amprolium should be monitored for development of secondary polioencephalomalacia. Pen mates of affected animals should be considered exposed and should be treated to control early stages of infection. Mechanisms of immunity have not been well defined but appear to be correlated with the particular coccidial species and their characteristics (for example, the extent of intracellular penetration). Immunity may result when low numbers are ingested and there is only mild disease. Immunity also may develop after more severe infections. iv. Cryptosporidiosis Etiology. Cryptosporidium organisms are a very common cause of diarrhea in young ruminants. Four Cryptosporidium species have been described in vertebrates: C. baileyi and C. meleagridis in birds and C. parvum and C. muris in mammals. Cryptosporidium parvum is the species affecting sheep ( Rings and Rings, 1996 ). Debate continues regarding whether there are definite host-specific variants. Clinical signs and diagnosis. Cryptosporidiosis is characterized by protracted, watery diarrhea and debilitation. The diarrhea may last only 6–10 days or may be persistent and fatal. The diarrhea is watery and yellow, and blood, mucus, bile, and undigested milk may also be present. Infected animals will display tenesmus, anorexia and weight loss, dehydration, and depression. In relapsing cases, animals become cachectic. Overall, morbidity will be high, and mortality variable. Mucosal scrapings or fixed stained tissue sections may be useful in diagnosis. The disease is also diagnosed by detecting the oocysts in iodine-stained feces or in tissues stained with periodic acid-Schiff stain or methenamine silver. Cryptosporidium also stains red on acid-fast stains such as Kinyoun or Ziehl-Neelsen. Fecal flotations should be performed without sugar solutions or with sugar solutions at specific gravity of 1.27 (Foryet, 1990). Fecal immunofluorescent antibody (IFA) techniques have also been described. Epizootiology and transmission. Younger ruminants are commonly affected: lambs, kids (especially kids between the ages of 5 and 10 days old), and calves less than 30 days old. Like other coccidians, Cryptosporidium is transmitted via the fecal-oral route. In addition to local contamination, water supplies have also been sources of the infecting oocysts. The oocysts are extremely resistant to desiccation in the environment and may survive in the soil and manure for many months. Necropsy findings. The lesions caused by Cryptosporidium are nonspecific. Animals will be emaciated. Moderate enteritis and hyperplasia of the crypt epithelial cells with villous atrophy as well as villous fusion, primarily in the lower small intestines, will be present. Cecal and colonic mucosae may sometimes be involved. Gastrointestinal smears may be made at necropsy and stained as described above. Pathogenesis. Although Cryptosporidium infections are clinically similar to Eimeria infections ( Moore, 1989 ), Cryptosporidium, in contrast to Eimeria, invades just under the surface but does not invade the cytoplasm of enterocytes. There is no intermediate host. The oocysts are half the size of Eimeria oocysts and are shed sporulated; they are, therefore, immediately infective. Within 2–7 days of exposure, diarrhea and oocyst shedding occur. The diarrhea is the result of malabsorption and, in younger animals, intraluminal milk fermentation. Autoinfection within the lumen of the intestines may also occur and result in persistent infections. In addition, several other pathogens may be involved, such as concurrent coronavirus and rotavirus infections in calves. Environmental stressors such as cold weather increase mortality. Intensive housing arrangements increase morbidity and mortality. Differential diagnosis. Other causes of diarrhea in younger ruminants include rotavirus, coronavirus, and other enteric viral infections; enterotoxigenic Escherichia coli; Clostridium; other coccidial pathogens; and dietary causes (inappropriate use of milk replacers). In addition, these other agents may also be causing illness in the affected animals and may complicate the diagnosis and the treatment picture. Eimeria is more likely to cause diarrhea in calves and lambs at 3–4 weeks of age. Giardia organisms may be seen in fecal preparations from young ruminants but are not considered to play a significant role in enteric disease. Prevention and control. Precautions should be taken when handling infected animals. Affected animals must be removed and isolated as soon as possible. Animal housing areas should be disinfected with undiluted commercial bleach or 5% ammonia. Formalin (10%) fumigation has proven successful (Foryet, 1990). After being cleaned, areas should be allowed to dry thoroughly and should remain unpopulated for a period of time. Because enteric disease often is multifactorial, other pathogens should also be considered, and management and husbandry should be examined. Treatment. No known drug treatment is available. The disease is generally self-limiting, so symptomatic, supportive therapy aimed at rehydrating, correcting electrolyte and acid-base balance, and providing energy is often effective. Supplementation with vitamin A may be helpful. Age resistance begins to develop when the animals are about 1 month old. Research complications. Cryptosporidiosis is a zoonotic disease. It is easily spread from calves to humans, for example, even as the result of simply handling clothing soiled by calf diarrhea. Adult immunocompetent humans are reported to experience watery diarrhea, cramping, flatulence, and headache. The disease can be life-threatening in immunocompromised individuals. i. Anaplasmosis Etiology. Anaplasmosis is an infectious, hemolytic, noncontagious, transmissible disease of cattle caused by the protozoan Anaplasma marginale. Anaplasma is a member of the Anaplas-matacae family within the order Rickettsiales. In sheep and goats, the disease is caused by A. ovis and is an uncommon cause of hemolytic disease. Anaplasmosis has not been reported in goats in the United States. Some controversy exists regarding the classification. Most recently it is classified as a protozoal disease because of similarities to babesiosis. It has also been classified as a rickettsial pathogen. This summary addresses the disease in cattle with limited reference to A. ovis infections, but there are many similarities to the disease in cattle. Clinical signs and diagnosis. Acute anemia is the predominant sign in anaplasmosis, and fever coincides with parasitemia. Weakness, pallor, lethargy, dehydration, and anorexia are the result of the anemia. Four disease stages—incubation, developmental, convalescent, and carrier—are recognized. The incubation stage may be long, 3–8 weeks, and is characterized by a rise in body temperature as the infection moves to the next stage. Most clinical signs occur during the 4- to 9-day developmental stage, with hemolytic anemia being common. Death is most likely to occur at this stage or at the beginning of the convalescent stage. Death may also occur from anoxia, because of the animal's inability to handle any exertion or stress, especially if treatment is initiated when severe anemia exists. Reticulocytosis characterizes the convalescent stage, which may continue for many weeks. Morbidity is high, and mortality is low. The carrier stage is defined as the time in the convalescent stage when the animal host becomes a reservoir of the disease, and Anaplasma organisms and any parasitemia are not discernible. Common serologic tests are the complement fixation test and the rapid card test. These become positive after the incubation phase and do not distinguish between the later three stages of disease. Definitive diagnosis is made by clinical and necropsy findings. Staining of thin blood smears with Wright's or Giemsa stain allows detection of basophilic, spherical A. marginale bodies near the red blood cell peripheries. Evidence will most likely be found before a hemolytic episode. A negative finding should not eliminate the pathogen from consideration. Epizootiology and transmission. The disease is common in cattle in the southern and western United States. Anaplasma organisms are spread biologically or mechanically. Mechanical transmission occurs when infected red blood cells are passed from one host to another on the mouthparts of seasonal biting flies. Sometimes mosquitoes or instruments such as dehorners or hypodermic needles may facilitate transfer of infected red cells from one animal to another. Biological transmission occurs when the tick stage of the organism is passed by Dermacentor andersoni and D. occidentalis ticks. The carrier stage covers the time when discernible Anaplasma organisms can be found on host blood smears. Recovered animals serve as immune carriers and disease reservoirs. Necropsy. Pale tissues and watery, thin blood are typical findings. Splenomegaly, hepatomegaly, and gallbladder distension are common findings. Pathogenesis. The parasites infect the host's red blood cells, and acute hemolysis occurs during the parasites' developmental stage. The four stages of the parasite's life cycle are described above because these are closely linked to the clinical stages. Differential diagnosis. The clinical disease closely resembles the protozoal disease babesiosis. Prevention and control. Offspring of immune carriers resist infection up to 6 months of age because of passive immunity. Vector control and attention to hygiene are essential, such as between-animal rinsing in disinfectant of mechanical vectors such as dehorners. There is no entirely effective means, however, to prevent and control the disease. Vaccination (killed whole organism) programs are not entirely effective, and vaccine should not be administered to pregnant cows. Neonatal isoerythrolysis may occur because of the antierythrocyte antibodies stimulated by one vaccine product. Vaccinated animals can still become infected and become carriers. The cattle vaccine has shown no efficacy in smaller ruminants, and there is no A. ovis vaccine. Identifying carriers serologically and treating with tetracycline during and/or after vector seasons may be an option. Removing carriers to a separate herd is also an approach. Interstate movement of infected animals is regulated. Treatment. Oxytetracycline, administered once, helps reduce the severity of the infection during the developmental stage. Other tetracycline treatment programs have been described to help control carriers. Etiology. Anaplasmosis is an infectious, hemolytic, noncontagious, transmissible disease of cattle caused by the protozoan Anaplasma marginale. Anaplasma is a member of the Anaplas-matacae family within the order Rickettsiales. In sheep and goats, the disease is caused by A. ovis and is an uncommon cause of hemolytic disease. Anaplasmosis has not been reported in goats in the United States. Some controversy exists regarding the classification. Most recently it is classified as a protozoal disease because of similarities to babesiosis. It has also been classified as a rickettsial pathogen. This summary addresses the disease in cattle with limited reference to A. ovis infections, but there are many similarities to the disease in cattle. Clinical signs and diagnosis. Acute anemia is the predominant sign in anaplasmosis, and fever coincides with parasitemia. Weakness, pallor, lethargy, dehydration, and anorexia are the result of the anemia. Four disease stages—incubation, developmental, convalescent, and carrier—are recognized. The incubation stage may be long, 3–8 weeks, and is characterized by a rise in body temperature as the infection moves to the next stage. Most clinical signs occur during the 4- to 9-day developmental stage, with hemolytic anemia being common. Death is most likely to occur at this stage or at the beginning of the convalescent stage. Death may also occur from anoxia, because of the animal's inability to handle any exertion or stress, especially if treatment is initiated when severe anemia exists. Reticulocytosis characterizes the convalescent stage, which may continue for many weeks. Morbidity is high, and mortality is low. The carrier stage is defined as the time in the convalescent stage when the animal host becomes a reservoir of the disease, and Anaplasma organisms and any parasitemia are not discernible. Common serologic tests are the complement fixation test and the rapid card test. These become positive after the incubation phase and do not distinguish between the later three stages of disease. Definitive diagnosis is made by clinical and necropsy findings. Staining of thin blood smears with Wright's or Giemsa stain allows detection of basophilic, spherical A. marginale bodies near the red blood cell peripheries. Evidence will most likely be found before a hemolytic episode. A negative finding should not eliminate the pathogen from consideration. Epizootiology and transmission. The disease is common in cattle in the southern and western United States. Anaplasma organisms are spread biologically or mechanically. Mechanical transmission occurs when infected red blood cells are passed from one host to another on the mouthparts of seasonal biting flies. Sometimes mosquitoes or instruments such as dehorners or hypodermic needles may facilitate transfer of infected red cells from one animal to another. Biological transmission occurs when the tick stage of the organism is passed by Dermacentor andersoni and D. occidentalis ticks. The carrier stage covers the time when discernible Anaplasma organisms can be found on host blood smears. Recovered animals serve as immune carriers and disease reservoirs. Necropsy. Pale tissues and watery, thin blood are typical findings. Splenomegaly, hepatomegaly, and gallbladder distension are common findings. Pathogenesis. The parasites infect the host's red blood cells, and acute hemolysis occurs during the parasites' developmental stage. The four stages of the parasite's life cycle are described above because these are closely linked to the clinical stages. Differential diagnosis. The clinical disease closely resembles the protozoal disease babesiosis. Prevention and control. Offspring of immune carriers resist infection up to 6 months of age because of passive immunity. Vector control and attention to hygiene are essential, such as between-animal rinsing in disinfectant of mechanical vectors such as dehorners. There is no entirely effective means, however, to prevent and control the disease. Vaccination (killed whole organism) programs are not entirely effective, and vaccine should not be administered to pregnant cows. Neonatal isoerythrolysis may occur because of the antierythrocyte antibodies stimulated by one vaccine product. Vaccinated animals can still become infected and become carriers. The cattle vaccine has shown no efficacy in smaller ruminants, and there is no A. ovis vaccine. Identifying carriers serologically and treating with tetracycline during and/or after vector seasons may be an option. Removing carriers to a separate herd is also an approach. Interstate movement of infected animals is regulated. Treatment. Oxytetracycline, administered once, helps reduce the severity of the infection during the developmental stage. Other tetracycline treatment programs have been described to help control carriers. ii. Babesiosis (red water, Texas cattle fever, cattle tick fever) Etiology. Babesia bovis and Ba. bigemina are protozoa that cause subclinical infections or disease in cattle. These are intraerythrocytic parasites. Babesia bovis is regarded as the more virulent of the two organisms. This disease is not seen in the smaller ruminants in the United States. Clinical signs and diagnosis. The more common presentation is liver and kidney failure due to hemolysis with icterus, hemoglobinuria, and fever. Hemoglobinuria indicates a poor prognosis. Acute encephalitis is a less common presentation and begins acutely with fever, ataxia, depression, deficits in conscious proprioception, mania, convulsions, and coma. The encephalitic form generally also has a poor prognosis. Sudden death may occur. Thin blood smears stained with Giemsa will show Babesia trophozoites at some stages of the disease, but lack of these cannot be interpreted as a negative. The trophozoites occur in a variety of shapes, such as piriform, round, or rod. Complement fixation, immunofluorescent antibody, and enzyme immunoassay are the most favored of the available serologic tests. Epizootiology and transmission. Babesiosis is present on several continents, including the Americas. In addition to domestic cattle, some wild ruminants, such as white-tailed deer and American buffalo, are also susceptible. Bos indicus breeds have resistance to the disease and the tick vectors. Innate resistance factors have been found in all calves. If infected, these animals will not show many signs of disease during the first year of life and will become carriers. Stress can cause disease development. Necropsy findings. Signs of acute hemolytic crisis are the most common findings, including hepatomegaly, splenomegaly, dark and distended gallbladder, pale tissues, thin blood, scattered hemorrhages, and petechiation. Animals dying after a longer course of disease will be emaciated and icteric, with thin blood, pale kidneys, and enlarged liver. Pathogenesis. The protozoon is transmitted by the cattle fever ticks Boophilus annulatus, B. microplus, and B. decoloratus; these one-host ticks acquire the protozoon from infected animals. It is passed transovarially, and both nymph and adult ticks may transmit to other cattle. Only B. ovis is transmitted by the larval stage. Clinical signs develop about 2 weeks after tick infestations or mechanical transmission but may develop sooner with the mechanical transmission. Hemolysis is due to intracellular reproduction of the parasites and occurs intra- and extravascularly. In addition to the release of merozoites, proteolytic enzymes are also released, and these contribute to the clinical metabolic acidosis and anoxia. The development of the encephalitis form is believed to be the result of direct invasion of the central nervous system, disseminated intravascular coagulation, capillary thrombosis by the parasites and infarction, and/or tissue anoxia. Differential diagnosis. In addition to anaplasmosis, other differentials for the hemolytic form of the disease are leptospirosis, chronic copper toxicity, and bacillary hemoglobinuria. Several differentials in the United States for the encephalitic presentation include rabies, nervous system coccidiosis, polioencephalomalacia, lead poisoning, infectious bovine rhinotracheitis, salt poisoning, and chlorinated hydrocarbon toxicity. Prevention and control. Control or eradication of ticks and cleaning of equipment to prevent mechanical transmission, as noted in Section III,A,3,a,i, are important preventive measures. Some vaccination approaches have been effective, but a commercial product is not available. Treatment. Supportive care is indicated, including blood transfusions, fluids, and antibiotics. Medications such as diminazene diaceturate, phenamidine diisethionate, imidocarb diprionate, or amicarbalide diisethionate are most commonly used. Treatment outcomes will be either elimination of the parasite or development of a chronic carrier state immune to further disease. Research complications. This is a reportable disease in the United States. Etiology. Babesia bovis and Ba. bigemina are protozoa that cause subclinical infections or disease in cattle. These are intraerythrocytic parasites. Babesia bovis is regarded as the more virulent of the two organisms. This disease is not seen in the smaller ruminants in the United States. Clinical signs and diagnosis. The more common presentation is liver and kidney failure due to hemolysis with icterus, hemoglobinuria, and fever. Hemoglobinuria indicates a poor prognosis. Acute encephalitis is a less common presentation and begins acutely with fever, ataxia, depression, deficits in conscious proprioception, mania, convulsions, and coma. The encephalitic form generally also has a poor prognosis. Sudden death may occur. Thin blood smears stained with Giemsa will show Babesia trophozoites at some stages of the disease, but lack of these cannot be interpreted as a negative. The trophozoites occur in a variety of shapes, such as piriform, round, or rod. Complement fixation, immunofluorescent antibody, and enzyme immunoassay are the most favored of the available serologic tests. Epizootiology and transmission. Babesiosis is present on several continents, including the Americas. In addition to domestic cattle, some wild ruminants, such as white-tailed deer and American buffalo, are also susceptible. Bos indicus breeds have resistance to the disease and the tick vectors. Innate resistance factors have been found in all calves. If infected, these animals will not show many signs of disease during the first year of life and will become carriers. Stress can cause disease development. Necropsy findings. Signs of acute hemolytic crisis are the most common findings, including hepatomegaly, splenomegaly, dark and distended gallbladder, pale tissues, thin blood, scattered hemorrhages, and petechiation. Animals dying after a longer course of disease will be emaciated and icteric, with thin blood, pale kidneys, and enlarged liver. Pathogenesis. The protozoon is transmitted by the cattle fever ticks Boophilus annulatus, B. microplus, and B. decoloratus; these one-host ticks acquire the protozoon from infected animals. It is passed transovarially, and both nymph and adult ticks may transmit to other cattle. Only B. ovis is transmitted by the larval stage. Clinical signs develop about 2 weeks after tick infestations or mechanical transmission but may develop sooner with the mechanical transmission. Hemolysis is due to intracellular reproduction of the parasites and occurs intra- and extravascularly. In addition to the release of merozoites, proteolytic enzymes are also released, and these contribute to the clinical metabolic acidosis and anoxia. The development of the encephalitis form is believed to be the result of direct invasion of the central nervous system, disseminated intravascular coagulation, capillary thrombosis by the parasites and infarction, and/or tissue anoxia. Differential diagnosis. In addition to anaplasmosis, other differentials for the hemolytic form of the disease are leptospirosis, chronic copper toxicity, and bacillary hemoglobinuria. Several differentials in the United States for the encephalitic presentation include rabies, nervous system coccidiosis, polioencephalomalacia, lead poisoning, infectious bovine rhinotracheitis, salt poisoning, and chlorinated hydrocarbon toxicity. Prevention and control. Control or eradication of ticks and cleaning of equipment to prevent mechanical transmission, as noted in Section III,A,3,a,i, are important preventive measures. Some vaccination approaches have been effective, but a commercial product is not available. Treatment. Supportive care is indicated, including blood transfusions, fluids, and antibiotics. Medications such as diminazene diaceturate, phenamidine diisethionate, imidocarb diprionate, or amicarbalide diisethionate are most commonly used. Treatment outcomes will be either elimination of the parasite or development of a chronic carrier state immune to further disease. Research complications. This is a reportable disease in the United States. iii. Coccidiosis Etiology. Coccidiosis is an important acute and chronic protozoal disease of ruminants. In young ruminants, it is characterized primarily by hemorrhagic diarrhea. Adult ruminants may carry and shed the protozoa, but they rarely display clinical signs. Intensive rearing and housing conditions and stress increase the severity of the disease in all age groups. Coccidia are protozoal organisms of the phylum Apicomplexa, members of which are obligatory intracellular parasites. There are at least 11 reported species of coccidia in sheep, of which several are considered pathogenic: Eimeria ashata, E. crandallis, and E. ovinoidalis ( Schillhorn van Veen, 1986 ). At least 9 species of Eimeria have been recognized in the goat ( Foreyt, 1990 ). Eimeria ninakohlyakimovae, E. arloingi, and E. christenseni are regarded as the most pathogenic. Eimeria bovis and E. zuernii (highly pathogenic), and E. auburnensis and E. alabamensis (moderately pathogenic), are among the 13 species known to infect cattle. Eimeria zuernii is more commonly seen in older cattle and is the agent of "winter coccidiosis." Clinical signs and diagnosis. Hemorrhagic diarrhea develops 10 days to 3 weeks after infection. Fecal staining of the tail and perineum will be present. Animals will frequently display tenesmus; rectal prolapses may also develop. Anorexia, weight loss, dehydration, anemia, fever (infrequently), depression, and weakness may also be seen in all ruminants. The diarrhea is watery and malodorous and will contain variable amounts of blood and fibrinous, necrotic tissues. The intestinal hemorrhage may subsequently lead to anemia and hypoproteinemia. Depending on the predilection of the coccidial species for small and/or large intestines, malabsorption of nutrients or water may occur, and electrolyte imbalances may be severe. Concurrent disease with other enteropathogens may also be part of the clinical picture. In sheep, secondary bacterial infection with organisms such as Fusobacterium necrophorum may ensue. Young goats may die peracutely or suffer severe anemia from blood loss into the bowel. Older goats may lose the pelleted form of feces. Cattle may have explosive diarrhea and develop anal paralysis. The disease is usually diagnosed by history and clinical signs. Numerous oocysts will frequently be observed in fresh fecal flotation (salt or sugar solution) samples as the diarrhea begins. Laboratory results are usually reported as number of oocysts per gram of feces. Coccidia seen on routine fecal evaluations reflect shedding, possibly of nonpathogenic species, without necessarily being indicative of impending or resolving mild disease. Epizootiology and transmission. As noted, coccidiosis is a common disease in young ruminants. In goats, young animals aged 3 weeks to 5 months are primarily affected, but isolated outbreaks in adults may occur after stressful conditions such as transportation or diet changes. Coccidia are host-specific and also host cell-specific. The disease is transmitted via ingestion of sporulated oocysts. Coccidial oocysts remain viable for long periods of time when in moist, shady conditions. Necropsy. Necropsies provide information on specific locations and severity of lesions that correlate with the species involved. Ileitis, typhlitis, and colitis with associated necrosis and hemorrhage will be observed. Mucosal scrapings will frequently yield oocysts. Various coccidial stages associated with schizogony or gametogony may be observed in histopathological sections of the intestines. Fibrin and cellular infiltrates will be found in the lamina propria. Pathogenesis. This parasite has a complex life cycle in which sexual and asexual reproduction occurs in gastrointestinal enterocytes ( Speer, 1996 ). The severity of the disease is correlated primarily with the number of ingested oocysts. Specifics of life cycles vary with the species, and those characteristics contribute to the pathogenicity. In most cases, the disease is well established by the time clinical signs are seen. Oocysts must undergo sporulation over a 3- to 10-day period in the environment. After ingestion of the sporulated oocysts, sporozoites are released and penetrate the intestinal mucosa and form schizonts. Schizonts initially undergo replication by fission to form merozoites and eventually undergo sexual reproduction, forming new oocysts. The organisms cause edema and hyperemia; penetration into the lamina propria may lead to necrosis of capillaries and hemorrhage. Differential diagnosis. Differential diagnoses include the many enteropathogens associated with acute diarrhea in young ruminants: cryptosporidia, colibacilli, salmonella, enterotoxins, Yersinia, viruses, and other intestinal parasites such as helminths. In cattle, for example, bovine viral diarrhea virus and helminthiasis caused by Ostergia must be considered. Management factors, such as dietary-induced diarrheas, are also differentials. In older animals, differentials in addition to stress are malnutrition, grain engorgement, and other intestinal parasitisms. Prevention and control. Good management practices will help prevent the disease. Oocysts are resistant to disinfectants but are susceptible to dry or freezing conditions. Proper sanitation of animal housing and minimizing overcrowding are essential. Coccidiostats added to the feed and water are helpful in preventing the disease in areas of high exposure. Treatment. Affected animals should be isolated. On an individual basis, treatment should also include provision of a dry, warm environment, fluids, electrolytes (orally or intravenously), antibiotics (to prevent bacterial invasion and septicemia), and administration of coccidiostats. Coccidiostats are preferred to coccidiocidals because the former allow immunity to develop. Although many coccidial infections tend to be self-limiting, sulfonamides and amprolium may be used to aid in the treatment of disease. Other anticoccidial drugs include decoquinate, lasalocid, and monensin; labels should be checked for specific approval in a species or specific indications. Animals treated with amprolium should be monitored for development of secondary polioencephalomalacia. Pen mates of affected animals should be considered exposed and should be treated to control early stages of infection. Mechanisms of immunity have not been well defined but appear to be correlated with the particular coccidial species and their characteristics (for example, the extent of intracellular penetration). Immunity may result when low numbers are ingested and there is only mild disease. Immunity also may develop after more severe infections. Etiology. Coccidiosis is an important acute and chronic protozoal disease of ruminants. In young ruminants, it is characterized primarily by hemorrhagic diarrhea. Adult ruminants may carry and shed the protozoa, but they rarely display clinical signs. Intensive rearing and housing conditions and stress increase the severity of the disease in all age groups. Coccidia are protozoal organisms of the phylum Apicomplexa, members of which are obligatory intracellular parasites. There are at least 11 reported species of coccidia in sheep, of which several are considered pathogenic: Eimeria ashata, E. crandallis, and E. ovinoidalis ( Schillhorn van Veen, 1986 ). At least 9 species of Eimeria have been recognized in the goat ( Foreyt, 1990 ). Eimeria ninakohlyakimovae, E. arloingi, and E. christenseni are regarded as the most pathogenic. Eimeria bovis and E. zuernii (highly pathogenic), and E. auburnensis and E. alabamensis (moderately pathogenic), are among the 13 species known to infect cattle. Eimeria zuernii is more commonly seen in older cattle and is the agent of "winter coccidiosis." Clinical signs and diagnosis. Hemorrhagic diarrhea develops 10 days to 3 weeks after infection. Fecal staining of the tail and perineum will be present. Animals will frequently display tenesmus; rectal prolapses may also develop. Anorexia, weight loss, dehydration, anemia, fever (infrequently), depression, and weakness may also be seen in all ruminants. The diarrhea is watery and malodorous and will contain variable amounts of blood and fibrinous, necrotic tissues. The intestinal hemorrhage may subsequently lead to anemia and hypoproteinemia. Depending on the predilection of the coccidial species for small and/or large intestines, malabsorption of nutrients or water may occur, and electrolyte imbalances may be severe. Concurrent disease with other enteropathogens may also be part of the clinical picture. In sheep, secondary bacterial infection with organisms such as Fusobacterium necrophorum may ensue. Young goats may die peracutely or suffer severe anemia from blood loss into the bowel. Older goats may lose the pelleted form of feces. Cattle may have explosive diarrhea and develop anal paralysis. The disease is usually diagnosed by history and clinical signs. Numerous oocysts will frequently be observed in fresh fecal flotation (salt or sugar solution) samples as the diarrhea begins. Laboratory results are usually reported as number of oocysts per gram of feces. Coccidia seen on routine fecal evaluations reflect shedding, possibly of nonpathogenic species, without necessarily being indicative of impending or resolving mild disease. Epizootiology and transmission. As noted, coccidiosis is a common disease in young ruminants. In goats, young animals aged 3 weeks to 5 months are primarily affected, but isolated outbreaks in adults may occur after stressful conditions such as transportation or diet changes. Coccidia are host-specific and also host cell-specific. The disease is transmitted via ingestion of sporulated oocysts. Coccidial oocysts remain viable for long periods of time when in moist, shady conditions. Necropsy. Necropsies provide information on specific locations and severity of lesions that correlate with the species involved. Ileitis, typhlitis, and colitis with associated necrosis and hemorrhage will be observed. Mucosal scrapings will frequently yield oocysts. Various coccidial stages associated with schizogony or gametogony may be observed in histopathological sections of the intestines. Fibrin and cellular infiltrates will be found in the lamina propria. Pathogenesis. This parasite has a complex life cycle in which sexual and asexual reproduction occurs in gastrointestinal enterocytes ( Speer, 1996 ). The severity of the disease is correlated primarily with the number of ingested oocysts. Specifics of life cycles vary with the species, and those characteristics contribute to the pathogenicity. In most cases, the disease is well established by the time clinical signs are seen. Oocysts must undergo sporulation over a 3- to 10-day period in the environment. After ingestion of the sporulated oocysts, sporozoites are released and penetrate the intestinal mucosa and form schizonts. Schizonts initially undergo replication by fission to form merozoites and eventually undergo sexual reproduction, forming new oocysts. The organisms cause edema and hyperemia; penetration into the lamina propria may lead to necrosis of capillaries and hemorrhage. Differential diagnosis. Differential diagnoses include the many enteropathogens associated with acute diarrhea in young ruminants: cryptosporidia, colibacilli, salmonella, enterotoxins, Yersinia, viruses, and other intestinal parasites such as helminths. In cattle, for example, bovine viral diarrhea virus and helminthiasis caused by Ostergia must be considered. Management factors, such as dietary-induced diarrheas, are also differentials. In older animals, differentials in addition to stress are malnutrition, grain engorgement, and other intestinal parasitisms. Prevention and control. Good management practices will help prevent the disease. Oocysts are resistant to disinfectants but are susceptible to dry or freezing conditions. Proper sanitation of animal housing and minimizing overcrowding are essential. Coccidiostats added to the feed and water are helpful in preventing the disease in areas of high exposure. Treatment. Affected animals should be isolated. On an individual basis, treatment should also include provision of a dry, warm environment, fluids, electrolytes (orally or intravenously), antibiotics (to prevent bacterial invasion and septicemia), and administration of coccidiostats. Coccidiostats are preferred to coccidiocidals because the former allow immunity to develop. Although many coccidial infections tend to be self-limiting, sulfonamides and amprolium may be used to aid in the treatment of disease. Other anticoccidial drugs include decoquinate, lasalocid, and monensin; labels should be checked for specific approval in a species or specific indications. Animals treated with amprolium should be monitored for development of secondary polioencephalomalacia. Pen mates of affected animals should be considered exposed and should be treated to control early stages of infection. Mechanisms of immunity have not been well defined but appear to be correlated with the particular coccidial species and their characteristics (for example, the extent of intracellular penetration). Immunity may result when low numbers are ingested and there is only mild disease. Immunity also may develop after more severe infections. iv. Cryptosporidiosis Etiology. Cryptosporidium organisms are a very common cause of diarrhea in young ruminants. Four Cryptosporidium species have been described in vertebrates: C. baileyi and C. meleagridis in birds and C. parvum and C. muris in mammals. Cryptosporidium parvum is the species affecting sheep ( Rings and Rings, 1996 ). Debate continues regarding whether there are definite host-specific variants. Clinical signs and diagnosis. Cryptosporidiosis is characterized by protracted, watery diarrhea and debilitation. The diarrhea may last only 6–10 days or may be persistent and fatal. The diarrhea is watery and yellow, and blood, mucus, bile, and undigested milk may also be present. Infected animals will display tenesmus, anorexia and weight loss, dehydration, and depression. In relapsing cases, animals become cachectic. Overall, morbidity will be high, and mortality variable. Mucosal scrapings or fixed stained tissue sections may be useful in diagnosis. The disease is also diagnosed by detecting the oocysts in iodine-stained feces or in tissues stained with periodic acid-Schiff stain or methenamine silver. Cryptosporidium also stains red on acid-fast stains such as Kinyoun or Ziehl-Neelsen. Fecal flotations should be performed without sugar solutions or with sugar solutions at specific gravity of 1.27 (Foryet, 1990). Fecal immunofluorescent antibody (IFA) techniques have also been described. Epizootiology and transmission. Younger ruminants are commonly affected: lambs, kids (especially kids between the ages of 5 and 10 days old), and calves less than 30 days old. Like other coccidians, Cryptosporidium is transmitted via the fecal-oral route. In addition to local contamination, water supplies have also been sources of the infecting oocysts. The oocysts are extremely resistant to desiccation in the environment and may survive in the soil and manure for many months. Necropsy findings. The lesions caused by Cryptosporidium are nonspecific. Animals will be emaciated. Moderate enteritis and hyperplasia of the crypt epithelial cells with villous atrophy as well as villous fusion, primarily in the lower small intestines, will be present. Cecal and colonic mucosae may sometimes be involved. Gastrointestinal smears may be made at necropsy and stained as described above. Pathogenesis. Although Cryptosporidium infections are clinically similar to Eimeria infections ( Moore, 1989 ), Cryptosporidium, in contrast to Eimeria, invades just under the surface but does not invade the cytoplasm of enterocytes. There is no intermediate host. The oocysts are half the size of Eimeria oocysts and are shed sporulated; they are, therefore, immediately infective. Within 2–7 days of exposure, diarrhea and oocyst shedding occur. The diarrhea is the result of malabsorption and, in younger animals, intraluminal milk fermentation. Autoinfection within the lumen of the intestines may also occur and result in persistent infections. In addition, several other pathogens may be involved, such as concurrent coronavirus and rotavirus infections in calves. Environmental stressors such as cold weather increase mortality. Intensive housing arrangements increase morbidity and mortality. Differential diagnosis. Other causes of diarrhea in younger ruminants include rotavirus, coronavirus, and other enteric viral infections; enterotoxigenic Escherichia coli; Clostridium; other coccidial pathogens; and dietary causes (inappropriate use of milk replacers). In addition, these other agents may also be causing illness in the affected animals and may complicate the diagnosis and the treatment picture. Eimeria is more likely to cause diarrhea in calves and lambs at 3–4 weeks of age. Giardia organisms may be seen in fecal preparations from young ruminants but are not considered to play a significant role in enteric disease. Prevention and control. Precautions should be taken when handling infected animals. Affected animals must be removed and isolated as soon as possible. Animal housing areas should be disinfected with undiluted commercial bleach or 5% ammonia. Formalin (10%) fumigation has proven successful (Foryet, 1990). After being cleaned, areas should be allowed to dry thoroughly and should remain unpopulated for a period of time. Because enteric disease often is multifactorial, other pathogens should also be considered, and management and husbandry should be examined. Treatment. No known drug treatment is available. The disease is generally self-limiting, so symptomatic, supportive therapy aimed at rehydrating, correcting electrolyte and acid-base balance, and providing energy is often effective. Supplementation with vitamin A may be helpful. Age resistance begins to develop when the animals are about 1 month old. Research complications. Cryptosporidiosis is a zoonotic disease. It is easily spread from calves to humans, for example, even as the result of simply handling clothing soiled by calf diarrhea. Adult immunocompetent humans are reported to experience watery diarrhea, cramping, flatulence, and headache. The disease can be life-threatening in immunocompromised individuals. Etiology. Cryptosporidium organisms are a very common cause of diarrhea in young ruminants. Four Cryptosporidium species have been described in vertebrates: C. baileyi and C. meleagridis in birds and C. parvum and C. muris in mammals. Cryptosporidium parvum is the species affecting sheep ( Rings and Rings, 1996 ). Debate continues regarding whether there are definite host-specific variants. Clinical signs and diagnosis. Cryptosporidiosis is characterized by protracted, watery diarrhea and debilitation. The diarrhea may last only 6–10 days or may be persistent and fatal. The diarrhea is watery and yellow, and blood, mucus, bile, and undigested milk may also be present. Infected animals will display tenesmus, anorexia and weight loss, dehydration, and depression. In relapsing cases, animals become cachectic. Overall, morbidity will be high, and mortality variable. Mucosal scrapings or fixed stained tissue sections may be useful in diagnosis. The disease is also diagnosed by detecting the oocysts in iodine-stained feces or in tissues stained with periodic acid-Schiff stain or methenamine silver. Cryptosporidium also stains red on acid-fast stains such as Kinyoun or Ziehl-Neelsen. Fecal flotations should be performed without sugar solutions or with sugar solutions at specific gravity of 1.27 (Foryet, 1990). Fecal immunofluorescent antibody (IFA) techniques have also been described. Epizootiology and transmission. Younger ruminants are commonly affected: lambs, kids (especially kids between the ages of 5 and 10 days old), and calves less than 30 days old. Like other coccidians, Cryptosporidium is transmitted via the fecal-oral route. In addition to local contamination, water supplies have also been sources of the infecting oocysts. The oocysts are extremely resistant to desiccation in the environment and may survive in the soil and manure for many months. Necropsy findings. The lesions caused by Cryptosporidium are nonspecific. Animals will be emaciated. Moderate enteritis and hyperplasia of the crypt epithelial cells with villous atrophy as well as villous fusion, primarily in the lower small intestines, will be present. Cecal and colonic mucosae may sometimes be involved. Gastrointestinal smears may be made at necropsy and stained as described above. Pathogenesis. Although Cryptosporidium infections are clinically similar to Eimeria infections ( Moore, 1989 ), Cryptosporidium, in contrast to Eimeria, invades just under the surface but does not invade the cytoplasm of enterocytes. There is no intermediate host. The oocysts are half the size of Eimeria oocysts and are shed sporulated; they are, therefore, immediately infective. Within 2–7 days of exposure, diarrhea and oocyst shedding occur. The diarrhea is the result of malabsorption and, in younger animals, intraluminal milk fermentation. Autoinfection within the lumen of the intestines may also occur and result in persistent infections. In addition, several other pathogens may be involved, such as concurrent coronavirus and rotavirus infections in calves. Environmental stressors such as cold weather increase mortality. Intensive housing arrangements increase morbidity and mortality. Differential diagnosis. Other causes of diarrhea in younger ruminants include rotavirus, coronavirus, and other enteric viral infections; enterotoxigenic Escherichia coli; Clostridium; other coccidial pathogens; and dietary causes (inappropriate use of milk replacers). In addition, these other agents may also be causing illness in the affected animals and may complicate the diagnosis and the treatment picture. Eimeria is more likely to cause diarrhea in calves and lambs at 3–4 weeks of age. Giardia organisms may be seen in fecal preparations from young ruminants but are not considered to play a significant role in enteric disease. Prevention and control. Precautions should be taken when handling infected animals. Affected animals must be removed and isolated as soon as possible. Animal housing areas should be disinfected with undiluted commercial bleach or 5% ammonia. Formalin (10%) fumigation has proven successful (Foryet, 1990). After being cleaned, areas should be allowed to dry thoroughly and should remain unpopulated for a period of time. Because enteric disease often is multifactorial, other pathogens should also be considered, and management and husbandry should be examined. Treatment. No known drug treatment is available. The disease is generally self-limiting, so symptomatic, supportive therapy aimed at rehydrating, correcting electrolyte and acid-base balance, and providing energy is often effective. Supplementation with vitamin A may be helpful. Age resistance begins to develop when the animals are about 1 month old. Research complications. Cryptosporidiosis is a zoonotic disease. It is easily spread from calves to humans, for example, even as the result of simply handling clothing soiled by calf diarrhea. Adult immunocompetent humans are reported to experience watery diarrhea, cramping, flatulence, and headache. The disease can be life-threatening in immunocompromised individuals. v. Giardiasis Etiology. Giardia lamblia (also called G. intestinalis and G. duodenalis) is a flagellate protozoon. Giardiasis is a worldwide protozoal-induced diarrheal disease of mammals and some birds ( Kirkpatrick, 1989 ), but it not considered to be a significant pathogen in ruminants. Clinical signs and diagnosis. Diarrhea may be continuous or intermittent, is pasty to watery, is yellow, and may contain blood. Animals exhibit fever, dehydration, and depression. Chronic cases may result in a "poor doer" syndrome with weight loss and unthriftiness. Giardia can be diagnosed by identifying the motile piriform trophozoites in fresh fecal mounts. Oval cysts can be floated with zinc sulfate solution (33%). Standard solutions tend to be too hyperosmotic and to distort the cysts. Newer enzyme-linked immunosorbent assay (ELISA) and IFA tests are sensitive and specific. Epizootiology and transmission. Giardia infection may occur at any age, but young animals are predisposed. Chronic oocyst shedding is common. Transmission of the cyst stage is fecal-oral. Wild animals may serve as reservoirs. Necropsy findings. Gross lesions may not be evident. Villous atrophy and cuboidal enterocytes may be evident histologically. Pathogenesis. Following ingestion, each Giardia cyst releases four trophozoites, which attach to the enterocytes of the duodenum and proximal jejunum and subsequently divide by binary fission or encyst. The organism causes little intestinal pathology, and the cause of diarrhea is unknown but is thought to be related to disruption of digestive enzyme function, leading to malabsorption. Disturbances in intestinal motility may also occur ( Rings and Rings, 1996 ). Prevention and control. Intensive housing and warm environments should be minimized. Cysts can survive in the environment for long periods of time but are susceptible to desiccation. Effective disinfectants include quaternary ammonium compounds, bleach-water solution (1:16 or 1:32), steam, or boiling water. After cleaning, areas should be left empty and allowed to dry completely. Treatment. Giardia has been successfully treated with oral metronidazole. Benzimidazole anthelmintics are also effective, but these are not approved for use in animals for this purpose. Research complications. Giardia is zoonotic. Precautions should be taken when handling infected animals. vi. Neosporosis Etiology. Neosporosis is a common, worldwide cause of bovine abortion caused by the protozoal species Neospora caninum. Abortions have also been reported in sheep and goats. Neonatal disease is seen in lambs, kids, and calves. Until 1988, these infections were misdiagnosed as caused by Toxoplasma gondii. Some similarities exist between the life cycles and pathogeneses of both organisms. Clinical signs and diagnosis. Abortion is the only clinical sign seen in adult cattle and occurs sporadically, endemically, or as abortion storms. Bovine abortions occur between the third and seventh month of gestation; fetal age at abortion correlates with the parity of the dam as well as with pattern of abortion in the herd. Although cows that abort tend to be culled after the first or second abortion, repeated N. caninum- caused abortions will occur progressively later in gestation (up to about 6 months) and within a shorter time frame in the same cow ( Thurmond and Hietala, 1997 ). Although infections in adults are asymptomatic other than the abortions, decreased milk production has been noted in congenitally infected cows. Many Neospo ra-infected calves will be born asymptomatic. Weakness will be evident in some infected calves, but this resolves. Rare clinical signs include exophthalmos or asymmetric eyes, weight loss, ataxia, hyperflexion or hyperextension of all limbs, decreased patellar reflexes, and loss of conscious proprioception. Some fetal deaths will occur, and resorption, mummification, autolysis, or stillbirth will follow. Immunohistochemistry and histopathology of fetal tissue are the most efficient and reliable means of establishing a postmortem diagnosis. Serology (IFA and ELISA) is useful, including precolostral levels in weak neonates, but this indicates only exposure. Titers of dams will not be elevated at the time of abortion; fetal serology is influenced by the stage of gestation and course of infection. Earlier and rapid infections are less likely to yield antibodies against Neospora. None of the currently available tests is predictive of disease. Epizootiology and transmission. The parasite is now acknowledged to be widespread in dairy and cattle herds. The life cycle of N. caninum is complex, and many aspects remain to be clarified. The definitive host is the dog ( McAllister et al., 1998 ). Placental or aborted tissues are the most likely sources of infection for the definitive host and play a minor role in transmission to the intermediate hosts. The many intermediate hosts include ruminants, deer, and horses. Transplacental transmission is the major mode of transmission in dairy cattle and is the means by which a herd's infection is perpetuated. A less significant mode of transmission is by ingestion of oocysts, which sporulate in the environment or in the intermediate host's body. Reactivation in a chronically infected animal's body is the result of rupture of tissue cysts in neural tissue. Seropositive immunity does not protect a cow from future abortions. Many seropositive cows and calves will never abort or show clinical signs, respectively. Some immunological cross-reactivity may exist among Neospora, Cryptosporidia, and Coccidium. Necropsy findings. Aborted fetuses will usually be autolysed. In those from which tissue can be recovered, tissue cysts are most commonly found in the brain. Spinal cord is also useful. Histological lesions include mild to moderate gliosis, nonsuppurative encephalitis, and perivascular infiltration by mixed mononuclear cells. Pathogenesis. As with Toxoplasma, cell death is the result of intracellular multiplication of Neospora tachyzoites. Neospora undergoes sexual replication in the dog's intestinal tract, and oocysts are shed in the feces. The intermediate hosts develop nonclinical systemic infections, with tachyzoites in several organs, and parasites then localize and become encysted in particular tissues, especially the brain. Infections of this type are latent and lifelong. Except when immunocompromised, most cattle do not usually develop clinical signs and do not have fetal loss. Fetuses become infected, leading to fetal death, mid-gestation abortions, or live calves with latent infections or congenital brain disease. It usually takes 2–4 weeks for a fetus to die and to be expelled. Many aspects of the role of the maternal immune response and pregnancy-associated immunodeficiency in the patterns of Neospora abortions remain to be elucidated. Differential diagnosis. Even when there is a herd history of confirmed Neospora abortions, leptospirosis, bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), salmonellosis, and campylobacteriosis should be considered. BVDV in particular should be considered for abortion storms. Differentials for weak calves are BVDV, perinatal hypoxia following dystocia (immediate postpartum time), bluetongue virus, Toxoplasma, exposure to teratogens, or congenital defects. Prevention and control. The primary preventive measure is preventing contact with contaminated feces. Oocysts will not survive dry environments or extremes of temperature. Dog populations should be controlled, and dogs and other canids should not have access to placentas or aborted fetuses. Dogs should also be restricted from feed bunks and other feed storage areas. Preventive culling is not economically practical for most producers. A vaccine recently became available. If embryo transfer is practiced, recipients should be screened serologically before use. Treatment. There is no known treatment or immunoprophylaxis. vii. Sarcocystosis Etiology. Sarcocystosis is the disease caused by the cyst-forming sporozoon Sarcocystis. Sarcocystis capricanus, S. ovi-canus, and S. tenella are the species that infect sheep and goats. Sarcocystis cruzi, S. hirsuta, and S. hominis are the species that infect cattle. Definitive hosts are carnivores, and all ruminant species are intermediate hosts. Clinical signs and diagnosis. Clinical signs of sarcocystosis infection are seen in cattle during the stage when the parasite encysts in soft tissues. Often the infections are asymptomatic. Fever, anemia, ataxia, symmetric lameness, tremors, tail-switch hair loss, excessive salivation, diarrhea, and weight loss are clinical signs. Abortions in cattle occur during the second trimester and in smaller ruminants 28 days after ingestion of the sporulated oocysts. Definitive diagnosis is based on finding merozoites and meronts in neural tissue lesions. Clinical hematology results include decreased hematocrit, decreased serum protein, and prolonged prothrombin times. Sarcocystis-specific IgG will increase dramatically by 5–6 weeks after infection. There is no cross-reaction between Sarcocystis and Toxoplasma. Epizootiology and transmission. Infection rates among cattle in the United States are estimated to be very high. Transmission is by ingestion of feed and water contaminated by feces of the definitive hosts. Dogs are the definitive hosts for the species that infect the smaller ruminants. Cats, dogs, and primates (including humans when S. hominis is involved) are the definitive hosts for the species that infect cattle. Necropsy. Aborted fetuses may be autolysed. Lesions in neural tissues, including meningoencephalomyelitis, focal malacia, perivascular cuffing, neuronal degeneration, and gliosis, are most marked in the cerebellum and midbrain. Lesions may be found in other tissues, such as lymphadenopathy, and hemorrhages may be found in muscles and on serous surfaces. Cysts in cardiac and skeletal muscles are common incidental findings during necropsies. Pathogenesis. Ingestion of muscle flesh from an infected ruminant results in Sarcocystis cysts' being broken down in the carnivore's digestive system, release of bradyzoites, infection of intestinal mucosal cells by the bradyzoites, differentiation into sexual stages, fusion of the male and female gametes to form oocysts, and shedding as sporocysts by the definitive hosts. The sporocysts are eaten by the ruminant and penetrate the bowel walls; several stages of development occur in endothelial cells of arteries. Merozoites are the form that enters soft tissues, such as muscle, and subsequently encysts. Prevention and control. Feed supplies of ruminants must be protected from fecal contamination by domestic and wild carnivores. These animals should be controlled and must also not have access to carcasses. In larger production situations, monensin may be fed as a prophylactic measure. Treatment. Monensin fed during incubation is prophylactic, but the efficacy in clinically affected cattle is not known. viii. Toxoplasmosis Etiology. Toxoplasmosis is caused by the obligate intracellular protozoon Toxoplasma gondii, a coccidial parasite of the family Eimeridae. Cats are the only definitive hosts, and several warm-blooded animals, including ruminants, have been shown to be intermediate hosts. The disease is a major cause of abortion in sheep and goats and less common in cattle. Clinical signs and diagnosis. Clinical signs depend on the organ or tissue parasitized. Toxoplasmosis is typically associated with placentitis, abortion, stillbirths, or birth of weak young ( Underwood and Rook, 1992 ; Buxton, 1998 ). It has also been shown to cause pneumonia and nonsuppurative encephalitis. The enteritis at the early stage of infection may be fatal in some hosts. Hydrocephalus does not occur in animals as it does in human fetal Toxoplasma infections. Rare clinical presentations in ruminants include retinitis and chorioretinitis; these are usually asymptomatic. Infection of the ewe during the first trimester usually leads to fetal resorption, during the second trimester leads to abortion, and during the third trimester leads to birth of weak to normal lambs with subsequent high perinatal mortality. Congenitally infected lambs may display encephalitic signs of circling, incoordination, muscular paresis, and prostration. In sheep, weak young will develop normally if they survive the first week after birth. Infected adult sheep show no systemic illness. Infected adult goats, however, may die. Diagnosis may be difficult, and biological, serological, and histological methods are helpful. Serological tests are the most readily available. Complement fixation and the Sabin-Feldman antibody test may assist in diagnosis. Antibodies found in fetuses are indicative of congenital infection and are typically detectable 35 days after infection; fetal thoracic fluid is especially useful in demonstrating serological evidence of exposure. Biological methods, such as tissue culture or inoculation of mice with maternal body fluids, or with postmortem or necropsy tissues, are more time-consuming and expensive. Epizootiology and transmission. This protozoon is considered ubiquitous. Fifty percent (50%) of adult western sheep and 20% of feedlot lambs have positive hemagglutination titers (1:64 or higher) ( Jensen and Swift, 1982 ). Transmission among the definitive host is by ingestion of tissue cysts. Necropsy findings. At necropsy, placental cotyledons contain multiple small white areas that are sites of necrosis, edema, and calcification. Fetal brains may show nonspecific lesions such as coagulative necrosis, nonsuppurative encephalomyelitis, pneumonia, myocarditis, and hepatitis. Histologically, granulomas with Toxoplasma organisms may be seen in the retina, myocardium, liver, kidney, brain, and other tissues. Impression smears of these tissues, stained appropriately (e.g., with Giemsa), provide a rapid means of diagnosis. Identification of the organism in tissue sections (especially of the heart and the brain) also confirms the findings. Toxoplasma gondii is crescent-shaped, with a clearly visible nuclei, and will be found within macrophages. Pathogenesis. The protozoon has three infectious stages: the tachyzoite, the bradyzoite, and the sporozoite within the oocyst. The definitive hosts, felids, become infected by ingesting cyst stages in mammalian tissues, by ingesting oocysts in feces, and by transplacental transfer. Ingested zoites invade epithelial cells and eventually undergo sexual reproduction, resulting in new oocysts, which the cats will shed in the feces. Cats rarely show clinical signs of infection. One cat can shed millions of oocysts in 1 gm of feces, but the asymptomatic shedding takes place for only a few weeks in its life. Oocysts sporulate in cat feces after 1 day. Ruminants are intermediate hosts of toxoplasmosis and become infected by ingesting sporulated oocyst-contaminated water or feed. As in the definitive host, the ingested sporozoite invades epithelial cells within the intestine but also further invades the bloodstream and is transported throughout the host. The organism migrates to tissues such as the brain, liver, muscles, and placenta. Placental infection develops about 14 days after ingestion of the oocysts. The damage caused by an infection is due to multiplication within cells. Toxoplasma does not produce any toxin. Differential diagnosis. Differentials for abortion include Campylobacter, Chlamydia, and Q fever. Prevention and control. Feline populations on source farms should be controlled. Eliminating contamination of feed and water with cat feces is the best preventive measure. Sporulated oocysts can survive in soil and other places for long periods of time and are resistant to desiccation and freezing. Vaccines for abortion prevention in sheep are available in New Zealand and Europe. Treatment. Toxoplasmosis treatment is ineffective, although feeding monensin during pregnancy may be helpful ( Underwood and Rook, 1992 ). (Monensin is not approved for this use in the Unites States.) Weak lambs that survive the first week after birth will mature normally and will not deliver Toxoplasma- infected young. Research complications. Because toxoplasmosis is zoonotic, precautions must be taken when handling tissues from any abortions or neurological cases. Infections in immunocompromised humans have been fatal. ix. Trichomoniasis Etiology. Trichomoniasis is an insidious venereal disease of cattle caused by Tritrichomonas (also referred to as Trichomonas) fetus, a large, pear-shaped, flagellated protozoon. The organism is an obligate parasite of the reproductive tract, and it requires a microaerophilic environment to establish chronic infections. In the United States, it is now primarily a disease seen in western beef herds. There are many similarities between trichomoniasis and campylobacteriosis; both diseases cause herd infertility problems. Clinical signs and diagnosis. Clinical signs include infertility manifested by high nonpregnancy rates as well as periodic pyometras and abortions during the first half of gestation. Often the problem is not recognized until herd pregnancy checks indicate many "open," delayed-estrus, late-bred cows, or cows with postcoital pyometras. The abortion rate varies from 5% to 30%, and placentas will be expelled or retained. Tritrichomonas fetus also causes mild salpingitis but this does not result in permanent damage. Other than these manifestations, infection with T. fetus causes no systemic signs. Diagnosis is based on patterns of infertility and pyometras. For example, pyometras in postcoital heifers or cows are suggestive of this pathogen. Diagnostic methods include identifying or culturing the trichomonads from preputial smegma, cervicovaginal mucus, uterine exudates, placental fluids, or abomasal contents of aborted fetuses. Other nonpathogenic protozoa from fecal contamination may be present in the sample. The trichomonad has three anterior flagellae, one posterior flagella, and an undulating membrane; it travels in fluids with a characteristic jerky movement. Culturing must be done on specific media, such as Diamond's or modified Pastridge. Epizootiology and transmission. All transmission is by venereal exposure from breeding bulls or cows or, in some cases, contaminated breeding equipment. Necropsy findings. Nonspecific lesions, such as pyogranulomatous bronchopneumonia of fetuses and placentitis, may be seen in aborted material; some cases will have no gross lesions. Histologically, trichomonads may be visible in the fetal lung lesions and the placenta; those tissues are also the most useful for culturing. Pathogenesis. Tritrichomonas fetus colonizes the female reproductive tract, and subsequent clinical manifestations may be related to the size of the initial infecting dose. Tritrichomonas fetus does not interfere with conception. Embryonic death occurs within the first 2 months of infection. Affected cows will clear the infection over a span of months and maintain immunity for about 6 months. Infections in younger bulls are transient; apparently organisms are cleared by the bulls' immune systems and are dependent on exposure to infected females. Older bulls become chronic carriers, probably because of the ability of T. fetus to colonize deeper epithelial crypts of the prepuce and penis. Differential diagnosis. Campylobacteriosis is the other primary differential for reduced reproductive efficiency of a herd. Other venereal diseases should be considered when infertility problems are noted in a herd: brucellosis, mycoplasmosis, ureaplasmosis, and infectious pustular vulvovaginitis. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. A bacterin vaccine is available. Heifers, cows, and breeding bulls are vaccinated subcutaneously twice at 2 to 4 week intervals, with the booster dose administered 4 weeks before breeding season starts. Similar timing is recommended for administration of the annual booster; a long, anamnestic response does not occur. Bulls used for artificial insemination (AI) are screened routinely for T. fetus (and Campylobacter). AI reduces but does not eliminate the disease. The use of younger, vaccinated bulls is recommended in all circumstances. New animals should be tested before introduction to the herd. Control measures also include culling affected cows or else removing them from the breeding herd for 3 months to rest and clear the infection. Culling chronically infected bulls is strongly recommended. Treatment. Imidazole compounds have been effective, but the use of these is not permitted in food animals in the United States. Therapeutic immunizations are worthwhile when a positive diagnosis has been made. These will not curtail fetal losses but will shorten the convalescence of the affected cows and improve immunity of breeding bulls. Research complications. Trichomoniasis should be considered whenever natural service is used and fertility problems are encountered. Etiology. Giardia lamblia (also called G. intestinalis and G. duodenalis) is a flagellate protozoon. Giardiasis is a worldwide protozoal-induced diarrheal disease of mammals and some birds ( Kirkpatrick, 1989 ), but it not considered to be a significant pathogen in ruminants. Clinical signs and diagnosis. Diarrhea may be continuous or intermittent, is pasty to watery, is yellow, and may contain blood. Animals exhibit fever, dehydration, and depression. Chronic cases may result in a "poor doer" syndrome with weight loss and unthriftiness. Giardia can be diagnosed by identifying the motile piriform trophozoites in fresh fecal mounts. Oval cysts can be floated with zinc sulfate solution (33%). Standard solutions tend to be too hyperosmotic and to distort the cysts. Newer enzyme-linked immunosorbent assay (ELISA) and IFA tests are sensitive and specific. Epizootiology and transmission. Giardia infection may occur at any age, but young animals are predisposed. Chronic oocyst shedding is common. Transmission of the cyst stage is fecal-oral. Wild animals may serve as reservoirs. Necropsy findings. Gross lesions may not be evident. Villous atrophy and cuboidal enterocytes may be evident histologically. Pathogenesis. Following ingestion, each Giardia cyst releases four trophozoites, which attach to the enterocytes of the duodenum and proximal jejunum and subsequently divide by binary fission or encyst. The organism causes little intestinal pathology, and the cause of diarrhea is unknown but is thought to be related to disruption of digestive enzyme function, leading to malabsorption. Disturbances in intestinal motility may also occur ( Rings and Rings, 1996 ). Prevention and control. Intensive housing and warm environments should be minimized. Cysts can survive in the environment for long periods of time but are susceptible to desiccation. Effective disinfectants include quaternary ammonium compounds, bleach-water solution (1:16 or 1:32), steam, or boiling water. After cleaning, areas should be left empty and allowed to dry completely. Treatment. Giardia has been successfully treated with oral metronidazole. Benzimidazole anthelmintics are also effective, but these are not approved for use in animals for this purpose. Research complications. Giardia is zoonotic. Precautions should be taken when handling infected animals. Etiology. Giardia lamblia (also called G. intestinalis and G. duodenalis) is a flagellate protozoon. Giardiasis is a worldwide protozoal-induced diarrheal disease of mammals and some birds ( Kirkpatrick, 1989 ), but it not considered to be a significant pathogen in ruminants. Clinical signs and diagnosis. Diarrhea may be continuous or intermittent, is pasty to watery, is yellow, and may contain blood. Animals exhibit fever, dehydration, and depression. Chronic cases may result in a "poor doer" syndrome with weight loss and unthriftiness. Giardia can be diagnosed by identifying the motile piriform trophozoites in fresh fecal mounts. Oval cysts can be floated with zinc sulfate solution (33%). Standard solutions tend to be too hyperosmotic and to distort the cysts. Newer enzyme-linked immunosorbent assay (ELISA) and IFA tests are sensitive and specific. Epizootiology and transmission. Giardia infection may occur at any age, but young animals are predisposed. Chronic oocyst shedding is common. Transmission of the cyst stage is fecal-oral. Wild animals may serve as reservoirs. Necropsy findings. Gross lesions may not be evident. Villous atrophy and cuboidal enterocytes may be evident histologically. Pathogenesis. Following ingestion, each Giardia cyst releases four trophozoites, which attach to the enterocytes of the duodenum and proximal jejunum and subsequently divide by binary fission or encyst. The organism causes little intestinal pathology, and the cause of diarrhea is unknown but is thought to be related to disruption of digestive enzyme function, leading to malabsorption. Disturbances in intestinal motility may also occur ( Rings and Rings, 1996 ). Prevention and control. Intensive housing and warm environments should be minimized. Cysts can survive in the environment for long periods of time but are susceptible to desiccation. Effective disinfectants include quaternary ammonium compounds, bleach-water solution (1:16 or 1:32), steam, or boiling water. After cleaning, areas should be left empty and allowed to dry completely. Treatment. Giardia has been successfully treated with oral metronidazole. Benzimidazole anthelmintics are also effective, but these are not approved for use in animals for this purpose. Research complications. Giardia is zoonotic. Precautions should be taken when handling infected animals. vi. Neosporosis Etiology. Neosporosis is a common, worldwide cause of bovine abortion caused by the protozoal species Neospora caninum. Abortions have also been reported in sheep and goats. Neonatal disease is seen in lambs, kids, and calves. Until 1988, these infections were misdiagnosed as caused by Toxoplasma gondii. Some similarities exist between the life cycles and pathogeneses of both organisms. Clinical signs and diagnosis. Abortion is the only clinical sign seen in adult cattle and occurs sporadically, endemically, or as abortion storms. Bovine abortions occur between the third and seventh month of gestation; fetal age at abortion correlates with the parity of the dam as well as with pattern of abortion in the herd. Although cows that abort tend to be culled after the first or second abortion, repeated N. caninum- caused abortions will occur progressively later in gestation (up to about 6 months) and within a shorter time frame in the same cow ( Thurmond and Hietala, 1997 ). Although infections in adults are asymptomatic other than the abortions, decreased milk production has been noted in congenitally infected cows. Many Neospo ra-infected calves will be born asymptomatic. Weakness will be evident in some infected calves, but this resolves. Rare clinical signs include exophthalmos or asymmetric eyes, weight loss, ataxia, hyperflexion or hyperextension of all limbs, decreased patellar reflexes, and loss of conscious proprioception. Some fetal deaths will occur, and resorption, mummification, autolysis, or stillbirth will follow. Immunohistochemistry and histopathology of fetal tissue are the most efficient and reliable means of establishing a postmortem diagnosis. Serology (IFA and ELISA) is useful, including precolostral levels in weak neonates, but this indicates only exposure. Titers of dams will not be elevated at the time of abortion; fetal serology is influenced by the stage of gestation and course of infection. Earlier and rapid infections are less likely to yield antibodies against Neospora. None of the currently available tests is predictive of disease. Epizootiology and transmission. The parasite is now acknowledged to be widespread in dairy and cattle herds. The life cycle of N. caninum is complex, and many aspects remain to be clarified. The definitive host is the dog ( McAllister et al., 1998 ). Placental or aborted tissues are the most likely sources of infection for the definitive host and play a minor role in transmission to the intermediate hosts. The many intermediate hosts include ruminants, deer, and horses. Transplacental transmission is the major mode of transmission in dairy cattle and is the means by which a herd's infection is perpetuated. A less significant mode of transmission is by ingestion of oocysts, which sporulate in the environment or in the intermediate host's body. Reactivation in a chronically infected animal's body is the result of rupture of tissue cysts in neural tissue. Seropositive immunity does not protect a cow from future abortions. Many seropositive cows and calves will never abort or show clinical signs, respectively. Some immunological cross-reactivity may exist among Neospora, Cryptosporidia, and Coccidium. Necropsy findings. Aborted fetuses will usually be autolysed. In those from which tissue can be recovered, tissue cysts are most commonly found in the brain. Spinal cord is also useful. Histological lesions include mild to moderate gliosis, nonsuppurative encephalitis, and perivascular infiltration by mixed mononuclear cells. Pathogenesis. As with Toxoplasma, cell death is the result of intracellular multiplication of Neospora tachyzoites. Neospora undergoes sexual replication in the dog's intestinal tract, and oocysts are shed in the feces. The intermediate hosts develop nonclinical systemic infections, with tachyzoites in several organs, and parasites then localize and become encysted in particular tissues, especially the brain. Infections of this type are latent and lifelong. Except when immunocompromised, most cattle do not usually develop clinical signs and do not have fetal loss. Fetuses become infected, leading to fetal death, mid-gestation abortions, or live calves with latent infections or congenital brain disease. It usually takes 2–4 weeks for a fetus to die and to be expelled. Many aspects of the role of the maternal immune response and pregnancy-associated immunodeficiency in the patterns of Neospora abortions remain to be elucidated. Differential diagnosis. Even when there is a herd history of confirmed Neospora abortions, leptospirosis, bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), salmonellosis, and campylobacteriosis should be considered. BVDV in particular should be considered for abortion storms. Differentials for weak calves are BVDV, perinatal hypoxia following dystocia (immediate postpartum time), bluetongue virus, Toxoplasma, exposure to teratogens, or congenital defects. Prevention and control. The primary preventive measure is preventing contact with contaminated feces. Oocysts will not survive dry environments or extremes of temperature. Dog populations should be controlled, and dogs and other canids should not have access to placentas or aborted fetuses. Dogs should also be restricted from feed bunks and other feed storage areas. Preventive culling is not economically practical for most producers. A vaccine recently became available. If embryo transfer is practiced, recipients should be screened serologically before use. Treatment. There is no known treatment or immunoprophylaxis. Etiology. Neosporosis is a common, worldwide cause of bovine abortion caused by the protozoal species Neospora caninum. Abortions have also been reported in sheep and goats. Neonatal disease is seen in lambs, kids, and calves. Until 1988, these infections were misdiagnosed as caused by Toxoplasma gondii. Some similarities exist between the life cycles and pathogeneses of both organisms. Clinical signs and diagnosis. Abortion is the only clinical sign seen in adult cattle and occurs sporadically, endemically, or as abortion storms. Bovine abortions occur between the third and seventh month of gestation; fetal age at abortion correlates with the parity of the dam as well as with pattern of abortion in the herd. Although cows that abort tend to be culled after the first or second abortion, repeated N. caninum- caused abortions will occur progressively later in gestation (up to about 6 months) and within a shorter time frame in the same cow ( Thurmond and Hietala, 1997 ). Although infections in adults are asymptomatic other than the abortions, decreased milk production has been noted in congenitally infected cows. Many Neospo ra-infected calves will be born asymptomatic. Weakness will be evident in some infected calves, but this resolves. Rare clinical signs include exophthalmos or asymmetric eyes, weight loss, ataxia, hyperflexion or hyperextension of all limbs, decreased patellar reflexes, and loss of conscious proprioception. Some fetal deaths will occur, and resorption, mummification, autolysis, or stillbirth will follow. Immunohistochemistry and histopathology of fetal tissue are the most efficient and reliable means of establishing a postmortem diagnosis. Serology (IFA and ELISA) is useful, including precolostral levels in weak neonates, but this indicates only exposure. Titers of dams will not be elevated at the time of abortion; fetal serology is influenced by the stage of gestation and course of infection. Earlier and rapid infections are less likely to yield antibodies against Neospora. None of the currently available tests is predictive of disease. Epizootiology and transmission. The parasite is now acknowledged to be widespread in dairy and cattle herds. The life cycle of N. caninum is complex, and many aspects remain to be clarified. The definitive host is the dog ( McAllister et al., 1998 ). Placental or aborted tissues are the most likely sources of infection for the definitive host and play a minor role in transmission to the intermediate hosts. The many intermediate hosts include ruminants, deer, and horses. Transplacental transmission is the major mode of transmission in dairy cattle and is the means by which a herd's infection is perpetuated. A less significant mode of transmission is by ingestion of oocysts, which sporulate in the environment or in the intermediate host's body. Reactivation in a chronically infected animal's body is the result of rupture of tissue cysts in neural tissue. Seropositive immunity does not protect a cow from future abortions. Many seropositive cows and calves will never abort or show clinical signs, respectively. Some immunological cross-reactivity may exist among Neospora, Cryptosporidia, and Coccidium. Necropsy findings. Aborted fetuses will usually be autolysed. In those from which tissue can be recovered, tissue cysts are most commonly found in the brain. Spinal cord is also useful. Histological lesions include mild to moderate gliosis, nonsuppurative encephalitis, and perivascular infiltration by mixed mononuclear cells. Pathogenesis. As with Toxoplasma, cell death is the result of intracellular multiplication of Neospora tachyzoites. Neospora undergoes sexual replication in the dog's intestinal tract, and oocysts are shed in the feces. The intermediate hosts develop nonclinical systemic infections, with tachyzoites in several organs, and parasites then localize and become encysted in particular tissues, especially the brain. Infections of this type are latent and lifelong. Except when immunocompromised, most cattle do not usually develop clinical signs and do not have fetal loss. Fetuses become infected, leading to fetal death, mid-gestation abortions, or live calves with latent infections or congenital brain disease. It usually takes 2–4 weeks for a fetus to die and to be expelled. Many aspects of the role of the maternal immune response and pregnancy-associated immunodeficiency in the patterns of Neospora abortions remain to be elucidated. Differential diagnosis. Even when there is a herd history of confirmed Neospora abortions, leptospirosis, bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), salmonellosis, and campylobacteriosis should be considered. BVDV in particular should be considered for abortion storms. Differentials for weak calves are BVDV, perinatal hypoxia following dystocia (immediate postpartum time), bluetongue virus, Toxoplasma, exposure to teratogens, or congenital defects. Prevention and control. The primary preventive measure is preventing contact with contaminated feces. Oocysts will not survive dry environments or extremes of temperature. Dog populations should be controlled, and dogs and other canids should not have access to placentas or aborted fetuses. Dogs should also be restricted from feed bunks and other feed storage areas. Preventive culling is not economically practical for most producers. A vaccine recently became available. If embryo transfer is practiced, recipients should be screened serologically before use. Treatment. There is no known treatment or immunoprophylaxis. vii. Sarcocystosis Etiology. Sarcocystosis is the disease caused by the cyst-forming sporozoon Sarcocystis. Sarcocystis capricanus, S. ovi-canus, and S. tenella are the species that infect sheep and goats. Sarcocystis cruzi, S. hirsuta, and S. hominis are the species that infect cattle. Definitive hosts are carnivores, and all ruminant species are intermediate hosts. Clinical signs and diagnosis. Clinical signs of sarcocystosis infection are seen in cattle during the stage when the parasite encysts in soft tissues. Often the infections are asymptomatic. Fever, anemia, ataxia, symmetric lameness, tremors, tail-switch hair loss, excessive salivation, diarrhea, and weight loss are clinical signs. Abortions in cattle occur during the second trimester and in smaller ruminants 28 days after ingestion of the sporulated oocysts. Definitive diagnosis is based on finding merozoites and meronts in neural tissue lesions. Clinical hematology results include decreased hematocrit, decreased serum protein, and prolonged prothrombin times. Sarcocystis-specific IgG will increase dramatically by 5–6 weeks after infection. There is no cross-reaction between Sarcocystis and Toxoplasma. Epizootiology and transmission. Infection rates among cattle in the United States are estimated to be very high. Transmission is by ingestion of feed and water contaminated by feces of the definitive hosts. Dogs are the definitive hosts for the species that infect the smaller ruminants. Cats, dogs, and primates (including humans when S. hominis is involved) are the definitive hosts for the species that infect cattle. Necropsy. Aborted fetuses may be autolysed. Lesions in neural tissues, including meningoencephalomyelitis, focal malacia, perivascular cuffing, neuronal degeneration, and gliosis, are most marked in the cerebellum and midbrain. Lesions may be found in other tissues, such as lymphadenopathy, and hemorrhages may be found in muscles and on serous surfaces. Cysts in cardiac and skeletal muscles are common incidental findings during necropsies. Pathogenesis. Ingestion of muscle flesh from an infected ruminant results in Sarcocystis cysts' being broken down in the carnivore's digestive system, release of bradyzoites, infection of intestinal mucosal cells by the bradyzoites, differentiation into sexual stages, fusion of the male and female gametes to form oocysts, and shedding as sporocysts by the definitive hosts. The sporocysts are eaten by the ruminant and penetrate the bowel walls; several stages of development occur in endothelial cells of arteries. Merozoites are the form that enters soft tissues, such as muscle, and subsequently encysts. Prevention and control. Feed supplies of ruminants must be protected from fecal contamination by domestic and wild carnivores. These animals should be controlled and must also not have access to carcasses. In larger production situations, monensin may be fed as a prophylactic measure. Treatment. Monensin fed during incubation is prophylactic, but the efficacy in clinically affected cattle is not known. Etiology. Sarcocystosis is the disease caused by the cyst-forming sporozoon Sarcocystis. Sarcocystis capricanus, S. ovi-canus, and S. tenella are the species that infect sheep and goats. Sarcocystis cruzi, S. hirsuta, and S. hominis are the species that infect cattle. Definitive hosts are carnivores, and all ruminant species are intermediate hosts. Clinical signs and diagnosis. Clinical signs of sarcocystosis infection are seen in cattle during the stage when the parasite encysts in soft tissues. Often the infections are asymptomatic. Fever, anemia, ataxia, symmetric lameness, tremors, tail-switch hair loss, excessive salivation, diarrhea, and weight loss are clinical signs. Abortions in cattle occur during the second trimester and in smaller ruminants 28 days after ingestion of the sporulated oocysts. Definitive diagnosis is based on finding merozoites and meronts in neural tissue lesions. Clinical hematology results include decreased hematocrit, decreased serum protein, and prolonged prothrombin times. Sarcocystis-specific IgG will increase dramatically by 5–6 weeks after infection. There is no cross-reaction between Sarcocystis and Toxoplasma. Epizootiology and transmission. Infection rates among cattle in the United States are estimated to be very high. Transmission is by ingestion of feed and water contaminated by feces of the definitive hosts. Dogs are the definitive hosts for the species that infect the smaller ruminants. Cats, dogs, and primates (including humans when S. hominis is involved) are the definitive hosts for the species that infect cattle. Necropsy. Aborted fetuses may be autolysed. Lesions in neural tissues, including meningoencephalomyelitis, focal malacia, perivascular cuffing, neuronal degeneration, and gliosis, are most marked in the cerebellum and midbrain. Lesions may be found in other tissues, such as lymphadenopathy, and hemorrhages may be found in muscles and on serous surfaces. Cysts in cardiac and skeletal muscles are common incidental findings during necropsies. Pathogenesis. Ingestion of muscle flesh from an infected ruminant results in Sarcocystis cysts' being broken down in the carnivore's digestive system, release of bradyzoites, infection of intestinal mucosal cells by the bradyzoites, differentiation into sexual stages, fusion of the male and female gametes to form oocysts, and shedding as sporocysts by the definitive hosts. The sporocysts are eaten by the ruminant and penetrate the bowel walls; several stages of development occur in endothelial cells of arteries. Merozoites are the form that enters soft tissues, such as muscle, and subsequently encysts. Prevention and control. Feed supplies of ruminants must be protected from fecal contamination by domestic and wild carnivores. These animals should be controlled and must also not have access to carcasses. In larger production situations, monensin may be fed as a prophylactic measure. Treatment. Monensin fed during incubation is prophylactic, but the efficacy in clinically affected cattle is not known. viii. Toxoplasmosis Etiology. Toxoplasmosis is caused by the obligate intracellular protozoon Toxoplasma gondii, a coccidial parasite of the family Eimeridae. Cats are the only definitive hosts, and several warm-blooded animals, including ruminants, have been shown to be intermediate hosts. The disease is a major cause of abortion in sheep and goats and less common in cattle. Clinical signs and diagnosis. Clinical signs depend on the organ or tissue parasitized. Toxoplasmosis is typically associated with placentitis, abortion, stillbirths, or birth of weak young ( Underwood and Rook, 1992 ; Buxton, 1998 ). It has also been shown to cause pneumonia and nonsuppurative encephalitis. The enteritis at the early stage of infection may be fatal in some hosts. Hydrocephalus does not occur in animals as it does in human fetal Toxoplasma infections. Rare clinical presentations in ruminants include retinitis and chorioretinitis; these are usually asymptomatic. Infection of the ewe during the first trimester usually leads to fetal resorption, during the second trimester leads to abortion, and during the third trimester leads to birth of weak to normal lambs with subsequent high perinatal mortality. Congenitally infected lambs may display encephalitic signs of circling, incoordination, muscular paresis, and prostration. In sheep, weak young will develop normally if they survive the first week after birth. Infected adult sheep show no systemic illness. Infected adult goats, however, may die. Diagnosis may be difficult, and biological, serological, and histological methods are helpful. Serological tests are the most readily available. Complement fixation and the Sabin-Feldman antibody test may assist in diagnosis. Antibodies found in fetuses are indicative of congenital infection and are typically detectable 35 days after infection; fetal thoracic fluid is especially useful in demonstrating serological evidence of exposure. Biological methods, such as tissue culture or inoculation of mice with maternal body fluids, or with postmortem or necropsy tissues, are more time-consuming and expensive. Epizootiology and transmission. This protozoon is considered ubiquitous. Fifty percent (50%) of adult western sheep and 20% of feedlot lambs have positive hemagglutination titers (1:64 or higher) ( Jensen and Swift, 1982 ). Transmission among the definitive host is by ingestion of tissue cysts. Necropsy findings. At necropsy, placental cotyledons contain multiple small white areas that are sites of necrosis, edema, and calcification. Fetal brains may show nonspecific lesions such as coagulative necrosis, nonsuppurative encephalomyelitis, pneumonia, myocarditis, and hepatitis. Histologically, granulomas with Toxoplasma organisms may be seen in the retina, myocardium, liver, kidney, brain, and other tissues. Impression smears of these tissues, stained appropriately (e.g., with Giemsa), provide a rapid means of diagnosis. Identification of the organism in tissue sections (especially of the heart and the brain) also confirms the findings. Toxoplasma gondii is crescent-shaped, with a clearly visible nuclei, and will be found within macrophages. Pathogenesis. The protozoon has three infectious stages: the tachyzoite, the bradyzoite, and the sporozoite within the oocyst. The definitive hosts, felids, become infected by ingesting cyst stages in mammalian tissues, by ingesting oocysts in feces, and by transplacental transfer. Ingested zoites invade epithelial cells and eventually undergo sexual reproduction, resulting in new oocysts, which the cats will shed in the feces. Cats rarely show clinical signs of infection. One cat can shed millions of oocysts in 1 gm of feces, but the asymptomatic shedding takes place for only a few weeks in its life. Oocysts sporulate in cat feces after 1 day. Ruminants are intermediate hosts of toxoplasmosis and become infected by ingesting sporulated oocyst-contaminated water or feed. As in the definitive host, the ingested sporozoite invades epithelial cells within the intestine but also further invades the bloodstream and is transported throughout the host. The organism migrates to tissues such as the brain, liver, muscles, and placenta. Placental infection develops about 14 days after ingestion of the oocysts. The damage caused by an infection is due to multiplication within cells. Toxoplasma does not produce any toxin. Differential diagnosis. Differentials for abortion include Campylobacter, Chlamydia, and Q fever. Prevention and control. Feline populations on source farms should be controlled. Eliminating contamination of feed and water with cat feces is the best preventive measure. Sporulated oocysts can survive in soil and other places for long periods of time and are resistant to desiccation and freezing. Vaccines for abortion prevention in sheep are available in New Zealand and Europe. Treatment. Toxoplasmosis treatment is ineffective, although feeding monensin during pregnancy may be helpful ( Underwood and Rook, 1992 ). (Monensin is not approved for this use in the Unites States.) Weak lambs that survive the first week after birth will mature normally and will not deliver Toxoplasma- infected young. Research complications. Because toxoplasmosis is zoonotic, precautions must be taken when handling tissues from any abortions or neurological cases. Infections in immunocompromised humans have been fatal. Etiology. Toxoplasmosis is caused by the obligate intracellular protozoon Toxoplasma gondii, a coccidial parasite of the family Eimeridae. Cats are the only definitive hosts, and several warm-blooded animals, including ruminants, have been shown to be intermediate hosts. The disease is a major cause of abortion in sheep and goats and less common in cattle. Clinical signs and diagnosis. Clinical signs depend on the organ or tissue parasitized. Toxoplasmosis is typically associated with placentitis, abortion, stillbirths, or birth of weak young ( Underwood and Rook, 1992 ; Buxton, 1998 ). It has also been shown to cause pneumonia and nonsuppurative encephalitis. The enteritis at the early stage of infection may be fatal in some hosts. Hydrocephalus does not occur in animals as it does in human fetal Toxoplasma infections. Rare clinical presentations in ruminants include retinitis and chorioretinitis; these are usually asymptomatic. Infection of the ewe during the first trimester usually leads to fetal resorption, during the second trimester leads to abortion, and during the third trimester leads to birth of weak to normal lambs with subsequent high perinatal mortality. Congenitally infected lambs may display encephalitic signs of circling, incoordination, muscular paresis, and prostration. In sheep, weak young will develop normally if they survive the first week after birth. Infected adult sheep show no systemic illness. Infected adult goats, however, may die. Diagnosis may be difficult, and biological, serological, and histological methods are helpful. Serological tests are the most readily available. Complement fixation and the Sabin-Feldman antibody test may assist in diagnosis. Antibodies found in fetuses are indicative of congenital infection and are typically detectable 35 days after infection; fetal thoracic fluid is especially useful in demonstrating serological evidence of exposure. Biological methods, such as tissue culture or inoculation of mice with maternal body fluids, or with postmortem or necropsy tissues, are more time-consuming and expensive. Epizootiology and transmission. This protozoon is considered ubiquitous. Fifty percent (50%) of adult western sheep and 20% of feedlot lambs have positive hemagglutination titers (1:64 or higher) ( Jensen and Swift, 1982 ). Transmission among the definitive host is by ingestion of tissue cysts. Necropsy findings. At necropsy, placental cotyledons contain multiple small white areas that are sites of necrosis, edema, and calcification. Fetal brains may show nonspecific lesions such as coagulative necrosis, nonsuppurative encephalomyelitis, pneumonia, myocarditis, and hepatitis. Histologically, granulomas with Toxoplasma organisms may be seen in the retina, myocardium, liver, kidney, brain, and other tissues. Impression smears of these tissues, stained appropriately (e.g., with Giemsa), provide a rapid means of diagnosis. Identification of the organism in tissue sections (especially of the heart and the brain) also confirms the findings. Toxoplasma gondii is crescent-shaped, with a clearly visible nuclei, and will be found within macrophages. Pathogenesis. The protozoon has three infectious stages: the tachyzoite, the bradyzoite, and the sporozoite within the oocyst. The definitive hosts, felids, become infected by ingesting cyst stages in mammalian tissues, by ingesting oocysts in feces, and by transplacental transfer. Ingested zoites invade epithelial cells and eventually undergo sexual reproduction, resulting in new oocysts, which the cats will shed in the feces. Cats rarely show clinical signs of infection. One cat can shed millions of oocysts in 1 gm of feces, but the asymptomatic shedding takes place for only a few weeks in its life. Oocysts sporulate in cat feces after 1 day. Ruminants are intermediate hosts of toxoplasmosis and become infected by ingesting sporulated oocyst-contaminated water or feed. As in the definitive host, the ingested sporozoite invades epithelial cells within the intestine but also further invades the bloodstream and is transported throughout the host. The organism migrates to tissues such as the brain, liver, muscles, and placenta. Placental infection develops about 14 days after ingestion of the oocysts. The damage caused by an infection is due to multiplication within cells. Toxoplasma does not produce any toxin. Differential diagnosis. Differentials for abortion include Campylobacter, Chlamydia, and Q fever. Prevention and control. Feline populations on source farms should be controlled. Eliminating contamination of feed and water with cat feces is the best preventive measure. Sporulated oocysts can survive in soil and other places for long periods of time and are resistant to desiccation and freezing. Vaccines for abortion prevention in sheep are available in New Zealand and Europe. Treatment. Toxoplasmosis treatment is ineffective, although feeding monensin during pregnancy may be helpful ( Underwood and Rook, 1992 ). (Monensin is not approved for this use in the Unites States.) Weak lambs that survive the first week after birth will mature normally and will not deliver Toxoplasma- infected young. Research complications. Because toxoplasmosis is zoonotic, precautions must be taken when handling tissues from any abortions or neurological cases. Infections in immunocompromised humans have been fatal. ix. Trichomoniasis Etiology. Trichomoniasis is an insidious venereal disease of cattle caused by Tritrichomonas (also referred to as Trichomonas) fetus, a large, pear-shaped, flagellated protozoon. The organism is an obligate parasite of the reproductive tract, and it requires a microaerophilic environment to establish chronic infections. In the United States, it is now primarily a disease seen in western beef herds. There are many similarities between trichomoniasis and campylobacteriosis; both diseases cause herd infertility problems. Clinical signs and diagnosis. Clinical signs include infertility manifested by high nonpregnancy rates as well as periodic pyometras and abortions during the first half of gestation. Often the problem is not recognized until herd pregnancy checks indicate many "open," delayed-estrus, late-bred cows, or cows with postcoital pyometras. The abortion rate varies from 5% to 30%, and placentas will be expelled or retained. Tritrichomonas fetus also causes mild salpingitis but this does not result in permanent damage. Other than these manifestations, infection with T. fetus causes no systemic signs. Diagnosis is based on patterns of infertility and pyometras. For example, pyometras in postcoital heifers or cows are suggestive of this pathogen. Diagnostic methods include identifying or culturing the trichomonads from preputial smegma, cervicovaginal mucus, uterine exudates, placental fluids, or abomasal contents of aborted fetuses. Other nonpathogenic protozoa from fecal contamination may be present in the sample. The trichomonad has three anterior flagellae, one posterior flagella, and an undulating membrane; it travels in fluids with a characteristic jerky movement. Culturing must be done on specific media, such as Diamond's or modified Pastridge. Epizootiology and transmission. All transmission is by venereal exposure from breeding bulls or cows or, in some cases, contaminated breeding equipment. Necropsy findings. Nonspecific lesions, such as pyogranulomatous bronchopneumonia of fetuses and placentitis, may be seen in aborted material; some cases will have no gross lesions. Histologically, trichomonads may be visible in the fetal lung lesions and the placenta; those tissues are also the most useful for culturing. Pathogenesis. Tritrichomonas fetus colonizes the female reproductive tract, and subsequent clinical manifestations may be related to the size of the initial infecting dose. Tritrichomonas fetus does not interfere with conception. Embryonic death occurs within the first 2 months of infection. Affected cows will clear the infection over a span of months and maintain immunity for about 6 months. Infections in younger bulls are transient; apparently organisms are cleared by the bulls' immune systems and are dependent on exposure to infected females. Older bulls become chronic carriers, probably because of the ability of T. fetus to colonize deeper epithelial crypts of the prepuce and penis. Differential diagnosis. Campylobacteriosis is the other primary differential for reduced reproductive efficiency of a herd. Other venereal diseases should be considered when infertility problems are noted in a herd: brucellosis, mycoplasmosis, ureaplasmosis, and infectious pustular vulvovaginitis. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. A bacterin vaccine is available. Heifers, cows, and breeding bulls are vaccinated subcutaneously twice at 2 to 4 week intervals, with the booster dose administered 4 weeks before breeding season starts. Similar timing is recommended for administration of the annual booster; a long, anamnestic response does not occur. Bulls used for artificial insemination (AI) are screened routinely for T. fetus (and Campylobacter). AI reduces but does not eliminate the disease. The use of younger, vaccinated bulls is recommended in all circumstances. New animals should be tested before introduction to the herd. Control measures also include culling affected cows or else removing them from the breeding herd for 3 months to rest and clear the infection. Culling chronically infected bulls is strongly recommended. Treatment. Imidazole compounds have been effective, but the use of these is not permitted in food animals in the United States. Therapeutic immunizations are worthwhile when a positive diagnosis has been made. These will not curtail fetal losses but will shorten the convalescence of the affected cows and improve immunity of breeding bulls. Research complications. Trichomoniasis should be considered whenever natural service is used and fertility problems are encountered. Etiology. Trichomoniasis is an insidious venereal disease of cattle caused by Tritrichomonas (also referred to as Trichomonas) fetus, a large, pear-shaped, flagellated protozoon. The organism is an obligate parasite of the reproductive tract, and it requires a microaerophilic environment to establish chronic infections. In the United States, it is now primarily a disease seen in western beef herds. There are many similarities between trichomoniasis and campylobacteriosis; both diseases cause herd infertility problems. Clinical signs and diagnosis. Clinical signs include infertility manifested by high nonpregnancy rates as well as periodic pyometras and abortions during the first half of gestation. Often the problem is not recognized until herd pregnancy checks indicate many "open," delayed-estrus, late-bred cows, or cows with postcoital pyometras. The abortion rate varies from 5% to 30%, and placentas will be expelled or retained. Tritrichomonas fetus also causes mild salpingitis but this does not result in permanent damage. Other than these manifestations, infection with T. fetus causes no systemic signs. Diagnosis is based on patterns of infertility and pyometras. For example, pyometras in postcoital heifers or cows are suggestive of this pathogen. Diagnostic methods include identifying or culturing the trichomonads from preputial smegma, cervicovaginal mucus, uterine exudates, placental fluids, or abomasal contents of aborted fetuses. Other nonpathogenic protozoa from fecal contamination may be present in the sample. The trichomonad has three anterior flagellae, one posterior flagella, and an undulating membrane; it travels in fluids with a characteristic jerky movement. Culturing must be done on specific media, such as Diamond's or modified Pastridge. Epizootiology and transmission. All transmission is by venereal exposure from breeding bulls or cows or, in some cases, contaminated breeding equipment. Necropsy findings. Nonspecific lesions, such as pyogranulomatous bronchopneumonia of fetuses and placentitis, may be seen in aborted material; some cases will have no gross lesions. Histologically, trichomonads may be visible in the fetal lung lesions and the placenta; those tissues are also the most useful for culturing. Pathogenesis. Tritrichomonas fetus colonizes the female reproductive tract, and subsequent clinical manifestations may be related to the size of the initial infecting dose. Tritrichomonas fetus does not interfere with conception. Embryonic death occurs within the first 2 months of infection. Affected cows will clear the infection over a span of months and maintain immunity for about 6 months. Infections in younger bulls are transient; apparently organisms are cleared by the bulls' immune systems and are dependent on exposure to infected females. Older bulls become chronic carriers, probably because of the ability of T. fetus to colonize deeper epithelial crypts of the prepuce and penis. Differential diagnosis. Campylobacteriosis is the other primary differential for reduced reproductive efficiency of a herd. Other venereal diseases should be considered when infertility problems are noted in a herd: brucellosis, mycoplasmosis, ureaplasmosis, and infectious pustular vulvovaginitis. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. Prevention and control. A bacterin vaccine is available. Heifers, cows, and breeding bulls are vaccinated subcutaneously twice at 2 to 4 week intervals, with the booster dose administered 4 weeks before breeding season starts. Similar timing is recommended for administration of the annual booster; a long, anamnestic response does not occur. Bulls used for artificial insemination (AI) are screened routinely for T. fetus (and Campylobacter). AI reduces but does not eliminate the disease. The use of younger, vaccinated bulls is recommended in all circumstances. New animals should be tested before introduction to the herd. Control measures also include culling affected cows or else removing them from the breeding herd for 3 months to rest and clear the infection. Culling chronically infected bulls is strongly recommended. Treatment. Imidazole compounds have been effective, but the use of these is not permitted in food animals in the United States. Therapeutic immunizations are worthwhile when a positive diagnosis has been made. These will not curtail fetal losses but will shorten the convalescence of the affected cows and improve immunity of breeding bulls. Research complications. Trichomoniasis should be considered whenever natural service is used and fertility problems are encountered. b. Nematodes Nematodes are important ruminant pathogens that cause acute, chronic, subclinical, and clinical disease in adults and adolescents. The major helminths may cause gastroenteritis associated with intestinal hemorrhage and malnutrition. Nematodiasis is associated with grazing exposure to infective larvae; animals procured for research may have had exposure to these helminths. Mixed infections of these parasites are common. Generally, older animals develop resistance to some of the species; thus, animals between about 2 months and 2 years of age are most susceptible to infection. Because of the parasites' effects on the animals' physiology, infection in these younger animals is a major contributor to a cycle of poor nutrition and digestion, compromised immune responses, and impaired growth and development. Diagnosis is primarily based on fecal flotation techniques; however, because many of these nematodes have similar-appearing ova, hatching the ova and identifying the larvae are often required (Baermann technique). A number of anthelmintics can be used to interrupt nematode life cycles. See Zajac and Moore (1993) and Pugh et al. (1998 ) for comprehensive reviews of treatment and control of nematodiasis. i. Haemonchus contortus, H. placei (barber's pole worm, large stomach worm). Haemonchus contortus is the most important internal parasite of sheep and goats, and the brief description here focuses on the disease in the smaller ruminants. Haemonchus contortus and H. placei infections do occur in younger cattle and are similar to the disease in sheep. Haemonchus is extremely pathogenic, and the adults feed by sucking blood from the mucosa of the abomasum. Severe anemia may lead to death. Weight loss, decreased milk production, poor wool growth, and intermandibular and cervical edema due to hypoproteinemia ("bottle jaw") are also common clinical signs. Diarrhea is not seen in all cases but may sometimes be severe or chronic. The life cycle is direct. Under optimal conditions, a complete life cycle, from ingestion of larvae to eggs passed in the feces, occurs in 3 weeks. Embryonated eggs may develop into infective larvae within a week. Hypobiotic (arrested) larvae may exist for several months in animal tissues, serving as a reservoir for future pasture contamination. Periparturient increases in egg shedding by ewes contribute to large numbers of eggs spread on spring pastures ("spring rise"). Resistance to common anthelmintics has developed; currently ivermectin or benzimidazole products are used, with a minimum of 2 dosings given 2–3 weeks apart. Levamisole is also used. In severe cases, animals may benefit from blood transfusions and iron supplementation. Because animals may easily acquire infective larvae from ingestion of contaminated feed and from contaminated pastures, general facility sanitation and pasture management and rotation are important preventive and control measures. Haemonchus contortus is susceptible to destruction by freezing temperatures and dry conditions. ii. Ostertagia (Teladorsagia) circumcincta (medium stomach worm). Ostertagia circumcincta is also highly pathogenic for sheep and goats and, like Haemonchus, attaches to the abomasal mucosa and ingests blood. The life cycle is comparable to that of Haemonchus, including the phenomenon of hypobiosis. Larvae are especially resistant to cool temperatures, however, and will overwinter on pastures. Larvae-induced hyperplasia of abomasal epithelial glands results in a change of gastric pH from about 2.0 to near 7.0, leading to decreased digestive enzyme activity and malnutrition. Clinical syndromes are categorized as type 1 or type 2. The former type is associated with infections acquired in fall or spring and is seen in younger animals. The latter type is associated with emergence of the arrested larvae during spring or fall. Clinical signs include anemia, weight loss, decreased milk production, and unthriftiness. Diarrhea is usually seen in type 1 only; the symptoms of type 2 are comparable to those of Haemonchus infections. Anthelmintic drug therapy is comparable to that for Haemonchus, and drug resistance is also a problem with Ostertagia. iii. Ostertagia ostertagi (cattle stomach worm). Ostertagia ostertagi is the most pathogenic and most costly of the cattle nematodes. Ostertagia leptospicularis and O. bisonis also cause disease. The life cycle is direct, and egg shedding by the cattle may occur within 3–4 weeks of ingestion of infective larvae. Hypobiosis is also a characteristic of O. ostertagi. In the initial steps of infection, the normal processes of the abomasum are profoundly disrupted and cells are destroyed as the larvae develop within and emerge from the glands. Moroccan leather appearance is the term to describe the result of cellular hyperplasia and loss of cell differentiation. Cycles of infection and morbidity depend on geographic location, climate, and production cycles. Type 1 cattle ostertagiasis is associated with ingestion of large numbers of infective larvae, occurs in animals less than 2 years old, and causes diarrhea and anorexia. Type 2 ostertagiasis occurs in cattle 2–4 years old and older adults, is the result of the emergence and development of hypobiotic larvae, and in addition to signs seen with type 1, hypoproteinemia with development of submandibular edema, fever, and anemia is a clinical sign. Treatment options include ivermectin, fenbendazole, and levamisole; all are effective against the arrested larvae. Ostertagia is susceptible to desiccation but is resistant to freezing. iv. Trichostrongylus vitrinus, T. axei, T. colubriformis (hair worms). Trichostrongylus species favor cooler conditions, and some larvae may overwinter. Although the different species may affect different segments of the gastrointestinal tract, the nematode attaches to the mucosa and affects secretion and/or absorption. Trichostrongylus vitrinus and T. colubriformis infect the small intestine of sheep and goats. Trichostrongylus axei infects the abomasum of cattle, sheep, and goats and causes increases in abomasal pH similar to those seen with Ostertagia. Mucosal hyperplasia is not seen. The prepatent period is about 3 weeks. Affected animals display unthriftiness, anorexia, decreased milk production, weight loss, diarrhea, and dehydration. These worms show intermediate resistance to freezing temperatures and dry conditions. v. Nematodirus spathiger, N. battus (thread-necked worms). Nematodirus has lower pathogenicity compared with other gastrointestinal nematodes. The larvae cause small-intestinal necrosis and inflammation. The larvae are especially resistant to desiccation and freezing. Clinical signs include depression, weight loss, anorexia, and diarrhea. vi. Cooperia (small intestinal worms). Cooperia primarily affects younger animals less than 1 year of age. Cooperia curticei infects the small intestine of sheep and goats; C. punctata and C. oncophora infect the small intestines of cattle, sheep, and goats. Cooperia pectinata infects the stomach of cattle. Large numbers lead to clinical infection, and the prepatent period is about 3 weeks. Cooperia and Osteragia infections, like infections of some other nematode species, may act synergistically. Because these nematodes suck blood, clinical signs include anemia, gastrointestinal hemorrhage, and malnutrition. Animals exhibit weight loss, diarrhea, and depression. Cooperia species are intermediate to resistant to the effects of cold temperatures. vii. Strongyloides papillosus. Strongyloides papillosus is a small-intestinal parasite of sheep and cattle. Strongyloides has a different life cycle from that of many nematodes. The eggs, expelled in the feces, are larvated, and when they hatch, they form both free-living males and females or parasitic females only. The parasitic females may enter the gastrointestinal tract through oral ingestion, such as in milk during nursing, or through direct penetration of the skin. Penetrating larvae enter the bloodstream and are transported to the lungs, where they penetrate the alveoli, are coughed up, and then swallowed to ultimately enter the gastrointestinal tract. Adult females may reproduce in the small intestines by parthenogenesis. Clinical signs associated with Strongyloides include weight loss, diarrhea, unthriftiness, and dermatitis in cases where large numbers migrate through the skin. The current broad-spectrum anthelmintics are effective against Strongyloides. viii. Bunostomum trigonocephalum (hookworm). Bunostomum trigonocephalum is a hookworm that occasionally infects sheep in locales in the southwestern United States. Like Strongyloides, Bunostomum infection may involve oral ingestion or direct penetration of the skin (followed by tracheal migration and swallowing). The larvae mature in the small intestines and suck blood. Larvae are susceptible to desiccation and freezing. Heavy infection with Bunostomum may result in anemia, diarrhea, intestinal hemorrhage, edema, and weight loss. ix. Oesophagostomum columbianum, O. venulosum (nodule worms). Oesophagostomum spp. primarily infect the large intestine and occasionally the distal small intestine, causing nodule worm disease, or simply gut. Oesophagostomum columbianum and O. venulosum infect sheep and cattle. These nematodes may affect sheep from 3 months to 2 years of age, and the prepatent period is about 6 weeks. Larvae are highly sensitive to freezing and desiccation and rarely overwinter. Larvae penetrate the large-intestinal mucosa but occasionally move into the deeper areas of the intestinal wall near the serosa. The resultant inflammatory reaction may lead to the formation of a caseous nodule that may mineralize over time. Intestinal lesions may accelerate peristalsis, leading to diarrhea, or may inhibit peristalsis (later stages), resulting in constipation. Clinical signs include weakness, unthriftiness, alternating episodes of diarrhea and constipation, and severe weight loss. Nodular lesions are typical at necropsy. x. Chabertia ovis (large-mouth bowel worm). Chabertia ovis is a minor colon parasite of sheep, goats, and cattle and is seen primarily in sheep. Signs of infection are not usually seen in cattle. Prepatent periods are up to 50 days. Heavy infection, which may result from as few as 100 worms located at the proximal end of the colon, may lead to hemorrhagic mucoid diarrhea, weight loss, weakness, colitis, and mild anemia. xi. Trichuris (whipworms). Trichuris spp. are mildly pathogenic nematodes and are usually attached to the cecal mucosa. Trichuris has a rather long prepatent period, extending from 1 to 3 months. The oval eggs are double-operculated and survive well in pasture environmental extremes. The adult worms also have a characterisitic morphology, with one thicker end appearing as a whip handle. The nematodes cause a minor cecitis and will feed on blood. Clinical infection is rare and results in diarrhea with mucus and blood. Treatment and prevention methods are similar to those for other nematodes. xii. Dictyocaulus (lungworms). Dictyocaulus spp., or lungworms, are nematodes that cause varying clinical signs in ruminants. In sheep, Dictyocaulus filaria, Protostrongylus rufescens, and Muellerius capillaris cause disease; Dictyocaulus is the most pathogenic. Goats are infected by the same species as sheep, but infections are uncommon. Dictyocaulus viviparus is the only lungworm found in cattle, causing "fog fever." Infections with these parasites in the United States tend to be associated with cooler, moister climates. Lungworms induce a severe parasitic bronchitis (known as husk, or verminous pneumonia) in sheep between approximately 2 and 18 months of age. Sheep infected with any of the lungworm species may display coughing, dyspnea, nasal discharge, weight loss, unthriftiness, and occasionally fever. Coughing and dyspnea are symptoms in goats. Diagnosis is suggested by persistent coughing and nasal discharge and is confirmed by identifying larvae in the feces or adults in pathological samples. The Baermann technique, involving prompt examination of room-temperature feces, is usually used; zinc sulfate flotation is also used. Dictyocaulus has a direct life cycle. The adult worms reside in the large bronchi. Dictyocaulus produces embryonated eggs that are coughed up and swallowed; the eggs then hatch in the intestines, and larvae are expelled in the feces. The expelled larvae are infectious in about 7–10 days and, after ingestion, penetrate the intestinal mucosa and move through the lymphatics and blood into the lungs, where they develop into adults in about 5 weeks. Dictyocaulus filaria causes an especially severe bronchitis in sheep. Protostrongylus inhabits smaller bronchioles. Muellerius is of minor pathogenicity. Protostrongylus and Muellerius require the snail or slug as an intermediate host. Infection occurs through ingestion of infected snails; infections are less likely than those caused by the direct ingestion of Dictyocaulus larvae. Immunity wanes over a year. Viral and bacterial respiratory tract infections may be associated with the parasitic infection. Dictyocaulus viviparus causes the obvious signs in cattle. More severe illness is seen after infections with Cooperia and Ostertagia, because of a synergism between the nematodes even if the cattle are not currently infected with those parasites. Hypobiosis (arrested development of immature worms in lung tissue) is associated with Dictyocaulus infections; cattle will be silent carriers, showing no clinical signs and serving as a means for the infection to survive over winter or a dry season. Pastures can be heavily contaminated during the next grazing season. Necropsy lesions include bronchiolitis and bronchitis, atelectasis, and hyperplasia of peribronchiolar lymphoid tissue. Nematodes frequently reside in the bronchi of the diaphragmatic lung lobes and are frequently enmeshed with frothy exudate. Prevention and control of the disease involve appropriate pasture management. Elimination of intermediate hosts is important in sheep and goat pastures. In a laboratory setting, animals may be procured that are already harboring the disease. Infected animals can be treated with anthelmintics such as ivermectin or levamisole. Muellerius tends to be resistant to levamisole. There is no anthelmintic currently approved for goats, but fenbendazole, administered 2 weeks apart, has been effective for all three nematodes. Treating D. viviparus depends on the type and stage of life of the cattle; label directions must be followed. There is no vaccine for D. viviparus in the United States. Even if infections are not severe and do resolve with treatment, permanent lesions may be inflicted on the lung tissue. i. Haemonchus contortus, H. placei (barber's pole worm, large stomach worm). Haemonchus contortus is the most important internal parasite of sheep and goats, and the brief description here focuses on the disease in the smaller ruminants. Haemonchus contortus and H. placei infections do occur in younger cattle and are similar to the disease in sheep. Haemonchus is extremely pathogenic, and the adults feed by sucking blood from the mucosa of the abomasum. Severe anemia may lead to death. Weight loss, decreased milk production, poor wool growth, and intermandibular and cervical edema due to hypoproteinemia ("bottle jaw") are also common clinical signs. Diarrhea is not seen in all cases but may sometimes be severe or chronic. The life cycle is direct. Under optimal conditions, a complete life cycle, from ingestion of larvae to eggs passed in the feces, occurs in 3 weeks. Embryonated eggs may develop into infective larvae within a week. Hypobiotic (arrested) larvae may exist for several months in animal tissues, serving as a reservoir for future pasture contamination. Periparturient increases in egg shedding by ewes contribute to large numbers of eggs spread on spring pastures ("spring rise"). Resistance to common anthelmintics has developed; currently ivermectin or benzimidazole products are used, with a minimum of 2 dosings given 2–3 weeks apart. Levamisole is also used. In severe cases, animals may benefit from blood transfusions and iron supplementation. Because animals may easily acquire infective larvae from ingestion of contaminated feed and from contaminated pastures, general facility sanitation and pasture management and rotation are important preventive and control measures. Haemonchus contortus is susceptible to destruction by freezing temperatures and dry conditions. ii. Ostertagia (Teladorsagia) circumcincta (medium stomach worm). Ostertagia circumcincta is also highly pathogenic for sheep and goats and, like Haemonchus, attaches to the abomasal mucosa and ingests blood. The life cycle is comparable to that of Haemonchus, including the phenomenon of hypobiosis. Larvae are especially resistant to cool temperatures, however, and will overwinter on pastures. Larvae-induced hyperplasia of abomasal epithelial glands results in a change of gastric pH from about 2.0 to near 7.0, leading to decreased digestive enzyme activity and malnutrition. Clinical syndromes are categorized as type 1 or type 2. The former type is associated with infections acquired in fall or spring and is seen in younger animals. The latter type is associated with emergence of the arrested larvae during spring or fall. Clinical signs include anemia, weight loss, decreased milk production, and unthriftiness. Diarrhea is usually seen in type 1 only; the symptoms of type 2 are comparable to those of Haemonchus infections. Anthelmintic drug therapy is comparable to that for Haemonchus, and drug resistance is also a problem with Ostertagia. iii. Ostertagia ostertagi (cattle stomach worm). Ostertagia ostertagi is the most pathogenic and most costly of the cattle nematodes. Ostertagia leptospicularis and O. bisonis also cause disease. The life cycle is direct, and egg shedding by the cattle may occur within 3–4 weeks of ingestion of infective larvae. Hypobiosis is also a characteristic of O. ostertagi. In the initial steps of infection, the normal processes of the abomasum are profoundly disrupted and cells are destroyed as the larvae develop within and emerge from the glands. Moroccan leather appearance is the term to describe the result of cellular hyperplasia and loss of cell differentiation. Cycles of infection and morbidity depend on geographic location, climate, and production cycles. Type 1 cattle ostertagiasis is associated with ingestion of large numbers of infective larvae, occurs in animals less than 2 years old, and causes diarrhea and anorexia. Type 2 ostertagiasis occurs in cattle 2–4 years old and older adults, is the result of the emergence and development of hypobiotic larvae, and in addition to signs seen with type 1, hypoproteinemia with development of submandibular edema, fever, and anemia is a clinical sign. Treatment options include ivermectin, fenbendazole, and levamisole; all are effective against the arrested larvae. Ostertagia is susceptible to desiccation but is resistant to freezing. iv. Trichostrongylus vitrinus, T. axei, T. colubriformis (hair worms). Trichostrongylus species favor cooler conditions, and some larvae may overwinter. Although the different species may affect different segments of the gastrointestinal tract, the nematode attaches to the mucosa and affects secretion and/or absorption. Trichostrongylus vitrinus and T. colubriformis infect the small intestine of sheep and goats. Trichostrongylus axei infects the abomasum of cattle, sheep, and goats and causes increases in abomasal pH similar to those seen with Ostertagia. Mucosal hyperplasia is not seen. The prepatent period is about 3 weeks. Affected animals display unthriftiness, anorexia, decreased milk production, weight loss, diarrhea, and dehydration. These worms show intermediate resistance to freezing temperatures and dry conditions. v. Nematodirus spathiger, N. battus (thread-necked worms). Nematodirus has lower pathogenicity compared with other gastrointestinal nematodes. The larvae cause small-intestinal necrosis and inflammation. The larvae are especially resistant to desiccation and freezing. Clinical signs include depression, weight loss, anorexia, and diarrhea. vi. Cooperia (small intestinal worms). Cooperia primarily affects younger animals less than 1 year of age. Cooperia curticei infects the small intestine of sheep and goats; C. punctata and C. oncophora infect the small intestines of cattle, sheep, and goats. Cooperia pectinata infects the stomach of cattle. Large numbers lead to clinical infection, and the prepatent period is about 3 weeks. Cooperia and Osteragia infections, like infections of some other nematode species, may act synergistically. Because these nematodes suck blood, clinical signs include anemia, gastrointestinal hemorrhage, and malnutrition. Animals exhibit weight loss, diarrhea, and depression. Cooperia species are intermediate to resistant to the effects of cold temperatures. vii. Strongyloides papillosus. Strongyloides papillosus is a small-intestinal parasite of sheep and cattle. Strongyloides has a different life cycle from that of many nematodes. The eggs, expelled in the feces, are larvated, and when they hatch, they form both free-living males and females or parasitic females only. The parasitic females may enter the gastrointestinal tract through oral ingestion, such as in milk during nursing, or through direct penetration of the skin. Penetrating larvae enter the bloodstream and are transported to the lungs, where they penetrate the alveoli, are coughed up, and then swallowed to ultimately enter the gastrointestinal tract. Adult females may reproduce in the small intestines by parthenogenesis. Clinical signs associated with Strongyloides include weight loss, diarrhea, unthriftiness, and dermatitis in cases where large numbers migrate through the skin. The current broad-spectrum anthelmintics are effective against Strongyloides. viii. Bunostomum trigonocephalum (hookworm). Bunostomum trigonocephalum is a hookworm that occasionally infects sheep in locales in the southwestern United States. Like Strongyloides, Bunostomum infection may involve oral ingestion or direct penetration of the skin (followed by tracheal migration and swallowing). The larvae mature in the small intestines and suck blood. Larvae are susceptible to desiccation and freezing. Heavy infection with Bunostomum may result in anemia, diarrhea, intestinal hemorrhage, edema, and weight loss. ix. Oesophagostomum columbianum, O. venulosum (nodule worms). Oesophagostomum spp. primarily infect the large intestine and occasionally the distal small intestine, causing nodule worm disease, or simply gut. Oesophagostomum columbianum and O. venulosum infect sheep and cattle. These nematodes may affect sheep from 3 months to 2 years of age, and the prepatent period is about 6 weeks. Larvae are highly sensitive to freezing and desiccation and rarely overwinter. Larvae penetrate the large-intestinal mucosa but occasionally move into the deeper areas of the intestinal wall near the serosa. The resultant inflammatory reaction may lead to the formation of a caseous nodule that may mineralize over time. Intestinal lesions may accelerate peristalsis, leading to diarrhea, or may inhibit peristalsis (later stages), resulting in constipation. Clinical signs include weakness, unthriftiness, alternating episodes of diarrhea and constipation, and severe weight loss. Nodular lesions are typical at necropsy. x. Chabertia ovis (large-mouth bowel worm). Chabertia ovis is a minor colon parasite of sheep, goats, and cattle and is seen primarily in sheep. Signs of infection are not usually seen in cattle. Prepatent periods are up to 50 days. Heavy infection, which may result from as few as 100 worms located at the proximal end of the colon, may lead to hemorrhagic mucoid diarrhea, weight loss, weakness, colitis, and mild anemia. xi. Trichuris (whipworms). Trichuris spp. are mildly pathogenic nematodes and are usually attached to the cecal mucosa. Trichuris has a rather long prepatent period, extending from 1 to 3 months. The oval eggs are double-operculated and survive well in pasture environmental extremes. The adult worms also have a characterisitic morphology, with one thicker end appearing as a whip handle. The nematodes cause a minor cecitis and will feed on blood. Clinical infection is rare and results in diarrhea with mucus and blood. Treatment and prevention methods are similar to those for other nematodes. xii. Dictyocaulus (lungworms). Dictyocaulus spp., or lungworms, are nematodes that cause varying clinical signs in ruminants. In sheep, Dictyocaulus filaria, Protostrongylus rufescens, and Muellerius capillaris cause disease; Dictyocaulus is the most pathogenic. Goats are infected by the same species as sheep, but infections are uncommon. Dictyocaulus viviparus is the only lungworm found in cattle, causing "fog fever." Infections with these parasites in the United States tend to be associated with cooler, moister climates. Lungworms induce a severe parasitic bronchitis (known as husk, or verminous pneumonia) in sheep between approximately 2 and 18 months of age. Sheep infected with any of the lungworm species may display coughing, dyspnea, nasal discharge, weight loss, unthriftiness, and occasionally fever. Coughing and dyspnea are symptoms in goats. Diagnosis is suggested by persistent coughing and nasal discharge and is confirmed by identifying larvae in the feces or adults in pathological samples. The Baermann technique, involving prompt examination of room-temperature feces, is usually used; zinc sulfate flotation is also used. Dictyocaulus has a direct life cycle. The adult worms reside in the large bronchi. Dictyocaulus produces embryonated eggs that are coughed up and swallowed; the eggs then hatch in the intestines, and larvae are expelled in the feces. The expelled larvae are infectious in about 7–10 days and, after ingestion, penetrate the intestinal mucosa and move through the lymphatics and blood into the lungs, where they develop into adults in about 5 weeks. Dictyocaulus filaria causes an especially severe bronchitis in sheep. Protostrongylus inhabits smaller bronchioles. Muellerius is of minor pathogenicity. Protostrongylus and Muellerius require the snail or slug as an intermediate host. Infection occurs through ingestion of infected snails; infections are less likely than those caused by the direct ingestion of Dictyocaulus larvae. Immunity wanes over a year. Viral and bacterial respiratory tract infections may be associated with the parasitic infection. Dictyocaulus viviparus causes the obvious signs in cattle. More severe illness is seen after infections with Cooperia and Ostertagia, because of a synergism between the nematodes even if the cattle are not currently infected with those parasites. Hypobiosis (arrested development of immature worms in lung tissue) is associated with Dictyocaulus infections; cattle will be silent carriers, showing no clinical signs and serving as a means for the infection to survive over winter or a dry season. Pastures can be heavily contaminated during the next grazing season. Necropsy lesions include bronchiolitis and bronchitis, atelectasis, and hyperplasia of peribronchiolar lymphoid tissue. Nematodes frequently reside in the bronchi of the diaphragmatic lung lobes and are frequently enmeshed with frothy exudate. Prevention and control of the disease involve appropriate pasture management. Elimination of intermediate hosts is important in sheep and goat pastures. In a laboratory setting, animals may be procured that are already harboring the disease. Infected animals can be treated with anthelmintics such as ivermectin or levamisole. Muellerius tends to be resistant to levamisole. There is no anthelmintic currently approved for goats, but fenbendazole, administered 2 weeks apart, has been effective for all three nematodes. Treating D. viviparus depends on the type and stage of life of the cattle; label directions must be followed. There is no vaccine for D. viviparus in the United States. Even if infections are not severe and do resolve with treatment, permanent lesions may be inflicted on the lung tissue. i. Haemonchus contortus, H. placei (barber's pole worm, large stomach worm). Haemonchus contortus is the most important internal parasite of sheep and goats, and the brief description here focuses on the disease in the smaller ruminants. Haemonchus contortus and H. placei infections do occur in younger cattle and are similar to the disease in sheep. Haemonchus is extremely pathogenic, and the adults feed by sucking blood from the mucosa of the abomasum. Severe anemia may lead to death. Weight loss, decreased milk production, poor wool growth, and intermandibular and cervical edema due to hypoproteinemia ("bottle jaw") are also common clinical signs. Diarrhea is not seen in all cases but may sometimes be severe or chronic. The life cycle is direct. Under optimal conditions, a complete life cycle, from ingestion of larvae to eggs passed in the feces, occurs in 3 weeks. Embryonated eggs may develop into infective larvae within a week. Hypobiotic (arrested) larvae may exist for several months in animal tissues, serving as a reservoir for future pasture contamination. Periparturient increases in egg shedding by ewes contribute to large numbers of eggs spread on spring pastures ("spring rise"). Resistance to common anthelmintics has developed; currently ivermectin or benzimidazole products are used, with a minimum of 2 dosings given 2–3 weeks apart. Levamisole is also used. In severe cases, animals may benefit from blood transfusions and iron supplementation. Because animals may easily acquire infective larvae from ingestion of contaminated feed and from contaminated pastures, general facility sanitation and pasture management and rotation are important preventive and control measures. Haemonchus contortus is susceptible to destruction by freezing temperatures and dry conditions. ii. Ostertagia (Teladorsagia) circumcincta (medium stomach worm). Ostertagia circumcincta is also highly pathogenic for sheep and goats and, like Haemonchus, attaches to the abomasal mucosa and ingests blood. The life cycle is comparable to that of Haemonchus, including the phenomenon of hypobiosis. Larvae are especially resistant to cool temperatures, however, and will overwinter on pastures. Larvae-induced hyperplasia of abomasal epithelial glands results in a change of gastric pH from about 2.0 to near 7.0, leading to decreased digestive enzyme activity and malnutrition. Clinical syndromes are categorized as type 1 or type 2. The former type is associated with infections acquired in fall or spring and is seen in younger animals. The latter type is associated with emergence of the arrested larvae during spring or fall. Clinical signs include anemia, weight loss, decreased milk production, and unthriftiness. Diarrhea is usually seen in type 1 only; the symptoms of type 2 are comparable to those of Haemonchus infections. Anthelmintic drug therapy is comparable to that for Haemonchus, and drug resistance is also a problem with Ostertagia. iii. Ostertagia ostertagi (cattle stomach worm). Ostertagia ostertagi is the most pathogenic and most costly of the cattle nematodes. Ostertagia leptospicularis and O. bisonis also cause disease. The life cycle is direct, and egg shedding by the cattle may occur within 3–4 weeks of ingestion of infective larvae. Hypobiosis is also a characteristic of O. ostertagi. In the initial steps of infection, the normal processes of the abomasum are profoundly disrupted and cells are destroyed as the larvae develop within and emerge from the glands. Moroccan leather appearance is the term to describe the result of cellular hyperplasia and loss of cell differentiation. Cycles of infection and morbidity depend on geographic location, climate, and production cycles. Type 1 cattle ostertagiasis is associated with ingestion of large numbers of infective larvae, occurs in animals less than 2 years old, and causes diarrhea and anorexia. Type 2 ostertagiasis occurs in cattle 2–4 years old and older adults, is the result of the emergence and development of hypobiotic larvae, and in addition to signs seen with type 1, hypoproteinemia with development of submandibular edema, fever, and anemia is a clinical sign. Treatment options include ivermectin, fenbendazole, and levamisole; all are effective against the arrested larvae. Ostertagia is susceptible to desiccation but is resistant to freezing. iv. Trichostrongylus vitrinus, T. axei, T. colubriformis (hair worms). Trichostrongylus species favor cooler conditions, and some larvae may overwinter. Although the different species may affect different segments of the gastrointestinal tract, the nematode attaches to the mucosa and affects secretion and/or absorption. Trichostrongylus vitrinus and T. colubriformis infect the small intestine of sheep and goats. Trichostrongylus axei infects the abomasum of cattle, sheep, and goats and causes increases in abomasal pH similar to those seen with Ostertagia. Mucosal hyperplasia is not seen. The prepatent period is about 3 weeks. Affected animals display unthriftiness, anorexia, decreased milk production, weight loss, diarrhea, and dehydration. These worms show intermediate resistance to freezing temperatures and dry conditions. v. Nematodirus spathiger, N. battus (thread-necked worms). Nematodirus has lower pathogenicity compared with other gastrointestinal nematodes. The larvae cause small-intestinal necrosis and inflammation. The larvae are especially resistant to desiccation and freezing. Clinical signs include depression, weight loss, anorexia, and diarrhea. vi. Cooperia (small intestinal worms). Cooperia primarily affects younger animals less than 1 year of age. Cooperia curticei infects the small intestine of sheep and goats; C. punctata and C. oncophora infect the small intestines of cattle, sheep, and goats. Cooperia pectinata infects the stomach of cattle. Large numbers lead to clinical infection, and the prepatent period is about 3 weeks. Cooperia and Osteragia infections, like infections of some other nematode species, may act synergistically. Because these nematodes suck blood, clinical signs include anemia, gastrointestinal hemorrhage, and malnutrition. Animals exhibit weight loss, diarrhea, and depression. Cooperia species are intermediate to resistant to the effects of cold temperatures. vii. Strongyloides papillosus. Strongyloides papillosus is a small-intestinal parasite of sheep and cattle. Strongyloides has a different life cycle from that of many nematodes. The eggs, expelled in the feces, are larvated, and when they hatch, they form both free-living males and females or parasitic females only. The parasitic females may enter the gastrointestinal tract through oral ingestion, such as in milk during nursing, or through direct penetration of the skin. Penetrating larvae enter the bloodstream and are transported to the lungs, where they penetrate the alveoli, are coughed up, and then swallowed to ultimately enter the gastrointestinal tract. Adult females may reproduce in the small intestines by parthenogenesis. Clinical signs associated with Strongyloides include weight loss, diarrhea, unthriftiness, and dermatitis in cases where large numbers migrate through the skin. The current broad-spectrum anthelmintics are effective against Strongyloides. viii. Bunostomum trigonocephalum (hookworm). Bunostomum trigonocephalum is a hookworm that occasionally infects sheep in locales in the southwestern United States. Like Strongyloides, Bunostomum infection may involve oral ingestion or direct penetration of the skin (followed by tracheal migration and swallowing). The larvae mature in the small intestines and suck blood. Larvae are susceptible to desiccation and freezing. Heavy infection with Bunostomum may result in anemia, diarrhea, intestinal hemorrhage, edema, and weight loss. ix. Oesophagostomum columbianum, O. venulosum (nodule worms). Oesophagostomum spp. primarily infect the large intestine and occasionally the distal small intestine, causing nodule worm disease, or simply gut. Oesophagostomum columbianum and O. venulosum infect sheep and cattle. These nematodes may affect sheep from 3 months to 2 years of age, and the prepatent period is about 6 weeks. Larvae are highly sensitive to freezing and desiccation and rarely overwinter. Larvae penetrate the large-intestinal mucosa but occasionally move into the deeper areas of the intestinal wall near the serosa. The resultant inflammatory reaction may lead to the formation of a caseous nodule that may mineralize over time. Intestinal lesions may accelerate peristalsis, leading to diarrhea, or may inhibit peristalsis (later stages), resulting in constipation. Clinical signs include weakness, unthriftiness, alternating episodes of diarrhea and constipation, and severe weight loss. Nodular lesions are typical at necropsy. x. Chabertia ovis (large-mouth bowel worm). Chabertia ovis is a minor colon parasite of sheep, goats, and cattle and is seen primarily in sheep. Signs of infection are not usually seen in cattle. Prepatent periods are up to 50 days. Heavy infection, which may result from as few as 100 worms located at the proximal end of the colon, may lead to hemorrhagic mucoid diarrhea, weight loss, weakness, colitis, and mild anemia. xi. Trichuris (whipworms). Trichuris spp. are mildly pathogenic nematodes and are usually attached to the cecal mucosa. Trichuris has a rather long prepatent period, extending from 1 to 3 months. The oval eggs are double-operculated and survive well in pasture environmental extremes. The adult worms also have a characterisitic morphology, with one thicker end appearing as a whip handle. The nematodes cause a minor cecitis and will feed on blood. Clinical infection is rare and results in diarrhea with mucus and blood. Treatment and prevention methods are similar to those for other nematodes. xii. Dictyocaulus (lungworms). Dictyocaulus spp., or lungworms, are nematodes that cause varying clinical signs in ruminants. In sheep, Dictyocaulus filaria, Protostrongylus rufescens, and Muellerius capillaris cause disease; Dictyocaulus is the most pathogenic. Goats are infected by the same species as sheep, but infections are uncommon. Dictyocaulus viviparus is the only lungworm found in cattle, causing "fog fever." Infections with these parasites in the United States tend to be associated with cooler, moister climates. Lungworms induce a severe parasitic bronchitis (known as husk, or verminous pneumonia) in sheep between approximately 2 and 18 months of age. Sheep infected with any of the lungworm species may display coughing, dyspnea, nasal discharge, weight loss, unthriftiness, and occasionally fever. Coughing and dyspnea are symptoms in goats. Diagnosis is suggested by persistent coughing and nasal discharge and is confirmed by identifying larvae in the feces or adults in pathological samples. The Baermann technique, involving prompt examination of room-temperature feces, is usually used; zinc sulfate flotation is also used. Dictyocaulus has a direct life cycle. The adult worms reside in the large bronchi. Dictyocaulus produces embryonated eggs that are coughed up and swallowed; the eggs then hatch in the intestines, and larvae are expelled in the feces. The expelled larvae are infectious in about 7–10 days and, after ingestion, penetrate the intestinal mucosa and move through the lymphatics and blood into the lungs, where they develop into adults in about 5 weeks. Dictyocaulus filaria causes an especially severe bronchitis in sheep. Protostrongylus inhabits smaller bronchioles. Muellerius is of minor pathogenicity. Protostrongylus and Muellerius require the snail or slug as an intermediate host. Infection occurs through ingestion of infected snails; infections are less likely than those caused by the direct ingestion of Dictyocaulus larvae. Immunity wanes over a year. Viral and bacterial respiratory tract infections may be associated with the parasitic infection. Dictyocaulus viviparus causes the obvious signs in cattle. More severe illness is seen after infections with Cooperia and Ostertagia, because of a synergism between the nematodes even if the cattle are not currently infected with those parasites. Hypobiosis (arrested development of immature worms in lung tissue) is associated with Dictyocaulus infections; cattle will be silent carriers, showing no clinical signs and serving as a means for the infection to survive over winter or a dry season. Pastures can be heavily contaminated during the next grazing season. Necropsy lesions include bronchiolitis and bronchitis, atelectasis, and hyperplasia of peribronchiolar lymphoid tissue. Nematodes frequently reside in the bronchi of the diaphragmatic lung lobes and are frequently enmeshed with frothy exudate. Prevention and control of the disease involve appropriate pasture management. Elimination of intermediate hosts is important in sheep and goat pastures. In a laboratory setting, animals may be procured that are already harboring the disease. Infected animals can be treated with anthelmintics such as ivermectin or levamisole. Muellerius tends to be resistant to levamisole. There is no anthelmintic currently approved for goats, but fenbendazole, administered 2 weeks apart, has been effective for all three nematodes. Treating D. viviparus depends on the type and stage of life of the cattle; label directions must be followed. There is no vaccine for D. viviparus in the United States. Even if infections are not severe and do resolve with treatment, permanent lesions may be inflicted on the lung tissue. c. Cestodes (Tapeworms) i. Moniezia expansa and Thysanosoma actinoides infections. Tapeworms are rarely of clinical or economic importance. In younger animals, heavy infections result in potbellies, constipation or mild diarrhea, poor growth, rough coat, and anemia. Moniezia expansa, and less commonly Moniezia benedini, inhabit the small intestines of grazing ruminants. Moniezia expansa has the widest distribution of the tapeworm species in North America. Soil mites (Galumna spp. and Oribatula spp.) contribute to the life cycle as intermediate hosts, a period that lasts up to 16 weeks. Cysticercoids released from the mites are grazed, pass into the small intestines, and mature. No clinical or pathological sign is usually observed with Moniezia infection; diagnosis is made by observing the characteristic triangular-shaped eggs in fecal flotation examinations. Infection is treated with cestocides. Thysanosoma actinoides, or the fringed tapeworm, is a cestode that resides in the duodenum, bile duct, and pancreatic duct of sheep and cattle raised primarily west of the Mississippi River in the United States. Thysanosoma is of the family Anoplocephalidae. The life cycle is indirect, and the intermediate host is the psocid louse. Larval forms, or cysticercoids, are ingested by grazing animals, and the prepatent period is several months. Typically, no clinical signs are observed with Thysanosoma infection; nonetheless, liver damage, resulting in liver condemnation at slaughter, occurs. Necropsy lesions include bile and/or ductal hyperplasia and fibrosis. Thysanosoma is diagnosed premortem by identifying the gravid segments in the feces. ii. Abdominal or visceral cysticercosis. Abdominal or visceral cysticercosis is an occasional finding at slaughter. The so-called bladder worms typically affect the liver or peritoneal cavity and are the larval form of Taenia hydatigena, the common tapeworm of the dog family. Taenia hydatigena resides in the small intestines of canids, and its gravid segments, oncospheres, contaminate feed and water sources. After ingestion, the larvae penetrate the intestinal mucosa, are transported via the bloodstream to the liver, and cause migration tracts throughout the liver parenchyma. The larvae may leave the liver and migrate into the peritoneal cavity, where they attach and develop over the next 1–9 months into small fluid-filled bladders. The life cycle is completed only after these bladders are ingested by a carnivore, thus completing the maturation of the adult tapeworms. Although larval migration may cause nonspecific signs such as anorexia, hyperthermia, and weight loss, affected animals are usually asymptomatic. At necropsy, the bladder worms will be observed attached to the peritoneal or organ surfaces. Migration tracts may result in fibrosis and inflammation. Diagnosis is usually made at necropsy. Because of the migration through the liver, Fasciola hepatica is a differential diagnosis. Minimizing exposure to canine feces-contaminated feeds and water effectively interrupts the life cycle. Research animals may have been exposed prior to purchase. iii. Echinococcosis (hydatidosis, hydatid cyst disease). Echinococcosis, like cysticercosis, is an occasional finding at slaughter or necropsy. The hydatid cyst is the larval intermediate of the adult tapeworm Echinococcus granulosus, which resides in the small intestines of dogs and wild canids. Embryonated ova are expelled in the feces of the primary host and are ingested by herbivores, swine, and potentially humans. The eggs hatch in the gastrointestinal tract, and the oncospheres penetrate the mucosal lining, enter the bloodstream, and are transported to various organs such as the liver and lungs. The cystic structure develops and potentially ruptures, forming new cystic structures. Clinically, echinococcosis presents minimal clinical signs; unthriftiness or pneumonic lesions may be associated with infected organs. Cysts are typically observed at necropsy. Prevention should be aimed at decreasing fecal contamination of feed and water by canids. Additionally, tapeworm-infected dogs can be treated with standard tapeworm therapies. Treatment of infected ruminants is uncommon. iv. Gid. Coenuris cerebralis, the larval form of the canid tapeworm Taenia (Multiceps) multiceps, is the causative agent of the rare condition called gid. The disease occurs in ruminants as well as many other mammalian species. The larval parasite, ingested from fecal-contaminated food and water, invades the brain and spinal cord and develops as a bladder worm that causes pressure necrosis of the nervous tissues. The resultant signs of hyperesthesia, meningitis, paresis, paralysis, ataxia, and convulsions are observed. Diagnosis is usually made at necropsy. Eliminating transfer from the canid hosts prevents the disease. i. Moniezia expansa and Thysanosoma actinoides infections. Tapeworms are rarely of clinical or economic importance. In younger animals, heavy infections result in potbellies, constipation or mild diarrhea, poor growth, rough coat, and anemia. Moniezia expansa, and less commonly Moniezia benedini, inhabit the small intestines of grazing ruminants. Moniezia expansa has the widest distribution of the tapeworm species in North America. Soil mites (Galumna spp. and Oribatula spp.) contribute to the life cycle as intermediate hosts, a period that lasts up to 16 weeks. Cysticercoids released from the mites are grazed, pass into the small intestines, and mature. No clinical or pathological sign is usually observed with Moniezia infection; diagnosis is made by observing the characteristic triangular-shaped eggs in fecal flotation examinations. Infection is treated with cestocides. Thysanosoma actinoides, or the fringed tapeworm, is a cestode that resides in the duodenum, bile duct, and pancreatic duct of sheep and cattle raised primarily west of the Mississippi River in the United States. Thysanosoma is of the family Anoplocephalidae. The life cycle is indirect, and the intermediate host is the psocid louse. Larval forms, or cysticercoids, are ingested by grazing animals, and the prepatent period is several months. Typically, no clinical signs are observed with Thysanosoma infection; nonetheless, liver damage, resulting in liver condemnation at slaughter, occurs. Necropsy lesions include bile and/or ductal hyperplasia and fibrosis. Thysanosoma is diagnosed premortem by identifying the gravid segments in the feces. ii. Abdominal or visceral cysticercosis. Abdominal or visceral cysticercosis is an occasional finding at slaughter. The so-called bladder worms typically affect the liver or peritoneal cavity and are the larval form of Taenia hydatigena, the common tapeworm of the dog family. Taenia hydatigena resides in the small intestines of canids, and its gravid segments, oncospheres, contaminate feed and water sources. After ingestion, the larvae penetrate the intestinal mucosa, are transported via the bloodstream to the liver, and cause migration tracts throughout the liver parenchyma. The larvae may leave the liver and migrate into the peritoneal cavity, where they attach and develop over the next 1–9 months into small fluid-filled bladders. The life cycle is completed only after these bladders are ingested by a carnivore, thus completing the maturation of the adult tapeworms. Although larval migration may cause nonspecific signs such as anorexia, hyperthermia, and weight loss, affected animals are usually asymptomatic. At necropsy, the bladder worms will be observed attached to the peritoneal or organ surfaces. Migration tracts may result in fibrosis and inflammation. Diagnosis is usually made at necropsy. Because of the migration through the liver, Fasciola hepatica is a differential diagnosis. Minimizing exposure to canine feces-contaminated feeds and water effectively interrupts the life cycle. Research animals may have been exposed prior to purchase. iii. Echinococcosis (hydatidosis, hydatid cyst disease). Echinococcosis, like cysticercosis, is an occasional finding at slaughter or necropsy. The hydatid cyst is the larval intermediate of the adult tapeworm Echinococcus granulosus, which resides in the small intestines of dogs and wild canids. Embryonated ova are expelled in the feces of the primary host and are ingested by herbivores, swine, and potentially humans. The eggs hatch in the gastrointestinal tract, and the oncospheres penetrate the mucosal lining, enter the bloodstream, and are transported to various organs such as the liver and lungs. The cystic structure develops and potentially ruptures, forming new cystic structures. Clinically, echinococcosis presents minimal clinical signs; unthriftiness or pneumonic lesions may be associated with infected organs. Cysts are typically observed at necropsy. Prevention should be aimed at decreasing fecal contamination of feed and water by canids. Additionally, tapeworm-infected dogs can be treated with standard tapeworm therapies. Treatment of infected ruminants is uncommon. iv. Gid. Coenuris cerebralis, the larval form of the canid tapeworm Taenia (Multiceps) multiceps, is the causative agent of the rare condition called gid. The disease occurs in ruminants as well as many other mammalian species. The larval parasite, ingested from fecal-contaminated food and water, invades the brain and spinal cord and develops as a bladder worm that causes pressure necrosis of the nervous tissues. The resultant signs of hyperesthesia, meningitis, paresis, paralysis, ataxia, and convulsions are observed. Diagnosis is usually made at necropsy. Eliminating transfer from the canid hosts prevents the disease. i. Moniezia expansa and Thysanosoma actinoides infections. Tapeworms are rarely of clinical or economic importance. In younger animals, heavy infections result in potbellies, constipation or mild diarrhea, poor growth, rough coat, and anemia. Moniezia expansa, and less commonly Moniezia benedini, inhabit the small intestines of grazing ruminants. Moniezia expansa has the widest distribution of the tapeworm species in North America. Soil mites (Galumna spp. and Oribatula spp.) contribute to the life cycle as intermediate hosts, a period that lasts up to 16 weeks. Cysticercoids released from the mites are grazed, pass into the small intestines, and mature. No clinical or pathological sign is usually observed with Moniezia infection; diagnosis is made by observing the characteristic triangular-shaped eggs in fecal flotation examinations. Infection is treated with cestocides. Thysanosoma actinoides, or the fringed tapeworm, is a cestode that resides in the duodenum, bile duct, and pancreatic duct of sheep and cattle raised primarily west of the Mississippi River in the United States. Thysanosoma is of the family Anoplocephalidae. The life cycle is indirect, and the intermediate host is the psocid louse. Larval forms, or cysticercoids, are ingested by grazing animals, and the prepatent period is several months. Typically, no clinical signs are observed with Thysanosoma infection; nonetheless, liver damage, resulting in liver condemnation at slaughter, occurs. Necropsy lesions include bile and/or ductal hyperplasia and fibrosis. Thysanosoma is diagnosed premortem by identifying the gravid segments in the feces. ii. Abdominal or visceral cysticercosis. Abdominal or visceral cysticercosis is an occasional finding at slaughter. The so-called bladder worms typically affect the liver or peritoneal cavity and are the larval form of Taenia hydatigena, the common tapeworm of the dog family. Taenia hydatigena resides in the small intestines of canids, and its gravid segments, oncospheres, contaminate feed and water sources. After ingestion, the larvae penetrate the intestinal mucosa, are transported via the bloodstream to the liver, and cause migration tracts throughout the liver parenchyma. The larvae may leave the liver and migrate into the peritoneal cavity, where they attach and develop over the next 1–9 months into small fluid-filled bladders. The life cycle is completed only after these bladders are ingested by a carnivore, thus completing the maturation of the adult tapeworms. Although larval migration may cause nonspecific signs such as anorexia, hyperthermia, and weight loss, affected animals are usually asymptomatic. At necropsy, the bladder worms will be observed attached to the peritoneal or organ surfaces. Migration tracts may result in fibrosis and inflammation. Diagnosis is usually made at necropsy. Because of the migration through the liver, Fasciola hepatica is a differential diagnosis. Minimizing exposure to canine feces-contaminated feeds and water effectively interrupts the life cycle. Research animals may have been exposed prior to purchase. iii. Echinococcosis (hydatidosis, hydatid cyst disease). Echinococcosis, like cysticercosis, is an occasional finding at slaughter or necropsy. The hydatid cyst is the larval intermediate of the adult tapeworm Echinococcus granulosus, which resides in the small intestines of dogs and wild canids. Embryonated ova are expelled in the feces of the primary host and are ingested by herbivores, swine, and potentially humans. The eggs hatch in the gastrointestinal tract, and the oncospheres penetrate the mucosal lining, enter the bloodstream, and are transported to various organs such as the liver and lungs. The cystic structure develops and potentially ruptures, forming new cystic structures. Clinically, echinococcosis presents minimal clinical signs; unthriftiness or pneumonic lesions may be associated with infected organs. Cysts are typically observed at necropsy. Prevention should be aimed at decreasing fecal contamination of feed and water by canids. Additionally, tapeworm-infected dogs can be treated with standard tapeworm therapies. Treatment of infected ruminants is uncommon. iv. Gid. Coenuris cerebralis, the larval form of the canid tapeworm Taenia (Multiceps) multiceps, is the causative agent of the rare condition called gid. The disease occurs in ruminants as well as many other mammalian species. The larval parasite, ingested from fecal-contaminated food and water, invades the brain and spinal cord and develops as a bladder worm that causes pressure necrosis of the nervous tissues. The resultant signs of hyperesthesia, meningitis, paresis, paralysis, ataxia, and convulsions are observed. Diagnosis is usually made at necropsy. Eliminating transfer from the canid hosts prevents the disease. d. Trematodes i. Fascioliasis (liver fluke disease). Liver flukes are an important cause of acute and chronic disease in grazing sheep and cattle. There are three common species of flukes in ruminants of the continental United States: Fasciola hepatica, Fascioloides magna, and Dicrocoelium dendriticum. Fasciola hepática infections are primarily seen in Gulf Coast and western states. Fascioloides magna infections are typically seen in Gulf, Great Lake, and northwestern states, where ruminants share pasture with deer, elk, and moose. Dicrocoelium dendriticum infections occur only in New York State. Liver fluke eggs are passed in the bile and feces and hatch in 2–3 weeks to form the free-swimming miracidia. It is important to note that each fluke egg represents the source of eventually thousands of cercariae or metacercariae. The miracidia penetrate the body of an intermediate host (usually freshwater snails) and develop through sporocyst and redia stages, finally forming cercariae. (Dicrocoelium is unique because it utilizes a land snail that expels slime balls, each containing several hundred cercariae. These are eaten by a second intermediate host, the ant Formica fusca.) The cercariae leave the intermediate host, swim to grassy vegetation, lose their tail, and become a cystlike metacercaria. The metacercariae may remain in a dormant stage on the grass for 6 months or longer until ingested by a ruminant. The ingested metacercariae penetrate the small-intestinal wall and migrate through the abdominal cavity to the liver. There they locate in a bile duct, mature, and remain for up to 4 years. Acute liver fluke disease is related to the damage caused by the migration of immature flukes. Migratory flukes may lead to liver inflammation, hemorrhage, necrosis, and fibrosis. Fascioloides magna infections in sheep and goats can be fatal as the result of just one fluke tunneling through hepatic tissue. In cattle, infections are often asymptomatic because of the host's encapsulation of the parasite. Liver fluke damage may predispose to invasion by anaerobic Clostridium species such as C. novyi that could lead to fatal black disease or bacillary hemoglobinuria. Chronic disease may result from fluke-induced physical damage to the bile ducts and cholangiohepatitis. Blood loss into the bile may lead to anemia and hypoproteinemia. Liver damage also is evidenced by increases in liver enzymes such as γ-glutamyl transpeptidase (GGT). Persistent eosinophilia is also seen with liver fluke disease. Other clinical signs of liver fluke disease include anorexia, weight loss, unthriftiness, edema, and ascites. At necropsy, livers will be pale and friable and may have distinct migration tunnels along the serosal surfaces. Bile ducts will be enlarged, and areas of fibrosis will be evident. Diagnosis can be made from clinical signs and postmortem analyses. Blood chemistries suggestive of liver disease and eosinophilia support the diagnosis. Liver fluke control involves removal of the intermediate hosts. In a laboratory setting, liver fluke infection is unlikely. Nonetheless, incoming animals from pasture environments may be infected. Liver flukes can be treated by using the anthelmintic albendazole. ii. Rumen fluke infections (paramphistomosis). Paramphistomosis is an uncommon disease found in sheep and cattle in southern states. Paramphistomum microbothrioides and P. cervi inhabit the duodenum and rumen of affected sheep. Eggs are passed in the feces and hatch in approximately 1 month, and the miracidia penetrate the intermediate snail hosts. Cercariae develop in the snail over the next month, emerge, and encyst on grasses as metacercariae. When eaten, the metacercariae develop into adult flukes and attach to the mucosal lining. The life cycle is complete in approximately 100 days. The flukes cause localized injury to the mucosa and, by interfering with digestive processes, cause diarrhea and protein loss. Clinically, animals may experience anorexia, dehydration, weight loss, and diarrhea with or without blood. Mortality may reach 25%. Diagnosis is based on clinical findings as well as the identification of flukes or eggs in the feces. Animals can be treated with fluki-cides. Eliminating the intermediate host prevents the disease. i. Fascioliasis (liver fluke disease). Liver flukes are an important cause of acute and chronic disease in grazing sheep and cattle. There are three common species of flukes in ruminants of the continental United States: Fasciola hepatica, Fascioloides magna, and Dicrocoelium dendriticum. Fasciola hepática infections are primarily seen in Gulf Coast and western states. Fascioloides magna infections are typically seen in Gulf, Great Lake, and northwestern states, where ruminants share pasture with deer, elk, and moose. Dicrocoelium dendriticum infections occur only in New York State. Liver fluke eggs are passed in the bile and feces and hatch in 2–3 weeks to form the free-swimming miracidia. It is important to note that each fluke egg represents the source of eventually thousands of cercariae or metacercariae. The miracidia penetrate the body of an intermediate host (usually freshwater snails) and develop through sporocyst and redia stages, finally forming cercariae. (Dicrocoelium is unique because it utilizes a land snail that expels slime balls, each containing several hundred cercariae. These are eaten by a second intermediate host, the ant Formica fusca.) The cercariae leave the intermediate host, swim to grassy vegetation, lose their tail, and become a cystlike metacercaria. The metacercariae may remain in a dormant stage on the grass for 6 months or longer until ingested by a ruminant. The ingested metacercariae penetrate the small-intestinal wall and migrate through the abdominal cavity to the liver. There they locate in a bile duct, mature, and remain for up to 4 years. Acute liver fluke disease is related to the damage caused by the migration of immature flukes. Migratory flukes may lead to liver inflammation, hemorrhage, necrosis, and fibrosis. Fascioloides magna infections in sheep and goats can be fatal as the result of just one fluke tunneling through hepatic tissue. In cattle, infections are often asymptomatic because of the host's encapsulation of the parasite. Liver fluke damage may predispose to invasion by anaerobic Clostridium species such as C. novyi that could lead to fatal black disease or bacillary hemoglobinuria. Chronic disease may result from fluke-induced physical damage to the bile ducts and cholangiohepatitis. Blood loss into the bile may lead to anemia and hypoproteinemia. Liver damage also is evidenced by increases in liver enzymes such as γ-glutamyl transpeptidase (GGT). Persistent eosinophilia is also seen with liver fluke disease. Other clinical signs of liver fluke disease include anorexia, weight loss, unthriftiness, edema, and ascites. At necropsy, livers will be pale and friable and may have distinct migration tunnels along the serosal surfaces. Bile ducts will be enlarged, and areas of fibrosis will be evident. Diagnosis can be made from clinical signs and postmortem analyses. Blood chemistries suggestive of liver disease and eosinophilia support the diagnosis. Liver fluke control involves removal of the intermediate hosts. In a laboratory setting, liver fluke infection is unlikely. Nonetheless, incoming animals from pasture environments may be infected. Liver flukes can be treated by using the anthelmintic albendazole. ii. Rumen fluke infections (paramphistomosis). Paramphistomosis is an uncommon disease found in sheep and cattle in southern states. Paramphistomum microbothrioides and P. cervi inhabit the duodenum and rumen of affected sheep. Eggs are passed in the feces and hatch in approximately 1 month, and the miracidia penetrate the intermediate snail hosts. Cercariae develop in the snail over the next month, emerge, and encyst on grasses as metacercariae. When eaten, the metacercariae develop into adult flukes and attach to the mucosal lining. The life cycle is complete in approximately 100 days. The flukes cause localized injury to the mucosa and, by interfering with digestive processes, cause diarrhea and protein loss. Clinically, animals may experience anorexia, dehydration, weight loss, and diarrhea with or without blood. Mortality may reach 25%. Diagnosis is based on clinical findings as well as the identification of flukes or eggs in the feces. Animals can be treated with fluki-cides. Eliminating the intermediate host prevents the disease. i. Fascioliasis (liver fluke disease). Liver flukes are an important cause of acute and chronic disease in grazing sheep and cattle. There are three common species of flukes in ruminants of the continental United States: Fasciola hepatica, Fascioloides magna, and Dicrocoelium dendriticum. Fasciola hepática infections are primarily seen in Gulf Coast and western states. Fascioloides magna infections are typically seen in Gulf, Great Lake, and northwestern states, where ruminants share pasture with deer, elk, and moose. Dicrocoelium dendriticum infections occur only in New York State. Liver fluke eggs are passed in the bile and feces and hatch in 2–3 weeks to form the free-swimming miracidia. It is important to note that each fluke egg represents the source of eventually thousands of cercariae or metacercariae. The miracidia penetrate the body of an intermediate host (usually freshwater snails) and develop through sporocyst and redia stages, finally forming cercariae. (Dicrocoelium is unique because it utilizes a land snail that expels slime balls, each containing several hundred cercariae. These are eaten by a second intermediate host, the ant Formica fusca.) The cercariae leave the intermediate host, swim to grassy vegetation, lose their tail, and become a cystlike metacercaria. The metacercariae may remain in a dormant stage on the grass for 6 months or longer until ingested by a ruminant. The ingested metacercariae penetrate the small-intestinal wall and migrate through the abdominal cavity to the liver. There they locate in a bile duct, mature, and remain for up to 4 years. Acute liver fluke disease is related to the damage caused by the migration of immature flukes. Migratory flukes may lead to liver inflammation, hemorrhage, necrosis, and fibrosis. Fascioloides magna infections in sheep and goats can be fatal as the result of just one fluke tunneling through hepatic tissue. In cattle, infections are often asymptomatic because of the host's encapsulation of the parasite. Liver fluke damage may predispose to invasion by anaerobic Clostridium species such as C. novyi that could lead to fatal black disease or bacillary hemoglobinuria. Chronic disease may result from fluke-induced physical damage to the bile ducts and cholangiohepatitis. Blood loss into the bile may lead to anemia and hypoproteinemia. Liver damage also is evidenced by increases in liver enzymes such as γ-glutamyl transpeptidase (GGT). Persistent eosinophilia is also seen with liver fluke disease. Other clinical signs of liver fluke disease include anorexia, weight loss, unthriftiness, edema, and ascites. At necropsy, livers will be pale and friable and may have distinct migration tunnels along the serosal surfaces. Bile ducts will be enlarged, and areas of fibrosis will be evident. Diagnosis can be made from clinical signs and postmortem analyses. Blood chemistries suggestive of liver disease and eosinophilia support the diagnosis. Liver fluke control involves removal of the intermediate hosts. In a laboratory setting, liver fluke infection is unlikely. Nonetheless, incoming animals from pasture environments may be infected. Liver flukes can be treated by using the anthelmintic albendazole. ii. Rumen fluke infections (paramphistomosis). Paramphistomosis is an uncommon disease found in sheep and cattle in southern states. Paramphistomum microbothrioides and P. cervi inhabit the duodenum and rumen of affected sheep. Eggs are passed in the feces and hatch in approximately 1 month, and the miracidia penetrate the intermediate snail hosts. Cercariae develop in the snail over the next month, emerge, and encyst on grasses as metacercariae. When eaten, the metacercariae develop into adult flukes and attach to the mucosal lining. The life cycle is complete in approximately 100 days. The flukes cause localized injury to the mucosa and, by interfering with digestive processes, cause diarrhea and protein loss. Clinically, animals may experience anorexia, dehydration, weight loss, and diarrhea with or without blood. Mortality may reach 25%. Diagnosis is based on clinical findings as well as the identification of flukes or eggs in the feces. Animals can be treated with fluki-cides. Eliminating the intermediate host prevents the disease. e. Mites (Mange) Mites cause a chronic dermatitis. The principal symptom of these infections is intense pruritus. In addition, papules, crusts, alopecia, and secondary dermatitis are seen. Anemia, disruption of reproductive cycles, and increased susceptibility to other diseases may also occur. Mites are rare in ruminants in the United States, but infections of Sarcoptes and Psorergates mange must be reported to animal health officials. Ruminants in poorly managed facilities are generally the most susceptible to infection, and infections are more frequent during winter months. Diagnosis is based on signs, examination of skin scrapings, and response to therapy. No effective treatment for demodectic mange in large animals has been found. The differential for mite infestations is pediculosis. Several genera of mites may affect sheep. These have been eradicated from flocks in the United States or are very rare and include Psoroptes ovis (common scabies), Sarcoptes scabiei (head scabies, barn itch), Psorergates ovis (sheep itch mite), Chorioptes ovis (foot scabies, tail mange), and Demodex ovis (follicular mange). Goats can also be infected by sarcoptic, chorioptic, and psoroptic mange. The scabies mite Sarcoptes rupicaprae invades epidermal tissue and causes focal pruritic areas around the head and neck. The chorioptic mite, either Chorioptes bovis or C. caprae, does not invade epidermal tissue but rather feeds on dead skin tissue. The chorioptic mite prefers distal limbs, the udder, and the scrotum and can be a significant cause of pruritus. The psoroptic mite Psoroptes cuniculi commonly occurs in the ear canal and causes head shaking and scratching. Repeated treatments of lime sulfur, amitraz, or ivermectin may be effective ( Smith and Sherman, 1994 ). Goats are also susceptible to demodectic mange caused by Demodex caprae. Adult mites invade hair follicles and sebaceous glands. Pustules may develop with secondary bacterial infection. Psoroptes bovis continues to be present in cattle in the United States, although it has been eradicated from sheep. Chorioptes bovis typically infects lower hindlimbs, perineum, tail, and scrotum but can become generalized. The sarcoptic mange mite S. scabei can survive off the host, so fomite transmission is a factor. The mange usually begins around the head but then spreads. This parasite can be transmitted to humans. Demodex bovis infects cattle; nodules on the face and neck are typical. Demodex bovis infections may resolve without treatment. Lindane, coumaphos, malathion, and lime sulfur are used to treat Psoroptes and Psorergates. Ivermectin is effective against Sarcoptes and is approved for use in cattle. f. Lice (Pediculosis) Lice that infect ruminants are of the orders Mallophaga, biting or chewing lice, and Anoplura, sucking lice. These are wingless insects. Members of the Mallophaga are colored yellow to red; members of the Anoplura are blue gray. Lice produce a seasonal (winter-to-spring), chronic dermatitis. In sheep, biting lice include Damalinia (Bovicola) ovis (sheep body louse). Sucking lice that infect sheep include Linognathus ovillus (blue body louse) and L. pedalis (sheep foot louse). In goats, biting lice infection are caused by D. caprae (goat biting louse), D. limbatus (Angora goat biting louse), and D. crassipes. Sucking louse infections in goats are caused by L. stenopis and L. africanus. Damalinia bovis is the cattle biting louse. Sucking lice include L. vituli, Solenopotes capillatus, Haematopinus eurysternus, and H. quadripertusus. Pruritus is the most common sign and often results in alopecia and excoriation. The host's rubbing and grooming may not correlate with the extent of infestation. Hairballs can result from overgrooming in cattle. In severe cases, the organisms can lead to anemia, weight loss, and damaged wool in sheep and damaged pelts in other ruminants. Young animals with severe infestations of sucking lice may become anemic or even die. Pregnant animals with heavy infestations may abort. In sheep infected with the foot louse, lameness may result. Lice are generally species-specific. Those infecting ruminants are usually smaller than 5 mm. Goats may serve as a source of infection for sheep by harboring Damalinia ovis. Transmission is primarily by direct contact between animals. Transmission can also occur by attachment to flies or by fomites. Some animals are identified as carriers and seem to be particularly susceptible to infestations. Biting or chewing lice inhabit the host's face, lower legs, and flanks and feed on epidermal debris and sebaceous secretions. Sucking lice inhabit the host's neck, back, and body region and feed on blood. Lice eggs or nits are attached to hairs near the skin. Three nymphal stages, or instars, occur between egg and adult, and the growth cycle takes about 1 month for all species. Lice cannot survive for more than a few days off the host. All ruminant mite infestations are differentials for the clinical signs seen with pediculosis. Animals that are carriers should be culled, because these individuals may perpetuate the infection in the group. Lice are effectively treated with a variety of insecticides, including coumaphos, dichlorvos, crotoxyphos, avermectin, and pyrethroids. Label directions should be read and adhered to, including withdrawal times. Products should not be used on female dairy animals. Treatments must be repeated at least twice at intervals appropriate for nit hatches (about every 16 days) because nits will not be killed. Fall treatments are useful in managing the infections. Systemic treatments in cattle are contraindicated when there may be concurrent larvae of cattle grubs (Hypoderma lineatum and H. bovis). Back rubbers with insecticides, capitalizing on self-treatment, are useful for cattle. Sustained-release insecticide-containing ear tags are approved for use in cattle. g. Ticks Etiology. Ruminants are susceptible to many species of Ixodidae (hard-shell ticks) and Argasidae (softshell ticks). Many diseases, including anaplasmosis, babesiosis, and Q fever are transmitted by ticks. Clinical signs and diagnosis. Tick infestations are associated with decreased productivity, loss of blood and blood proteins, transmission of diseases, debilitation, and even death. Feeding sites on the host vary with the tick species. Ticks are associated with an acute paralytic syndrome called tick paralysis. This disease is characterized by ascending paralysis and may lead to death if the tick is not removed before the paralysis reaches the respiratory muscles. Diagnosis is based on identification of the species. Epizootiology and transmission. Ticks are not as host-specific as lice. Ticks are classified as one-host, two-host, or three-host; this refers to whether they drop off the host between larval and nymphal stages to molt. Pathogenesis of tick infestations. Patterns of feeding on the host differ between Argasidae and Ixodidae. The former feed repeatedly, whereas the latter feed once during each life stage. Pathogenesis of tick paralysis. Following a tick-feeding period of 4–6 days, the tick salivary toxin travels hematogenously to the myoneural junctions and spinal cord and inhibits nerve transmission. Removal of the ticks reverses the syndrome unless paralysis has migrated anteriorly to the respiratory centers of the medulla. In these cases, death due to respiratory failure occurs. Treatment. Ticks can be treated using systemic or topical insecticides. Etiology. Ruminants are susceptible to many species of Ixodidae (hard-shell ticks) and Argasidae (softshell ticks). Many diseases, including anaplasmosis, babesiosis, and Q fever are transmitted by ticks. Clinical signs and diagnosis. Tick infestations are associated with decreased productivity, loss of blood and blood proteins, transmission of diseases, debilitation, and even death. Feeding sites on the host vary with the tick species. Ticks are associated with an acute paralytic syndrome called tick paralysis. This disease is characterized by ascending paralysis and may lead to death if the tick is not removed before the paralysis reaches the respiratory muscles. Diagnosis is based on identification of the species. Epizootiology and transmission. Ticks are not as host-specific as lice. Ticks are classified as one-host, two-host, or three-host; this refers to whether they drop off the host between larval and nymphal stages to molt. Pathogenesis of tick infestations. Patterns of feeding on the host differ between Argasidae and Ixodidae. The former feed repeatedly, whereas the latter feed once during each life stage. Pathogenesis of tick paralysis. Following a tick-feeding period of 4–6 days, the tick salivary toxin travels hematogenously to the myoneural junctions and spinal cord and inhibits nerve transmission. Removal of the ticks reverses the syndrome unless paralysis has migrated anteriorly to the respiratory centers of the medulla. In these cases, death due to respiratory failure occurs. Treatment. Ticks can be treated using systemic or topical insecticides. Etiology. Ruminants are susceptible to many species of Ixodidae (hard-shell ticks) and Argasidae (softshell ticks). Many diseases, including anaplasmosis, babesiosis, and Q fever are transmitted by ticks. Clinical signs and diagnosis. Tick infestations are associated with decreased productivity, loss of blood and blood proteins, transmission of diseases, debilitation, and even death. Feeding sites on the host vary with the tick species. Ticks are associated with an acute paralytic syndrome called tick paralysis. This disease is characterized by ascending paralysis and may lead to death if the tick is not removed before the paralysis reaches the respiratory muscles. Diagnosis is based on identification of the species. Epizootiology and transmission. Ticks are not as host-specific as lice. Ticks are classified as one-host, two-host, or three-host; this refers to whether they drop off the host between larval and nymphal stages to molt. Pathogenesis of tick infestations. Patterns of feeding on the host differ between Argasidae and Ixodidae. The former feed repeatedly, whereas the latter feed once during each life stage. Pathogenesis of tick paralysis. Following a tick-feeding period of 4–6 days, the tick salivary toxin travels hematogenously to the myoneural junctions and spinal cord and inhibits nerve transmission. Removal of the ticks reverses the syndrome unless paralysis has migrated anteriorly to the respiratory centers of the medulla. In these cases, death due to respiratory failure occurs. Treatment. Ticks can be treated using systemic or topical insecticides. h. Other Parasites i. Nasal bots (nasal myiasis, head grubs). Nasal myiasis causes a chronic rhinitis and sinusitis. The disease is caused by the larval forms of the botfly Oestrus ovis. The botfly deposits eggs around the nostrils of sheep. The ova hatch, and the larvae migrate throughout the nasal cavity and sinuses, feeding on mucus and debris. In 2–10 months, the larvae complete their growing phase, migrate back to the nasal cavity, and are sneezed out. The mature larvae penetrate the soil and pupate for 1–1.5 months and emerge as botflies. Clinically, early in the disease course, animals display unique behaviors such as stamping, snorting, sneezing, and rubbing their noses against each other or objects. Hypersensitivity to the larvae occurs ( Dorchies et al., 1998 ). Later, mucopurulent nasal discharges associated with the larval-induced inflammation of mucosal linings will be observed. At necropsy, larvae will be observed in the nasal cavity or sinuses. Mild inflammatory reactions, mucosal thickening, and exudates will accompany the larvae. The disease is diagnosed by observing the behaviors or identifying organisms at necropsy. Up to 80% of a flock will potentially be infected; treatment should be employed on the rest of the flock. Ivermectins and other insecticides will eliminate the larvae; but treatment should be done in the early fall, when larvae are small. Fly repellents may be helpful at preventing additional infections. ii. Screwworm flies. Cochliomyia hominivorax (Callitroga americana) is the the screwworm that causes occasional disease in the southwestern United States along the Mexico border. Eradication programs have been pursued, and the disease is reportable. Large greenish flies lay large numbers of white eggs as shinglelike layers at the edges of open wounds (including docking and castration sites), soiled skin, or abrasions. Eggs hatch within 24 hr. Larvae are obligate parasites of living tissue, and the cycle is perpetuated because the increasingly large wound continues to be attractive to the next generation of flies. Larvae eventually drop off, pupate best in hot climates, and hatch in 3 weeks. Large cavities in parasitized tissue are formed, and lesions are characterized by malodor, large volumes of brown exudate, and necrosis. Single animals or entire herds may be affected. Treatment is intensive, with dressings and larvicidal applications. If there is no intervention, the host succumbs to secondary infections and fluid loss. Effective current control regimens include subcutaneous injection of ivermectin and programs that release sterile male flies. iii. Sheep keds ("sheep ticks"). In sheep and goats, sheep keds produce a chronic irritation and dermatitis with associated pruritus. The disease is caused by Melophagus ovinus, which is a flat, brown, blood-sucking, wingless fly; the term sheep tick is incorrectly used. The adult fly lives entirely on the skin of sheep. Females mate and produce 10–15 larvae following a gestation of about 10–12 days. The larvae attach to the wool or hair and then pupate for about 3 weeks. The adult female feeds on blood and lives for 4–5 months; the life cycle is completed in about 5–6 weeks. Infection is highest in fall and winter. Pruritus develops around the neck, sides, abdomen, and rump. In severe cases, anemia may occur. Keds can transmit bluetongue virus. Keds are diagnosed by gross or microscopic identification. Ivermectin or other insecticides are useful treatment agents. i. Nasal bots (nasal myiasis, head grubs). Nasal myiasis causes a chronic rhinitis and sinusitis. The disease is caused by the larval forms of the botfly Oestrus ovis. The botfly deposits eggs around the nostrils of sheep. The ova hatch, and the larvae migrate throughout the nasal cavity and sinuses, feeding on mucus and debris. In 2–10 months, the larvae complete their growing phase, migrate back to the nasal cavity, and are sneezed out. The mature larvae penetrate the soil and pupate for 1–1.5 months and emerge as botflies. Clinically, early in the disease course, animals display unique behaviors such as stamping, snorting, sneezing, and rubbing their noses against each other or objects. Hypersensitivity to the larvae occurs ( Dorchies et al., 1998 ). Later, mucopurulent nasal discharges associated with the larval-induced inflammation of mucosal linings will be observed. At necropsy, larvae will be observed in the nasal cavity or sinuses. Mild inflammatory reactions, mucosal thickening, and exudates will accompany the larvae. The disease is diagnosed by observing the behaviors or identifying organisms at necropsy. Up to 80% of a flock will potentially be infected; treatment should be employed on the rest of the flock. Ivermectins and other insecticides will eliminate the larvae; but treatment should be done in the early fall, when larvae are small. Fly repellents may be helpful at preventing additional infections. ii. Screwworm flies. Cochliomyia hominivorax (Callitroga americana) is the the screwworm that causes occasional disease in the southwestern United States along the Mexico border. Eradication programs have been pursued, and the disease is reportable. Large greenish flies lay large numbers of white eggs as shinglelike layers at the edges of open wounds (including docking and castration sites), soiled skin, or abrasions. Eggs hatch within 24 hr. Larvae are obligate parasites of living tissue, and the cycle is perpetuated because the increasingly large wound continues to be attractive to the next generation of flies. Larvae eventually drop off, pupate best in hot climates, and hatch in 3 weeks. Large cavities in parasitized tissue are formed, and lesions are characterized by malodor, large volumes of brown exudate, and necrosis. Single animals or entire herds may be affected. Treatment is intensive, with dressings and larvicidal applications. If there is no intervention, the host succumbs to secondary infections and fluid loss. Effective current control regimens include subcutaneous injection of ivermectin and programs that release sterile male flies. iii. Sheep keds ("sheep ticks"). In sheep and goats, sheep keds produce a chronic irritation and dermatitis with associated pruritus. The disease is caused by Melophagus ovinus, which is a flat, brown, blood-sucking, wingless fly; the term sheep tick is incorrectly used. The adult fly lives entirely on the skin of sheep. Females mate and produce 10–15 larvae following a gestation of about 10–12 days. The larvae attach to the wool or hair and then pupate for about 3 weeks. The adult female feeds on blood and lives for 4–5 months; the life cycle is completed in about 5–6 weeks. Infection is highest in fall and winter. Pruritus develops around the neck, sides, abdomen, and rump. In severe cases, anemia may occur. Keds can transmit bluetongue virus. Keds are diagnosed by gross or microscopic identification. Ivermectin or other insecticides are useful treatment agents. i. Nasal bots (nasal myiasis, head grubs). Nasal myiasis causes a chronic rhinitis and sinusitis. The disease is caused by the larval forms of the botfly Oestrus ovis. The botfly deposits eggs around the nostrils of sheep. The ova hatch, and the larvae migrate throughout the nasal cavity and sinuses, feeding on mucus and debris. In 2–10 months, the larvae complete their growing phase, migrate back to the nasal cavity, and are sneezed out. The mature larvae penetrate the soil and pupate for 1–1.5 months and emerge as botflies. Clinically, early in the disease course, animals display unique behaviors such as stamping, snorting, sneezing, and rubbing their noses against each other or objects. Hypersensitivity to the larvae occurs ( Dorchies et al., 1998 ). Later, mucopurulent nasal discharges associated with the larval-induced inflammation of mucosal linings will be observed. At necropsy, larvae will be observed in the nasal cavity or sinuses. Mild inflammatory reactions, mucosal thickening, and exudates will accompany the larvae. The disease is diagnosed by observing the behaviors or identifying organisms at necropsy. Up to 80% of a flock will potentially be infected; treatment should be employed on the rest of the flock. Ivermectins and other insecticides will eliminate the larvae; but treatment should be done in the early fall, when larvae are small. Fly repellents may be helpful at preventing additional infections. ii. Screwworm flies. Cochliomyia hominivorax (Callitroga americana) is the the screwworm that causes occasional disease in the southwestern United States along the Mexico border. Eradication programs have been pursued, and the disease is reportable. Large greenish flies lay large numbers of white eggs as shinglelike layers at the edges of open wounds (including docking and castration sites), soiled skin, or abrasions. Eggs hatch within 24 hr. Larvae are obligate parasites of living tissue, and the cycle is perpetuated because the increasingly large wound continues to be attractive to the next generation of flies. Larvae eventually drop off, pupate best in hot climates, and hatch in 3 weeks. Large cavities in parasitized tissue are formed, and lesions are characterized by malodor, large volumes of brown exudate, and necrosis. Single animals or entire herds may be affected. Treatment is intensive, with dressings and larvicidal applications. If there is no intervention, the host succumbs to secondary infections and fluid loss. Effective current control regimens include subcutaneous injection of ivermectin and programs that release sterile male flies. iii. Sheep keds ("sheep ticks"). In sheep and goats, sheep keds produce a chronic irritation and dermatitis with associated pruritus. The disease is caused by Melophagus ovinus, which is a flat, brown, blood-sucking, wingless fly; the term sheep tick is incorrectly used. The adult fly lives entirely on the skin of sheep. Females mate and produce 10–15 larvae following a gestation of about 10–12 days. The larvae attach to the wool or hair and then pupate for about 3 weeks. The adult female feeds on blood and lives for 4–5 months; the life cycle is completed in about 5–6 weeks. Infection is highest in fall and winter. Pruritus develops around the neck, sides, abdomen, and rump. In severe cases, anemia may occur. Keds can transmit bluetongue virus. Keds are diagnosed by gross or microscopic identification. Ivermectin or other insecticides are useful treatment agents. 5. Fungal Disease: Dermatophytes (Ringworm) Etiology. Dermatophytosis, or infection of the keratinized layers of skin, is caused mostly by species of the genera Trichophyton and Microsporum. The primary causes in sheep are T. mentagrophytes and T. verrucosum. In goats, the agents are T. mentagrophytes, M. canis, M. gypseum, T. verrucosum, T. schoenleinii, and Epidermophyton floccosum. In cattle, T. verrucosum is the primary causative agent. Dermatophytosis is a common fungal infection of the epidermis of cattle and is less common in sheep and goats. Clinical signs and diagnosis. Multiple, gray, crusty, circumscribed, hyperkeratotic lesions are characteristic of infection. Lesions will vary in size. In all ruminants, lesions will be around the head, neck, and ears. In goats and cattle, lesions will extend down the neck, and in cattle, lesions develop particularly around the eyes and on the thorax. Cattle lesions are unique in the marked crustiness, which progressively appears wartlike. Hair shafts become brittle and break off. Intense pruritus is often associated with the alopecic lesions. The disease can be diagnosed by microscopic identification of hyphae and conidia on the hairs following skin scraping and 20% potassium hydroxide digestion. Dermatophyte test media (DTM) cultures are the most reliable means to diagnose the fungus. Broken hairs from the periphery of the lesion are the best sources of the fungus. Epizootiology and transmission. Younger animals are more susceptible, and factors such as crowding, indoor housing, warm and humid conditions, and poor nutrition are also important. Transmission is by direct contact or by contact with contaminated fomites, such as equipment, fencing, or feed bunks. Pathogenesis. Incubation can be as long as 6 weeks. The organisms invade and multiply in hair shafts. Treatment. Spontaneous recovery occurs in all species in 1–4 months. Although cell-mediated immunity is considered important, other immune mechanisms are not well understood. Immunity may not be of long duration. Recovery is enhanced by correcting nutritional deficiencies and improving housing and ventilation problems. A number of topical treatments, such as 2–5% lime-sulfur solution, 3% captan, iodophors, thiabendazole, and 0.5% sodium hypochlorite, can be used. In severe cases, systemic therapy with griseofulvin may be successful. Prevention and control. The animals' environment and overall physical condition should be reassessed with particular attention to ventilation, crowding, sanitation, and nutrition. Pens should be thoroughly cleaned and disinfected. Research complications. Ringworm is a zoonotic disease. Etiology. Dermatophytosis, or infection of the keratinized layers of skin, is caused mostly by species of the genera Trichophyton and Microsporum. The primary causes in sheep are T. mentagrophytes and T. verrucosum. In goats, the agents are T. mentagrophytes, M. canis, M. gypseum, T. verrucosum, T. schoenleinii, and Epidermophyton floccosum. In cattle, T. verrucosum is the primary causative agent. Dermatophytosis is a common fungal infection of the epidermis of cattle and is less common in sheep and goats. Clinical signs and diagnosis. Multiple, gray, crusty, circumscribed, hyperkeratotic lesions are characteristic of infection. Lesions will vary in size. In all ruminants, lesions will be around the head, neck, and ears. In goats and cattle, lesions will extend down the neck, and in cattle, lesions develop particularly around the eyes and on the thorax. Cattle lesions are unique in the marked crustiness, which progressively appears wartlike. Hair shafts become brittle and break off. Intense pruritus is often associated with the alopecic lesions. The disease can be diagnosed by microscopic identification of hyphae and conidia on the hairs following skin scraping and 20% potassium hydroxide digestion. Dermatophyte test media (DTM) cultures are the most reliable means to diagnose the fungus. Broken hairs from the periphery of the lesion are the best sources of the fungus. Epizootiology and transmission. Younger animals are more susceptible, and factors such as crowding, indoor housing, warm and humid conditions, and poor nutrition are also important. Transmission is by direct contact or by contact with contaminated fomites, such as equipment, fencing, or feed bunks. Pathogenesis. Incubation can be as long as 6 weeks. The organisms invade and multiply in hair shafts. Treatment. Spontaneous recovery occurs in all species in 1–4 months. Although cell-mediated immunity is considered important, other immune mechanisms are not well understood. Immunity may not be of long duration. Recovery is enhanced by correcting nutritional deficiencies and improving housing and ventilation problems. A number of topical treatments, such as 2–5% lime-sulfur solution, 3% captan, iodophors, thiabendazole, and 0.5% sodium hypochlorite, can be used. In severe cases, systemic therapy with griseofulvin may be successful. Prevention and control. The animals' environment and overall physical condition should be reassessed with particular attention to ventilation, crowding, sanitation, and nutrition. Pens should be thoroughly cleaned and disinfected. Research complications. Ringworm is a zoonotic disease. Etiology. Dermatophytosis, or infection of the keratinized layers of skin, is caused mostly by species of the genera Trichophyton and Microsporum. The primary causes in sheep are T. mentagrophytes and T. verrucosum. In goats, the agents are T. mentagrophytes, M. canis, M. gypseum, T. verrucosum, T. schoenleinii, and Epidermophyton floccosum. In cattle, T. verrucosum is the primary causative agent. Dermatophytosis is a common fungal infection of the epidermis of cattle and is less common in sheep and goats. Clinical signs and diagnosis. Multiple, gray, crusty, circumscribed, hyperkeratotic lesions are characteristic of infection. Lesions will vary in size. In all ruminants, lesions will be around the head, neck, and ears. In goats and cattle, lesions will extend down the neck, and in cattle, lesions develop particularly around the eyes and on the thorax. Cattle lesions are unique in the marked crustiness, which progressively appears wartlike. Hair shafts become brittle and break off. Intense pruritus is often associated with the alopecic lesions. The disease can be diagnosed by microscopic identification of hyphae and conidia on the hairs following skin scraping and 20% potassium hydroxide digestion. Dermatophyte test media (DTM) cultures are the most reliable means to diagnose the fungus. Broken hairs from the periphery of the lesion are the best sources of the fungus. Epizootiology and transmission. Younger animals are more susceptible, and factors such as crowding, indoor housing, warm and humid conditions, and poor nutrition are also important. Transmission is by direct contact or by contact with contaminated fomites, such as equipment, fencing, or feed bunks. Pathogenesis. Incubation can be as long as 6 weeks. The organisms invade and multiply in hair shafts. Treatment. Spontaneous recovery occurs in all species in 1–4 months. Although cell-mediated immunity is considered important, other immune mechanisms are not well understood. Immunity may not be of long duration. Recovery is enhanced by correcting nutritional deficiencies and improving housing and ventilation problems. A number of topical treatments, such as 2–5% lime-sulfur solution, 3% captan, iodophors, thiabendazole, and 0.5% sodium hypochlorite, can be used. In severe cases, systemic therapy with griseofulvin may be successful. Prevention and control. The animals' environment and overall physical condition should be reassessed with particular attention to ventilation, crowding, sanitation, and nutrition. Pens should be thoroughly cleaned and disinfected. Research complications. Ringworm is a zoonotic disease. Etiology. Dermatophytosis, or infection of the keratinized layers of skin, is caused mostly by species of the genera Trichophyton and Microsporum. The primary causes in sheep are T. mentagrophytes and T. verrucosum. In goats, the agents are T. mentagrophytes, M. canis, M. gypseum, T. verrucosum, T. schoenleinii, and Epidermophyton floccosum. In cattle, T. verrucosum is the primary causative agent. Dermatophytosis is a common fungal infection of the epidermis of cattle and is less common in sheep and goats. Clinical signs and diagnosis. Multiple, gray, crusty, circumscribed, hyperkeratotic lesions are characteristic of infection. Lesions will vary in size. In all ruminants, lesions will be around the head, neck, and ears. In goats and cattle, lesions will extend down the neck, and in cattle, lesions develop particularly around the eyes and on the thorax. Cattle lesions are unique in the marked crustiness, which progressively appears wartlike. Hair shafts become brittle and break off. Intense pruritus is often associated with the alopecic lesions. The disease can be diagnosed by microscopic identification of hyphae and conidia on the hairs following skin scraping and 20% potassium hydroxide digestion. Dermatophyte test media (DTM) cultures are the most reliable means to diagnose the fungus. Broken hairs from the periphery of the lesion are the best sources of the fungus. Epizootiology and transmission. Younger animals are more susceptible, and factors such as crowding, indoor housing, warm and humid conditions, and poor nutrition are also important. Transmission is by direct contact or by contact with contaminated fomites, such as equipment, fencing, or feed bunks. Pathogenesis. Incubation can be as long as 6 weeks. The organisms invade and multiply in hair shafts. Treatment. Spontaneous recovery occurs in all species in 1–4 months. Although cell-mediated immunity is considered important, other immune mechanisms are not well understood. Immunity may not be of long duration. Recovery is enhanced by correcting nutritional deficiencies and improving housing and ventilation problems. A number of topical treatments, such as 2–5% lime-sulfur solution, 3% captan, iodophors, thiabendazole, and 0.5% sodium hypochlorite, can be used. In severe cases, systemic therapy with griseofulvin may be successful. Prevention and control. The animals' environment and overall physical condition should be reassessed with particular attention to ventilation, crowding, sanitation, and nutrition. Pens should be thoroughly cleaned and disinfected. Research complications. Ringworm is a zoonotic disease. B. Genetic, Metabolic, Nutritional, and Management-Related Diseases 1. Genetic Diseases a. Entropion Inverted eyelids are a common inherited disorder of lambs and kids of most breeds. Generally, the lower eyelid is affected and turns inward, causing various degrees of trauma to the conjunctiva and cornea. Young animals will display tearing, blepharospasm, and photophobia initially. If the disorder is left uncorrected, corneal ulcers, perforating ulcers, uveitis, and blindness may occur. Placing a suture or a surgical staple in the lower eyelid and the cheek, effectively anchoring the lid in an everted position, successfully treats the condition. The procedure likely results in the formation of some degree of scar tissue within the lower lid, because when the suture eventually is removed, the condition rarely returns. Other treatments include the injection of a "bleb" of penicillin in the lid, regular manual correction over a 2-day period early in the animal's life, and application of ophthalmic ointments, powders, and solutions. Boric acid or 10% Argyrol solutions have been used as treatments. Because of the genetic predisposition, prevention of the condition requires removal of maternal or paternal carriers. b. β-Mannosidosis of Goats β-Mannosidosis is an autosomal recessive lysosomal storage disease of goats. The disease affects kids of the Nubian breed and is identified by intention tremors and difficulty or inability of newborns to stand. Cells of affected animals are vacuolated because of a lack of lysosomal hydroxylase, which results in accumulation of oligosaccharides. Newborn kids are unable to rise, and they have characteristic flexion of the carpal joint and hyperextension of the pastern joint. Kids are born deaf and with musculoskeletal deformities such as domed skull, small narrow muzzle, small palpebral fissures, enophthalmos, and depressed nasal bridge ( Smith and Sherman, 1994 ). Carrier adults can be identified by plasma measurements of β-mannosidase activity. c. Congenital Myotonia of Goats Caprine congenital myotonia is an inherited autosomal dominant disease that affects voluntary striated skeletal muscles. Goats with this disease are commonly known as fainting goats. "Fainting" is actually transient spasms of skeletal musculature brought about by visual, tactile, or auditory stimuli ( Smith and Sherman, 1994 ). Muscle fiber membranes appear to have fewer chloride channels than normal, resulting in decreased chloride conduction across the membrane, with subsequent increased membrane excitability and repetitive firing ( Smith and Sherman, 1994 ). Contractions of skeletal muscle are sustained for up to 1 min. Kids exhibit the condition by 6 weeks of age, and males appear to exhibit more severe clinical signs than females ( Smith and Sherman, 1994 ). Electromyographic studies produce an audible "dive-bomber" sound characteristic of hyper-excitable cell membranes ( Smith and Sherman, 1994 ). d. Inherited Conditions of Cattle i. Congenital erythropoietic porphyria. Congenital erythropoietic porphyria (CEP) is an autosomal recessive disease of cattle seen primarily in Holsteins, Herefords, and Shorthorns. The disease also occurs in Limousin cattle, humans, and some other species. In the homozygous recessive animal, symptoms of the disease may vary from mild to severe and occur at different times of the year and in different ages of animals. A reddish brown discoloration of teeth and bones is a characteristic of the disease, as is discolored urine, general weakness and failure to thrive, photosensitization, and photophobia. Bones are more fragile compared with bones of normal animals. A regenerative anemia occurs as the result of the shortened life span of erythrocytes, due to accumulations of porphyrins. The genetic defect is associated with low activity of an essential enzyme, uroporphyrinogen III synthase, in the porphyrin–heme synthesis pathway in erythrocytic tissue. The ranges in the presentation of the disease are believed to be related to varying cycles of porphyrin synthesis. Porphyrins are excreted in varying amounts in the urine and the discoloration fluoresces under a Wood's lamp. Diagnosis is based on these clinical and visible signs of porphyria; skin biopsy provides definitive diagnosis. Heterozygotes may have milder symptoms. Many other genetic defects, in all major organ systems, have been described in numerous breeds of cattle and are described in detail elsewhere ( "Large Animal Internal Medicine," 1996 ). In many cases, the genetic basis has been clarified, and associated defects also noted. Many defects are reported in particular breeds, but as crossbreeding increases and new breeds are developed, these traits are appearing in these animals. The bovine genome continues to be further characterized, and more linkage maps and gene locations are forthcoming ( Womack, 1998 ). Some bovine genetic defects are also regarded as models of genetic disease, such as leukocyte adhesion deficiency of Holstein cattle. Some of the more commonly reported defects include syndactyly in Holsteins and other breeds and Polydactyly in Simmentals; lysosomal storage diseases such as α-mannosidosis in some beef breeds; enzyme deficiencies such as citrulline-mia in Holsteins; and progressive degenerative myeloencephalopathy ("weaver") in Brown Swiss. ii. Goiter of sheep. A defect in the synthesis of thyroid hormone has been identified in Merino sheep ( Radostits et al., 1994 ). Lambs born with the defect have enlargement of the thyroid gland, a silky appearance to the wool, and a high degree of mortality. Edema, bowing of the legs, and facial abnormalities have also been noted in animals with this disorder. Immaturity of the lungs at birth causes neonatal respiratory distress and results in dyspnea and respiratory failure. iii. Spider lamb syndrome (hereditary chondrodysplasia). Spider lamb syndrome is an inherited, often lethal, musculoskeletal disorder primarily occurring in Suffolk and Hampshire breeds. Severely affected lambs die shortly after birth. Animals that survive the perinatal period develop angular limb deformities, scoliosis, and facial deformities. With time, affected animals become debilitated, exhibit joint pain, and develop neurological problems associated with the spinal abnormalities. Radiologically, secondary ossification centers—especially the physis, subchondral areas, and cuboidal bones—are affected. Abnormal endochondral ossification leads to excess cartilage formation, notably apparent in the elbows. Lambs will typically display abnormally long limbs, medial deviation of the carpus and tarsus, flattening of the sternum, scoliosis/kyphosis of the vertebrae, and a rounded nose. Muscle atrophy is common. Diagnosis can be based on typical clinical signs, which are similar to those seen with Marfan syndrome in humans ( Rook et al., 1986 ). Long-term survival is rare; treatment is unsuccessful. 2. Metabolic Diseases a. Abomasal Disorders i. Abomasal and duodenal ulcers. Abomasal and duodenal ulcers occur more frequently in calves and adult cattle than in sheep and goats. Like rumenitis, abomasal and duodenal ulcers may be associated with lactic acidosis. Concurrent disease, such as salmonellosis, bluetongue, or overuse of anti-inflammatory drugs, or recent shipping or environmental stresses may also lead to ulcer formation. Copper deficiency, dietary changes, mycotic infections, Clostridium perfringens abomasitis, and abomasal bezoars are associated with this disease in calves. In older adult cattle, abomasal lymphosarcoma may be the underlying condition. Gastric acid hypersecretion in conjunction with insufficient gastric mucous secretion will physically destroy the gastric epithelium. Deep ulceration may cause serious hemorrhage and/or perforation with peritonitis. Chronic hemorrhage may lead to anemia. Although ulcers are often asymptomatic in calves, perforation with peritonitis is more common than hemorrhage. Dark feces or melena and abdominal pain may be observed. Arched back, restlessness, kicking at the abdomen, bruxism, and anorexia are common signs of abdominal pain. Fecal occult blood is as an easy diagnostic test. Treatment includes gastrointestinal protectants and histamine antagonists. Anemia may be symptomatically treated with parenteral iron injections and anabolic steroids. Preventive measures in cattle herds include ensuring optimal passive immunity for calves, minimizing stress to calves, and striving for a herd free of bovine leukosis virus. ii. Abomasal emptying defect. Abomasal emptying defect of sheep is a sporadic syndrome associated with abomasal distension and weight loss. Suffolks tend to be especially predisposed, although the disease has been diagnosed in Hampshires, Columbias, and Corriedales. The mechanism of the disease is unknown. Affected animals will exhibit a gradual weight loss with a history of normal appetites. Feces will continue to be normal. Ventral abdominal distension associated with abomasal accumulation of feedstuffs will be apparent in many of the animals. Diagnosis is primarily based on history and clinical signs. Elevations in rumen chloride concentrations (>15 mEq/liter) are commonly found. Radiography or ultrasonography may be helpful at identifying the distended abomasum. Abomasal emptying defect is usually eventually fatal. Medical treatment with metoclopramide and mineral oil may be helpful in early disease. iii. Abomasal displacement. Displaced abomasum (DA) is a sporadic disorder usually associated with multiparous 4- to 7-year-old dairy cows in early lactation, but the condition can occur even in young calves. Displacement to the right (RDA) may be further complicated by torsion (RTA), a surgical emergency. Left displacement (LDA) is more common than RDA. Clinical signs include anorexia, lack of cud chewing, decreased frequency of ruminal contractions, shallow respirations, increased heart rate, treading, and decreased milk production. Diagnosis is based on characteristic areas of tympanic resonance during auscultation-percussion of the lateral to lateral-ventral abdomen ("pings"), ruminal displacement palpated per rectum, and clinical signs. Cow-side clinical chemistry findings include hypoglycemia and ketonuria; more extensive evaluations will often indicate moderate to severe electrolyte and acid-base abnormalities. DA occurs because of gas accumulation within the viscus, and the abomasum "floats" up from its normal ventral location to the lateral abdominal wall. No exact cause of DA has been identified, but it is commonly associated with stress; high levels of concentrate in the diet, leading to forestomach atony; and many disorders, including lack of regular exercise, mastitis, hypocalcemia, retained placenta, metritis, or twins. Factors such as body size and conformation indicate the possibility of genetic predisposition. Treatments include surgical and nonsurgical techniques for LDA; the former has a better chance of permanent correction. Emergency surgery is necessary for RTA; the disorder is fatal within 72 hr. Recurrence is rare after surgical correction. Electrolyte and acid-base imbalances are likely in severe cases and especially with RTA. Prevention includes reducing stress, taking greater care in the introduction and feeding of concentrates, and reducing incidence of predisposing diseases noted above ( Rohrbach et al., 1999 ). b. Fat Cow Syndrome, Hepatic Lipidosis Fat cow syndrome is seen in peri- or postparturient overconditioned or obese multiparous dairy cows. Factors in the development of the condition include negative energy balance related to the normal decreased dry matter intake as parturition approaches; hormonal changes associated with parturition; and concurrent diseases of parturition that decrease feed intake and increase energy needs. The possible concurrent diseases include metritis, retained fetal membranes, mastitis, parturient paresis, and displaced abomasum. Signs are nonspecific and include depression, anorexia, and weakness. Prognosis is usually guarded. Diagnosis is based on herd management, the animal's condition, ketonuria, and clinical signs. In prepartum cattle and in lactating cows, blood levels of nonesterified fatty acids (NEFA) greater than 1000 μEq/liter and 325–400 μEq/liter, respectively, are abnormal ( Gerloff and Herdt, 1999 ). Triglyceride analysis of liver biposy specimens are useful. In affected cows, body fat is mobilized, in the form of NEFA in response to the energy demands. Hepatic lipidosis occurs rapidly as the NEFA are converted into hepatic triglycerides. The ability of the liver to extract the albumin-bound NEFA from the blood is better than that of other tissues that need and can also use NEFA as an energy source. Treatment for any concurrent diseases must be pursued aggressively, as well as measures to increase and stabilize blood glucose, decrease NEFA production, and increase forestomach digestion to improve production of normally metabolized volatile fatty acids. Therapeutic measures include intravenous glucose drips, insulin (NPH or Lente) injections every 12 hr, and transfaunation of ruminal fluid from a normal cow. Prevention includes minimizing stress to late-gestation cows. Dry and lactating cows should be maintained separately; their energy, protein, and dry matter requirements are very different. Cows with prolonged lactation or delayed breeding should be managed to prevent weight gain. c. Rumen and Reticulum Disorders i. Bloat. Bloat or tympanites refers to an excessive accumulation of gas in the rumen. The condition most frequently occurs in animals that have been recently fed abundant quantities of succulent forages or grains. Bloat is classified into two broad categories: frothy bloat and free-gas bloat. Frothy bloat is associated with ingestion of feeds that produce a stable froth that is not easily expelled from the rumen. Fermentation gases such as CO 2 , CH 4 , and minor gases such as N 2 , O 2 , H 2 , and H 2 S incorporate into the froth, overdistend the rumen, and eventually compromise respiration by limiting diaphragm movement. The froth is often derived from a combination of salivary mucoproteins, protozoal or bacterial proteins, and proteins, pectins, saponins, or hemicellulose associated with ingested leaves or grain. Typical foodstuffs that cause frothy bloat include green legumes, leguminous hay (alfalfa, clover), or grain (especially barley, corn, and soybean meal). Free-gas bloat is less related to feeds ingested; rather, it is caused by rumen atony or by physical or pathological problems that prevent normal gas eructation. Some examples of causes of free-gas bloat are esophageal obstructions (foreign bodies, tumors, abscesses, and enlarged cervical or thoracic lymph nodes), vagal nerve paralysis or injury, and central nervous system conditions that affect eructation reflexes. Clinically, the animal will exhibit rumen distension, and tympany will be observed in the left paralumbar fossa. Additional signs may include colic-like pain of the abdomen and dyspnea. Passage of a stomach tube helps to differentiate between free-gas bloat and frothy bloat; and with free-gas bloat, expulsion of gas through the stomach tube aids in treatment of the disorder. Once rumen distension is alleviated with free-gas bloat, the underlying cause must be investigated to prevent recurrence. Frothy bloat is more difficult to treat, because the foam blocks the stomach tube. Addition of mineral oil, household detergents, or antifermentative compounds via the tube may help break down the surface tension, allowing the gas to be expelled. In acute, life-threatening cases of bloat, treatment should be aimed at alleviating rumen distension by placing a trocar or surgical rumenotomy into the rumen via the paralumbar fossa. Limiting the consumption of feedstuffs prone to induce bloat can prevent the disease. Additionally, poloxalene or monensin will decrease the incidence of frothy bloat. ii. Lactic acidosis. Lactic acidosis, or rumen acidosis, is an acute metabolic disease caused by engorgement of grains or other highly fermentable carbohydrate sources. The disease is most frequently related to a rapid change in diet from one containing high roughage to one containing excessive carbohydrates. Diet components that predispose to acidosis include common feed grains; feedstuffs such as sugar beets, molasses, and potatoes; by-products such as brewer's grains; and bakery products. Biochemically, ingestion of large amounts of the carbohydrate-rich diet causes the normally gram-negative rumen bacterial populations to shift to gram-positive Streptococcus and Lactobacillus species. The gram-positive organisms efficiently convert the starches to lactic acid. The lactic acid acidifies the rumen contents, leading to rumen mucosal inflammation, and increases the osmolality of rumen fluids, leading to sequestration of fluids and osmotic attraction of plasma and tissue fluid to the rumen. Lactic acid-induced rumenitis predisposes the animal to ulcers, to liver abscesses from "absorbed" bacterial pathogens, to laminitis from absorbed toxins, and to polioencephalomalacia from the inability of the new rumen bacterial populations to produce sufficient thiamine needed to maintain normal nervous system function. Clinically, animals will become anorexic, depressed, and weak within 1–3 days after the initial insult. Incoordination, ataxia, dehydration, hemoconcentration, rapid pulse and respiration, diarrhea, abdominal pain, and lameness will also be noted. Rumen distension and an acetone-like odor to the breath, milk, or urine may also be observed. Diagnosis is based on history and clinical signs. Blood, urine, or milk ketones can be detected ( Moore and Ishler, 1997 ). Additionally, rumen pH, which is normally above 6.0, will drop to less than 5.0 and in severe cases may achieve levels as low as 3.8. Similarly, urine pH will become acidic, blood pH will drop below 7.4, and hematocrit will appear to increase due to the relative hemoconcentration. Necropsy findings will be determined by secondary conditions. The primary lactic acidosis will cause swelling and necrosis of rumen papillae and abomasal hemorrhages and ulcers. Treatment must be applied early in the syndrome. In early hours of severe carbohydrate engorgement, rumenotomy and evacuation of the contents are appropriate. The patient should be given mineral oil and antifermentatives to prevent the continued conversion of starches to acids and the absorption of metabolic products. Bicarbonate or other antacids like magnesium carbonate or magnesium hydroxide introduced into the rumen will aid in adjusting rumen pH. Furthermore, animals can be given oral tetracycline or penicillin, which will decrease the gram-positive bacterial population. iii. Rumen parakeratosis. Parakeratosis is a degenerative condition of the rumen mucosa that leads to keratinization of the papillary epithelium. Excessive and continuous feeding of diets low in roughage causes the mucosal changes. Generally, this condition is seen in feedlot lambs and steers that are fed an all-grain diet. Clinically, animals may exhibit only poor rates of gain, due to changes in the absorptive capacity of the injured mucosa. At necropsy, papillae will be thickened and rough. They will frequently be dark in color, and multiple papillae will clump together. Abscessation may be observed. Histopathologically, papilla surfaces will have hyperkeratinization of the squamous epithelium. Chronic laminitis may be observed. However, diagnosis of parakeratosis is generally made at necropsy. Feeding adequate roughage, such as stemmy hay, will prevent the disease. Antibiotics may be administered to prevent secondary liver abscess formation. iv. Rumenitis. Rumenitis is an acute or chronic inflammation of the rumen, which occurs most commonly as a sequela to lactic acidosis. In addition to concentrate feeding, inadequate roughage in the diet is also associated with this disorder. Rumenitis may occur with contagious ecthyma infection or following ingestion of poisons or other irritants. Because rumenitis is often associated with lactic acidosis, it tends to occur in feedlot animals. The inflamed ruminal epithelium becomes necrotic and sloughs, creating ulcers. Endogenous rumen bacteria such as Fusobacterium necrophorum may invade the ulcers, penetrate the circulatory system, and induce abscesses of the liver. Clinically, the animals will appear depressed and anorexic. Rumen motility will be decreased, and animals will lose weight. The disease may resolve in a week to 10 days; mortality may reach 20%. Necropsy lesions include rumen inflammation and ulcers in the anteroventral sac. Granulation tissue and scarring may be observed following healing. Rumenitis is not typically diagnosed clinically; thus, specific treatment is not commonly done. The disease can be prevented by minimizing the incidence of lactic acidosis. d. Traumatic Reticulitis-Reticuloperitonitis (Hardware Disease) Etiology. Traumatic reticulitis–reticuloperitonitis is a disease of cattle related to their exploratory tendencies and ingestion of many different, nonvegetative materials. The disease is rarely seen in smaller ruminants. Clinical signs. Clinical signs range from asymptomatic to severe, depending on the penetration and damage by the foreign object after settling in the animal's forestomach. Many signs during the early, acute stages will be nonspecific, ranging from arched back, listlessness, anorexia, fever, decrease in production, ketosis, regurgitation, decrease or cessation of ruminal contractions, bloat, tachypnea, tachycardia, and grunts when urinating, defecating, or being forced to move. The prognosis is poor when peritonitis becomes diffuse. Sudden death can occur if the heart, coronary vessels, or other large vessels are punctured by the migrating object. Epizootiology and transmission. This is a noncontagious disease. The occurrence is directly related to sharp or metallic indigestible items in the feed or environment that the cattle mouth and swallow. Necropsy findings. In severe cases, necropsy findings include extensive inflammation throughout the cranial abdomen, malodorous peritoneal fluid accumulations, and lesions at the reticular sites of migration of the foreign objects. Cardiac puncture will be present in those animals succumbing to sudden death. Pathogenesis. Consumed objects initially settle in the rumen but are dumped into the reticulum during the digestive process, and normal contraction may eventually lead to puncture of the reticular wall. This sets off a localized inflammation or a localized or more generalized peritonitis. The inflammation may also temporarily or permanently affect innervation of local tissues and organs. Further damage may result from migration and penetration of the diaphragm, pericardium, and heart. Diagnosis is based on clinical signs, knowledge of herd management techniques in terms of placement of forestomach magnets, and reflection of acute or chronic infection on the hemogram. Radiographs and abdominocentesis may be useful. Differential diagnosis. Differentials include abomasal ulcers, hepatic ulcers, neoplasia (such as lymphosarcoma, usually in older animals, or intestinal carcinoma), laminitis, and cor pulmonale. Infectious diseases that are differentials include systemic leptospirosis and internal parasitism. Diseases causing sudden death may need to be considered. Prevention and control. This problem can be prevented entirely by elimination of sharp objects in cattle feed and in the housing and pasture environments. Adequately sized magnets placed in feed handling equipment and forestomach magnets (placed per os with a balling gun in young stock at 6–8 months of age) are also significant prevention measures. Treatment. Provision of a forestomach magnet, confinement, and nursing care, including antibiotics, are the initial treatments. In severe cases, rumenotomy may be considered. e. Pregnancy Toxemia (Ketosis), Protein Energy Malnutrition Etiology. Pregnancy toxemia is a primary metabolic disease of ewes and does in advanced pregnancy. Beef heifers are susceptible to protein energy malnutrition (PEM) syndrome, which is also referred to as pregnancy toxemia. Clinical signs. In sheep, this disease is characterized by hypoglycemia, ketonemia, ketonuria, weakness, and blindness. Hypoglycemic and ketotic ewes begin to wander aimlessly and to move away from the flock. They become anorexic and act uncoordinated, frequently leaning against objects. Advanced signs may include blindness, muscle tremors, teeth grinding, convulsions, and coma. Body temperature, heart rate, respiratory rate, and rumen motility continue normally. Up to 80% of infected ewes may die from the disease. The course of the disease may last up to a week. In goats, the disease usually occurs in the last 6 weeks of gestation, especially in does carrying triplets. Pregnancy toxemia should be considered with any goat showing signs of illness in late gestation. The doe may separate herself from the herd, stagger, or circle and may appear blind. Appetite is poor, and tremors may be evident. A rapid metabolic acidosis results in subsequent recumbency. Urinalysis will readily reveal ketonuria. If fetal death occurs, acute toxemia and death of the doe may result. In beef heifers, weight loss and thin body condition, weakness and inability to stand, and depression are clinical signs. Some cows develop diarrhea. Because the catabolic state is often so advanced, most affected heifers die even if treated. Pregnancy toxemia is diagnosed by evidence of typical clinical signs. Sodium nitroprusside tablets or ketosis dipsticks may be used to identify ketones in the urine or plasma of ewes and does. Blood glucose levels found to be below 25 mg/dl and ketonuria are good diagnostic indicators. In cattle, ketonuria is not a typical finding; hypocalcemia and anemia may be present. Epizootiology. Pregnancy toxemia occurs primarily in ewes that are obese or bearing twins or triplets. The disease develops during the last 6 weeks of pregnancy. PEM most frequently occurs in heifers during the final trimester of pregnancy. Necropsy findings. At necropsy, affected ewes will often have multiple fetuses, which may have died and decomposed. The liver will be enlarged, yellow, and friable, with fatty degeneration. The adrenal gland may also be enlarged. In cattle, heifers will be very thin, and in addition to a fatty liver, signs of concurrent diseases may be present. Pathogenesis. Rapid fetal growth, a decline in maternal nutrition, and a voluntary decrease in food intake in overfat ewes result in an inadequate supply of glucose needed for both maternal and fetal tissues. The ewe develops a severe hypoglycemia in early stages of the disease. The ruminant absorbs little dietary glucose; rather, it produces and absorbs volatile fatty acids (acetic, propionic, and butyric acids) from consumed feedstuffs. Propionic acid is absorbed and selectively converted to glucose through gluconeogenesis. When the animal is in a state of negative energy balance, it hydrolyzes fats to glycerol and fatty acids. Glycerol is converted to glucose while the fatty acids are metabolized for energy. The oxidation of fatty acids in the face of declining oxaloacetate levels (required for normal Krebs cycle function) results in the formation of ketone bodies (acetone, acetoacetic acid, and β-hydroxybutyric acid), thus causing the condition ketoacidosis. Heifer cattle have high energy requirements for completing normal body growth and supporting a pregnancy. Additional energy requirements are needed during pregnancy for winter conditions and during concurrent diseases. Marginal diets and poor-quality forage will place the cows in a negative energy balance. Differential diagnosis. Hypocalcemia is a common differential diagnosis. In cattle, differentials include chronic or untreated diseases such as Johne's disease, lymphosarcoma, parasitism, and chronic respiratory diseases. Prevention and control. Pregnancy toxemia can be prevented by providing adequate nutrition during late gestation and by maintaining animals in appropriate nonfat condition during pregnancy. In late pregnancy, the dietary energy and protein should be increased 1.5–2 times the maintenance level. PEM can be prevented by maintaining appropriate body condition earlier in pregnancy and supplying good-quality forage for the last trimester. Treatment. In sheep, because the morbidity may be as high as 20%, treatment should be directed at the flock rather than the individual. Treating the individual is usually unsuccessful. Oral administration of 200 ml of propylene glycol or 50% glucose twice a day, anabolic steroids, and high doses of adrenocorti-costeroids may be helpful. If ewes are still responsive and not severely acidotic or in renal failure, cesarean section may be successful by rapidly removing the fetus, which is the dietary drain for the ewe. In goats, pregnancy toxemia is best treated by removal of the fetuses either by cesarean section or induction of parturition. Parturition can be induced in does by either dexamethasone (10 mg) or PGF 2a (10 pg). In addition, goats may be treated with 10% dextrose (100 to 200 ml iv) or propylene glycol (60 ml per os 2 or 3 times a day). Adjunctive therapy includes normalizing acid base and hydration status, administration of vitamin B 12 and transfaunation. Heifers may be force-fed alfalfa gruels, given propylene glycol per os, placed on IV 50% glucose drips, and treated for concurrent disease. Research complications. In research requiring pregnant ewes in late stages of gestation, for example, this disease should be considered if the animals are likely to bear twins and will be transported or stressed in other ways during that time. f. Hypocalcemia (Parturient Paresis, Milk Fever) Etiology. Hypocalcemia is an acute metabolic disease of ruminants that requires emergency treatment; the presentation is slightly different in ewes, does, and cows. Clinical signs and diagnosis. In sheep, the disease is seen in ewes during the last 6 weeks of pregnancy and is characterized by muscle tetany, incoordination, paralysis, and finally coma. As calcium levels drop, ewes begin to show early signs such as stiffness and incoordination of movements, especially in the hindlimbs. Later, muscular tremors, muscular weakness, and recumbency will ensue. Animals will frequently be found breathing rapidly despite a normal body temperature. Morbidity may approach 30%, and mortality may reach as high as 90% in untreated animals. Affected does become bloated, weak, unsteady, and eventually recumbent. Cows are affected within 24–48 hr before or after parturition. Cows initially are weak and show evidence of muscle tremors, then deteriorate to sternal recumbency, with the head usually tucked to the abdomen, and an inability to stand. Tachycardia, dilated pupils, anorexia, hypothermia, depression, ruminal stasis, bloat, uterine inertia, and loss of anal tone are also seen at this stage. The terminal stage of disease is a rapid progression from coma to death. Heart rates will be high, but pulse may not be detectable. Hypocalcemia is diagnosed based on the pregnancy stage of the female and on clinical signs. It is later confirmed by laboratory findings of low serum calcium. With hypocalcemia in ewes, the plasma concentrations of calcium drop from normal values of 8–12 mg/dl to values of 3–6 mg/dl. In cattle, plasma levels below 7.5 mg/dl are hypocalcemic; at the terminal stages levels may be 2 mg/dl. Epizootiology. Hypocalcemia occurs primarily in overweight ewes during the last 6 weeks of pregnancy or during the first few weeks of lactation. The disease is not as common in the dairy goat as in the dairy cow. High-producing, older, multiparous dairy cows are the most susceptible, and the Jersey breed is considered susceptible. Cows that have survived one episode are prone to recurrence. In addition, dry cows must be managed carefully regarding limiting dietary calcium. The disease is not common in beef cattle unless there is an overall poor nutrition program. Necropsy findings. There is no pathognomonic or typical finding at necropsy. Pathogenesis. During the periparturient period, calcium requirements for fetal skeletal growth exceed calcium absorbed from the diet and from bone metabolism. Additionally, dietary calcium intake is thought to be compromised because, in advanced pregnancy, animals may not be able to eat enough to sustain adequate nutrient levels, and intestinal absorption capabilities do not respond as quickly as needed. After parturition, calcium needs increase dramatically because of calcium levels in colostrum and milk. Recent information suggests that legume and grass forages, high in potassium and low in magnesium, create a slight physiological alkalosis (at least in cattle), which antagonizes normal calcium regulation ( Rings et al., 1997 ). Thus, bone resorption, renal resorption, and gastrointestinal absorption of calcium are less than maximal. Prevention and control. Maintaining appropriate nutrition during the last trimester is helpful in preventing the disease. In cows and does, for example, limiting calcium intake by removing alfalfa from the diet is helpful. Treatment. Hypocalcemia must be treated quickly based on clinical signs; pretreatment blood samples can be saved for later confirmation. Twenty percent calcium borogluconate solution should be administered by slow intravenous infusion. Response will often be rapid, with the resolution of the animal's dull mentation. Less severely affected animals will often try to stand in a short time. Relapses are common, however, in sheep and cattle. Hypermagnesemia and hypophosphatemia often coincide with hypocalcemia. These imbalances should be considered when animals appear to be unresponsive to treatment. Hypocalcemia in the goat can be treated with 50–100 ml of calcium borogluconate. Heart rate should be monitored closely throughout calcium administration. If an irregular or rapid heart rate is detected, then calcium treatment should be slowed or discontinued. Calcium gels and boluses are also available for treatment ( Rings et al., 1997 ). Prognosis is generally good if the animal is treated early in the disease, but the prognosis will often be poor when treatment is initiated in later stages of the disease. g. Urinary Calculi (Obstructive Urolithiasis, Water Belly) Etiology. Urolithiasis is a metabolic disease of intact and castrated male sheep, goats, and cattle that is characterized by the formation of bladder and urethral crystals, urethral blockage, and anuria (Murray, 1985). The disease occurs rarely in female ruminants. Clinical signs and diagnosis. Affected animals will vocalize and begin to show signs of uneasiness, such as treading, straining postures, arched backs, raised tails, and squatting while attempting to urinate. These postures may be mistaken for tenesmus. Male cattle may develop swelling along the ventral perineal area. Affected animals will not stay with the herd or flock. Small amounts of urine may be discharged, and crystal deposits may be visible attached to the preputial hairs. Additionally, in smaller ruminants, the filiform urethral appendage (pizzle) often becomes dark purple to black in color. The pulsing pelvic urethra may be detected by manual or digital rectal palpation, and bladder distention may be noticeable in cattle by the same means. As the disease progresses to complete urethral blockage, the animal will become anorexic and show signs of abdominal pain, such as kicking at the belly. The abdomen will swell as the bladder enlarges, and rupture can occur within 36 hr after development of clinical signs. Bladder or urethral rupture may cause a short-lived period of apparent pain relief; subsequent development of uremia will eventually lead to death. The disease may progress over a period of 1–2 weeks, and the mortality is high unless the blockages are reversed. Diagnosis is made by the typical clinical signs. Abdominal taps may yield urine. Calculi are usually composed of calcium phosphate or ammonium phosphate matrices. Epizootiology and transmission. Clinical disease is usually seen in growing intact or castrated males. The disease may be sporadic or there may be clusters of cases in the flock or herd. Necropsy findings. Necropsy findings include urine in the abdomen with or without bladder or urethral rupture. Renal hydronephrosis may be evident. Calculi or struvite crystal sediment will be observed in the bladder and urethra. Histologically, trauma to the urethra and ureters will be present. Pathogenesis. Urolithiasis is multifactorial and involves dietary, anatomical, hormonal, and environmental factors. Male sheep and goats have a urethral process that predisposes them to entrapment of calculi. In cattle, the urethra narrows at the sigmoid flexure, and calculi lodge there most frequently. Additionally, the removal of testosterone by early castration is thought to result in hypoplasia of the urethra and penis. This physical reduction in the size of the excretory tube may predispose to the precipitation of and blockage by the struvite minerals. Grains fed to growing animals tend to be high in phosphorus and magnesium content. These calculogenic diets lead to the formation of struvite (magnesium ammonium phosphate) crystals. Other minerals associated with urolithiasis include silica (range grasses), carbonates (some grasses and clover pastures), calcium (exclusively alfalfa hay), and oxalates (fescue grasses). Differential diagnosis. Grain engorgement colic, gastrointestinal blockage, and causes of tenemus, such as enteritis or trauma, are differentials. Trauma to the urethral process should be considered. Urinary tract infections are uncommon in ruminants. Prevention and control. One case often is indicative of a potential problem in the group. Urolithiasis can be minimized by monitoring the calcium:phosphorus ratio in the diet. The normal ratio should be 2:1. Additionally, increasing the amount of dietary roughage will help balance the mineral intake. Increasing the amount of salt (sodium chloride, 2–4%) in the diet to increase water consumption, or adding ammonium chloride to the diet, at 10 gm/head/day or 2% of the ration, to acidify the urine, will aid in the prevention of this disease. Palatability of and accessibility to water should be assessed as well as functioning of automatic watering equipment. Treatment. Treatment is primarily surgical (Van Metre et al. 1996). Initially, amputation of the filiform urethral appendage may alleviate the disease since urethral blockage often begins here. As the disease progresses, urethral blockage in the sigmoid flexure as well as throughout the urethra may occur. In more advanced stages, perineal urethrostomy may yield good results. The prognosis is poor when the condition becomes chronic, reoccurs, or surgery is required. Research complications. Young castrated and intact male ruminants used in the laboratory setting will be the susceptible age group for this disorder. h. Rickets Rickets is a disease of young, growing animals but rarely occurs in goats. It is a metabolic disease characterized by a failure of bone matrix mineralization at the epiphysis of long bones due to lack of phosphorus. The condition can occur as an absolute deficiency in vitamin D 2 , an inadequate dietary supply of phosphorus, or a long-term dietary imbalance of calcium and phosphorus. The syndrome must be differentiated from epiphisitis (unequal growth of the epiphyses of long bones in young, rapidly growing kids fed diets with excess calcium). Clinical signs include poor growth, enlarged costochondral junctions, narrow chests, painful joints, and reluctance to move. Spontaneous fractures of long bones may occur. Animals will recover when dietary phosphorus is provided and if joint damage is not severe. 3. Nutritional Diseases a. Copper Deficiency (Enzootic Ataxia, Swayback) Etiology. Chronic copper deficiency in pregnant ewes and does may produce a metabolic disorder in their lambs and kids called enzootic ataxia. In goats, this deficiency also causes swayback in the fetuses. Clinical signs and diagnosis. This disease results in a progressive hindlimb ataxia and apparent blindness in lambs up to about 3 months of age. Additionally, because copper is essential for osteogenesis, hematopoiesis, myelination, and pigmentation of wool and hair, ewes may appear unthrifty, may be anemic, and may have poor, depigmented wool with a decrease in wool crimp. Affected kids are born weak, tremble, and have a characteristic concavity to the spinal cord, leading to the name swayback. When the deficiency occurs later during gestation, demyelination is limited to the spinal cord and brain stem. Kids are born normally but develop a progressive ataxia, leading to paralysis, muscle atrophy, and depressed spinal reflexes with lower motor neuron signs. Diagnosis is based on low copper levels found in feedstuffs and tissues at necropsy. Diagnosis is based on clinical signs, feed analysis, and pathological findings. Epizootiology and transmission. Enzootic ataxia is rarely seen in western states; most North American diets have sufficient copper levels to prevent this disease. Copper antagonists in the feed or forage at sufficient levels, such as molybdenum, sulfate, and cadmium, however, may predispose to copper deficiencies. Pathogenesis. The maternal copper deficiency leads to a disturbance early in the embryonic development of myelination in the central nervous system and the spinal cord. Copper is part of the cytochrome oxidase system and other enzyme complexes and is important in myelination, osteogenesis, hematopoiesis (iron absorption and hemoglobin formation), immune system development, and maintenance and normal growth ( Smith and Sherman, 1994 ). Differential diagnosis. The differential diagnosis for newborns includes β-mannosidosis, hypoglycemia, and hypothermia. For older animals the differential should include caprine arthritis encephalitis (goats), enzootic muscular dystrophy, listeriosis, spinal trauma or abscessation, and cerebrospinal nematodiasis. Prevention and control. Copper deficiency can be prevented by providing balanced nutrition for pregnant animals. Necropsy findings. Gross encephalomalacia has been noted. Histopathologically, white matter of the brain and spinal cord displays gelatinization and cavitation. Extensive nerve demyelination and necrosis are evident. Postmortem lesions include extensive demyelination and neuronal degeneration. Treatment. Because the condition is developmental, supplemental copper may improve clinical signs but not eliminate them. b. Copper Toxicosis Etiology: Acute or chronic copper ingestion or liver injury often causes a severe, acute hemolytic anemia in weanling to adult sheep and in calves and adult dairy cattle. Growing lambs may be the most susceptible. Copper toxicosis is rare in goats. Clinical signs and diagnosis. The clinical course in sheep can be as short as 1–4 days, and mortality may reach 75%. Hemolysis, anemia, hemoglobinuria, and icterus characterize the acute hemolytic crisis, associated with copper released from the overloaded liver. Some clinical signs are related to direct irritation to the gastrointestinal tract mucosa. Weakness, vomiting, abdominal pain, bruxism, diarrhea, respiratory difficulty, and circulatory collapse are followed by recumbency and death. Hepatic biopsy is currently considered the best diagnostic approach; serum or plasma levels of copper and hepatic enzymes such as aspartate aminotransferase (AST) and γ-glutamyltransferase (GGT) may provide some information, but it is generally believed that these will not accurately reflect total copper load or hepatic damage. Epizootiology and transmission. A single toxic dose for sheep and goats is the range of 20–100 mg/kg, and for cattle it is 220–880 mg/kg. Chronic poisoning in sheep may occur when 3.5 mg/kg is ingested. Copper-containing pesticides, soil additives, therapeutics, and improperly formulated feeds may potentially lead to copper toxicity. Phytogenous sources include certain pastures such as subterranean clover. Feed low in molybdenum, zinc, or calcium may lead to increased uptake of copper from properly balanced rations. A common cause of the disease in sheep is feeding concentrates balanced for cattle; cattle feeds and mineral blocks contain much higher quantities of copper than are required for sheep. Chronic ingestion of these feedstuffs leads to copper accumulation and toxicity. Copper toxicosis has been reported in calves given regular oral or parenteral copper supplements, and in adult dairy cattle given copper supplements to compensate for copper-deficient pasture. Pregnant dairy cattle may be more susceptible to copper toxicity. Rare sources of copper ingestion may include copper sulfate footbaths. Necropsy findings. Common findings at necropsy include icterus; a soft, dark, friable, enlarged spleen; an enlarged, yellow-brown friable liver; and "gun-barrel" black kidneys. Hemoglobin-stained urine will be visible in the bladder. Copper accumulations in the liver reaching 1000–3000 ppm are toxic. Pathogenesis. Hemolysis occurs when sufficient amounts of copper are ingested or released suddenly from the liver and is believed to be due direct interaction of the copper with red-cell surface molecules. Stresses such as transportation, lactation, and poor nutrition or exercise may precipitate the hemolysis. Differential diagnosis. Other causes of hemolytic disease include babesiosis, trypanosomiasis, and plant poisonings such as kale. Arsenic ingestion, organophosphate toxicity, and cyanide or nitrate poisoning should also be considered as the source of poisoning. Urethral obstruction and gastrointestinal emergencies should be considered for the abdominal pain. Control and prevention. The disease is prevented by carefully monitoring copper access in sheep and copper supplementation in cattle. Sheep and goats should not be fed feedstuffs formulated for cattle, and dairy calf milk replacer should not be used for lambs and kids. Molybdenum may be administered to animals considered at high risk. Molybdenum-deficient pastures may be treated with molybdenum superphosphate. Herd copper supplementation should be undertaken with the knowledge of existing hepatic copper levels, and existing copper and molybdenum levels, in the feedstuffs. Treatment. Oral treatment for sheep consists of ammonium or sodium molybdenate (50–100 mg/day), and sodium thiosulfate (0.5–1.0 mg/day) for 3 weeks aids in excretion of copper. Oral D-penicillamine daily for 6 days (50 mg/kg) has also been shown to increase copper excretion in sheep. Ammonium molybdenate has been administered intravenously to goats at 1.7 mg/kg for 3 treatments on alternate days. Cattle have been treated orally with sodium molybdenate (3 gm/day) or sodium thiosulfate (5 gm/day). Treatment for anemia and nephrosis may be necessary in severe cases. Research complications. Some breeds of sheep, such as Merino crosses and the British breeds, may be more susceptible to copper toxicosis caused by phytogenous sources. c. Selenium/Vitamin E Deficiency (Nutritional Muscular Dystrophy, Nutritional Myodegeneration, White Muscle Disease, Stiff Lamb Disease) Etiology. White muscle disease, also known as stiff lamb disease, is a nutritional muscular dystrophy caused by a deficiency of selenium or vitamin E. Clinical signs and diagnosis. Clinically two forms of the disease have been identified: cardiac and skeletal. The cardiac form occurs most commonly in neonates. In these, respiratory difficulty will be a manifestation of damage to cardiac, diaphragmatic, and intercostal muscles. Young will be able to nurse when assisted. In slightly older animals, the disease is characterized by locomotor disturbances and/or circulatory failure. Clinically, animals may display paresis, stiffness or inability to stand, rapid but weak pulse, and acute death. Mortality may reach 70% ( Jensen and Swift, 1982 ). Paresis and sudden death in neonates with associated pathological signs are frequently diagnostic. With the skeletal form, affected animals are stiff and reluctant to move, and muscles of affected animals are painful. Young will be reluctant to get up but will readily nurse when assisted. Peracute to acute myocardial degeneration may occur in the cardiac form, and animals may simply be found dead. Serum selenium levels are usually below 50 ppb (normal is 158–160 ppb) ( Nelson, 1983 ). Diagnosis may also include determination of antemortem whole blood levels of selenium and plasma levels of vitamin E. Glutathione peroxidase levels in red blood cells can be measured as an indirect test. Clinical biochemistry findings of significant elevations of aspartate aminotransferase (AST) in creatinine kinase (CK) are also supportive of the diagnosis. Epizootiology and transmission. Selenium deficiency has been associated with formulated diets deficient in selenium, forages grown on selenium-deficient soils in certain geographic regions, and forages such as alfalfa and clover that have an inability to efficiently extract available selenium from the soils. Rumen bacterial reduction of selenium compounds to unavailable elemental selenium may also contribute to the disease. Necropsy findings. Necropsy lesions include petechial hemorrhages and muscle edema. Hallmarks are pale white streaking of affected skeletal and cardiac muscle. These are due to coagulation necrosis. Pale striated muscles of the limb, diaphragm, and tongue are also seen. Pathogenesis. Selenium and vitamin E function together as antioxidants that protect lipid membranes from oxidative destruction. Selenium is a cofactor for glutathione peroxidase, which converts hydrogen peroxide to water and other nontoxic compounds. Lack of one or both results in loss of membrane integrity. Differential diagnosis. In neonatal ruminants presenting with respiratory and cardiac dysfunction, differentials include congenital cardiac anomalies. Differentials generally for weak neonates or sudden or peracute neonatal deaths should include septicemia, pneumonia, toxicity, diarrhea, and dehydration. Prevention and control. Awareness of regional selenium deficiencies is important. Control involves providing goodquality roughage, vitamin E and selenium supplementation, and parenteral injections prior to parturition and weaning. Treatment. Affected animals may be treated by administering vitamin E or selenium injections. Administering vitamin E or selenium to ewes in late pregnancy can prevent white muscle disease ( Kott et al., 1998 ). The label dose for selenium is 2.5–3 mg/45 kg of body weight. Combination products are available and can be used in goats at the sheep dose ( Smith and Sherman, 1994 ). Proper mineral balance in the diet is critical. d. Selenium Toxicity Selenium toxicity occurs most frequently as the result of excessive dosing to prevent or correct selenium deficiency or as the result of ingestion of selenium-converting plants. The main preventive measure for the former is the use of the appropriate product for the species. Secondarily, the concentration of the available product should be double-checked. In the United States, ruminants in the Midwest and western areas may be subject to selenium toxicity when pastured in areas containing selenium-converting plants. Signs of overdosing include weakness, dyspnea, bloating, and diarrhea. Shock, paresis, and death may occur. Initial clinical signs of excessive selenium intake from plants are observed in the distal limb, with cracked hoof walls and subsequent infection and irregular hoof growth. e. Thiamin Deficiency (Polioencephalomalacia) Etiology. Polioencephalomalacia (PEM) is a noninfectious, noncontagious disease characterized by neurological signs. Growing and adult ruminants on high-concentrate diets are typically affected. Animals exposed to toxic plants or moldy feed containing thiaminases, feed high in sulfates, or unusually high doses of some medications are also at risk. Clinical signs and diagnosis. An early sign may be mild diarrhea. Acute clinical signs include bruxism, hyperesthesia, involuntary muscle contractions, depression, partial or complete opisthotonus, nystagmus, dorsomedial strabismus, seizures, and death. In subacute cases of the disease, animals may appear to walk aimlessly as if blind or may display head-pressing postures. Hypersalivation may be present, but body temperatures and ocular reflexes are normal. Morbidity and mortality may be high, especially in younger animals. Diagnosis is suggestive from clinical signs and from response to intensive parental thiamine hydrochloride. Epizootiology and transmission. PEM is caused by a thiamin deficiency. The disease tends to be seen more frequently in cattle and sheep feedlots where the concentrates fed are high in fermentable carbohydrates. Pastured animals are also vulnerable if grain is feed. Thiaminase-containing plants, such as bracken fern, are often unpalatable so will less likely be a contributing factor. Recent studies have also indicated that high levels of sulfate in the diet, such as in the fermentable, low-fiber concentrates, may play an important role. Medications such as as amprolium, levamisole, and thiabendazole have thiaminantagonizing activity when given in excessive doses. Necropsy signs. Cerebral lesions characterized by softening and discoloration are grossly observed in the gray matter. Microscopically, neurons will exhibit edema, chromatolysis, and shrinkage. Gliosis and cerebral capillary proliferation may be observed. Pathogenesis. A lack of thiamin results in inappropriate carbohydrate metabolism and accumulation of pyruvate and other intermediaries that lead to cerebral edema and neuronal degeneration. Differential diagnosis. Several important differentials include acute lead poisoning, nitrofuran toxicity, hypomagnesemia, vitamin A deficiency, listeriosis, pregnancy toxemia, infectious thromboembolic meningoencephalitis, and type D clostridial enterotoxemia. Prevention and control. The disease can be prevented by monitoring the diet and by providing adequate roughage necessary to prevent overgrowth of thiaminase-producing ruminal flora and to maximize ruminal production of B vitamins. If excess sulfur is the primary factor, immediate removal of the source is critical. Treatment. Early aggressive treatment is essential to save animals. The disease is treated by frequent parenteral administration of thiamine hydrochloride, the first dose being administered intravenously. Dexamethasone, B vitamins, and diazepam may also be required. Treatment is less successful when sulfur plays a prominent role in the etiology. Research complications. This disease is preventable. Although the disease is less likely to occur in smaller groups of confined ruminants, the risks of feeding concentrates or moldy feed, for example, with minimal good-quality roughage, should be kept in mind. f. Vitamin D Toxicity Vitamin D toxicity can result either from iatrogenic overadministration or ingestion of the plant Trisetum flavescens. Serum calcium levels may be high enough that blood in EDTA tubes will clot. g. Nutritional Deficiencies In goats, nutritional deficiencies often manifest as a generalized poor coat that is dry, scaly, thin, and erectile. Zinc-responsive dermatitis has been reported in goats ( Smith and Sherman, 1994 ). Vitamin A deficiencies associated with hyperkeratosis have been reported, as well as vitamin E-responsive and selenium-responsive dermatitis. 4. Management-Related Diseases a. Failure of Passive Transfer Neonatal ruminants are born without immunoglobulins and must receive colostrum by 24 hr after birth. The morbidity and mortality associated with failure of or inadequate passive transfer, such as enteric and respiratory illnesses, can be severe. Measures to assure passive immunity for neonatal ruminants are covered in Section II,B,5, and clinical signs of illness associated with lack of immunity are addressed in the discussions of bacterial diseases (e.g., Escherichia coli infections) and, of viral diseases (e.g., diarrheas) in Section III,A,1 and III,A,2. Generally, transfer of less than 600 mg/dl of immunoglobulins in the serum is classified as failure of transfer, 600–1600 mg/dl is partial, and above 1600 mg/dl is complete transfer. Methods to determine success of transfer should be performed within a week of birth and include single radial immunodiffusion (quantitates immunogloblin classes); zinc sulfate turbidity (semiquantitative); sodium sulfite precipitation (semiquantitative); glutaraldehyde coagulation (coagulates above specific level); and, γ-glutamyltransferase (assays enzyme in high concentration in colostrum and absorbed simultaneously with colostrum). b. Laminitis Laminitis is common in ruminants and can be caused by sudden changes in diet, excess dietary energy, and grain overload (or overeating). Laminitis is also associated with mastitis and metritis. Facility conditions, such as concrete flooring, poor manure management, and inadequate resting areas may also contribute to the pathogenesis of the disease. The complete pathogenesis of laminitis is poorly understood; however, it is thought that changes in the diet cause changes in rumen microbial populations, resulting in acidosis and endotoxemia. Dramatic changes in the vascular endothelium result in chronic inflammation of the sensitive laminae of the hoof, separation of corium and hoof wall, and rotation of the third phalanx. Affected animals may be reluctant to get up or walk, will shift their weight frequently, and will grind teeth or walk on carpi. Chronically, the hoof wall takes on a "slipper" appearance. Treatment consists of identifying the underlying cause, administering antiinflammatories (phenylbutazone, flunixin meglumin), feeding good-quality forages only, and regular foot trimming. c. Nutritional Diarrhea Otherwise normal, well-managed lambs, kids, and calves can develop loose, pasty feces due to a nutritional imbalance caused by overfeeding and/or improper mixing of milk replacers. Only milk replacer formulated for the particular species should be used. Once nutritional imbalances are corrected, the feces readily return to normal. Sudden changes in diet can also result in loose feces. d. Photosensitization (Bighead) Photosensitization is an acute dermatitis associated with an interaction between photosensitive chemicals and sunlight. The photosensitive chemicals are usually ingested, but in some cases exposure may be by contact. Animals with a lack of pigment are more susceptible to the disease. Three types of photosensitization occur: primary; secondary, or hepatogenous; and aberrant. Primary photosensitization is related to uncommon plant pigments or to drugs such as phenothiazine, sulfonamides, or tetracyclines. Secondary photosensitization is more common in large animals and is specifically related to the plant pigment phylloerythrin. Phylloerythrin, a porphyrin compound, is a degradation product of chlorophyll released by rumen microbial digestion. Liver disease or injury, which prevents normal conjugation of phylloerythrin and excretion through the biliary system, predisposes to photosensitization. The only example of aberrant photosensitization is congenital porphyria of cattle (see Section III,B,1). Pathologically, the photosensitive chemical is deposited in the skin and is activated by absorbed sunlight. The activated pigments transfer their energy to local proteins and amino acids, which, in the presence of oxygen, are converted to vasoactive substances. The vasoactive substances increase the permeability of capillaries, leading to fluid and plasma protein losses and eventually to local tissue necrosis. Photosensitization can occur within hours to days after sun exposure and produces lesions of the face, vulva, and coronary bands; lesions are most likely to occur on white-haired areas. Initially, edema of the lips, corneas, eyelids, nasal planum, face, vulva, or coronary bands occurs. The facial edema, nostril constriction, and swollen lips potentially lead to difficulty in breathing. With secondary photosensitization, icterus is also common. Necrosis and gangrene may occur. Diagnosis is based on clinical lesions and exposure to the photosensitive chemicals and sunlight. Treatment is symptomatic. The prognosis for hepatogenous type may be guarded if hepatic disease is severe. e. Reproductive Prolapses (Vaginal, Uterine) Vaginal and uterine prolapses occur in ewes, does, and cows. The conditions are not common in does. Vaginal prolapses usually occur during late gestation and may be related to relaxation of the pelvic ligaments in response to hormone levels. In sheep, these are most common in overconditioned ewes that are also carrying twins or triplets. Overconsumption of roughages, which distends the rumen, and lack of exercise leading to intra-abdominal fat may predispose an animal to vaginal prolapse by increasing intra-abdominal pressure. The condition may result from excessive straining associated with dysuria from the pressure of the fetuses and/or abdominal contents on the bladder. If the prolapse obstructs subsequent urination, rupture of the bladder may occur. The vaginal prolapse can be reduced and repaired if discovered early, and techniques in small and large ruminants are comparable. The animal should be restrained, and the prolapsed tissue should be cleansed with disinfectants. Best done under epidural anesthesia, the vagina is replaced into the pelvic canal and the vulvar or vestibular opening is sutured closed (Buhner suture). Alternatively, a commercial device called a bearing retainer (or truss) can be placed into the reduced vagina and tied to the wool, thereby holding the vagina in proper orientation without interfering with subsequent lambing. Vaginal prolapses may have a hereditary basis in ewes and cows and may prolapse the following year. These animals should be culled. Vaginal prolapses may occur in nonpregnant animals that graze estrogenic plants or as a sequela to docking the tail too close to the body ( Ross, 1989 ). Uterine prolapses occur sporadically in postpartum ewes and cattle. The gravid horn invaginates after delivery and protrudes from the vulva. The cause is unknown, but excessive traction utilized to correct dystocia or retained placenta, uterine atony, hypocalcemia, and overconditioning or lack of exercise have been implicated. In cattle, the uterine prolapses usually develop within 1 week of calving, are more common in dairy cows than in beef cows, and are often associated with dystocia or hypocalcemia. Cows may also have concurrent parturient paresis. Initially, the tissue will appear normal, but edema and environmental contamination or injuries of the tissue develop quickly. Clinical signs will include increased pulse and respiratory rates, straining, restlessness, and anorexia. If identified early, the uterus can be replaced as for vaginal prolapses. Electrolyte imbalances should be corrected if present. Additional supportive therapy, including the use of antibiotics should always be considered. Tetanus prophylaxis should be included. Oxytocin should be administered to induce uterine reduction. Vaginal closures are less successful at retaining uterine prolapses. Preventive and control measures include regular exercise for breeding animals, and management of prepartum nutrition and body condition. f. Rectal Prolapse Rectal prolapse is common in growing, weaned lambs and in cattle from 6 months to 2 years old. The physical eversion of the rectum through the anal sphincter is usually secondary to other diseases or management-related circumstances. Rectal prolapses may occur secondary to gastrointestinal infection or inflammation, especially when the colon is involved. Diseases that cause tenesmus, such as coccidiosis, salmonellosis, and intestinal worms, may result in prolapse. Urolithiasis may result in prolapses as the animal strains to urinate. Any form of cystitis or urethritis, vaginal irritation, or vaginal prolapse and some forms of hepatic disease may lead to rectal prolapse. Abdominal enlargement related to advanced stages of pregnancy, excessive rumen filling or bloat, and overconditioning may cause prolapse. Finally, excessive coughing during respiratory tract infections, improper tail docking (too short), growth implants, prolonged recumbency, or overcrowded housing with animal piling may lead to prolapses. Diagnosis is based on clinical signs. Early prolapses may be corrected by holding the animal with the head down, while a colleague places a pursestring suture around the anus. The mucosa and underlying tissue of prolapses that have been present for longer periods of time will often become necrotic, dry, friable, and devitalized and will require surgical amputation or the placement of prolapse rings to remove the tissue. Rectal prolapse may also be accompanied by intestinal intussusceptions that will further complicate the treatment and increase mortality. Occasionally, acute rectal prolapse with evisceration will result in shock and prompt death of the animal. Prognosis depends on the cause and extent of the prolapse as well as the timeliness of intervention. In all cases of treatment, determination and elimination of the underlying cause are essential. g. Trichobezoars Gastrointestinal accumulations or obstructions of hair (and/or sometimes very coarse roughage, forming bezoars) occur in cattle and sheep. Cattle that are maintained on a low-roughage diet, that lick their coats frequently, that have long hair coats from outdoor housing, or that have heavy lice or mite infestations and associated pruritus will often develop bezoars. In addition, younger calves with abomasal ulcers have been found to be more likely to have abomasal trichobezoars as well. Clinical signs may be mild or severe according to size, number, and location. Ruminal trichobezoars rarely result in clinical signs. Obstruction will be accompanied by signs of pain, development of bloat, and decreased fecal production. Serum profiles will show hypochloridemia; other imbalances depend on the duration of the problem. Diagnosis is also based on abdominal auscultation, rectal palpation, and ultrasound (useful in calves and smaller ruminants). Treatment is surgical, such as paracostal laparotomy (for abomasal), paralumbar celiotomy with manual breakdown, or enterotomy. Supportive care should be administered as necessary to correct electrolyte imbalances and to prevent inflammation and sepsis. Prognosis is generally good if the condition is diagnosed and treated before dehydration and imbalances become severe and peritonitis develops. Prevention includes providing good-quality roughage and treating lice and mange infestations. 1. Genetic Diseases a. Entropion Inverted eyelids are a common inherited disorder of lambs and kids of most breeds. Generally, the lower eyelid is affected and turns inward, causing various degrees of trauma to the conjunctiva and cornea. Young animals will display tearing, blepharospasm, and photophobia initially. If the disorder is left uncorrected, corneal ulcers, perforating ulcers, uveitis, and blindness may occur. Placing a suture or a surgical staple in the lower eyelid and the cheek, effectively anchoring the lid in an everted position, successfully treats the condition. The procedure likely results in the formation of some degree of scar tissue within the lower lid, because when the suture eventually is removed, the condition rarely returns. Other treatments include the injection of a "bleb" of penicillin in the lid, regular manual correction over a 2-day period early in the animal's life, and application of ophthalmic ointments, powders, and solutions. Boric acid or 10% Argyrol solutions have been used as treatments. Because of the genetic predisposition, prevention of the condition requires removal of maternal or paternal carriers. b. β-Mannosidosis of Goats β-Mannosidosis is an autosomal recessive lysosomal storage disease of goats. The disease affects kids of the Nubian breed and is identified by intention tremors and difficulty or inability of newborns to stand. Cells of affected animals are vacuolated because of a lack of lysosomal hydroxylase, which results in accumulation of oligosaccharides. Newborn kids are unable to rise, and they have characteristic flexion of the carpal joint and hyperextension of the pastern joint. Kids are born deaf and with musculoskeletal deformities such as domed skull, small narrow muzzle, small palpebral fissures, enophthalmos, and depressed nasal bridge ( Smith and Sherman, 1994 ). Carrier adults can be identified by plasma measurements of β-mannosidase activity. c. Congenital Myotonia of Goats Caprine congenital myotonia is an inherited autosomal dominant disease that affects voluntary striated skeletal muscles. Goats with this disease are commonly known as fainting goats. "Fainting" is actually transient spasms of skeletal musculature brought about by visual, tactile, or auditory stimuli ( Smith and Sherman, 1994 ). Muscle fiber membranes appear to have fewer chloride channels than normal, resulting in decreased chloride conduction across the membrane, with subsequent increased membrane excitability and repetitive firing ( Smith and Sherman, 1994 ). Contractions of skeletal muscle are sustained for up to 1 min. Kids exhibit the condition by 6 weeks of age, and males appear to exhibit more severe clinical signs than females ( Smith and Sherman, 1994 ). Electromyographic studies produce an audible "dive-bomber" sound characteristic of hyper-excitable cell membranes ( Smith and Sherman, 1994 ). d. Inherited Conditions of Cattle i. Congenital erythropoietic porphyria. Congenital erythropoietic porphyria (CEP) is an autosomal recessive disease of cattle seen primarily in Holsteins, Herefords, and Shorthorns. The disease also occurs in Limousin cattle, humans, and some other species. In the homozygous recessive animal, symptoms of the disease may vary from mild to severe and occur at different times of the year and in different ages of animals. A reddish brown discoloration of teeth and bones is a characteristic of the disease, as is discolored urine, general weakness and failure to thrive, photosensitization, and photophobia. Bones are more fragile compared with bones of normal animals. A regenerative anemia occurs as the result of the shortened life span of erythrocytes, due to accumulations of porphyrins. The genetic defect is associated with low activity of an essential enzyme, uroporphyrinogen III synthase, in the porphyrin–heme synthesis pathway in erythrocytic tissue. The ranges in the presentation of the disease are believed to be related to varying cycles of porphyrin synthesis. Porphyrins are excreted in varying amounts in the urine and the discoloration fluoresces under a Wood's lamp. Diagnosis is based on these clinical and visible signs of porphyria; skin biopsy provides definitive diagnosis. Heterozygotes may have milder symptoms. Many other genetic defects, in all major organ systems, have been described in numerous breeds of cattle and are described in detail elsewhere ( "Large Animal Internal Medicine," 1996 ). In many cases, the genetic basis has been clarified, and associated defects also noted. Many defects are reported in particular breeds, but as crossbreeding increases and new breeds are developed, these traits are appearing in these animals. The bovine genome continues to be further characterized, and more linkage maps and gene locations are forthcoming ( Womack, 1998 ). Some bovine genetic defects are also regarded as models of genetic disease, such as leukocyte adhesion deficiency of Holstein cattle. Some of the more commonly reported defects include syndactyly in Holsteins and other breeds and Polydactyly in Simmentals; lysosomal storage diseases such as α-mannosidosis in some beef breeds; enzyme deficiencies such as citrulline-mia in Holsteins; and progressive degenerative myeloencephalopathy ("weaver") in Brown Swiss. ii. Goiter of sheep. A defect in the synthesis of thyroid hormone has been identified in Merino sheep ( Radostits et al., 1994 ). Lambs born with the defect have enlargement of the thyroid gland, a silky appearance to the wool, and a high degree of mortality. Edema, bowing of the legs, and facial abnormalities have also been noted in animals with this disorder. Immaturity of the lungs at birth causes neonatal respiratory distress and results in dyspnea and respiratory failure. iii. Spider lamb syndrome (hereditary chondrodysplasia). Spider lamb syndrome is an inherited, often lethal, musculoskeletal disorder primarily occurring in Suffolk and Hampshire breeds. Severely affected lambs die shortly after birth. Animals that survive the perinatal period develop angular limb deformities, scoliosis, and facial deformities. With time, affected animals become debilitated, exhibit joint pain, and develop neurological problems associated with the spinal abnormalities. Radiologically, secondary ossification centers—especially the physis, subchondral areas, and cuboidal bones—are affected. Abnormal endochondral ossification leads to excess cartilage formation, notably apparent in the elbows. Lambs will typically display abnormally long limbs, medial deviation of the carpus and tarsus, flattening of the sternum, scoliosis/kyphosis of the vertebrae, and a rounded nose. Muscle atrophy is common. Diagnosis can be based on typical clinical signs, which are similar to those seen with Marfan syndrome in humans ( Rook et al., 1986 ). Long-term survival is rare; treatment is unsuccessful. a. Entropion Inverted eyelids are a common inherited disorder of lambs and kids of most breeds. Generally, the lower eyelid is affected and turns inward, causing various degrees of trauma to the conjunctiva and cornea. Young animals will display tearing, blepharospasm, and photophobia initially. If the disorder is left uncorrected, corneal ulcers, perforating ulcers, uveitis, and blindness may occur. Placing a suture or a surgical staple in the lower eyelid and the cheek, effectively anchoring the lid in an everted position, successfully treats the condition. The procedure likely results in the formation of some degree of scar tissue within the lower lid, because when the suture eventually is removed, the condition rarely returns. Other treatments include the injection of a "bleb" of penicillin in the lid, regular manual correction over a 2-day period early in the animal's life, and application of ophthalmic ointments, powders, and solutions. Boric acid or 10% Argyrol solutions have been used as treatments. Because of the genetic predisposition, prevention of the condition requires removal of maternal or paternal carriers. b. β-Mannosidosis of Goats β-Mannosidosis is an autosomal recessive lysosomal storage disease of goats. The disease affects kids of the Nubian breed and is identified by intention tremors and difficulty or inability of newborns to stand. Cells of affected animals are vacuolated because of a lack of lysosomal hydroxylase, which results in accumulation of oligosaccharides. Newborn kids are unable to rise, and they have characteristic flexion of the carpal joint and hyperextension of the pastern joint. Kids are born deaf and with musculoskeletal deformities such as domed skull, small narrow muzzle, small palpebral fissures, enophthalmos, and depressed nasal bridge ( Smith and Sherman, 1994 ). Carrier adults can be identified by plasma measurements of β-mannosidase activity. c. Congenital Myotonia of Goats Caprine congenital myotonia is an inherited autosomal dominant disease that affects voluntary striated skeletal muscles. Goats with this disease are commonly known as fainting goats. "Fainting" is actually transient spasms of skeletal musculature brought about by visual, tactile, or auditory stimuli ( Smith and Sherman, 1994 ). Muscle fiber membranes appear to have fewer chloride channels than normal, resulting in decreased chloride conduction across the membrane, with subsequent increased membrane excitability and repetitive firing ( Smith and Sherman, 1994 ). Contractions of skeletal muscle are sustained for up to 1 min. Kids exhibit the condition by 6 weeks of age, and males appear to exhibit more severe clinical signs than females ( Smith and Sherman, 1994 ). Electromyographic studies produce an audible "dive-bomber" sound characteristic of hyper-excitable cell membranes ( Smith and Sherman, 1994 ). d. Inherited Conditions of Cattle i. Congenital erythropoietic porphyria. Congenital erythropoietic porphyria (CEP) is an autosomal recessive disease of cattle seen primarily in Holsteins, Herefords, and Shorthorns. The disease also occurs in Limousin cattle, humans, and some other species. In the homozygous recessive animal, symptoms of the disease may vary from mild to severe and occur at different times of the year and in different ages of animals. A reddish brown discoloration of teeth and bones is a characteristic of the disease, as is discolored urine, general weakness and failure to thrive, photosensitization, and photophobia. Bones are more fragile compared with bones of normal animals. A regenerative anemia occurs as the result of the shortened life span of erythrocytes, due to accumulations of porphyrins. The genetic defect is associated with low activity of an essential enzyme, uroporphyrinogen III synthase, in the porphyrin–heme synthesis pathway in erythrocytic tissue. The ranges in the presentation of the disease are believed to be related to varying cycles of porphyrin synthesis. Porphyrins are excreted in varying amounts in the urine and the discoloration fluoresces under a Wood's lamp. Diagnosis is based on these clinical and visible signs of porphyria; skin biopsy provides definitive diagnosis. Heterozygotes may have milder symptoms. Many other genetic defects, in all major organ systems, have been described in numerous breeds of cattle and are described in detail elsewhere ( "Large Animal Internal Medicine," 1996 ). In many cases, the genetic basis has been clarified, and associated defects also noted. Many defects are reported in particular breeds, but as crossbreeding increases and new breeds are developed, these traits are appearing in these animals. The bovine genome continues to be further characterized, and more linkage maps and gene locations are forthcoming ( Womack, 1998 ). Some bovine genetic defects are also regarded as models of genetic disease, such as leukocyte adhesion deficiency of Holstein cattle. Some of the more commonly reported defects include syndactyly in Holsteins and other breeds and Polydactyly in Simmentals; lysosomal storage diseases such as α-mannosidosis in some beef breeds; enzyme deficiencies such as citrulline-mia in Holsteins; and progressive degenerative myeloencephalopathy ("weaver") in Brown Swiss. ii. Goiter of sheep. A defect in the synthesis of thyroid hormone has been identified in Merino sheep ( Radostits et al., 1994 ). Lambs born with the defect have enlargement of the thyroid gland, a silky appearance to the wool, and a high degree of mortality. Edema, bowing of the legs, and facial abnormalities have also been noted in animals with this disorder. Immaturity of the lungs at birth causes neonatal respiratory distress and results in dyspnea and respiratory failure. iii. Spider lamb syndrome (hereditary chondrodysplasia). Spider lamb syndrome is an inherited, often lethal, musculoskeletal disorder primarily occurring in Suffolk and Hampshire breeds. Severely affected lambs die shortly after birth. Animals that survive the perinatal period develop angular limb deformities, scoliosis, and facial deformities. With time, affected animals become debilitated, exhibit joint pain, and develop neurological problems associated with the spinal abnormalities. Radiologically, secondary ossification centers—especially the physis, subchondral areas, and cuboidal bones—are affected. Abnormal endochondral ossification leads to excess cartilage formation, notably apparent in the elbows. Lambs will typically display abnormally long limbs, medial deviation of the carpus and tarsus, flattening of the sternum, scoliosis/kyphosis of the vertebrae, and a rounded nose. Muscle atrophy is common. Diagnosis can be based on typical clinical signs, which are similar to those seen with Marfan syndrome in humans ( Rook et al., 1986 ). Long-term survival is rare; treatment is unsuccessful. i. Congenital erythropoietic porphyria. Congenital erythropoietic porphyria (CEP) is an autosomal recessive disease of cattle seen primarily in Holsteins, Herefords, and Shorthorns. The disease also occurs in Limousin cattle, humans, and some other species. In the homozygous recessive animal, symptoms of the disease may vary from mild to severe and occur at different times of the year and in different ages of animals. A reddish brown discoloration of teeth and bones is a characteristic of the disease, as is discolored urine, general weakness and failure to thrive, photosensitization, and photophobia. Bones are more fragile compared with bones of normal animals. A regenerative anemia occurs as the result of the shortened life span of erythrocytes, due to accumulations of porphyrins. The genetic defect is associated with low activity of an essential enzyme, uroporphyrinogen III synthase, in the porphyrin–heme synthesis pathway in erythrocytic tissue. The ranges in the presentation of the disease are believed to be related to varying cycles of porphyrin synthesis. Porphyrins are excreted in varying amounts in the urine and the discoloration fluoresces under a Wood's lamp. Diagnosis is based on these clinical and visible signs of porphyria; skin biopsy provides definitive diagnosis. Heterozygotes may have milder symptoms. Many other genetic defects, in all major organ systems, have been described in numerous breeds of cattle and are described in detail elsewhere ( "Large Animal Internal Medicine," 1996 ). In many cases, the genetic basis has been clarified, and associated defects also noted. Many defects are reported in particular breeds, but as crossbreeding increases and new breeds are developed, these traits are appearing in these animals. The bovine genome continues to be further characterized, and more linkage maps and gene locations are forthcoming ( Womack, 1998 ). Some bovine genetic defects are also regarded as models of genetic disease, such as leukocyte adhesion deficiency of Holstein cattle. Some of the more commonly reported defects include syndactyly in Holsteins and other breeds and Polydactyly in Simmentals; lysosomal storage diseases such as α-mannosidosis in some beef breeds; enzyme deficiencies such as citrulline-mia in Holsteins; and progressive degenerative myeloencephalopathy ("weaver") in Brown Swiss. ii. Goiter of sheep. A defect in the synthesis of thyroid hormone has been identified in Merino sheep ( Radostits et al., 1994 ). Lambs born with the defect have enlargement of the thyroid gland, a silky appearance to the wool, and a high degree of mortality. Edema, bowing of the legs, and facial abnormalities have also been noted in animals with this disorder. Immaturity of the lungs at birth causes neonatal respiratory distress and results in dyspnea and respiratory failure. iii. Spider lamb syndrome (hereditary chondrodysplasia). Spider lamb syndrome is an inherited, often lethal, musculoskeletal disorder primarily occurring in Suffolk and Hampshire breeds. Severely affected lambs die shortly after birth. Animals that survive the perinatal period develop angular limb deformities, scoliosis, and facial deformities. With time, affected animals become debilitated, exhibit joint pain, and develop neurological problems associated with the spinal abnormalities. Radiologically, secondary ossification centers—especially the physis, subchondral areas, and cuboidal bones—are affected. Abnormal endochondral ossification leads to excess cartilage formation, notably apparent in the elbows. Lambs will typically display abnormally long limbs, medial deviation of the carpus and tarsus, flattening of the sternum, scoliosis/kyphosis of the vertebrae, and a rounded nose. Muscle atrophy is common. Diagnosis can be based on typical clinical signs, which are similar to those seen with Marfan syndrome in humans ( Rook et al., 1986 ). Long-term survival is rare; treatment is unsuccessful. i. Congenital erythropoietic porphyria. Congenital erythropoietic porphyria (CEP) is an autosomal recessive disease of cattle seen primarily in Holsteins, Herefords, and Shorthorns. The disease also occurs in Limousin cattle, humans, and some other species. In the homozygous recessive animal, symptoms of the disease may vary from mild to severe and occur at different times of the year and in different ages of animals. A reddish brown discoloration of teeth and bones is a characteristic of the disease, as is discolored urine, general weakness and failure to thrive, photosensitization, and photophobia. Bones are more fragile compared with bones of normal animals. A regenerative anemia occurs as the result of the shortened life span of erythrocytes, due to accumulations of porphyrins. The genetic defect is associated with low activity of an essential enzyme, uroporphyrinogen III synthase, in the porphyrin–heme synthesis pathway in erythrocytic tissue. The ranges in the presentation of the disease are believed to be related to varying cycles of porphyrin synthesis. Porphyrins are excreted in varying amounts in the urine and the discoloration fluoresces under a Wood's lamp. Diagnosis is based on these clinical and visible signs of porphyria; skin biopsy provides definitive diagnosis. Heterozygotes may have milder symptoms. Many other genetic defects, in all major organ systems, have been described in numerous breeds of cattle and are described in detail elsewhere ( "Large Animal Internal Medicine," 1996 ). In many cases, the genetic basis has been clarified, and associated defects also noted. Many defects are reported in particular breeds, but as crossbreeding increases and new breeds are developed, these traits are appearing in these animals. The bovine genome continues to be further characterized, and more linkage maps and gene locations are forthcoming ( Womack, 1998 ). Some bovine genetic defects are also regarded as models of genetic disease, such as leukocyte adhesion deficiency of Holstein cattle. Some of the more commonly reported defects include syndactyly in Holsteins and other breeds and Polydactyly in Simmentals; lysosomal storage diseases such as α-mannosidosis in some beef breeds; enzyme deficiencies such as citrulline-mia in Holsteins; and progressive degenerative myeloencephalopathy ("weaver") in Brown Swiss. ii. Goiter of sheep. A defect in the synthesis of thyroid hormone has been identified in Merino sheep ( Radostits et al., 1994 ). Lambs born with the defect have enlargement of the thyroid gland, a silky appearance to the wool, and a high degree of mortality. Edema, bowing of the legs, and facial abnormalities have also been noted in animals with this disorder. Immaturity of the lungs at birth causes neonatal respiratory distress and results in dyspnea and respiratory failure. iii. Spider lamb syndrome (hereditary chondrodysplasia). Spider lamb syndrome is an inherited, often lethal, musculoskeletal disorder primarily occurring in Suffolk and Hampshire breeds. Severely affected lambs die shortly after birth. Animals that survive the perinatal period develop angular limb deformities, scoliosis, and facial deformities. With time, affected animals become debilitated, exhibit joint pain, and develop neurological problems associated with the spinal abnormalities. Radiologically, secondary ossification centers—especially the physis, subchondral areas, and cuboidal bones—are affected. Abnormal endochondral ossification leads to excess cartilage formation, notably apparent in the elbows. Lambs will typically display abnormally long limbs, medial deviation of the carpus and tarsus, flattening of the sternum, scoliosis/kyphosis of the vertebrae, and a rounded nose. Muscle atrophy is common. Diagnosis can be based on typical clinical signs, which are similar to those seen with Marfan syndrome in humans ( Rook et al., 1986 ). Long-term survival is rare; treatment is unsuccessful. 2. Metabolic Diseases a. Abomasal Disorders i. Abomasal and duodenal ulcers. Abomasal and duodenal ulcers occur more frequently in calves and adult cattle than in sheep and goats. Like rumenitis, abomasal and duodenal ulcers may be associated with lactic acidosis. Concurrent disease, such as salmonellosis, bluetongue, or overuse of anti-inflammatory drugs, or recent shipping or environmental stresses may also lead to ulcer formation. Copper deficiency, dietary changes, mycotic infections, Clostridium perfringens abomasitis, and abomasal bezoars are associated with this disease in calves. In older adult cattle, abomasal lymphosarcoma may be the underlying condition. Gastric acid hypersecretion in conjunction with insufficient gastric mucous secretion will physically destroy the gastric epithelium. Deep ulceration may cause serious hemorrhage and/or perforation with peritonitis. Chronic hemorrhage may lead to anemia. Although ulcers are often asymptomatic in calves, perforation with peritonitis is more common than hemorrhage. Dark feces or melena and abdominal pain may be observed. Arched back, restlessness, kicking at the abdomen, bruxism, and anorexia are common signs of abdominal pain. Fecal occult blood is as an easy diagnostic test. Treatment includes gastrointestinal protectants and histamine antagonists. Anemia may be symptomatically treated with parenteral iron injections and anabolic steroids. Preventive measures in cattle herds include ensuring optimal passive immunity for calves, minimizing stress to calves, and striving for a herd free of bovine leukosis virus. ii. Abomasal emptying defect. Abomasal emptying defect of sheep is a sporadic syndrome associated with abomasal distension and weight loss. Suffolks tend to be especially predisposed, although the disease has been diagnosed in Hampshires, Columbias, and Corriedales. The mechanism of the disease is unknown. Affected animals will exhibit a gradual weight loss with a history of normal appetites. Feces will continue to be normal. Ventral abdominal distension associated with abomasal accumulation of feedstuffs will be apparent in many of the animals. Diagnosis is primarily based on history and clinical signs. Elevations in rumen chloride concentrations (>15 mEq/liter) are commonly found. Radiography or ultrasonography may be helpful at identifying the distended abomasum. Abomasal emptying defect is usually eventually fatal. Medical treatment with metoclopramide and mineral oil may be helpful in early disease. iii. Abomasal displacement. Displaced abomasum (DA) is a sporadic disorder usually associated with multiparous 4- to 7-year-old dairy cows in early lactation, but the condition can occur even in young calves. Displacement to the right (RDA) may be further complicated by torsion (RTA), a surgical emergency. Left displacement (LDA) is more common than RDA. Clinical signs include anorexia, lack of cud chewing, decreased frequency of ruminal contractions, shallow respirations, increased heart rate, treading, and decreased milk production. Diagnosis is based on characteristic areas of tympanic resonance during auscultation-percussion of the lateral to lateral-ventral abdomen ("pings"), ruminal displacement palpated per rectum, and clinical signs. Cow-side clinical chemistry findings include hypoglycemia and ketonuria; more extensive evaluations will often indicate moderate to severe electrolyte and acid-base abnormalities. DA occurs because of gas accumulation within the viscus, and the abomasum "floats" up from its normal ventral location to the lateral abdominal wall. No exact cause of DA has been identified, but it is commonly associated with stress; high levels of concentrate in the diet, leading to forestomach atony; and many disorders, including lack of regular exercise, mastitis, hypocalcemia, retained placenta, metritis, or twins. Factors such as body size and conformation indicate the possibility of genetic predisposition. Treatments include surgical and nonsurgical techniques for LDA; the former has a better chance of permanent correction. Emergency surgery is necessary for RTA; the disorder is fatal within 72 hr. Recurrence is rare after surgical correction. Electrolyte and acid-base imbalances are likely in severe cases and especially with RTA. Prevention includes reducing stress, taking greater care in the introduction and feeding of concentrates, and reducing incidence of predisposing diseases noted above ( Rohrbach et al., 1999 ). b. Fat Cow Syndrome, Hepatic Lipidosis Fat cow syndrome is seen in peri- or postparturient overconditioned or obese multiparous dairy cows. Factors in the development of the condition include negative energy balance related to the normal decreased dry matter intake as parturition approaches; hormonal changes associated with parturition; and concurrent diseases of parturition that decrease feed intake and increase energy needs. The possible concurrent diseases include metritis, retained fetal membranes, mastitis, parturient paresis, and displaced abomasum. Signs are nonspecific and include depression, anorexia, and weakness. Prognosis is usually guarded. Diagnosis is based on herd management, the animal's condition, ketonuria, and clinical signs. In prepartum cattle and in lactating cows, blood levels of nonesterified fatty acids (NEFA) greater than 1000 μEq/liter and 325–400 μEq/liter, respectively, are abnormal ( Gerloff and Herdt, 1999 ). Triglyceride analysis of liver biposy specimens are useful. In affected cows, body fat is mobilized, in the form of NEFA in response to the energy demands. Hepatic lipidosis occurs rapidly as the NEFA are converted into hepatic triglycerides. The ability of the liver to extract the albumin-bound NEFA from the blood is better than that of other tissues that need and can also use NEFA as an energy source. Treatment for any concurrent diseases must be pursued aggressively, as well as measures to increase and stabilize blood glucose, decrease NEFA production, and increase forestomach digestion to improve production of normally metabolized volatile fatty acids. Therapeutic measures include intravenous glucose drips, insulin (NPH or Lente) injections every 12 hr, and transfaunation of ruminal fluid from a normal cow. Prevention includes minimizing stress to late-gestation cows. Dry and lactating cows should be maintained separately; their energy, protein, and dry matter requirements are very different. Cows with prolonged lactation or delayed breeding should be managed to prevent weight gain. c. Rumen and Reticulum Disorders i. Bloat. Bloat or tympanites refers to an excessive accumulation of gas in the rumen. The condition most frequently occurs in animals that have been recently fed abundant quantities of succulent forages or grains. Bloat is classified into two broad categories: frothy bloat and free-gas bloat. Frothy bloat is associated with ingestion of feeds that produce a stable froth that is not easily expelled from the rumen. Fermentation gases such as CO 2 , CH 4 , and minor gases such as N 2 , O 2 , H 2 , and H 2 S incorporate into the froth, overdistend the rumen, and eventually compromise respiration by limiting diaphragm movement. The froth is often derived from a combination of salivary mucoproteins, protozoal or bacterial proteins, and proteins, pectins, saponins, or hemicellulose associated with ingested leaves or grain. Typical foodstuffs that cause frothy bloat include green legumes, leguminous hay (alfalfa, clover), or grain (especially barley, corn, and soybean meal). Free-gas bloat is less related to feeds ingested; rather, it is caused by rumen atony or by physical or pathological problems that prevent normal gas eructation. Some examples of causes of free-gas bloat are esophageal obstructions (foreign bodies, tumors, abscesses, and enlarged cervical or thoracic lymph nodes), vagal nerve paralysis or injury, and central nervous system conditions that affect eructation reflexes. Clinically, the animal will exhibit rumen distension, and tympany will be observed in the left paralumbar fossa. Additional signs may include colic-like pain of the abdomen and dyspnea. Passage of a stomach tube helps to differentiate between free-gas bloat and frothy bloat; and with free-gas bloat, expulsion of gas through the stomach tube aids in treatment of the disorder. Once rumen distension is alleviated with free-gas bloat, the underlying cause must be investigated to prevent recurrence. Frothy bloat is more difficult to treat, because the foam blocks the stomach tube. Addition of mineral oil, household detergents, or antifermentative compounds via the tube may help break down the surface tension, allowing the gas to be expelled. In acute, life-threatening cases of bloat, treatment should be aimed at alleviating rumen distension by placing a trocar or surgical rumenotomy into the rumen via the paralumbar fossa. Limiting the consumption of feedstuffs prone to induce bloat can prevent the disease. Additionally, poloxalene or monensin will decrease the incidence of frothy bloat. ii. Lactic acidosis. Lactic acidosis, or rumen acidosis, is an acute metabolic disease caused by engorgement of grains or other highly fermentable carbohydrate sources. The disease is most frequently related to a rapid change in diet from one containing high roughage to one containing excessive carbohydrates. Diet components that predispose to acidosis include common feed grains; feedstuffs such as sugar beets, molasses, and potatoes; by-products such as brewer's grains; and bakery products. Biochemically, ingestion of large amounts of the carbohydrate-rich diet causes the normally gram-negative rumen bacterial populations to shift to gram-positive Streptococcus and Lactobacillus species. The gram-positive organisms efficiently convert the starches to lactic acid. The lactic acid acidifies the rumen contents, leading to rumen mucosal inflammation, and increases the osmolality of rumen fluids, leading to sequestration of fluids and osmotic attraction of plasma and tissue fluid to the rumen. Lactic acid-induced rumenitis predisposes the animal to ulcers, to liver abscesses from "absorbed" bacterial pathogens, to laminitis from absorbed toxins, and to polioencephalomalacia from the inability of the new rumen bacterial populations to produce sufficient thiamine needed to maintain normal nervous system function. Clinically, animals will become anorexic, depressed, and weak within 1–3 days after the initial insult. Incoordination, ataxia, dehydration, hemoconcentration, rapid pulse and respiration, diarrhea, abdominal pain, and lameness will also be noted. Rumen distension and an acetone-like odor to the breath, milk, or urine may also be observed. Diagnosis is based on history and clinical signs. Blood, urine, or milk ketones can be detected ( Moore and Ishler, 1997 ). Additionally, rumen pH, which is normally above 6.0, will drop to less than 5.0 and in severe cases may achieve levels as low as 3.8. Similarly, urine pH will become acidic, blood pH will drop below 7.4, and hematocrit will appear to increase due to the relative hemoconcentration. Necropsy findings will be determined by secondary conditions. The primary lactic acidosis will cause swelling and necrosis of rumen papillae and abomasal hemorrhages and ulcers. Treatment must be applied early in the syndrome. In early hours of severe carbohydrate engorgement, rumenotomy and evacuation of the contents are appropriate. The patient should be given mineral oil and antifermentatives to prevent the continued conversion of starches to acids and the absorption of metabolic products. Bicarbonate or other antacids like magnesium carbonate or magnesium hydroxide introduced into the rumen will aid in adjusting rumen pH. Furthermore, animals can be given oral tetracycline or penicillin, which will decrease the gram-positive bacterial population. iii. Rumen parakeratosis. Parakeratosis is a degenerative condition of the rumen mucosa that leads to keratinization of the papillary epithelium. Excessive and continuous feeding of diets low in roughage causes the mucosal changes. Generally, this condition is seen in feedlot lambs and steers that are fed an all-grain diet. Clinically, animals may exhibit only poor rates of gain, due to changes in the absorptive capacity of the injured mucosa. At necropsy, papillae will be thickened and rough. They will frequently be dark in color, and multiple papillae will clump together. Abscessation may be observed. Histopathologically, papilla surfaces will have hyperkeratinization of the squamous epithelium. Chronic laminitis may be observed. However, diagnosis of parakeratosis is generally made at necropsy. Feeding adequate roughage, such as stemmy hay, will prevent the disease. Antibiotics may be administered to prevent secondary liver abscess formation. iv. Rumenitis. Rumenitis is an acute or chronic inflammation of the rumen, which occurs most commonly as a sequela to lactic acidosis. In addition to concentrate feeding, inadequate roughage in the diet is also associated with this disorder. Rumenitis may occur with contagious ecthyma infection or following ingestion of poisons or other irritants. Because rumenitis is often associated with lactic acidosis, it tends to occur in feedlot animals. The inflamed ruminal epithelium becomes necrotic and sloughs, creating ulcers. Endogenous rumen bacteria such as Fusobacterium necrophorum may invade the ulcers, penetrate the circulatory system, and induce abscesses of the liver. Clinically, the animals will appear depressed and anorexic. Rumen motility will be decreased, and animals will lose weight. The disease may resolve in a week to 10 days; mortality may reach 20%. Necropsy lesions include rumen inflammation and ulcers in the anteroventral sac. Granulation tissue and scarring may be observed following healing. Rumenitis is not typically diagnosed clinically; thus, specific treatment is not commonly done. The disease can be prevented by minimizing the incidence of lactic acidosis. d. Traumatic Reticulitis-Reticuloperitonitis (Hardware Disease) Etiology. Traumatic reticulitis–reticuloperitonitis is a disease of cattle related to their exploratory tendencies and ingestion of many different, nonvegetative materials. The disease is rarely seen in smaller ruminants. Clinical signs. Clinical signs range from asymptomatic to severe, depending on the penetration and damage by the foreign object after settling in the animal's forestomach. Many signs during the early, acute stages will be nonspecific, ranging from arched back, listlessness, anorexia, fever, decrease in production, ketosis, regurgitation, decrease or cessation of ruminal contractions, bloat, tachypnea, tachycardia, and grunts when urinating, defecating, or being forced to move. The prognosis is poor when peritonitis becomes diffuse. Sudden death can occur if the heart, coronary vessels, or other large vessels are punctured by the migrating object. Epizootiology and transmission. This is a noncontagious disease. The occurrence is directly related to sharp or metallic indigestible items in the feed or environment that the cattle mouth and swallow. Necropsy findings. In severe cases, necropsy findings include extensive inflammation throughout the cranial abdomen, malodorous peritoneal fluid accumulations, and lesions at the reticular sites of migration of the foreign objects. Cardiac puncture will be present in those animals succumbing to sudden death. Pathogenesis. Consumed objects initially settle in the rumen but are dumped into the reticulum during the digestive process, and normal contraction may eventually lead to puncture of the reticular wall. This sets off a localized inflammation or a localized or more generalized peritonitis. The inflammation may also temporarily or permanently affect innervation of local tissues and organs. Further damage may result from migration and penetration of the diaphragm, pericardium, and heart. Diagnosis is based on clinical signs, knowledge of herd management techniques in terms of placement of forestomach magnets, and reflection of acute or chronic infection on the hemogram. Radiographs and abdominocentesis may be useful. Differential diagnosis. Differentials include abomasal ulcers, hepatic ulcers, neoplasia (such as lymphosarcoma, usually in older animals, or intestinal carcinoma), laminitis, and cor pulmonale. Infectious diseases that are differentials include systemic leptospirosis and internal parasitism. Diseases causing sudden death may need to be considered. Prevention and control. This problem can be prevented entirely by elimination of sharp objects in cattle feed and in the housing and pasture environments. Adequately sized magnets placed in feed handling equipment and forestomach magnets (placed per os with a balling gun in young stock at 6–8 months of age) are also significant prevention measures. Treatment. Provision of a forestomach magnet, confinement, and nursing care, including antibiotics, are the initial treatments. In severe cases, rumenotomy may be considered. e. Pregnancy Toxemia (Ketosis), Protein Energy Malnutrition Etiology. Pregnancy toxemia is a primary metabolic disease of ewes and does in advanced pregnancy. Beef heifers are susceptible to protein energy malnutrition (PEM) syndrome, which is also referred to as pregnancy toxemia. Clinical signs. In sheep, this disease is characterized by hypoglycemia, ketonemia, ketonuria, weakness, and blindness. Hypoglycemic and ketotic ewes begin to wander aimlessly and to move away from the flock. They become anorexic and act uncoordinated, frequently leaning against objects. Advanced signs may include blindness, muscle tremors, teeth grinding, convulsions, and coma. Body temperature, heart rate, respiratory rate, and rumen motility continue normally. Up to 80% of infected ewes may die from the disease. The course of the disease may last up to a week. In goats, the disease usually occurs in the last 6 weeks of gestation, especially in does carrying triplets. Pregnancy toxemia should be considered with any goat showing signs of illness in late gestation. The doe may separate herself from the herd, stagger, or circle and may appear blind. Appetite is poor, and tremors may be evident. A rapid metabolic acidosis results in subsequent recumbency. Urinalysis will readily reveal ketonuria. If fetal death occurs, acute toxemia and death of the doe may result. In beef heifers, weight loss and thin body condition, weakness and inability to stand, and depression are clinical signs. Some cows develop diarrhea. Because the catabolic state is often so advanced, most affected heifers die even if treated. Pregnancy toxemia is diagnosed by evidence of typical clinical signs. Sodium nitroprusside tablets or ketosis dipsticks may be used to identify ketones in the urine or plasma of ewes and does. Blood glucose levels found to be below 25 mg/dl and ketonuria are good diagnostic indicators. In cattle, ketonuria is not a typical finding; hypocalcemia and anemia may be present. Epizootiology. Pregnancy toxemia occurs primarily in ewes that are obese or bearing twins or triplets. The disease develops during the last 6 weeks of pregnancy. PEM most frequently occurs in heifers during the final trimester of pregnancy. Necropsy findings. At necropsy, affected ewes will often have multiple fetuses, which may have died and decomposed. The liver will be enlarged, yellow, and friable, with fatty degeneration. The adrenal gland may also be enlarged. In cattle, heifers will be very thin, and in addition to a fatty liver, signs of concurrent diseases may be present. Pathogenesis. Rapid fetal growth, a decline in maternal nutrition, and a voluntary decrease in food intake in overfat ewes result in an inadequate supply of glucose needed for both maternal and fetal tissues. The ewe develops a severe hypoglycemia in early stages of the disease. The ruminant absorbs little dietary glucose; rather, it produces and absorbs volatile fatty acids (acetic, propionic, and butyric acids) from consumed feedstuffs. Propionic acid is absorbed and selectively converted to glucose through gluconeogenesis. When the animal is in a state of negative energy balance, it hydrolyzes fats to glycerol and fatty acids. Glycerol is converted to glucose while the fatty acids are metabolized for energy. The oxidation of fatty acids in the face of declining oxaloacetate levels (required for normal Krebs cycle function) results in the formation of ketone bodies (acetone, acetoacetic acid, and β-hydroxybutyric acid), thus causing the condition ketoacidosis. Heifer cattle have high energy requirements for completing normal body growth and supporting a pregnancy. Additional energy requirements are needed during pregnancy for winter conditions and during concurrent diseases. Marginal diets and poor-quality forage will place the cows in a negative energy balance. Differential diagnosis. Hypocalcemia is a common differential diagnosis. In cattle, differentials include chronic or untreated diseases such as Johne's disease, lymphosarcoma, parasitism, and chronic respiratory diseases. Prevention and control. Pregnancy toxemia can be prevented by providing adequate nutrition during late gestation and by maintaining animals in appropriate nonfat condition during pregnancy. In late pregnancy, the dietary energy and protein should be increased 1.5–2 times the maintenance level. PEM can be prevented by maintaining appropriate body condition earlier in pregnancy and supplying good-quality forage for the last trimester. Treatment. In sheep, because the morbidity may be as high as 20%, treatment should be directed at the flock rather than the individual. Treating the individual is usually unsuccessful. Oral administration of 200 ml of propylene glycol or 50% glucose twice a day, anabolic steroids, and high doses of adrenocorti-costeroids may be helpful. If ewes are still responsive and not severely acidotic or in renal failure, cesarean section may be successful by rapidly removing the fetus, which is the dietary drain for the ewe. In goats, pregnancy toxemia is best treated by removal of the fetuses either by cesarean section or induction of parturition. Parturition can be induced in does by either dexamethasone (10 mg) or PGF 2a (10 pg). In addition, goats may be treated with 10% dextrose (100 to 200 ml iv) or propylene glycol (60 ml per os 2 or 3 times a day). Adjunctive therapy includes normalizing acid base and hydration status, administration of vitamin B 12 and transfaunation. Heifers may be force-fed alfalfa gruels, given propylene glycol per os, placed on IV 50% glucose drips, and treated for concurrent disease. Research complications. In research requiring pregnant ewes in late stages of gestation, for example, this disease should be considered if the animals are likely to bear twins and will be transported or stressed in other ways during that time. f. Hypocalcemia (Parturient Paresis, Milk Fever) Etiology. Hypocalcemia is an acute metabolic disease of ruminants that requires emergency treatment; the presentation is slightly different in ewes, does, and cows. Clinical signs and diagnosis. In sheep, the disease is seen in ewes during the last 6 weeks of pregnancy and is characterized by muscle tetany, incoordination, paralysis, and finally coma. As calcium levels drop, ewes begin to show early signs such as stiffness and incoordination of movements, especially in the hindlimbs. Later, muscular tremors, muscular weakness, and recumbency will ensue. Animals will frequently be found breathing rapidly despite a normal body temperature. Morbidity may approach 30%, and mortality may reach as high as 90% in untreated animals. Affected does become bloated, weak, unsteady, and eventually recumbent. Cows are affected within 24–48 hr before or after parturition. Cows initially are weak and show evidence of muscle tremors, then deteriorate to sternal recumbency, with the head usually tucked to the abdomen, and an inability to stand. Tachycardia, dilated pupils, anorexia, hypothermia, depression, ruminal stasis, bloat, uterine inertia, and loss of anal tone are also seen at this stage. The terminal stage of disease is a rapid progression from coma to death. Heart rates will be high, but pulse may not be detectable. Hypocalcemia is diagnosed based on the pregnancy stage of the female and on clinical signs. It is later confirmed by laboratory findings of low serum calcium. With hypocalcemia in ewes, the plasma concentrations of calcium drop from normal values of 8–12 mg/dl to values of 3–6 mg/dl. In cattle, plasma levels below 7.5 mg/dl are hypocalcemic; at the terminal stages levels may be 2 mg/dl. Epizootiology. Hypocalcemia occurs primarily in overweight ewes during the last 6 weeks of pregnancy or during the first few weeks of lactation. The disease is not as common in the dairy goat as in the dairy cow. High-producing, older, multiparous dairy cows are the most susceptible, and the Jersey breed is considered susceptible. Cows that have survived one episode are prone to recurrence. In addition, dry cows must be managed carefully regarding limiting dietary calcium. The disease is not common in beef cattle unless there is an overall poor nutrition program. Necropsy findings. There is no pathognomonic or typical finding at necropsy. Pathogenesis. During the periparturient period, calcium requirements for fetal skeletal growth exceed calcium absorbed from the diet and from bone metabolism. Additionally, dietary calcium intake is thought to be compromised because, in advanced pregnancy, animals may not be able to eat enough to sustain adequate nutrient levels, and intestinal absorption capabilities do not respond as quickly as needed. After parturition, calcium needs increase dramatically because of calcium levels in colostrum and milk. Recent information suggests that legume and grass forages, high in potassium and low in magnesium, create a slight physiological alkalosis (at least in cattle), which antagonizes normal calcium regulation ( Rings et al., 1997 ). Thus, bone resorption, renal resorption, and gastrointestinal absorption of calcium are less than maximal. Prevention and control. Maintaining appropriate nutrition during the last trimester is helpful in preventing the disease. In cows and does, for example, limiting calcium intake by removing alfalfa from the diet is helpful. Treatment. Hypocalcemia must be treated quickly based on clinical signs; pretreatment blood samples can be saved for later confirmation. Twenty percent calcium borogluconate solution should be administered by slow intravenous infusion. Response will often be rapid, with the resolution of the animal's dull mentation. Less severely affected animals will often try to stand in a short time. Relapses are common, however, in sheep and cattle. Hypermagnesemia and hypophosphatemia often coincide with hypocalcemia. These imbalances should be considered when animals appear to be unresponsive to treatment. Hypocalcemia in the goat can be treated with 50–100 ml of calcium borogluconate. Heart rate should be monitored closely throughout calcium administration. If an irregular or rapid heart rate is detected, then calcium treatment should be slowed or discontinued. Calcium gels and boluses are also available for treatment ( Rings et al., 1997 ). Prognosis is generally good if the animal is treated early in the disease, but the prognosis will often be poor when treatment is initiated in later stages of the disease. g. Urinary Calculi (Obstructive Urolithiasis, Water Belly) Etiology. Urolithiasis is a metabolic disease of intact and castrated male sheep, goats, and cattle that is characterized by the formation of bladder and urethral crystals, urethral blockage, and anuria (Murray, 1985). The disease occurs rarely in female ruminants. Clinical signs and diagnosis. Affected animals will vocalize and begin to show signs of uneasiness, such as treading, straining postures, arched backs, raised tails, and squatting while attempting to urinate. These postures may be mistaken for tenesmus. Male cattle may develop swelling along the ventral perineal area. Affected animals will not stay with the herd or flock. Small amounts of urine may be discharged, and crystal deposits may be visible attached to the preputial hairs. Additionally, in smaller ruminants, the filiform urethral appendage (pizzle) often becomes dark purple to black in color. The pulsing pelvic urethra may be detected by manual or digital rectal palpation, and bladder distention may be noticeable in cattle by the same means. As the disease progresses to complete urethral blockage, the animal will become anorexic and show signs of abdominal pain, such as kicking at the belly. The abdomen will swell as the bladder enlarges, and rupture can occur within 36 hr after development of clinical signs. Bladder or urethral rupture may cause a short-lived period of apparent pain relief; subsequent development of uremia will eventually lead to death. The disease may progress over a period of 1–2 weeks, and the mortality is high unless the blockages are reversed. Diagnosis is made by the typical clinical signs. Abdominal taps may yield urine. Calculi are usually composed of calcium phosphate or ammonium phosphate matrices. Epizootiology and transmission. Clinical disease is usually seen in growing intact or castrated males. The disease may be sporadic or there may be clusters of cases in the flock or herd. Necropsy findings. Necropsy findings include urine in the abdomen with or without bladder or urethral rupture. Renal hydronephrosis may be evident. Calculi or struvite crystal sediment will be observed in the bladder and urethra. Histologically, trauma to the urethra and ureters will be present. Pathogenesis. Urolithiasis is multifactorial and involves dietary, anatomical, hormonal, and environmental factors. Male sheep and goats have a urethral process that predisposes them to entrapment of calculi. In cattle, the urethra narrows at the sigmoid flexure, and calculi lodge there most frequently. Additionally, the removal of testosterone by early castration is thought to result in hypoplasia of the urethra and penis. This physical reduction in the size of the excretory tube may predispose to the precipitation of and blockage by the struvite minerals. Grains fed to growing animals tend to be high in phosphorus and magnesium content. These calculogenic diets lead to the formation of struvite (magnesium ammonium phosphate) crystals. Other minerals associated with urolithiasis include silica (range grasses), carbonates (some grasses and clover pastures), calcium (exclusively alfalfa hay), and oxalates (fescue grasses). Differential diagnosis. Grain engorgement colic, gastrointestinal blockage, and causes of tenemus, such as enteritis or trauma, are differentials. Trauma to the urethral process should be considered. Urinary tract infections are uncommon in ruminants. Prevention and control. One case often is indicative of a potential problem in the group. Urolithiasis can be minimized by monitoring the calcium:phosphorus ratio in the diet. The normal ratio should be 2:1. Additionally, increasing the amount of dietary roughage will help balance the mineral intake. Increasing the amount of salt (sodium chloride, 2–4%) in the diet to increase water consumption, or adding ammonium chloride to the diet, at 10 gm/head/day or 2% of the ration, to acidify the urine, will aid in the prevention of this disease. Palatability of and accessibility to water should be assessed as well as functioning of automatic watering equipment. Treatment. Treatment is primarily surgical (Van Metre et al. 1996). Initially, amputation of the filiform urethral appendage may alleviate the disease since urethral blockage often begins here. As the disease progresses, urethral blockage in the sigmoid flexure as well as throughout the urethra may occur. In more advanced stages, perineal urethrostomy may yield good results. The prognosis is poor when the condition becomes chronic, reoccurs, or surgery is required. Research complications. Young castrated and intact male ruminants used in the laboratory setting will be the susceptible age group for this disorder. h. Rickets Rickets is a disease of young, growing animals but rarely occurs in goats. It is a metabolic disease characterized by a failure of bone matrix mineralization at the epiphysis of long bones due to lack of phosphorus. The condition can occur as an absolute deficiency in vitamin D 2 , an inadequate dietary supply of phosphorus, or a long-term dietary imbalance of calcium and phosphorus. The syndrome must be differentiated from epiphisitis (unequal growth of the epiphyses of long bones in young, rapidly growing kids fed diets with excess calcium). Clinical signs include poor growth, enlarged costochondral junctions, narrow chests, painful joints, and reluctance to move. Spontaneous fractures of long bones may occur. Animals will recover when dietary phosphorus is provided and if joint damage is not severe. a. Abomasal Disorders i. Abomasal and duodenal ulcers. Abomasal and duodenal ulcers occur more frequently in calves and adult cattle than in sheep and goats. Like rumenitis, abomasal and duodenal ulcers may be associated with lactic acidosis. Concurrent disease, such as salmonellosis, bluetongue, or overuse of anti-inflammatory drugs, or recent shipping or environmental stresses may also lead to ulcer formation. Copper deficiency, dietary changes, mycotic infections, Clostridium perfringens abomasitis, and abomasal bezoars are associated with this disease in calves. In older adult cattle, abomasal lymphosarcoma may be the underlying condition. Gastric acid hypersecretion in conjunction with insufficient gastric mucous secretion will physically destroy the gastric epithelium. Deep ulceration may cause serious hemorrhage and/or perforation with peritonitis. Chronic hemorrhage may lead to anemia. Although ulcers are often asymptomatic in calves, perforation with peritonitis is more common than hemorrhage. Dark feces or melena and abdominal pain may be observed. Arched back, restlessness, kicking at the abdomen, bruxism, and anorexia are common signs of abdominal pain. Fecal occult blood is as an easy diagnostic test. Treatment includes gastrointestinal protectants and histamine antagonists. Anemia may be symptomatically treated with parenteral iron injections and anabolic steroids. Preventive measures in cattle herds include ensuring optimal passive immunity for calves, minimizing stress to calves, and striving for a herd free of bovine leukosis virus. ii. Abomasal emptying defect. Abomasal emptying defect of sheep is a sporadic syndrome associated with abomasal distension and weight loss. Suffolks tend to be especially predisposed, although the disease has been diagnosed in Hampshires, Columbias, and Corriedales. The mechanism of the disease is unknown. Affected animals will exhibit a gradual weight loss with a history of normal appetites. Feces will continue to be normal. Ventral abdominal distension associated with abomasal accumulation of feedstuffs will be apparent in many of the animals. Diagnosis is primarily based on history and clinical signs. Elevations in rumen chloride concentrations (>15 mEq/liter) are commonly found. Radiography or ultrasonography may be helpful at identifying the distended abomasum. Abomasal emptying defect is usually eventually fatal. Medical treatment with metoclopramide and mineral oil may be helpful in early disease. iii. Abomasal displacement. Displaced abomasum (DA) is a sporadic disorder usually associated with multiparous 4- to 7-year-old dairy cows in early lactation, but the condition can occur even in young calves. Displacement to the right (RDA) may be further complicated by torsion (RTA), a surgical emergency. Left displacement (LDA) is more common than RDA. Clinical signs include anorexia, lack of cud chewing, decreased frequency of ruminal contractions, shallow respirations, increased heart rate, treading, and decreased milk production. Diagnosis is based on characteristic areas of tympanic resonance during auscultation-percussion of the lateral to lateral-ventral abdomen ("pings"), ruminal displacement palpated per rectum, and clinical signs. Cow-side clinical chemistry findings include hypoglycemia and ketonuria; more extensive evaluations will often indicate moderate to severe electrolyte and acid-base abnormalities. DA occurs because of gas accumulation within the viscus, and the abomasum "floats" up from its normal ventral location to the lateral abdominal wall. No exact cause of DA has been identified, but it is commonly associated with stress; high levels of concentrate in the diet, leading to forestomach atony; and many disorders, including lack of regular exercise, mastitis, hypocalcemia, retained placenta, metritis, or twins. Factors such as body size and conformation indicate the possibility of genetic predisposition. Treatments include surgical and nonsurgical techniques for LDA; the former has a better chance of permanent correction. Emergency surgery is necessary for RTA; the disorder is fatal within 72 hr. Recurrence is rare after surgical correction. Electrolyte and acid-base imbalances are likely in severe cases and especially with RTA. Prevention includes reducing stress, taking greater care in the introduction and feeding of concentrates, and reducing incidence of predisposing diseases noted above ( Rohrbach et al., 1999 ). i. Abomasal and duodenal ulcers. Abomasal and duodenal ulcers occur more frequently in calves and adult cattle than in sheep and goats. Like rumenitis, abomasal and duodenal ulcers may be associated with lactic acidosis. Concurrent disease, such as salmonellosis, bluetongue, or overuse of anti-inflammatory drugs, or recent shipping or environmental stresses may also lead to ulcer formation. Copper deficiency, dietary changes, mycotic infections, Clostridium perfringens abomasitis, and abomasal bezoars are associated with this disease in calves. In older adult cattle, abomasal lymphosarcoma may be the underlying condition. Gastric acid hypersecretion in conjunction with insufficient gastric mucous secretion will physically destroy the gastric epithelium. Deep ulceration may cause serious hemorrhage and/or perforation with peritonitis. Chronic hemorrhage may lead to anemia. Although ulcers are often asymptomatic in calves, perforation with peritonitis is more common than hemorrhage. Dark feces or melena and abdominal pain may be observed. Arched back, restlessness, kicking at the abdomen, bruxism, and anorexia are common signs of abdominal pain. Fecal occult blood is as an easy diagnostic test. Treatment includes gastrointestinal protectants and histamine antagonists. Anemia may be symptomatically treated with parenteral iron injections and anabolic steroids. Preventive measures in cattle herds include ensuring optimal passive immunity for calves, minimizing stress to calves, and striving for a herd free of bovine leukosis virus. ii. Abomasal emptying defect. Abomasal emptying defect of sheep is a sporadic syndrome associated with abomasal distension and weight loss. Suffolks tend to be especially predisposed, although the disease has been diagnosed in Hampshires, Columbias, and Corriedales. The mechanism of the disease is unknown. Affected animals will exhibit a gradual weight loss with a history of normal appetites. Feces will continue to be normal. Ventral abdominal distension associated with abomasal accumulation of feedstuffs will be apparent in many of the animals. Diagnosis is primarily based on history and clinical signs. Elevations in rumen chloride concentrations (>15 mEq/liter) are commonly found. Radiography or ultrasonography may be helpful at identifying the distended abomasum. Abomasal emptying defect is usually eventually fatal. Medical treatment with metoclopramide and mineral oil may be helpful in early disease. iii. Abomasal displacement. Displaced abomasum (DA) is a sporadic disorder usually associated with multiparous 4- to 7-year-old dairy cows in early lactation, but the condition can occur even in young calves. Displacement to the right (RDA) may be further complicated by torsion (RTA), a surgical emergency. Left displacement (LDA) is more common than RDA. Clinical signs include anorexia, lack of cud chewing, decreased frequency of ruminal contractions, shallow respirations, increased heart rate, treading, and decreased milk production. Diagnosis is based on characteristic areas of tympanic resonance during auscultation-percussion of the lateral to lateral-ventral abdomen ("pings"), ruminal displacement palpated per rectum, and clinical signs. Cow-side clinical chemistry findings include hypoglycemia and ketonuria; more extensive evaluations will often indicate moderate to severe electrolyte and acid-base abnormalities. DA occurs because of gas accumulation within the viscus, and the abomasum "floats" up from its normal ventral location to the lateral abdominal wall. No exact cause of DA has been identified, but it is commonly associated with stress; high levels of concentrate in the diet, leading to forestomach atony; and many disorders, including lack of regular exercise, mastitis, hypocalcemia, retained placenta, metritis, or twins. Factors such as body size and conformation indicate the possibility of genetic predisposition. Treatments include surgical and nonsurgical techniques for LDA; the former has a better chance of permanent correction. Emergency surgery is necessary for RTA; the disorder is fatal within 72 hr. Recurrence is rare after surgical correction. Electrolyte and acid-base imbalances are likely in severe cases and especially with RTA. Prevention includes reducing stress, taking greater care in the introduction and feeding of concentrates, and reducing incidence of predisposing diseases noted above ( Rohrbach et al., 1999 ). i. Abomasal and duodenal ulcers. Abomasal and duodenal ulcers occur more frequently in calves and adult cattle than in sheep and goats. Like rumenitis, abomasal and duodenal ulcers may be associated with lactic acidosis. Concurrent disease, such as salmonellosis, bluetongue, or overuse of anti-inflammatory drugs, or recent shipping or environmental stresses may also lead to ulcer formation. Copper deficiency, dietary changes, mycotic infections, Clostridium perfringens abomasitis, and abomasal bezoars are associated with this disease in calves. In older adult cattle, abomasal lymphosarcoma may be the underlying condition. Gastric acid hypersecretion in conjunction with insufficient gastric mucous secretion will physically destroy the gastric epithelium. Deep ulceration may cause serious hemorrhage and/or perforation with peritonitis. Chronic hemorrhage may lead to anemia. Although ulcers are often asymptomatic in calves, perforation with peritonitis is more common than hemorrhage. Dark feces or melena and abdominal pain may be observed. Arched back, restlessness, kicking at the abdomen, bruxism, and anorexia are common signs of abdominal pain. Fecal occult blood is as an easy diagnostic test. Treatment includes gastrointestinal protectants and histamine antagonists. Anemia may be symptomatically treated with parenteral iron injections and anabolic steroids. Preventive measures in cattle herds include ensuring optimal passive immunity for calves, minimizing stress to calves, and striving for a herd free of bovine leukosis virus. ii. Abomasal emptying defect. Abomasal emptying defect of sheep is a sporadic syndrome associated with abomasal distension and weight loss. Suffolks tend to be especially predisposed, although the disease has been diagnosed in Hampshires, Columbias, and Corriedales. The mechanism of the disease is unknown. Affected animals will exhibit a gradual weight loss with a history of normal appetites. Feces will continue to be normal. Ventral abdominal distension associated with abomasal accumulation of feedstuffs will be apparent in many of the animals. Diagnosis is primarily based on history and clinical signs. Elevations in rumen chloride concentrations (>15 mEq/liter) are commonly found. Radiography or ultrasonography may be helpful at identifying the distended abomasum. Abomasal emptying defect is usually eventually fatal. Medical treatment with metoclopramide and mineral oil may be helpful in early disease. iii. Abomasal displacement. Displaced abomasum (DA) is a sporadic disorder usually associated with multiparous 4- to 7-year-old dairy cows in early lactation, but the condition can occur even in young calves. Displacement to the right (RDA) may be further complicated by torsion (RTA), a surgical emergency. Left displacement (LDA) is more common than RDA. Clinical signs include anorexia, lack of cud chewing, decreased frequency of ruminal contractions, shallow respirations, increased heart rate, treading, and decreased milk production. Diagnosis is based on characteristic areas of tympanic resonance during auscultation-percussion of the lateral to lateral-ventral abdomen ("pings"), ruminal displacement palpated per rectum, and clinical signs. Cow-side clinical chemistry findings include hypoglycemia and ketonuria; more extensive evaluations will often indicate moderate to severe electrolyte and acid-base abnormalities. DA occurs because of gas accumulation within the viscus, and the abomasum "floats" up from its normal ventral location to the lateral abdominal wall. No exact cause of DA has been identified, but it is commonly associated with stress; high levels of concentrate in the diet, leading to forestomach atony; and many disorders, including lack of regular exercise, mastitis, hypocalcemia, retained placenta, metritis, or twins. Factors such as body size and conformation indicate the possibility of genetic predisposition. Treatments include surgical and nonsurgical techniques for LDA; the former has a better chance of permanent correction. Emergency surgery is necessary for RTA; the disorder is fatal within 72 hr. Recurrence is rare after surgical correction. Electrolyte and acid-base imbalances are likely in severe cases and especially with RTA. Prevention includes reducing stress, taking greater care in the introduction and feeding of concentrates, and reducing incidence of predisposing diseases noted above ( Rohrbach et al., 1999 ). b. Fat Cow Syndrome, Hepatic Lipidosis Fat cow syndrome is seen in peri- or postparturient overconditioned or obese multiparous dairy cows. Factors in the development of the condition include negative energy balance related to the normal decreased dry matter intake as parturition approaches; hormonal changes associated with parturition; and concurrent diseases of parturition that decrease feed intake and increase energy needs. The possible concurrent diseases include metritis, retained fetal membranes, mastitis, parturient paresis, and displaced abomasum. Signs are nonspecific and include depression, anorexia, and weakness. Prognosis is usually guarded. Diagnosis is based on herd management, the animal's condition, ketonuria, and clinical signs. In prepartum cattle and in lactating cows, blood levels of nonesterified fatty acids (NEFA) greater than 1000 μEq/liter and 325–400 μEq/liter, respectively, are abnormal ( Gerloff and Herdt, 1999 ). Triglyceride analysis of liver biposy specimens are useful. In affected cows, body fat is mobilized, in the form of NEFA in response to the energy demands. Hepatic lipidosis occurs rapidly as the NEFA are converted into hepatic triglycerides. The ability of the liver to extract the albumin-bound NEFA from the blood is better than that of other tissues that need and can also use NEFA as an energy source. Treatment for any concurrent diseases must be pursued aggressively, as well as measures to increase and stabilize blood glucose, decrease NEFA production, and increase forestomach digestion to improve production of normally metabolized volatile fatty acids. Therapeutic measures include intravenous glucose drips, insulin (NPH or Lente) injections every 12 hr, and transfaunation of ruminal fluid from a normal cow. Prevention includes minimizing stress to late-gestation cows. Dry and lactating cows should be maintained separately; their energy, protein, and dry matter requirements are very different. Cows with prolonged lactation or delayed breeding should be managed to prevent weight gain. c. Rumen and Reticulum Disorders i. Bloat. Bloat or tympanites refers to an excessive accumulation of gas in the rumen. The condition most frequently occurs in animals that have been recently fed abundant quantities of succulent forages or grains. Bloat is classified into two broad categories: frothy bloat and free-gas bloat. Frothy bloat is associated with ingestion of feeds that produce a stable froth that is not easily expelled from the rumen. Fermentation gases such as CO 2 , CH 4 , and minor gases such as N 2 , O 2 , H 2 , and H 2 S incorporate into the froth, overdistend the rumen, and eventually compromise respiration by limiting diaphragm movement. The froth is often derived from a combination of salivary mucoproteins, protozoal or bacterial proteins, and proteins, pectins, saponins, or hemicellulose associated with ingested leaves or grain. Typical foodstuffs that cause frothy bloat include green legumes, leguminous hay (alfalfa, clover), or grain (especially barley, corn, and soybean meal). Free-gas bloat is less related to feeds ingested; rather, it is caused by rumen atony or by physical or pathological problems that prevent normal gas eructation. Some examples of causes of free-gas bloat are esophageal obstructions (foreign bodies, tumors, abscesses, and enlarged cervical or thoracic lymph nodes), vagal nerve paralysis or injury, and central nervous system conditions that affect eructation reflexes. Clinically, the animal will exhibit rumen distension, and tympany will be observed in the left paralumbar fossa. Additional signs may include colic-like pain of the abdomen and dyspnea. Passage of a stomach tube helps to differentiate between free-gas bloat and frothy bloat; and with free-gas bloat, expulsion of gas through the stomach tube aids in treatment of the disorder. Once rumen distension is alleviated with free-gas bloat, the underlying cause must be investigated to prevent recurrence. Frothy bloat is more difficult to treat, because the foam blocks the stomach tube. Addition of mineral oil, household detergents, or antifermentative compounds via the tube may help break down the surface tension, allowing the gas to be expelled. In acute, life-threatening cases of bloat, treatment should be aimed at alleviating rumen distension by placing a trocar or surgical rumenotomy into the rumen via the paralumbar fossa. Limiting the consumption of feedstuffs prone to induce bloat can prevent the disease. Additionally, poloxalene or monensin will decrease the incidence of frothy bloat. ii. Lactic acidosis. Lactic acidosis, or rumen acidosis, is an acute metabolic disease caused by engorgement of grains or other highly fermentable carbohydrate sources. The disease is most frequently related to a rapid change in diet from one containing high roughage to one containing excessive carbohydrates. Diet components that predispose to acidosis include common feed grains; feedstuffs such as sugar beets, molasses, and potatoes; by-products such as brewer's grains; and bakery products. Biochemically, ingestion of large amounts of the carbohydrate-rich diet causes the normally gram-negative rumen bacterial populations to shift to gram-positive Streptococcus and Lactobacillus species. The gram-positive organisms efficiently convert the starches to lactic acid. The lactic acid acidifies the rumen contents, leading to rumen mucosal inflammation, and increases the osmolality of rumen fluids, leading to sequestration of fluids and osmotic attraction of plasma and tissue fluid to the rumen. Lactic acid-induced rumenitis predisposes the animal to ulcers, to liver abscesses from "absorbed" bacterial pathogens, to laminitis from absorbed toxins, and to polioencephalomalacia from the inability of the new rumen bacterial populations to produce sufficient thiamine needed to maintain normal nervous system function. Clinically, animals will become anorexic, depressed, and weak within 1–3 days after the initial insult. Incoordination, ataxia, dehydration, hemoconcentration, rapid pulse and respiration, diarrhea, abdominal pain, and lameness will also be noted. Rumen distension and an acetone-like odor to the breath, milk, or urine may also be observed. Diagnosis is based on history and clinical signs. Blood, urine, or milk ketones can be detected ( Moore and Ishler, 1997 ). Additionally, rumen pH, which is normally above 6.0, will drop to less than 5.0 and in severe cases may achieve levels as low as 3.8. Similarly, urine pH will become acidic, blood pH will drop below 7.4, and hematocrit will appear to increase due to the relative hemoconcentration. Necropsy findings will be determined by secondary conditions. The primary lactic acidosis will cause swelling and necrosis of rumen papillae and abomasal hemorrhages and ulcers. Treatment must be applied early in the syndrome. In early hours of severe carbohydrate engorgement, rumenotomy and evacuation of the contents are appropriate. The patient should be given mineral oil and antifermentatives to prevent the continued conversion of starches to acids and the absorption of metabolic products. Bicarbonate or other antacids like magnesium carbonate or magnesium hydroxide introduced into the rumen will aid in adjusting rumen pH. Furthermore, animals can be given oral tetracycline or penicillin, which will decrease the gram-positive bacterial population. iii. Rumen parakeratosis. Parakeratosis is a degenerative condition of the rumen mucosa that leads to keratinization of the papillary epithelium. Excessive and continuous feeding of diets low in roughage causes the mucosal changes. Generally, this condition is seen in feedlot lambs and steers that are fed an all-grain diet. Clinically, animals may exhibit only poor rates of gain, due to changes in the absorptive capacity of the injured mucosa. At necropsy, papillae will be thickened and rough. They will frequently be dark in color, and multiple papillae will clump together. Abscessation may be observed. Histopathologically, papilla surfaces will have hyperkeratinization of the squamous epithelium. Chronic laminitis may be observed. However, diagnosis of parakeratosis is generally made at necropsy. Feeding adequate roughage, such as stemmy hay, will prevent the disease. Antibiotics may be administered to prevent secondary liver abscess formation. iv. Rumenitis. Rumenitis is an acute or chronic inflammation of the rumen, which occurs most commonly as a sequela to lactic acidosis. In addition to concentrate feeding, inadequate roughage in the diet is also associated with this disorder. Rumenitis may occur with contagious ecthyma infection or following ingestion of poisons or other irritants. Because rumenitis is often associated with lactic acidosis, it tends to occur in feedlot animals. The inflamed ruminal epithelium becomes necrotic and sloughs, creating ulcers. Endogenous rumen bacteria such as Fusobacterium necrophorum may invade the ulcers, penetrate the circulatory system, and induce abscesses of the liver. Clinically, the animals will appear depressed and anorexic. Rumen motility will be decreased, and animals will lose weight. The disease may resolve in a week to 10 days; mortality may reach 20%. Necropsy lesions include rumen inflammation and ulcers in the anteroventral sac. Granulation tissue and scarring may be observed following healing. Rumenitis is not typically diagnosed clinically; thus, specific treatment is not commonly done. The disease can be prevented by minimizing the incidence of lactic acidosis. i. Bloat. Bloat or tympanites refers to an excessive accumulation of gas in the rumen. The condition most frequently occurs in animals that have been recently fed abundant quantities of succulent forages or grains. Bloat is classified into two broad categories: frothy bloat and free-gas bloat. Frothy bloat is associated with ingestion of feeds that produce a stable froth that is not easily expelled from the rumen. Fermentation gases such as CO 2 , CH 4 , and minor gases such as N 2 , O 2 , H 2 , and H 2 S incorporate into the froth, overdistend the rumen, and eventually compromise respiration by limiting diaphragm movement. The froth is often derived from a combination of salivary mucoproteins, protozoal or bacterial proteins, and proteins, pectins, saponins, or hemicellulose associated with ingested leaves or grain. Typical foodstuffs that cause frothy bloat include green legumes, leguminous hay (alfalfa, clover), or grain (especially barley, corn, and soybean meal). Free-gas bloat is less related to feeds ingested; rather, it is caused by rumen atony or by physical or pathological problems that prevent normal gas eructation. Some examples of causes of free-gas bloat are esophageal obstructions (foreign bodies, tumors, abscesses, and enlarged cervical or thoracic lymph nodes), vagal nerve paralysis or injury, and central nervous system conditions that affect eructation reflexes. Clinically, the animal will exhibit rumen distension, and tympany will be observed in the left paralumbar fossa. Additional signs may include colic-like pain of the abdomen and dyspnea. Passage of a stomach tube helps to differentiate between free-gas bloat and frothy bloat; and with free-gas bloat, expulsion of gas through the stomach tube aids in treatment of the disorder. Once rumen distension is alleviated with free-gas bloat, the underlying cause must be investigated to prevent recurrence. Frothy bloat is more difficult to treat, because the foam blocks the stomach tube. Addition of mineral oil, household detergents, or antifermentative compounds via the tube may help break down the surface tension, allowing the gas to be expelled. In acute, life-threatening cases of bloat, treatment should be aimed at alleviating rumen distension by placing a trocar or surgical rumenotomy into the rumen via the paralumbar fossa. Limiting the consumption of feedstuffs prone to induce bloat can prevent the disease. Additionally, poloxalene or monensin will decrease the incidence of frothy bloat. ii. Lactic acidosis. Lactic acidosis, or rumen acidosis, is an acute metabolic disease caused by engorgement of grains or other highly fermentable carbohydrate sources. The disease is most frequently related to a rapid change in diet from one containing high roughage to one containing excessive carbohydrates. Diet components that predispose to acidosis include common feed grains; feedstuffs such as sugar beets, molasses, and potatoes; by-products such as brewer's grains; and bakery products. Biochemically, ingestion of large amounts of the carbohydrate-rich diet causes the normally gram-negative rumen bacterial populations to shift to gram-positive Streptococcus and Lactobacillus species. The gram-positive organisms efficiently convert the starches to lactic acid. The lactic acid acidifies the rumen contents, leading to rumen mucosal inflammation, and increases the osmolality of rumen fluids, leading to sequestration of fluids and osmotic attraction of plasma and tissue fluid to the rumen. Lactic acid-induced rumenitis predisposes the animal to ulcers, to liver abscesses from "absorbed" bacterial pathogens, to laminitis from absorbed toxins, and to polioencephalomalacia from the inability of the new rumen bacterial populations to produce sufficient thiamine needed to maintain normal nervous system function. Clinically, animals will become anorexic, depressed, and weak within 1–3 days after the initial insult. Incoordination, ataxia, dehydration, hemoconcentration, rapid pulse and respiration, diarrhea, abdominal pain, and lameness will also be noted. Rumen distension and an acetone-like odor to the breath, milk, or urine may also be observed. Diagnosis is based on history and clinical signs. Blood, urine, or milk ketones can be detected ( Moore and Ishler, 1997 ). Additionally, rumen pH, which is normally above 6.0, will drop to less than 5.0 and in severe cases may achieve levels as low as 3.8. Similarly, urine pH will become acidic, blood pH will drop below 7.4, and hematocrit will appear to increase due to the relative hemoconcentration. Necropsy findings will be determined by secondary conditions. The primary lactic acidosis will cause swelling and necrosis of rumen papillae and abomasal hemorrhages and ulcers. Treatment must be applied early in the syndrome. In early hours of severe carbohydrate engorgement, rumenotomy and evacuation of the contents are appropriate. The patient should be given mineral oil and antifermentatives to prevent the continued conversion of starches to acids and the absorption of metabolic products. Bicarbonate or other antacids like magnesium carbonate or magnesium hydroxide introduced into the rumen will aid in adjusting rumen pH. Furthermore, animals can be given oral tetracycline or penicillin, which will decrease the gram-positive bacterial population. iii. Rumen parakeratosis. Parakeratosis is a degenerative condition of the rumen mucosa that leads to keratinization of the papillary epithelium. Excessive and continuous feeding of diets low in roughage causes the mucosal changes. Generally, this condition is seen in feedlot lambs and steers that are fed an all-grain diet. Clinically, animals may exhibit only poor rates of gain, due to changes in the absorptive capacity of the injured mucosa. At necropsy, papillae will be thickened and rough. They will frequently be dark in color, and multiple papillae will clump together. Abscessation may be observed. Histopathologically, papilla surfaces will have hyperkeratinization of the squamous epithelium. Chronic laminitis may be observed. However, diagnosis of parakeratosis is generally made at necropsy. Feeding adequate roughage, such as stemmy hay, will prevent the disease. Antibiotics may be administered to prevent secondary liver abscess formation. iv. Rumenitis. Rumenitis is an acute or chronic inflammation of the rumen, which occurs most commonly as a sequela to lactic acidosis. In addition to concentrate feeding, inadequate roughage in the diet is also associated with this disorder. Rumenitis may occur with contagious ecthyma infection or following ingestion of poisons or other irritants. Because rumenitis is often associated with lactic acidosis, it tends to occur in feedlot animals. The inflamed ruminal epithelium becomes necrotic and sloughs, creating ulcers. Endogenous rumen bacteria such as Fusobacterium necrophorum may invade the ulcers, penetrate the circulatory system, and induce abscesses of the liver. Clinically, the animals will appear depressed and anorexic. Rumen motility will be decreased, and animals will lose weight. The disease may resolve in a week to 10 days; mortality may reach 20%. Necropsy lesions include rumen inflammation and ulcers in the anteroventral sac. Granulation tissue and scarring may be observed following healing. Rumenitis is not typically diagnosed clinically; thus, specific treatment is not commonly done. The disease can be prevented by minimizing the incidence of lactic acidosis. i. Bloat. Bloat or tympanites refers to an excessive accumulation of gas in the rumen. The condition most frequently occurs in animals that have been recently fed abundant quantities of succulent forages or grains. Bloat is classified into two broad categories: frothy bloat and free-gas bloat. Frothy bloat is associated with ingestion of feeds that produce a stable froth that is not easily expelled from the rumen. Fermentation gases such as CO 2 , CH 4 , and minor gases such as N 2 , O 2 , H 2 , and H 2 S incorporate into the froth, overdistend the rumen, and eventually compromise respiration by limiting diaphragm movement. The froth is often derived from a combination of salivary mucoproteins, protozoal or bacterial proteins, and proteins, pectins, saponins, or hemicellulose associated with ingested leaves or grain. Typical foodstuffs that cause frothy bloat include green legumes, leguminous hay (alfalfa, clover), or grain (especially barley, corn, and soybean meal). Free-gas bloat is less related to feeds ingested; rather, it is caused by rumen atony or by physical or pathological problems that prevent normal gas eructation. Some examples of causes of free-gas bloat are esophageal obstructions (foreign bodies, tumors, abscesses, and enlarged cervical or thoracic lymph nodes), vagal nerve paralysis or injury, and central nervous system conditions that affect eructation reflexes. Clinically, the animal will exhibit rumen distension, and tympany will be observed in the left paralumbar fossa. Additional signs may include colic-like pain of the abdomen and dyspnea. Passage of a stomach tube helps to differentiate between free-gas bloat and frothy bloat; and with free-gas bloat, expulsion of gas through the stomach tube aids in treatment of the disorder. Once rumen distension is alleviated with free-gas bloat, the underlying cause must be investigated to prevent recurrence. Frothy bloat is more difficult to treat, because the foam blocks the stomach tube. Addition of mineral oil, household detergents, or antifermentative compounds via the tube may help break down the surface tension, allowing the gas to be expelled. In acute, life-threatening cases of bloat, treatment should be aimed at alleviating rumen distension by placing a trocar or surgical rumenotomy into the rumen via the paralumbar fossa. Limiting the consumption of feedstuffs prone to induce bloat can prevent the disease. Additionally, poloxalene or monensin will decrease the incidence of frothy bloat. ii. Lactic acidosis. Lactic acidosis, or rumen acidosis, is an acute metabolic disease caused by engorgement of grains or other highly fermentable carbohydrate sources. The disease is most frequently related to a rapid change in diet from one containing high roughage to one containing excessive carbohydrates. Diet components that predispose to acidosis include common feed grains; feedstuffs such as sugar beets, molasses, and potatoes; by-products such as brewer's grains; and bakery products. Biochemically, ingestion of large amounts of the carbohydrate-rich diet causes the normally gram-negative rumen bacterial populations to shift to gram-positive Streptococcus and Lactobacillus species. The gram-positive organisms efficiently convert the starches to lactic acid. The lactic acid acidifies the rumen contents, leading to rumen mucosal inflammation, and increases the osmolality of rumen fluids, leading to sequestration of fluids and osmotic attraction of plasma and tissue fluid to the rumen. Lactic acid-induced rumenitis predisposes the animal to ulcers, to liver abscesses from "absorbed" bacterial pathogens, to laminitis from absorbed toxins, and to polioencephalomalacia from the inability of the new rumen bacterial populations to produce sufficient thiamine needed to maintain normal nervous system function. Clinically, animals will become anorexic, depressed, and weak within 1–3 days after the initial insult. Incoordination, ataxia, dehydration, hemoconcentration, rapid pulse and respiration, diarrhea, abdominal pain, and lameness will also be noted. Rumen distension and an acetone-like odor to the breath, milk, or urine may also be observed. Diagnosis is based on history and clinical signs. Blood, urine, or milk ketones can be detected ( Moore and Ishler, 1997 ). Additionally, rumen pH, which is normally above 6.0, will drop to less than 5.0 and in severe cases may achieve levels as low as 3.8. Similarly, urine pH will become acidic, blood pH will drop below 7.4, and hematocrit will appear to increase due to the relative hemoconcentration. Necropsy findings will be determined by secondary conditions. The primary lactic acidosis will cause swelling and necrosis of rumen papillae and abomasal hemorrhages and ulcers. Treatment must be applied early in the syndrome. In early hours of severe carbohydrate engorgement, rumenotomy and evacuation of the contents are appropriate. The patient should be given mineral oil and antifermentatives to prevent the continued conversion of starches to acids and the absorption of metabolic products. Bicarbonate or other antacids like magnesium carbonate or magnesium hydroxide introduced into the rumen will aid in adjusting rumen pH. Furthermore, animals can be given oral tetracycline or penicillin, which will decrease the gram-positive bacterial population. iii. Rumen parakeratosis. Parakeratosis is a degenerative condition of the rumen mucosa that leads to keratinization of the papillary epithelium. Excessive and continuous feeding of diets low in roughage causes the mucosal changes. Generally, this condition is seen in feedlot lambs and steers that are fed an all-grain diet. Clinically, animals may exhibit only poor rates of gain, due to changes in the absorptive capacity of the injured mucosa. At necropsy, papillae will be thickened and rough. They will frequently be dark in color, and multiple papillae will clump together. Abscessation may be observed. Histopathologically, papilla surfaces will have hyperkeratinization of the squamous epithelium. Chronic laminitis may be observed. However, diagnosis of parakeratosis is generally made at necropsy. Feeding adequate roughage, such as stemmy hay, will prevent the disease. Antibiotics may be administered to prevent secondary liver abscess formation. iv. Rumenitis. Rumenitis is an acute or chronic inflammation of the rumen, which occurs most commonly as a sequela to lactic acidosis. In addition to concentrate feeding, inadequate roughage in the diet is also associated with this disorder. Rumenitis may occur with contagious ecthyma infection or following ingestion of poisons or other irritants. Because rumenitis is often associated with lactic acidosis, it tends to occur in feedlot animals. The inflamed ruminal epithelium becomes necrotic and sloughs, creating ulcers. Endogenous rumen bacteria such as Fusobacterium necrophorum may invade the ulcers, penetrate the circulatory system, and induce abscesses of the liver. Clinically, the animals will appear depressed and anorexic. Rumen motility will be decreased, and animals will lose weight. The disease may resolve in a week to 10 days; mortality may reach 20%. Necropsy lesions include rumen inflammation and ulcers in the anteroventral sac. Granulation tissue and scarring may be observed following healing. Rumenitis is not typically diagnosed clinically; thus, specific treatment is not commonly done. The disease can be prevented by minimizing the incidence of lactic acidosis. d. Traumatic Reticulitis-Reticuloperitonitis (Hardware Disease) Etiology. Traumatic reticulitis–reticuloperitonitis is a disease of cattle related to their exploratory tendencies and ingestion of many different, nonvegetative materials. The disease is rarely seen in smaller ruminants. Clinical signs. Clinical signs range from asymptomatic to severe, depending on the penetration and damage by the foreign object after settling in the animal's forestomach. Many signs during the early, acute stages will be nonspecific, ranging from arched back, listlessness, anorexia, fever, decrease in production, ketosis, regurgitation, decrease or cessation of ruminal contractions, bloat, tachypnea, tachycardia, and grunts when urinating, defecating, or being forced to move. The prognosis is poor when peritonitis becomes diffuse. Sudden death can occur if the heart, coronary vessels, or other large vessels are punctured by the migrating object. Epizootiology and transmission. This is a noncontagious disease. The occurrence is directly related to sharp or metallic indigestible items in the feed or environment that the cattle mouth and swallow. Necropsy findings. In severe cases, necropsy findings include extensive inflammation throughout the cranial abdomen, malodorous peritoneal fluid accumulations, and lesions at the reticular sites of migration of the foreign objects. Cardiac puncture will be present in those animals succumbing to sudden death. Pathogenesis. Consumed objects initially settle in the rumen but are dumped into the reticulum during the digestive process, and normal contraction may eventually lead to puncture of the reticular wall. This sets off a localized inflammation or a localized or more generalized peritonitis. The inflammation may also temporarily or permanently affect innervation of local tissues and organs. Further damage may result from migration and penetration of the diaphragm, pericardium, and heart. Diagnosis is based on clinical signs, knowledge of herd management techniques in terms of placement of forestomach magnets, and reflection of acute or chronic infection on the hemogram. Radiographs and abdominocentesis may be useful. Differential diagnosis. Differentials include abomasal ulcers, hepatic ulcers, neoplasia (such as lymphosarcoma, usually in older animals, or intestinal carcinoma), laminitis, and cor pulmonale. Infectious diseases that are differentials include systemic leptospirosis and internal parasitism. Diseases causing sudden death may need to be considered. Prevention and control. This problem can be prevented entirely by elimination of sharp objects in cattle feed and in the housing and pasture environments. Adequately sized magnets placed in feed handling equipment and forestomach magnets (placed per os with a balling gun in young stock at 6–8 months of age) are also significant prevention measures. Treatment. Provision of a forestomach magnet, confinement, and nursing care, including antibiotics, are the initial treatments. In severe cases, rumenotomy may be considered. Etiology. Traumatic reticulitis–reticuloperitonitis is a disease of cattle related to their exploratory tendencies and ingestion of many different, nonvegetative materials. The disease is rarely seen in smaller ruminants. Clinical signs. Clinical signs range from asymptomatic to severe, depending on the penetration and damage by the foreign object after settling in the animal's forestomach. Many signs during the early, acute stages will be nonspecific, ranging from arched back, listlessness, anorexia, fever, decrease in production, ketosis, regurgitation, decrease or cessation of ruminal contractions, bloat, tachypnea, tachycardia, and grunts when urinating, defecating, or being forced to move. The prognosis is poor when peritonitis becomes diffuse. Sudden death can occur if the heart, coronary vessels, or other large vessels are punctured by the migrating object. Epizootiology and transmission. This is a noncontagious disease. The occurrence is directly related to sharp or metallic indigestible items in the feed or environment that the cattle mouth and swallow. Necropsy findings. In severe cases, necropsy findings include extensive inflammation throughout the cranial abdomen, malodorous peritoneal fluid accumulations, and lesions at the reticular sites of migration of the foreign objects. Cardiac puncture will be present in those animals succumbing to sudden death. Pathogenesis. Consumed objects initially settle in the rumen but are dumped into the reticulum during the digestive process, and normal contraction may eventually lead to puncture of the reticular wall. This sets off a localized inflammation or a localized or more generalized peritonitis. The inflammation may also temporarily or permanently affect innervation of local tissues and organs. Further damage may result from migration and penetration of the diaphragm, pericardium, and heart. Diagnosis is based on clinical signs, knowledge of herd management techniques in terms of placement of forestomach magnets, and reflection of acute or chronic infection on the hemogram. Radiographs and abdominocentesis may be useful. Differential diagnosis. Differentials include abomasal ulcers, hepatic ulcers, neoplasia (such as lymphosarcoma, usually in older animals, or intestinal carcinoma), laminitis, and cor pulmonale. Infectious diseases that are differentials include systemic leptospirosis and internal parasitism. Diseases causing sudden death may need to be considered. Prevention and control. This problem can be prevented entirely by elimination of sharp objects in cattle feed and in the housing and pasture environments. Adequately sized magnets placed in feed handling equipment and forestomach magnets (placed per os with a balling gun in young stock at 6–8 months of age) are also significant prevention measures. Treatment. Provision of a forestomach magnet, confinement, and nursing care, including antibiotics, are the initial treatments. In severe cases, rumenotomy may be considered. Etiology. Traumatic reticulitis–reticuloperitonitis is a disease of cattle related to their exploratory tendencies and ingestion of many different, nonvegetative materials. The disease is rarely seen in smaller ruminants. Clinical signs. Clinical signs range from asymptomatic to severe, depending on the penetration and damage by the foreign object after settling in the animal's forestomach. Many signs during the early, acute stages will be nonspecific, ranging from arched back, listlessness, anorexia, fever, decrease in production, ketosis, regurgitation, decrease or cessation of ruminal contractions, bloat, tachypnea, tachycardia, and grunts when urinating, defecating, or being forced to move. The prognosis is poor when peritonitis becomes diffuse. Sudden death can occur if the heart, coronary vessels, or other large vessels are punctured by the migrating object. Epizootiology and transmission. This is a noncontagious disease. The occurrence is directly related to sharp or metallic indigestible items in the feed or environment that the cattle mouth and swallow. Necropsy findings. In severe cases, necropsy findings include extensive inflammation throughout the cranial abdomen, malodorous peritoneal fluid accumulations, and lesions at the reticular sites of migration of the foreign objects. Cardiac puncture will be present in those animals succumbing to sudden death. Pathogenesis. Consumed objects initially settle in the rumen but are dumped into the reticulum during the digestive process, and normal contraction may eventually lead to puncture of the reticular wall. This sets off a localized inflammation or a localized or more generalized peritonitis. The inflammation may also temporarily or permanently affect innervation of local tissues and organs. Further damage may result from migration and penetration of the diaphragm, pericardium, and heart. Diagnosis is based on clinical signs, knowledge of herd management techniques in terms of placement of forestomach magnets, and reflection of acute or chronic infection on the hemogram. Radiographs and abdominocentesis may be useful. Differential diagnosis. Differentials include abomasal ulcers, hepatic ulcers, neoplasia (such as lymphosarcoma, usually in older animals, or intestinal carcinoma), laminitis, and cor pulmonale. Infectious diseases that are differentials include systemic leptospirosis and internal parasitism. Diseases causing sudden death may need to be considered. Prevention and control. This problem can be prevented entirely by elimination of sharp objects in cattle feed and in the housing and pasture environments. Adequately sized magnets placed in feed handling equipment and forestomach magnets (placed per os with a balling gun in young stock at 6–8 months of age) are also significant prevention measures. Treatment. Provision of a forestomach magnet, confinement, and nursing care, including antibiotics, are the initial treatments. In severe cases, rumenotomy may be considered. e. Pregnancy Toxemia (Ketosis), Protein Energy Malnutrition Etiology. Pregnancy toxemia is a primary metabolic disease of ewes and does in advanced pregnancy. Beef heifers are susceptible to protein energy malnutrition (PEM) syndrome, which is also referred to as pregnancy toxemia. Clinical signs. In sheep, this disease is characterized by hypoglycemia, ketonemia, ketonuria, weakness, and blindness. Hypoglycemic and ketotic ewes begin to wander aimlessly and to move away from the flock. They become anorexic and act uncoordinated, frequently leaning against objects. Advanced signs may include blindness, muscle tremors, teeth grinding, convulsions, and coma. Body temperature, heart rate, respiratory rate, and rumen motility continue normally. Up to 80% of infected ewes may die from the disease. The course of the disease may last up to a week. In goats, the disease usually occurs in the last 6 weeks of gestation, especially in does carrying triplets. Pregnancy toxemia should be considered with any goat showing signs of illness in late gestation. The doe may separate herself from the herd, stagger, or circle and may appear blind. Appetite is poor, and tremors may be evident. A rapid metabolic acidosis results in subsequent recumbency. Urinalysis will readily reveal ketonuria. If fetal death occurs, acute toxemia and death of the doe may result. In beef heifers, weight loss and thin body condition, weakness and inability to stand, and depression are clinical signs. Some cows develop diarrhea. Because the catabolic state is often so advanced, most affected heifers die even if treated. Pregnancy toxemia is diagnosed by evidence of typical clinical signs. Sodium nitroprusside tablets or ketosis dipsticks may be used to identify ketones in the urine or plasma of ewes and does. Blood glucose levels found to be below 25 mg/dl and ketonuria are good diagnostic indicators. In cattle, ketonuria is not a typical finding; hypocalcemia and anemia may be present. Epizootiology. Pregnancy toxemia occurs primarily in ewes that are obese or bearing twins or triplets. The disease develops during the last 6 weeks of pregnancy. PEM most frequently occurs in heifers during the final trimester of pregnancy. Necropsy findings. At necropsy, affected ewes will often have multiple fetuses, which may have died and decomposed. The liver will be enlarged, yellow, and friable, with fatty degeneration. The adrenal gland may also be enlarged. In cattle, heifers will be very thin, and in addition to a fatty liver, signs of concurrent diseases may be present. Pathogenesis. Rapid fetal growth, a decline in maternal nutrition, and a voluntary decrease in food intake in overfat ewes result in an inadequate supply of glucose needed for both maternal and fetal tissues. The ewe develops a severe hypoglycemia in early stages of the disease. The ruminant absorbs little dietary glucose; rather, it produces and absorbs volatile fatty acids (acetic, propionic, and butyric acids) from consumed feedstuffs. Propionic acid is absorbed and selectively converted to glucose through gluconeogenesis. When the animal is in a state of negative energy balance, it hydrolyzes fats to glycerol and fatty acids. Glycerol is converted to glucose while the fatty acids are metabolized for energy. The oxidation of fatty acids in the face of declining oxaloacetate levels (required for normal Krebs cycle function) results in the formation of ketone bodies (acetone, acetoacetic acid, and β-hydroxybutyric acid), thus causing the condition ketoacidosis. Heifer cattle have high energy requirements for completing normal body growth and supporting a pregnancy. Additional energy requirements are needed during pregnancy for winter conditions and during concurrent diseases. Marginal diets and poor-quality forage will place the cows in a negative energy balance. Differential diagnosis. Hypocalcemia is a common differential diagnosis. In cattle, differentials include chronic or untreated diseases such as Johne's disease, lymphosarcoma, parasitism, and chronic respiratory diseases. Prevention and control. Pregnancy toxemia can be prevented by providing adequate nutrition during late gestation and by maintaining animals in appropriate nonfat condition during pregnancy. In late pregnancy, the dietary energy and protein should be increased 1.5–2 times the maintenance level. PEM can be prevented by maintaining appropriate body condition earlier in pregnancy and supplying good-quality forage for the last trimester. Treatment. In sheep, because the morbidity may be as high as 20%, treatment should be directed at the flock rather than the individual. Treating the individual is usually unsuccessful. Oral administration of 200 ml of propylene glycol or 50% glucose twice a day, anabolic steroids, and high doses of adrenocorti-costeroids may be helpful. If ewes are still responsive and not severely acidotic or in renal failure, cesarean section may be successful by rapidly removing the fetus, which is the dietary drain for the ewe. In goats, pregnancy toxemia is best treated by removal of the fetuses either by cesarean section or induction of parturition. Parturition can be induced in does by either dexamethasone (10 mg) or PGF 2a (10 pg). In addition, goats may be treated with 10% dextrose (100 to 200 ml iv) or propylene glycol (60 ml per os 2 or 3 times a day). Adjunctive therapy includes normalizing acid base and hydration status, administration of vitamin B 12 and transfaunation. Heifers may be force-fed alfalfa gruels, given propylene glycol per os, placed on IV 50% glucose drips, and treated for concurrent disease. Research complications. In research requiring pregnant ewes in late stages of gestation, for example, this disease should be considered if the animals are likely to bear twins and will be transported or stressed in other ways during that time. Etiology. Pregnancy toxemia is a primary metabolic disease of ewes and does in advanced pregnancy. Beef heifers are susceptible to protein energy malnutrition (PEM) syndrome, which is also referred to as pregnancy toxemia. Clinical signs. In sheep, this disease is characterized by hypoglycemia, ketonemia, ketonuria, weakness, and blindness. Hypoglycemic and ketotic ewes begin to wander aimlessly and to move away from the flock. They become anorexic and act uncoordinated, frequently leaning against objects. Advanced signs may include blindness, muscle tremors, teeth grinding, convulsions, and coma. Body temperature, heart rate, respiratory rate, and rumen motility continue normally. Up to 80% of infected ewes may die from the disease. The course of the disease may last up to a week. In goats, the disease usually occurs in the last 6 weeks of gestation, especially in does carrying triplets. Pregnancy toxemia should be considered with any goat showing signs of illness in late gestation. The doe may separate herself from the herd, stagger, or circle and may appear blind. Appetite is poor, and tremors may be evident. A rapid metabolic acidosis results in subsequent recumbency. Urinalysis will readily reveal ketonuria. If fetal death occurs, acute toxemia and death of the doe may result. In beef heifers, weight loss and thin body condition, weakness and inability to stand, and depression are clinical signs. Some cows develop diarrhea. Because the catabolic state is often so advanced, most affected heifers die even if treated. Pregnancy toxemia is diagnosed by evidence of typical clinical signs. Sodium nitroprusside tablets or ketosis dipsticks may be used to identify ketones in the urine or plasma of ewes and does. Blood glucose levels found to be below 25 mg/dl and ketonuria are good diagnostic indicators. In cattle, ketonuria is not a typical finding; hypocalcemia and anemia may be present. Epizootiology. Pregnancy toxemia occurs primarily in ewes that are obese or bearing twins or triplets. The disease develops during the last 6 weeks of pregnancy. PEM most frequently occurs in heifers during the final trimester of pregnancy. Necropsy findings. At necropsy, affected ewes will often have multiple fetuses, which may have died and decomposed. The liver will be enlarged, yellow, and friable, with fatty degeneration. The adrenal gland may also be enlarged. In cattle, heifers will be very thin, and in addition to a fatty liver, signs of concurrent diseases may be present. Pathogenesis. Rapid fetal growth, a decline in maternal nutrition, and a voluntary decrease in food intake in overfat ewes result in an inadequate supply of glucose needed for both maternal and fetal tissues. The ewe develops a severe hypoglycemia in early stages of the disease. The ruminant absorbs little dietary glucose; rather, it produces and absorbs volatile fatty acids (acetic, propionic, and butyric acids) from consumed feedstuffs. Propionic acid is absorbed and selectively converted to glucose through gluconeogenesis. When the animal is in a state of negative energy balance, it hydrolyzes fats to glycerol and fatty acids. Glycerol is converted to glucose while the fatty acids are metabolized for energy. The oxidation of fatty acids in the face of declining oxaloacetate levels (required for normal Krebs cycle function) results in the formation of ketone bodies (acetone, acetoacetic acid, and β-hydroxybutyric acid), thus causing the condition ketoacidosis. Heifer cattle have high energy requirements for completing normal body growth and supporting a pregnancy. Additional energy requirements are needed during pregnancy for winter conditions and during concurrent diseases. Marginal diets and poor-quality forage will place the cows in a negative energy balance. Differential diagnosis. Hypocalcemia is a common differential diagnosis. In cattle, differentials include chronic or untreated diseases such as Johne's disease, lymphosarcoma, parasitism, and chronic respiratory diseases. Prevention and control. Pregnancy toxemia can be prevented by providing adequate nutrition during late gestation and by maintaining animals in appropriate nonfat condition during pregnancy. In late pregnancy, the dietary energy and protein should be increased 1.5–2 times the maintenance level. PEM can be prevented by maintaining appropriate body condition earlier in pregnancy and supplying good-quality forage for the last trimester. Treatment. In sheep, because the morbidity may be as high as 20%, treatment should be directed at the flock rather than the individual. Treating the individual is usually unsuccessful. Oral administration of 200 ml of propylene glycol or 50% glucose twice a day, anabolic steroids, and high doses of adrenocorti-costeroids may be helpful. If ewes are still responsive and not severely acidotic or in renal failure, cesarean section may be successful by rapidly removing the fetus, which is the dietary drain for the ewe. In goats, pregnancy toxemia is best treated by removal of the fetuses either by cesarean section or induction of parturition. Parturition can be induced in does by either dexamethasone (10 mg) or PGF 2a (10 pg). In addition, goats may be treated with 10% dextrose (100 to 200 ml iv) or propylene glycol (60 ml per os 2 or 3 times a day). Adjunctive therapy includes normalizing acid base and hydration status, administration of vitamin B 12 and transfaunation. Heifers may be force-fed alfalfa gruels, given propylene glycol per os, placed on IV 50% glucose drips, and treated for concurrent disease. Research complications. In research requiring pregnant ewes in late stages of gestation, for example, this disease should be considered if the animals are likely to bear twins and will be transported or stressed in other ways during that time. Etiology. Pregnancy toxemia is a primary metabolic disease of ewes and does in advanced pregnancy. Beef heifers are susceptible to protein energy malnutrition (PEM) syndrome, which is also referred to as pregnancy toxemia. Clinical signs. In sheep, this disease is characterized by hypoglycemia, ketonemia, ketonuria, weakness, and blindness. Hypoglycemic and ketotic ewes begin to wander aimlessly and to move away from the flock. They become anorexic and act uncoordinated, frequently leaning against objects. Advanced signs may include blindness, muscle tremors, teeth grinding, convulsions, and coma. Body temperature, heart rate, respiratory rate, and rumen motility continue normally. Up to 80% of infected ewes may die from the disease. The course of the disease may last up to a week. In goats, the disease usually occurs in the last 6 weeks of gestation, especially in does carrying triplets. Pregnancy toxemia should be considered with any goat showing signs of illness in late gestation. The doe may separate herself from the herd, stagger, or circle and may appear blind. Appetite is poor, and tremors may be evident. A rapid metabolic acidosis results in subsequent recumbency. Urinalysis will readily reveal ketonuria. If fetal death occurs, acute toxemia and death of the doe may result. In beef heifers, weight loss and thin body condition, weakness and inability to stand, and depression are clinical signs. Some cows develop diarrhea. Because the catabolic state is often so advanced, most affected heifers die even if treated. Pregnancy toxemia is diagnosed by evidence of typical clinical signs. Sodium nitroprusside tablets or ketosis dipsticks may be used to identify ketones in the urine or plasma of ewes and does. Blood glucose levels found to be below 25 mg/dl and ketonuria are good diagnostic indicators. In cattle, ketonuria is not a typical finding; hypocalcemia and anemia may be present. Epizootiology. Pregnancy toxemia occurs primarily in ewes that are obese or bearing twins or triplets. The disease develops during the last 6 weeks of pregnancy. PEM most frequently occurs in heifers during the final trimester of pregnancy. Necropsy findings. At necropsy, affected ewes will often have multiple fetuses, which may have died and decomposed. The liver will be enlarged, yellow, and friable, with fatty degeneration. The adrenal gland may also be enlarged. In cattle, heifers will be very thin, and in addition to a fatty liver, signs of concurrent diseases may be present. Pathogenesis. Rapid fetal growth, a decline in maternal nutrition, and a voluntary decrease in food intake in overfat ewes result in an inadequate supply of glucose needed for both maternal and fetal tissues. The ewe develops a severe hypoglycemia in early stages of the disease. The ruminant absorbs little dietary glucose; rather, it produces and absorbs volatile fatty acids (acetic, propionic, and butyric acids) from consumed feedstuffs. Propionic acid is absorbed and selectively converted to glucose through gluconeogenesis. When the animal is in a state of negative energy balance, it hydrolyzes fats to glycerol and fatty acids. Glycerol is converted to glucose while the fatty acids are metabolized for energy. The oxidation of fatty acids in the face of declining oxaloacetate levels (required for normal Krebs cycle function) results in the formation of ketone bodies (acetone, acetoacetic acid, and β-hydroxybutyric acid), thus causing the condition ketoacidosis. Heifer cattle have high energy requirements for completing normal body growth and supporting a pregnancy. Additional energy requirements are needed during pregnancy for winter conditions and during concurrent diseases. Marginal diets and poor-quality forage will place the cows in a negative energy balance. Differential diagnosis. Hypocalcemia is a common differential diagnosis. In cattle, differentials include chronic or untreated diseases such as Johne's disease, lymphosarcoma, parasitism, and chronic respiratory diseases. Prevention and control. Pregnancy toxemia can be prevented by providing adequate nutrition during late gestation and by maintaining animals in appropriate nonfat condition during pregnancy. In late pregnancy, the dietary energy and protein should be increased 1.5–2 times the maintenance level. PEM can be prevented by maintaining appropriate body condition earlier in pregnancy and supplying good-quality forage for the last trimester. Treatment. In sheep, because the morbidity may be as high as 20%, treatment should be directed at the flock rather than the individual. Treating the individual is usually unsuccessful. Oral administration of 200 ml of propylene glycol or 50% glucose twice a day, anabolic steroids, and high doses of adrenocorti-costeroids may be helpful. If ewes are still responsive and not severely acidotic or in renal failure, cesarean section may be successful by rapidly removing the fetus, which is the dietary drain for the ewe. In goats, pregnancy toxemia is best treated by removal of the fetuses either by cesarean section or induction of parturition. Parturition can be induced in does by either dexamethasone (10 mg) or PGF 2a (10 pg). In addition, goats may be treated with 10% dextrose (100 to 200 ml iv) or propylene glycol (60 ml per os 2 or 3 times a day). Adjunctive therapy includes normalizing acid base and hydration status, administration of vitamin B 12 and transfaunation. Heifers may be force-fed alfalfa gruels, given propylene glycol per os, placed on IV 50% glucose drips, and treated for concurrent disease. Research complications. In research requiring pregnant ewes in late stages of gestation, for example, this disease should be considered if the animals are likely to bear twins and will be transported or stressed in other ways during that time. f. Hypocalcemia (Parturient Paresis, Milk Fever) Etiology. Hypocalcemia is an acute metabolic disease of ruminants that requires emergency treatment; the presentation is slightly different in ewes, does, and cows. Clinical signs and diagnosis. In sheep, the disease is seen in ewes during the last 6 weeks of pregnancy and is characterized by muscle tetany, incoordination, paralysis, and finally coma. As calcium levels drop, ewes begin to show early signs such as stiffness and incoordination of movements, especially in the hindlimbs. Later, muscular tremors, muscular weakness, and recumbency will ensue. Animals will frequently be found breathing rapidly despite a normal body temperature. Morbidity may approach 30%, and mortality may reach as high as 90% in untreated animals. Affected does become bloated, weak, unsteady, and eventually recumbent. Cows are affected within 24–48 hr before or after parturition. Cows initially are weak and show evidence of muscle tremors, then deteriorate to sternal recumbency, with the head usually tucked to the abdomen, and an inability to stand. Tachycardia, dilated pupils, anorexia, hypothermia, depression, ruminal stasis, bloat, uterine inertia, and loss of anal tone are also seen at this stage. The terminal stage of disease is a rapid progression from coma to death. Heart rates will be high, but pulse may not be detectable. Hypocalcemia is diagnosed based on the pregnancy stage of the female and on clinical signs. It is later confirmed by laboratory findings of low serum calcium. With hypocalcemia in ewes, the plasma concentrations of calcium drop from normal values of 8–12 mg/dl to values of 3–6 mg/dl. In cattle, plasma levels below 7.5 mg/dl are hypocalcemic; at the terminal stages levels may be 2 mg/dl. Epizootiology. Hypocalcemia occurs primarily in overweight ewes during the last 6 weeks of pregnancy or during the first few weeks of lactation. The disease is not as common in the dairy goat as in the dairy cow. High-producing, older, multiparous dairy cows are the most susceptible, and the Jersey breed is considered susceptible. Cows that have survived one episode are prone to recurrence. In addition, dry cows must be managed carefully regarding limiting dietary calcium. The disease is not common in beef cattle unless there is an overall poor nutrition program. Necropsy findings. There is no pathognomonic or typical finding at necropsy. Pathogenesis. During the periparturient period, calcium requirements for fetal skeletal growth exceed calcium absorbed from the diet and from bone metabolism. Additionally, dietary calcium intake is thought to be compromised because, in advanced pregnancy, animals may not be able to eat enough to sustain adequate nutrient levels, and intestinal absorption capabilities do not respond as quickly as needed. After parturition, calcium needs increase dramatically because of calcium levels in colostrum and milk. Recent information suggests that legume and grass forages, high in potassium and low in magnesium, create a slight physiological alkalosis (at least in cattle), which antagonizes normal calcium regulation ( Rings et al., 1997 ). Thus, bone resorption, renal resorption, and gastrointestinal absorption of calcium are less than maximal. Prevention and control. Maintaining appropriate nutrition during the last trimester is helpful in preventing the disease. In cows and does, for example, limiting calcium intake by removing alfalfa from the diet is helpful. Treatment. Hypocalcemia must be treated quickly based on clinical signs; pretreatment blood samples can be saved for later confirmation. Twenty percent calcium borogluconate solution should be administered by slow intravenous infusion. Response will often be rapid, with the resolution of the animal's dull mentation. Less severely affected animals will often try to stand in a short time. Relapses are common, however, in sheep and cattle. Hypermagnesemia and hypophosphatemia often coincide with hypocalcemia. These imbalances should be considered when animals appear to be unresponsive to treatment. Hypocalcemia in the goat can be treated with 50–100 ml of calcium borogluconate. Heart rate should be monitored closely throughout calcium administration. If an irregular or rapid heart rate is detected, then calcium treatment should be slowed or discontinued. Calcium gels and boluses are also available for treatment ( Rings et al., 1997 ). Prognosis is generally good if the animal is treated early in the disease, but the prognosis will often be poor when treatment is initiated in later stages of the disease. Etiology. Hypocalcemia is an acute metabolic disease of ruminants that requires emergency treatment; the presentation is slightly different in ewes, does, and cows. Clinical signs and diagnosis. In sheep, the disease is seen in ewes during the last 6 weeks of pregnancy and is characterized by muscle tetany, incoordination, paralysis, and finally coma. As calcium levels drop, ewes begin to show early signs such as stiffness and incoordination of movements, especially in the hindlimbs. Later, muscular tremors, muscular weakness, and recumbency will ensue. Animals will frequently be found breathing rapidly despite a normal body temperature. Morbidity may approach 30%, and mortality may reach as high as 90% in untreated animals. Affected does become bloated, weak, unsteady, and eventually recumbent. Cows are affected within 24–48 hr before or after parturition. Cows initially are weak and show evidence of muscle tremors, then deteriorate to sternal recumbency, with the head usually tucked to the abdomen, and an inability to stand. Tachycardia, dilated pupils, anorexia, hypothermia, depression, ruminal stasis, bloat, uterine inertia, and loss of anal tone are also seen at this stage. The terminal stage of disease is a rapid progression from coma to death. Heart rates will be high, but pulse may not be detectable. Hypocalcemia is diagnosed based on the pregnancy stage of the female and on clinical signs. It is later confirmed by laboratory findings of low serum calcium. With hypocalcemia in ewes, the plasma concentrations of calcium drop from normal values of 8–12 mg/dl to values of 3–6 mg/dl. In cattle, plasma levels below 7.5 mg/dl are hypocalcemic; at the terminal stages levels may be 2 mg/dl. Epizootiology. Hypocalcemia occurs primarily in overweight ewes during the last 6 weeks of pregnancy or during the first few weeks of lactation. The disease is not as common in the dairy goat as in the dairy cow. High-producing, older, multiparous dairy cows are the most susceptible, and the Jersey breed is considered susceptible. Cows that have survived one episode are prone to recurrence. In addition, dry cows must be managed carefully regarding limiting dietary calcium. The disease is not common in beef cattle unless there is an overall poor nutrition program. Necropsy findings. There is no pathognomonic or typical finding at necropsy. Pathogenesis. During the periparturient period, calcium requirements for fetal skeletal growth exceed calcium absorbed from the diet and from bone metabolism. Additionally, dietary calcium intake is thought to be compromised because, in advanced pregnancy, animals may not be able to eat enough to sustain adequate nutrient levels, and intestinal absorption capabilities do not respond as quickly as needed. After parturition, calcium needs increase dramatically because of calcium levels in colostrum and milk. Recent information suggests that legume and grass forages, high in potassium and low in magnesium, create a slight physiological alkalosis (at least in cattle), which antagonizes normal calcium regulation ( Rings et al., 1997 ). Thus, bone resorption, renal resorption, and gastrointestinal absorption of calcium are less than maximal. Prevention and control. Maintaining appropriate nutrition during the last trimester is helpful in preventing the disease. In cows and does, for example, limiting calcium intake by removing alfalfa from the diet is helpful. Treatment. Hypocalcemia must be treated quickly based on clinical signs; pretreatment blood samples can be saved for later confirmation. Twenty percent calcium borogluconate solution should be administered by slow intravenous infusion. Response will often be rapid, with the resolution of the animal's dull mentation. Less severely affected animals will often try to stand in a short time. Relapses are common, however, in sheep and cattle. Hypermagnesemia and hypophosphatemia often coincide with hypocalcemia. These imbalances should be considered when animals appear to be unresponsive to treatment. Hypocalcemia in the goat can be treated with 50–100 ml of calcium borogluconate. Heart rate should be monitored closely throughout calcium administration. If an irregular or rapid heart rate is detected, then calcium treatment should be slowed or discontinued. Calcium gels and boluses are also available for treatment ( Rings et al., 1997 ). Prognosis is generally good if the animal is treated early in the disease, but the prognosis will often be poor when treatment is initiated in later stages of the disease. Etiology. Hypocalcemia is an acute metabolic disease of ruminants that requires emergency treatment; the presentation is slightly different in ewes, does, and cows. Clinical signs and diagnosis. In sheep, the disease is seen in ewes during the last 6 weeks of pregnancy and is characterized by muscle tetany, incoordination, paralysis, and finally coma. As calcium levels drop, ewes begin to show early signs such as stiffness and incoordination of movements, especially in the hindlimbs. Later, muscular tremors, muscular weakness, and recumbency will ensue. Animals will frequently be found breathing rapidly despite a normal body temperature. Morbidity may approach 30%, and mortality may reach as high as 90% in untreated animals. Affected does become bloated, weak, unsteady, and eventually recumbent. Cows are affected within 24–48 hr before or after parturition. Cows initially are weak and show evidence of muscle tremors, then deteriorate to sternal recumbency, with the head usually tucked to the abdomen, and an inability to stand. Tachycardia, dilated pupils, anorexia, hypothermia, depression, ruminal stasis, bloat, uterine inertia, and loss of anal tone are also seen at this stage. The terminal stage of disease is a rapid progression from coma to death. Heart rates will be high, but pulse may not be detectable. Hypocalcemia is diagnosed based on the pregnancy stage of the female and on clinical signs. It is later confirmed by laboratory findings of low serum calcium. With hypocalcemia in ewes, the plasma concentrations of calcium drop from normal values of 8–12 mg/dl to values of 3–6 mg/dl. In cattle, plasma levels below 7.5 mg/dl are hypocalcemic; at the terminal stages levels may be 2 mg/dl. Epizootiology. Hypocalcemia occurs primarily in overweight ewes during the last 6 weeks of pregnancy or during the first few weeks of lactation. The disease is not as common in the dairy goat as in the dairy cow. High-producing, older, multiparous dairy cows are the most susceptible, and the Jersey breed is considered susceptible. Cows that have survived one episode are prone to recurrence. In addition, dry cows must be managed carefully regarding limiting dietary calcium. The disease is not common in beef cattle unless there is an overall poor nutrition program. Necropsy findings. There is no pathognomonic or typical finding at necropsy. Pathogenesis. During the periparturient period, calcium requirements for fetal skeletal growth exceed calcium absorbed from the diet and from bone metabolism. Additionally, dietary calcium intake is thought to be compromised because, in advanced pregnancy, animals may not be able to eat enough to sustain adequate nutrient levels, and intestinal absorption capabilities do not respond as quickly as needed. After parturition, calcium needs increase dramatically because of calcium levels in colostrum and milk. Recent information suggests that legume and grass forages, high in potassium and low in magnesium, create a slight physiological alkalosis (at least in cattle), which antagonizes normal calcium regulation ( Rings et al., 1997 ). Thus, bone resorption, renal resorption, and gastrointestinal absorption of calcium are less than maximal. Prevention and control. Maintaining appropriate nutrition during the last trimester is helpful in preventing the disease. In cows and does, for example, limiting calcium intake by removing alfalfa from the diet is helpful. Treatment. Hypocalcemia must be treated quickly based on clinical signs; pretreatment blood samples can be saved for later confirmation. Twenty percent calcium borogluconate solution should be administered by slow intravenous infusion. Response will often be rapid, with the resolution of the animal's dull mentation. Less severely affected animals will often try to stand in a short time. Relapses are common, however, in sheep and cattle. Hypermagnesemia and hypophosphatemia often coincide with hypocalcemia. These imbalances should be considered when animals appear to be unresponsive to treatment. Hypocalcemia in the goat can be treated with 50–100 ml of calcium borogluconate. Heart rate should be monitored closely throughout calcium administration. If an irregular or rapid heart rate is detected, then calcium treatment should be slowed or discontinued. Calcium gels and boluses are also available for treatment ( Rings et al., 1997 ). Prognosis is generally good if the animal is treated early in the disease, but the prognosis will often be poor when treatment is initiated in later stages of the disease. g. Urinary Calculi (Obstructive Urolithiasis, Water Belly) Etiology. Urolithiasis is a metabolic disease of intact and castrated male sheep, goats, and cattle that is characterized by the formation of bladder and urethral crystals, urethral blockage, and anuria (Murray, 1985). The disease occurs rarely in female ruminants. Clinical signs and diagnosis. Affected animals will vocalize and begin to show signs of uneasiness, such as treading, straining postures, arched backs, raised tails, and squatting while attempting to urinate. These postures may be mistaken for tenesmus. Male cattle may develop swelling along the ventral perineal area. Affected animals will not stay with the herd or flock. Small amounts of urine may be discharged, and crystal deposits may be visible attached to the preputial hairs. Additionally, in smaller ruminants, the filiform urethral appendage (pizzle) often becomes dark purple to black in color. The pulsing pelvic urethra may be detected by manual or digital rectal palpation, and bladder distention may be noticeable in cattle by the same means. As the disease progresses to complete urethral blockage, the animal will become anorexic and show signs of abdominal pain, such as kicking at the belly. The abdomen will swell as the bladder enlarges, and rupture can occur within 36 hr after development of clinical signs. Bladder or urethral rupture may cause a short-lived period of apparent pain relief; subsequent development of uremia will eventually lead to death. The disease may progress over a period of 1–2 weeks, and the mortality is high unless the blockages are reversed. Diagnosis is made by the typical clinical signs. Abdominal taps may yield urine. Calculi are usually composed of calcium phosphate or ammonium phosphate matrices. Epizootiology and transmission. Clinical disease is usually seen in growing intact or castrated males. The disease may be sporadic or there may be clusters of cases in the flock or herd. Necropsy findings. Necropsy findings include urine in the abdomen with or without bladder or urethral rupture. Renal hydronephrosis may be evident. Calculi or struvite crystal sediment will be observed in the bladder and urethra. Histologically, trauma to the urethra and ureters will be present. Pathogenesis. Urolithiasis is multifactorial and involves dietary, anatomical, hormonal, and environmental factors. Male sheep and goats have a urethral process that predisposes them to entrapment of calculi. In cattle, the urethra narrows at the sigmoid flexure, and calculi lodge there most frequently. Additionally, the removal of testosterone by early castration is thought to result in hypoplasia of the urethra and penis. This physical reduction in the size of the excretory tube may predispose to the precipitation of and blockage by the struvite minerals. Grains fed to growing animals tend to be high in phosphorus and magnesium content. These calculogenic diets lead to the formation of struvite (magnesium ammonium phosphate) crystals. Other minerals associated with urolithiasis include silica (range grasses), carbonates (some grasses and clover pastures), calcium (exclusively alfalfa hay), and oxalates (fescue grasses). Differential diagnosis. Grain engorgement colic, gastrointestinal blockage, and causes of tenemus, such as enteritis or trauma, are differentials. Trauma to the urethral process should be considered. Urinary tract infections are uncommon in ruminants. Prevention and control. One case often is indicative of a potential problem in the group. Urolithiasis can be minimized by monitoring the calcium:phosphorus ratio in the diet. The normal ratio should be 2:1. Additionally, increasing the amount of dietary roughage will help balance the mineral intake. Increasing the amount of salt (sodium chloride, 2–4%) in the diet to increase water consumption, or adding ammonium chloride to the diet, at 10 gm/head/day or 2% of the ration, to acidify the urine, will aid in the prevention of this disease. Palatability of and accessibility to water should be assessed as well as functioning of automatic watering equipment. Treatment. Treatment is primarily surgical (Van Metre et al. 1996). Initially, amputation of the filiform urethral appendage may alleviate the disease since urethral blockage often begins here. As the disease progresses, urethral blockage in the sigmoid flexure as well as throughout the urethra may occur. In more advanced stages, perineal urethrostomy may yield good results. The prognosis is poor when the condition becomes chronic, reoccurs, or surgery is required. Research complications. Young castrated and intact male ruminants used in the laboratory setting will be the susceptible age group for this disorder. Etiology. Urolithiasis is a metabolic disease of intact and castrated male sheep, goats, and cattle that is characterized by the formation of bladder and urethral crystals, urethral blockage, and anuria (Murray, 1985). The disease occurs rarely in female ruminants. Clinical signs and diagnosis. Affected animals will vocalize and begin to show signs of uneasiness, such as treading, straining postures, arched backs, raised tails, and squatting while attempting to urinate. These postures may be mistaken for tenesmus. Male cattle may develop swelling along the ventral perineal area. Affected animals will not stay with the herd or flock. Small amounts of urine may be discharged, and crystal deposits may be visible attached to the preputial hairs. Additionally, in smaller ruminants, the filiform urethral appendage (pizzle) often becomes dark purple to black in color. The pulsing pelvic urethra may be detected by manual or digital rectal palpation, and bladder distention may be noticeable in cattle by the same means. As the disease progresses to complete urethral blockage, the animal will become anorexic and show signs of abdominal pain, such as kicking at the belly. The abdomen will swell as the bladder enlarges, and rupture can occur within 36 hr after development of clinical signs. Bladder or urethral rupture may cause a short-lived period of apparent pain relief; subsequent development of uremia will eventually lead to death. The disease may progress over a period of 1–2 weeks, and the mortality is high unless the blockages are reversed. Diagnosis is made by the typical clinical signs. Abdominal taps may yield urine. Calculi are usually composed of calcium phosphate or ammonium phosphate matrices. Epizootiology and transmission. Clinical disease is usually seen in growing intact or castrated males. The disease may be sporadic or there may be clusters of cases in the flock or herd. Necropsy findings. Necropsy findings include urine in the abdomen with or without bladder or urethral rupture. Renal hydronephrosis may be evident. Calculi or struvite crystal sediment will be observed in the bladder and urethra. Histologically, trauma to the urethra and ureters will be present. Pathogenesis. Urolithiasis is multifactorial and involves dietary, anatomical, hormonal, and environmental factors. Male sheep and goats have a urethral process that predisposes them to entrapment of calculi. In cattle, the urethra narrows at the sigmoid flexure, and calculi lodge there most frequently. Additionally, the removal of testosterone by early castration is thought to result in hypoplasia of the urethra and penis. This physical reduction in the size of the excretory tube may predispose to the precipitation of and blockage by the struvite minerals. Grains fed to growing animals tend to be high in phosphorus and magnesium content. These calculogenic diets lead to the formation of struvite (magnesium ammonium phosphate) crystals. Other minerals associated with urolithiasis include silica (range grasses), carbonates (some grasses and clover pastures), calcium (exclusively alfalfa hay), and oxalates (fescue grasses). Differential diagnosis. Grain engorgement colic, gastrointestinal blockage, and causes of tenemus, such as enteritis or trauma, are differentials. Trauma to the urethral process should be considered. Urinary tract infections are uncommon in ruminants. Prevention and control. One case often is indicative of a potential problem in the group. Urolithiasis can be minimized by monitoring the calcium:phosphorus ratio in the diet. The normal ratio should be 2:1. Additionally, increasing the amount of dietary roughage will help balance the mineral intake. Increasing the amount of salt (sodium chloride, 2–4%) in the diet to increase water consumption, or adding ammonium chloride to the diet, at 10 gm/head/day or 2% of the ration, to acidify the urine, will aid in the prevention of this disease. Palatability of and accessibility to water should be assessed as well as functioning of automatic watering equipment. Treatment. Treatment is primarily surgical (Van Metre et al. 1996). Initially, amputation of the filiform urethral appendage may alleviate the disease since urethral blockage often begins here. As the disease progresses, urethral blockage in the sigmoid flexure as well as throughout the urethra may occur. In more advanced stages, perineal urethrostomy may yield good results. The prognosis is poor when the condition becomes chronic, reoccurs, or surgery is required. Research complications. Young castrated and intact male ruminants used in the laboratory setting will be the susceptible age group for this disorder. Etiology. Urolithiasis is a metabolic disease of intact and castrated male sheep, goats, and cattle that is characterized by the formation of bladder and urethral crystals, urethral blockage, and anuria (Murray, 1985). The disease occurs rarely in female ruminants. Clinical signs and diagnosis. Affected animals will vocalize and begin to show signs of uneasiness, such as treading, straining postures, arched backs, raised tails, and squatting while attempting to urinate. These postures may be mistaken for tenesmus. Male cattle may develop swelling along the ventral perineal area. Affected animals will not stay with the herd or flock. Small amounts of urine may be discharged, and crystal deposits may be visible attached to the preputial hairs. Additionally, in smaller ruminants, the filiform urethral appendage (pizzle) often becomes dark purple to black in color. The pulsing pelvic urethra may be detected by manual or digital rectal palpation, and bladder distention may be noticeable in cattle by the same means. As the disease progresses to complete urethral blockage, the animal will become anorexic and show signs of abdominal pain, such as kicking at the belly. The abdomen will swell as the bladder enlarges, and rupture can occur within 36 hr after development of clinical signs. Bladder or urethral rupture may cause a short-lived period of apparent pain relief; subsequent development of uremia will eventually lead to death. The disease may progress over a period of 1–2 weeks, and the mortality is high unless the blockages are reversed. Diagnosis is made by the typical clinical signs. Abdominal taps may yield urine. Calculi are usually composed of calcium phosphate or ammonium phosphate matrices. Epizootiology and transmission. Clinical disease is usually seen in growing intact or castrated males. The disease may be sporadic or there may be clusters of cases in the flock or herd. Necropsy findings. Necropsy findings include urine in the abdomen with or without bladder or urethral rupture. Renal hydronephrosis may be evident. Calculi or struvite crystal sediment will be observed in the bladder and urethra. Histologically, trauma to the urethra and ureters will be present. Pathogenesis. Urolithiasis is multifactorial and involves dietary, anatomical, hormonal, and environmental factors. Male sheep and goats have a urethral process that predisposes them to entrapment of calculi. In cattle, the urethra narrows at the sigmoid flexure, and calculi lodge there most frequently. Additionally, the removal of testosterone by early castration is thought to result in hypoplasia of the urethra and penis. This physical reduction in the size of the excretory tube may predispose to the precipitation of and blockage by the struvite minerals. Grains fed to growing animals tend to be high in phosphorus and magnesium content. These calculogenic diets lead to the formation of struvite (magnesium ammonium phosphate) crystals. Other minerals associated with urolithiasis include silica (range grasses), carbonates (some grasses and clover pastures), calcium (exclusively alfalfa hay), and oxalates (fescue grasses). Differential diagnosis. Grain engorgement colic, gastrointestinal blockage, and causes of tenemus, such as enteritis or trauma, are differentials. Trauma to the urethral process should be considered. Urinary tract infections are uncommon in ruminants. Prevention and control. One case often is indicative of a potential problem in the group. Urolithiasis can be minimized by monitoring the calcium:phosphorus ratio in the diet. The normal ratio should be 2:1. Additionally, increasing the amount of dietary roughage will help balance the mineral intake. Increasing the amount of salt (sodium chloride, 2–4%) in the diet to increase water consumption, or adding ammonium chloride to the diet, at 10 gm/head/day or 2% of the ration, to acidify the urine, will aid in the prevention of this disease. Palatability of and accessibility to water should be assessed as well as functioning of automatic watering equipment. Treatment. Treatment is primarily surgical (Van Metre et al. 1996). Initially, amputation of the filiform urethral appendage may alleviate the disease since urethral blockage often begins here. As the disease progresses, urethral blockage in the sigmoid flexure as well as throughout the urethra may occur. In more advanced stages, perineal urethrostomy may yield good results. The prognosis is poor when the condition becomes chronic, reoccurs, or surgery is required. Research complications. Young castrated and intact male ruminants used in the laboratory setting will be the susceptible age group for this disorder. h. Rickets Rickets is a disease of young, growing animals but rarely occurs in goats. It is a metabolic disease characterized by a failure of bone matrix mineralization at the epiphysis of long bones due to lack of phosphorus. The condition can occur as an absolute deficiency in vitamin D 2 , an inadequate dietary supply of phosphorus, or a long-term dietary imbalance of calcium and phosphorus. The syndrome must be differentiated from epiphisitis (unequal growth of the epiphyses of long bones in young, rapidly growing kids fed diets with excess calcium). Clinical signs include poor growth, enlarged costochondral junctions, narrow chests, painful joints, and reluctance to move. Spontaneous fractures of long bones may occur. Animals will recover when dietary phosphorus is provided and if joint damage is not severe. 3. Nutritional Diseases a. Copper Deficiency (Enzootic Ataxia, Swayback) Etiology. Chronic copper deficiency in pregnant ewes and does may produce a metabolic disorder in their lambs and kids called enzootic ataxia. In goats, this deficiency also causes swayback in the fetuses. Clinical signs and diagnosis. This disease results in a progressive hindlimb ataxia and apparent blindness in lambs up to about 3 months of age. Additionally, because copper is essential for osteogenesis, hematopoiesis, myelination, and pigmentation of wool and hair, ewes may appear unthrifty, may be anemic, and may have poor, depigmented wool with a decrease in wool crimp. Affected kids are born weak, tremble, and have a characteristic concavity to the spinal cord, leading to the name swayback. When the deficiency occurs later during gestation, demyelination is limited to the spinal cord and brain stem. Kids are born normally but develop a progressive ataxia, leading to paralysis, muscle atrophy, and depressed spinal reflexes with lower motor neuron signs. Diagnosis is based on low copper levels found in feedstuffs and tissues at necropsy. Diagnosis is based on clinical signs, feed analysis, and pathological findings. Epizootiology and transmission. Enzootic ataxia is rarely seen in western states; most North American diets have sufficient copper levels to prevent this disease. Copper antagonists in the feed or forage at sufficient levels, such as molybdenum, sulfate, and cadmium, however, may predispose to copper deficiencies. Pathogenesis. The maternal copper deficiency leads to a disturbance early in the embryonic development of myelination in the central nervous system and the spinal cord. Copper is part of the cytochrome oxidase system and other enzyme complexes and is important in myelination, osteogenesis, hematopoiesis (iron absorption and hemoglobin formation), immune system development, and maintenance and normal growth ( Smith and Sherman, 1994 ). Differential diagnosis. The differential diagnosis for newborns includes β-mannosidosis, hypoglycemia, and hypothermia. For older animals the differential should include caprine arthritis encephalitis (goats), enzootic muscular dystrophy, listeriosis, spinal trauma or abscessation, and cerebrospinal nematodiasis. Prevention and control. Copper deficiency can be prevented by providing balanced nutrition for pregnant animals. Necropsy findings. Gross encephalomalacia has been noted. Histopathologically, white matter of the brain and spinal cord displays gelatinization and cavitation. Extensive nerve demyelination and necrosis are evident. Postmortem lesions include extensive demyelination and neuronal degeneration. Treatment. Because the condition is developmental, supplemental copper may improve clinical signs but not eliminate them. b. Copper Toxicosis Etiology: Acute or chronic copper ingestion or liver injury often causes a severe, acute hemolytic anemia in weanling to adult sheep and in calves and adult dairy cattle. Growing lambs may be the most susceptible. Copper toxicosis is rare in goats. Clinical signs and diagnosis. The clinical course in sheep can be as short as 1–4 days, and mortality may reach 75%. Hemolysis, anemia, hemoglobinuria, and icterus characterize the acute hemolytic crisis, associated with copper released from the overloaded liver. Some clinical signs are related to direct irritation to the gastrointestinal tract mucosa. Weakness, vomiting, abdominal pain, bruxism, diarrhea, respiratory difficulty, and circulatory collapse are followed by recumbency and death. Hepatic biopsy is currently considered the best diagnostic approach; serum or plasma levels of copper and hepatic enzymes such as aspartate aminotransferase (AST) and γ-glutamyltransferase (GGT) may provide some information, but it is generally believed that these will not accurately reflect total copper load or hepatic damage. Epizootiology and transmission. A single toxic dose for sheep and goats is the range of 20–100 mg/kg, and for cattle it is 220–880 mg/kg. Chronic poisoning in sheep may occur when 3.5 mg/kg is ingested. Copper-containing pesticides, soil additives, therapeutics, and improperly formulated feeds may potentially lead to copper toxicity. Phytogenous sources include certain pastures such as subterranean clover. Feed low in molybdenum, zinc, or calcium may lead to increased uptake of copper from properly balanced rations. A common cause of the disease in sheep is feeding concentrates balanced for cattle; cattle feeds and mineral blocks contain much higher quantities of copper than are required for sheep. Chronic ingestion of these feedstuffs leads to copper accumulation and toxicity. Copper toxicosis has been reported in calves given regular oral or parenteral copper supplements, and in adult dairy cattle given copper supplements to compensate for copper-deficient pasture. Pregnant dairy cattle may be more susceptible to copper toxicity. Rare sources of copper ingestion may include copper sulfate footbaths. Necropsy findings. Common findings at necropsy include icterus; a soft, dark, friable, enlarged spleen; an enlarged, yellow-brown friable liver; and "gun-barrel" black kidneys. Hemoglobin-stained urine will be visible in the bladder. Copper accumulations in the liver reaching 1000–3000 ppm are toxic. Pathogenesis. Hemolysis occurs when sufficient amounts of copper are ingested or released suddenly from the liver and is believed to be due direct interaction of the copper with red-cell surface molecules. Stresses such as transportation, lactation, and poor nutrition or exercise may precipitate the hemolysis. Differential diagnosis. Other causes of hemolytic disease include babesiosis, trypanosomiasis, and plant poisonings such as kale. Arsenic ingestion, organophosphate toxicity, and cyanide or nitrate poisoning should also be considered as the source of poisoning. Urethral obstruction and gastrointestinal emergencies should be considered for the abdominal pain. Control and prevention. The disease is prevented by carefully monitoring copper access in sheep and copper supplementation in cattle. Sheep and goats should not be fed feedstuffs formulated for cattle, and dairy calf milk replacer should not be used for lambs and kids. Molybdenum may be administered to animals considered at high risk. Molybdenum-deficient pastures may be treated with molybdenum superphosphate. Herd copper supplementation should be undertaken with the knowledge of existing hepatic copper levels, and existing copper and molybdenum levels, in the feedstuffs. Treatment. Oral treatment for sheep consists of ammonium or sodium molybdenate (50–100 mg/day), and sodium thiosulfate (0.5–1.0 mg/day) for 3 weeks aids in excretion of copper. Oral D-penicillamine daily for 6 days (50 mg/kg) has also been shown to increase copper excretion in sheep. Ammonium molybdenate has been administered intravenously to goats at 1.7 mg/kg for 3 treatments on alternate days. Cattle have been treated orally with sodium molybdenate (3 gm/day) or sodium thiosulfate (5 gm/day). Treatment for anemia and nephrosis may be necessary in severe cases. Research complications. Some breeds of sheep, such as Merino crosses and the British breeds, may be more susceptible to copper toxicosis caused by phytogenous sources. c. Selenium/Vitamin E Deficiency (Nutritional Muscular Dystrophy, Nutritional Myodegeneration, White Muscle Disease, Stiff Lamb Disease) Etiology. White muscle disease, also known as stiff lamb disease, is a nutritional muscular dystrophy caused by a deficiency of selenium or vitamin E. Clinical signs and diagnosis. Clinically two forms of the disease have been identified: cardiac and skeletal. The cardiac form occurs most commonly in neonates. In these, respiratory difficulty will be a manifestation of damage to cardiac, diaphragmatic, and intercostal muscles. Young will be able to nurse when assisted. In slightly older animals, the disease is characterized by locomotor disturbances and/or circulatory failure. Clinically, animals may display paresis, stiffness or inability to stand, rapid but weak pulse, and acute death. Mortality may reach 70% ( Jensen and Swift, 1982 ). Paresis and sudden death in neonates with associated pathological signs are frequently diagnostic. With the skeletal form, affected animals are stiff and reluctant to move, and muscles of affected animals are painful. Young will be reluctant to get up but will readily nurse when assisted. Peracute to acute myocardial degeneration may occur in the cardiac form, and animals may simply be found dead. Serum selenium levels are usually below 50 ppb (normal is 158–160 ppb) ( Nelson, 1983 ). Diagnosis may also include determination of antemortem whole blood levels of selenium and plasma levels of vitamin E. Glutathione peroxidase levels in red blood cells can be measured as an indirect test. Clinical biochemistry findings of significant elevations of aspartate aminotransferase (AST) in creatinine kinase (CK) are also supportive of the diagnosis. Epizootiology and transmission. Selenium deficiency has been associated with formulated diets deficient in selenium, forages grown on selenium-deficient soils in certain geographic regions, and forages such as alfalfa and clover that have an inability to efficiently extract available selenium from the soils. Rumen bacterial reduction of selenium compounds to unavailable elemental selenium may also contribute to the disease. Necropsy findings. Necropsy lesions include petechial hemorrhages and muscle edema. Hallmarks are pale white streaking of affected skeletal and cardiac muscle. These are due to coagulation necrosis. Pale striated muscles of the limb, diaphragm, and tongue are also seen. Pathogenesis. Selenium and vitamin E function together as antioxidants that protect lipid membranes from oxidative destruction. Selenium is a cofactor for glutathione peroxidase, which converts hydrogen peroxide to water and other nontoxic compounds. Lack of one or both results in loss of membrane integrity. Differential diagnosis. In neonatal ruminants presenting with respiratory and cardiac dysfunction, differentials include congenital cardiac anomalies. Differentials generally for weak neonates or sudden or peracute neonatal deaths should include septicemia, pneumonia, toxicity, diarrhea, and dehydration. Prevention and control. Awareness of regional selenium deficiencies is important. Control involves providing goodquality roughage, vitamin E and selenium supplementation, and parenteral injections prior to parturition and weaning. Treatment. Affected animals may be treated by administering vitamin E or selenium injections. Administering vitamin E or selenium to ewes in late pregnancy can prevent white muscle disease ( Kott et al., 1998 ). The label dose for selenium is 2.5–3 mg/45 kg of body weight. Combination products are available and can be used in goats at the sheep dose ( Smith and Sherman, 1994 ). Proper mineral balance in the diet is critical. d. Selenium Toxicity Selenium toxicity occurs most frequently as the result of excessive dosing to prevent or correct selenium deficiency or as the result of ingestion of selenium-converting plants. The main preventive measure for the former is the use of the appropriate product for the species. Secondarily, the concentration of the available product should be double-checked. In the United States, ruminants in the Midwest and western areas may be subject to selenium toxicity when pastured in areas containing selenium-converting plants. Signs of overdosing include weakness, dyspnea, bloating, and diarrhea. Shock, paresis, and death may occur. Initial clinical signs of excessive selenium intake from plants are observed in the distal limb, with cracked hoof walls and subsequent infection and irregular hoof growth. e. Thiamin Deficiency (Polioencephalomalacia) Etiology. Polioencephalomalacia (PEM) is a noninfectious, noncontagious disease characterized by neurological signs. Growing and adult ruminants on high-concentrate diets are typically affected. Animals exposed to toxic plants or moldy feed containing thiaminases, feed high in sulfates, or unusually high doses of some medications are also at risk. Clinical signs and diagnosis. An early sign may be mild diarrhea. Acute clinical signs include bruxism, hyperesthesia, involuntary muscle contractions, depression, partial or complete opisthotonus, nystagmus, dorsomedial strabismus, seizures, and death. In subacute cases of the disease, animals may appear to walk aimlessly as if blind or may display head-pressing postures. Hypersalivation may be present, but body temperatures and ocular reflexes are normal. Morbidity and mortality may be high, especially in younger animals. Diagnosis is suggestive from clinical signs and from response to intensive parental thiamine hydrochloride. Epizootiology and transmission. PEM is caused by a thiamin deficiency. The disease tends to be seen more frequently in cattle and sheep feedlots where the concentrates fed are high in fermentable carbohydrates. Pastured animals are also vulnerable if grain is feed. Thiaminase-containing plants, such as bracken fern, are often unpalatable so will less likely be a contributing factor. Recent studies have also indicated that high levels of sulfate in the diet, such as in the fermentable, low-fiber concentrates, may play an important role. Medications such as as amprolium, levamisole, and thiabendazole have thiaminantagonizing activity when given in excessive doses. Necropsy signs. Cerebral lesions characterized by softening and discoloration are grossly observed in the gray matter. Microscopically, neurons will exhibit edema, chromatolysis, and shrinkage. Gliosis and cerebral capillary proliferation may be observed. Pathogenesis. A lack of thiamin results in inappropriate carbohydrate metabolism and accumulation of pyruvate and other intermediaries that lead to cerebral edema and neuronal degeneration. Differential diagnosis. Several important differentials include acute lead poisoning, nitrofuran toxicity, hypomagnesemia, vitamin A deficiency, listeriosis, pregnancy toxemia, infectious thromboembolic meningoencephalitis, and type D clostridial enterotoxemia. Prevention and control. The disease can be prevented by monitoring the diet and by providing adequate roughage necessary to prevent overgrowth of thiaminase-producing ruminal flora and to maximize ruminal production of B vitamins. If excess sulfur is the primary factor, immediate removal of the source is critical. Treatment. Early aggressive treatment is essential to save animals. The disease is treated by frequent parenteral administration of thiamine hydrochloride, the first dose being administered intravenously. Dexamethasone, B vitamins, and diazepam may also be required. Treatment is less successful when sulfur plays a prominent role in the etiology. Research complications. This disease is preventable. Although the disease is less likely to occur in smaller groups of confined ruminants, the risks of feeding concentrates or moldy feed, for example, with minimal good-quality roughage, should be kept in mind. f. Vitamin D Toxicity Vitamin D toxicity can result either from iatrogenic overadministration or ingestion of the plant Trisetum flavescens. Serum calcium levels may be high enough that blood in EDTA tubes will clot. g. Nutritional Deficiencies In goats, nutritional deficiencies often manifest as a generalized poor coat that is dry, scaly, thin, and erectile. Zinc-responsive dermatitis has been reported in goats ( Smith and Sherman, 1994 ). Vitamin A deficiencies associated with hyperkeratosis have been reported, as well as vitamin E-responsive and selenium-responsive dermatitis. a. Copper Deficiency (Enzootic Ataxia, Swayback) Etiology. Chronic copper deficiency in pregnant ewes and does may produce a metabolic disorder in their lambs and kids called enzootic ataxia. In goats, this deficiency also causes swayback in the fetuses. Clinical signs and diagnosis. This disease results in a progressive hindlimb ataxia and apparent blindness in lambs up to about 3 months of age. Additionally, because copper is essential for osteogenesis, hematopoiesis, myelination, and pigmentation of wool and hair, ewes may appear unthrifty, may be anemic, and may have poor, depigmented wool with a decrease in wool crimp. Affected kids are born weak, tremble, and have a characteristic concavity to the spinal cord, leading to the name swayback. When the deficiency occurs later during gestation, demyelination is limited to the spinal cord and brain stem. Kids are born normally but develop a progressive ataxia, leading to paralysis, muscle atrophy, and depressed spinal reflexes with lower motor neuron signs. Diagnosis is based on low copper levels found in feedstuffs and tissues at necropsy. Diagnosis is based on clinical signs, feed analysis, and pathological findings. Epizootiology and transmission. Enzootic ataxia is rarely seen in western states; most North American diets have sufficient copper levels to prevent this disease. Copper antagonists in the feed or forage at sufficient levels, such as molybdenum, sulfate, and cadmium, however, may predispose to copper deficiencies. Pathogenesis. The maternal copper deficiency leads to a disturbance early in the embryonic development of myelination in the central nervous system and the spinal cord. Copper is part of the cytochrome oxidase system and other enzyme complexes and is important in myelination, osteogenesis, hematopoiesis (iron absorption and hemoglobin formation), immune system development, and maintenance and normal growth ( Smith and Sherman, 1994 ). Differential diagnosis. The differential diagnosis for newborns includes β-mannosidosis, hypoglycemia, and hypothermia. For older animals the differential should include caprine arthritis encephalitis (goats), enzootic muscular dystrophy, listeriosis, spinal trauma or abscessation, and cerebrospinal nematodiasis. Prevention and control. Copper deficiency can be prevented by providing balanced nutrition for pregnant animals. Necropsy findings. Gross encephalomalacia has been noted. Histopathologically, white matter of the brain and spinal cord displays gelatinization and cavitation. Extensive nerve demyelination and necrosis are evident. Postmortem lesions include extensive demyelination and neuronal degeneration. Treatment. Because the condition is developmental, supplemental copper may improve clinical signs but not eliminate them. Etiology. Chronic copper deficiency in pregnant ewes and does may produce a metabolic disorder in their lambs and kids called enzootic ataxia. In goats, this deficiency also causes swayback in the fetuses. Clinical signs and diagnosis. This disease results in a progressive hindlimb ataxia and apparent blindness in lambs up to about 3 months of age. Additionally, because copper is essential for osteogenesis, hematopoiesis, myelination, and pigmentation of wool and hair, ewes may appear unthrifty, may be anemic, and may have poor, depigmented wool with a decrease in wool crimp. Affected kids are born weak, tremble, and have a characteristic concavity to the spinal cord, leading to the name swayback. When the deficiency occurs later during gestation, demyelination is limited to the spinal cord and brain stem. Kids are born normally but develop a progressive ataxia, leading to paralysis, muscle atrophy, and depressed spinal reflexes with lower motor neuron signs. Diagnosis is based on low copper levels found in feedstuffs and tissues at necropsy. Diagnosis is based on clinical signs, feed analysis, and pathological findings. Epizootiology and transmission. Enzootic ataxia is rarely seen in western states; most North American diets have sufficient copper levels to prevent this disease. Copper antagonists in the feed or forage at sufficient levels, such as molybdenum, sulfate, and cadmium, however, may predispose to copper deficiencies. Pathogenesis. The maternal copper deficiency leads to a disturbance early in the embryonic development of myelination in the central nervous system and the spinal cord. Copper is part of the cytochrome oxidase system and other enzyme complexes and is important in myelination, osteogenesis, hematopoiesis (iron absorption and hemoglobin formation), immune system development, and maintenance and normal growth ( Smith and Sherman, 1994 ). Differential diagnosis. The differential diagnosis for newborns includes β-mannosidosis, hypoglycemia, and hypothermia. For older animals the differential should include caprine arthritis encephalitis (goats), enzootic muscular dystrophy, listeriosis, spinal trauma or abscessation, and cerebrospinal nematodiasis. Prevention and control. Copper deficiency can be prevented by providing balanced nutrition for pregnant animals. Necropsy findings. Gross encephalomalacia has been noted. Histopathologically, white matter of the brain and spinal cord displays gelatinization and cavitation. Extensive nerve demyelination and necrosis are evident. Postmortem lesions include extensive demyelination and neuronal degeneration. Treatment. Because the condition is developmental, supplemental copper may improve clinical signs but not eliminate them. Etiology. Chronic copper deficiency in pregnant ewes and does may produce a metabolic disorder in their lambs and kids called enzootic ataxia. In goats, this deficiency also causes swayback in the fetuses. Clinical signs and diagnosis. This disease results in a progressive hindlimb ataxia and apparent blindness in lambs up to about 3 months of age. Additionally, because copper is essential for osteogenesis, hematopoiesis, myelination, and pigmentation of wool and hair, ewes may appear unthrifty, may be anemic, and may have poor, depigmented wool with a decrease in wool crimp. Affected kids are born weak, tremble, and have a characteristic concavity to the spinal cord, leading to the name swayback. When the deficiency occurs later during gestation, demyelination is limited to the spinal cord and brain stem. Kids are born normally but develop a progressive ataxia, leading to paralysis, muscle atrophy, and depressed spinal reflexes with lower motor neuron signs. Diagnosis is based on low copper levels found in feedstuffs and tissues at necropsy. Diagnosis is based on clinical signs, feed analysis, and pathological findings. Epizootiology and transmission. Enzootic ataxia is rarely seen in western states; most North American diets have sufficient copper levels to prevent this disease. Copper antagonists in the feed or forage at sufficient levels, such as molybdenum, sulfate, and cadmium, however, may predispose to copper deficiencies. Pathogenesis. The maternal copper deficiency leads to a disturbance early in the embryonic development of myelination in the central nervous system and the spinal cord. Copper is part of the cytochrome oxidase system and other enzyme complexes and is important in myelination, osteogenesis, hematopoiesis (iron absorption and hemoglobin formation), immune system development, and maintenance and normal growth ( Smith and Sherman, 1994 ). Differential diagnosis. The differential diagnosis for newborns includes β-mannosidosis, hypoglycemia, and hypothermia. For older animals the differential should include caprine arthritis encephalitis (goats), enzootic muscular dystrophy, listeriosis, spinal trauma or abscessation, and cerebrospinal nematodiasis. Prevention and control. Copper deficiency can be prevented by providing balanced nutrition for pregnant animals. Necropsy findings. Gross encephalomalacia has been noted. Histopathologically, white matter of the brain and spinal cord displays gelatinization and cavitation. Extensive nerve demyelination and necrosis are evident. Postmortem lesions include extensive demyelination and neuronal degeneration. Treatment. Because the condition is developmental, supplemental copper may improve clinical signs but not eliminate them. b. Copper Toxicosis Etiology: Acute or chronic copper ingestion or liver injury often causes a severe, acute hemolytic anemia in weanling to adult sheep and in calves and adult dairy cattle. Growing lambs may be the most susceptible. Copper toxicosis is rare in goats. Clinical signs and diagnosis. The clinical course in sheep can be as short as 1–4 days, and mortality may reach 75%. Hemolysis, anemia, hemoglobinuria, and icterus characterize the acute hemolytic crisis, associated with copper released from the overloaded liver. Some clinical signs are related to direct irritation to the gastrointestinal tract mucosa. Weakness, vomiting, abdominal pain, bruxism, diarrhea, respiratory difficulty, and circulatory collapse are followed by recumbency and death. Hepatic biopsy is currently considered the best diagnostic approach; serum or plasma levels of copper and hepatic enzymes such as aspartate aminotransferase (AST) and γ-glutamyltransferase (GGT) may provide some information, but it is generally believed that these will not accurately reflect total copper load or hepatic damage. Epizootiology and transmission. A single toxic dose for sheep and goats is the range of 20–100 mg/kg, and for cattle it is 220–880 mg/kg. Chronic poisoning in sheep may occur when 3.5 mg/kg is ingested. Copper-containing pesticides, soil additives, therapeutics, and improperly formulated feeds may potentially lead to copper toxicity. Phytogenous sources include certain pastures such as subterranean clover. Feed low in molybdenum, zinc, or calcium may lead to increased uptake of copper from properly balanced rations. A common cause of the disease in sheep is feeding concentrates balanced for cattle; cattle feeds and mineral blocks contain much higher quantities of copper than are required for sheep. Chronic ingestion of these feedstuffs leads to copper accumulation and toxicity. Copper toxicosis has been reported in calves given regular oral or parenteral copper supplements, and in adult dairy cattle given copper supplements to compensate for copper-deficient pasture. Pregnant dairy cattle may be more susceptible to copper toxicity. Rare sources of copper ingestion may include copper sulfate footbaths. Necropsy findings. Common findings at necropsy include icterus; a soft, dark, friable, enlarged spleen; an enlarged, yellow-brown friable liver; and "gun-barrel" black kidneys. Hemoglobin-stained urine will be visible in the bladder. Copper accumulations in the liver reaching 1000–3000 ppm are toxic. Pathogenesis. Hemolysis occurs when sufficient amounts of copper are ingested or released suddenly from the liver and is believed to be due direct interaction of the copper with red-cell surface molecules. Stresses such as transportation, lactation, and poor nutrition or exercise may precipitate the hemolysis. Differential diagnosis. Other causes of hemolytic disease include babesiosis, trypanosomiasis, and plant poisonings such as kale. Arsenic ingestion, organophosphate toxicity, and cyanide or nitrate poisoning should also be considered as the source of poisoning. Urethral obstruction and gastrointestinal emergencies should be considered for the abdominal pain. Control and prevention. The disease is prevented by carefully monitoring copper access in sheep and copper supplementation in cattle. Sheep and goats should not be fed feedstuffs formulated for cattle, and dairy calf milk replacer should not be used for lambs and kids. Molybdenum may be administered to animals considered at high risk. Molybdenum-deficient pastures may be treated with molybdenum superphosphate. Herd copper supplementation should be undertaken with the knowledge of existing hepatic copper levels, and existing copper and molybdenum levels, in the feedstuffs. Treatment. Oral treatment for sheep consists of ammonium or sodium molybdenate (50–100 mg/day), and sodium thiosulfate (0.5–1.0 mg/day) for 3 weeks aids in excretion of copper. Oral D-penicillamine daily for 6 days (50 mg/kg) has also been shown to increase copper excretion in sheep. Ammonium molybdenate has been administered intravenously to goats at 1.7 mg/kg for 3 treatments on alternate days. Cattle have been treated orally with sodium molybdenate (3 gm/day) or sodium thiosulfate (5 gm/day). Treatment for anemia and nephrosis may be necessary in severe cases. Research complications. Some breeds of sheep, such as Merino crosses and the British breeds, may be more susceptible to copper toxicosis caused by phytogenous sources. Clinical signs and diagnosis. The clinical course in sheep can be as short as 1–4 days, and mortality may reach 75%. Hemolysis, anemia, hemoglobinuria, and icterus characterize the acute hemolytic crisis, associated with copper released from the overloaded liver. Some clinical signs are related to direct irritation to the gastrointestinal tract mucosa. Weakness, vomiting, abdominal pain, bruxism, diarrhea, respiratory difficulty, and circulatory collapse are followed by recumbency and death. Hepatic biopsy is currently considered the best diagnostic approach; serum or plasma levels of copper and hepatic enzymes such as aspartate aminotransferase (AST) and γ-glutamyltransferase (GGT) may provide some information, but it is generally believed that these will not accurately reflect total copper load or hepatic damage. Epizootiology and transmission. A single toxic dose for sheep and goats is the range of 20–100 mg/kg, and for cattle it is 220–880 mg/kg. Chronic poisoning in sheep may occur when 3.5 mg/kg is ingested. Copper-containing pesticides, soil additives, therapeutics, and improperly formulated feeds may potentially lead to copper toxicity. Phytogenous sources include certain pastures such as subterranean clover. Feed low in molybdenum, zinc, or calcium may lead to increased uptake of copper from properly balanced rations. A common cause of the disease in sheep is feeding concentrates balanced for cattle; cattle feeds and mineral blocks contain much higher quantities of copper than are required for sheep. Chronic ingestion of these feedstuffs leads to copper accumulation and toxicity. Copper toxicosis has been reported in calves given regular oral or parenteral copper supplements, and in adult dairy cattle given copper supplements to compensate for copper-deficient pasture. Pregnant dairy cattle may be more susceptible to copper toxicity. Rare sources of copper ingestion may include copper sulfate footbaths. Necropsy findings. Common findings at necropsy include icterus; a soft, dark, friable, enlarged spleen; an enlarged, yellow-brown friable liver; and "gun-barrel" black kidneys. Hemoglobin-stained urine will be visible in the bladder. Copper accumulations in the liver reaching 1000–3000 ppm are toxic. Pathogenesis. Hemolysis occurs when sufficient amounts of copper are ingested or released suddenly from the liver and is believed to be due direct interaction of the copper with red-cell surface molecules. Stresses such as transportation, lactation, and poor nutrition or exercise may precipitate the hemolysis. Differential diagnosis. Other causes of hemolytic disease include babesiosis, trypanosomiasis, and plant poisonings such as kale. Arsenic ingestion, organophosphate toxicity, and cyanide or nitrate poisoning should also be considered as the source of poisoning. Urethral obstruction and gastrointestinal emergencies should be considered for the abdominal pain. Control and prevention. The disease is prevented by carefully monitoring copper access in sheep and copper supplementation in cattle. Sheep and goats should not be fed feedstuffs formulated for cattle, and dairy calf milk replacer should not be used for lambs and kids. Molybdenum may be administered to animals considered at high risk. Molybdenum-deficient pastures may be treated with molybdenum superphosphate. Herd copper supplementation should be undertaken with the knowledge of existing hepatic copper levels, and existing copper and molybdenum levels, in the feedstuffs. Treatment. Oral treatment for sheep consists of ammonium or sodium molybdenate (50–100 mg/day), and sodium thiosulfate (0.5–1.0 mg/day) for 3 weeks aids in excretion of copper. Oral D-penicillamine daily for 6 days (50 mg/kg) has also been shown to increase copper excretion in sheep. Ammonium molybdenate has been administered intravenously to goats at 1.7 mg/kg for 3 treatments on alternate days. Cattle have been treated orally with sodium molybdenate (3 gm/day) or sodium thiosulfate (5 gm/day). Treatment for anemia and nephrosis may be necessary in severe cases. Research complications. Some breeds of sheep, such as Merino crosses and the British breeds, may be more susceptible to copper toxicosis caused by phytogenous sources. Clinical signs and diagnosis. The clinical course in sheep can be as short as 1–4 days, and mortality may reach 75%. Hemolysis, anemia, hemoglobinuria, and icterus characterize the acute hemolytic crisis, associated with copper released from the overloaded liver. Some clinical signs are related to direct irritation to the gastrointestinal tract mucosa. Weakness, vomiting, abdominal pain, bruxism, diarrhea, respiratory difficulty, and circulatory collapse are followed by recumbency and death. Hepatic biopsy is currently considered the best diagnostic approach; serum or plasma levels of copper and hepatic enzymes such as aspartate aminotransferase (AST) and γ-glutamyltransferase (GGT) may provide some information, but it is generally believed that these will not accurately reflect total copper load or hepatic damage. Epizootiology and transmission. A single toxic dose for sheep and goats is the range of 20–100 mg/kg, and for cattle it is 220–880 mg/kg. Chronic poisoning in sheep may occur when 3.5 mg/kg is ingested. Copper-containing pesticides, soil additives, therapeutics, and improperly formulated feeds may potentially lead to copper toxicity. Phytogenous sources include certain pastures such as subterranean clover. Feed low in molybdenum, zinc, or calcium may lead to increased uptake of copper from properly balanced rations. A common cause of the disease in sheep is feeding concentrates balanced for cattle; cattle feeds and mineral blocks contain much higher quantities of copper than are required for sheep. Chronic ingestion of these feedstuffs leads to copper accumulation and toxicity. Copper toxicosis has been reported in calves given regular oral or parenteral copper supplements, and in adult dairy cattle given copper supplements to compensate for copper-deficient pasture. Pregnant dairy cattle may be more susceptible to copper toxicity. Rare sources of copper ingestion may include copper sulfate footbaths. Necropsy findings. Common findings at necropsy include icterus; a soft, dark, friable, enlarged spleen; an enlarged, yellow-brown friable liver; and "gun-barrel" black kidneys. Hemoglobin-stained urine will be visible in the bladder. Copper accumulations in the liver reaching 1000–3000 ppm are toxic. Pathogenesis. Hemolysis occurs when sufficient amounts of copper are ingested or released suddenly from the liver and is believed to be due direct interaction of the copper with red-cell surface molecules. Stresses such as transportation, lactation, and poor nutrition or exercise may precipitate the hemolysis. Differential diagnosis. Other causes of hemolytic disease include babesiosis, trypanosomiasis, and plant poisonings such as kale. Arsenic ingestion, organophosphate toxicity, and cyanide or nitrate poisoning should also be considered as the source of poisoning. Urethral obstruction and gastrointestinal emergencies should be considered for the abdominal pain. Control and prevention. The disease is prevented by carefully monitoring copper access in sheep and copper supplementation in cattle. Sheep and goats should not be fed feedstuffs formulated for cattle, and dairy calf milk replacer should not be used for lambs and kids. Molybdenum may be administered to animals considered at high risk. Molybdenum-deficient pastures may be treated with molybdenum superphosphate. Herd copper supplementation should be undertaken with the knowledge of existing hepatic copper levels, and existing copper and molybdenum levels, in the feedstuffs. Treatment. Oral treatment for sheep consists of ammonium or sodium molybdenate (50–100 mg/day), and sodium thiosulfate (0.5–1.0 mg/day) for 3 weeks aids in excretion of copper. Oral D-penicillamine daily for 6 days (50 mg/kg) has also been shown to increase copper excretion in sheep. Ammonium molybdenate has been administered intravenously to goats at 1.7 mg/kg for 3 treatments on alternate days. Cattle have been treated orally with sodium molybdenate (3 gm/day) or sodium thiosulfate (5 gm/day). Treatment for anemia and nephrosis may be necessary in severe cases. Research complications. Some breeds of sheep, such as Merino crosses and the British breeds, may be more susceptible to copper toxicosis caused by phytogenous sources. c. Selenium/Vitamin E Deficiency (Nutritional Muscular Dystrophy, Nutritional Myodegeneration, White Muscle Disease, Stiff Lamb Disease) Etiology. White muscle disease, also known as stiff lamb disease, is a nutritional muscular dystrophy caused by a deficiency of selenium or vitamin E. Clinical signs and diagnosis. Clinically two forms of the disease have been identified: cardiac and skeletal. The cardiac form occurs most commonly in neonates. In these, respiratory difficulty will be a manifestation of damage to cardiac, diaphragmatic, and intercostal muscles. Young will be able to nurse when assisted. In slightly older animals, the disease is characterized by locomotor disturbances and/or circulatory failure. Clinically, animals may display paresis, stiffness or inability to stand, rapid but weak pulse, and acute death. Mortality may reach 70% ( Jensen and Swift, 1982 ). Paresis and sudden death in neonates with associated pathological signs are frequently diagnostic. With the skeletal form, affected animals are stiff and reluctant to move, and muscles of affected animals are painful. Young will be reluctant to get up but will readily nurse when assisted. Peracute to acute myocardial degeneration may occur in the cardiac form, and animals may simply be found dead. Serum selenium levels are usually below 50 ppb (normal is 158–160 ppb) ( Nelson, 1983 ). Diagnosis may also include determination of antemortem whole blood levels of selenium and plasma levels of vitamin E. Glutathione peroxidase levels in red blood cells can be measured as an indirect test. Clinical biochemistry findings of significant elevations of aspartate aminotransferase (AST) in creatinine kinase (CK) are also supportive of the diagnosis. Epizootiology and transmission. Selenium deficiency has been associated with formulated diets deficient in selenium, forages grown on selenium-deficient soils in certain geographic regions, and forages such as alfalfa and clover that have an inability to efficiently extract available selenium from the soils. Rumen bacterial reduction of selenium compounds to unavailable elemental selenium may also contribute to the disease. Necropsy findings. Necropsy lesions include petechial hemorrhages and muscle edema. Hallmarks are pale white streaking of affected skeletal and cardiac muscle. These are due to coagulation necrosis. Pale striated muscles of the limb, diaphragm, and tongue are also seen. Pathogenesis. Selenium and vitamin E function together as antioxidants that protect lipid membranes from oxidative destruction. Selenium is a cofactor for glutathione peroxidase, which converts hydrogen peroxide to water and other nontoxic compounds. Lack of one or both results in loss of membrane integrity. Differential diagnosis. In neonatal ruminants presenting with respiratory and cardiac dysfunction, differentials include congenital cardiac anomalies. Differentials generally for weak neonates or sudden or peracute neonatal deaths should include septicemia, pneumonia, toxicity, diarrhea, and dehydration. Prevention and control. Awareness of regional selenium deficiencies is important. Control involves providing goodquality roughage, vitamin E and selenium supplementation, and parenteral injections prior to parturition and weaning. Treatment. Affected animals may be treated by administering vitamin E or selenium injections. Administering vitamin E or selenium to ewes in late pregnancy can prevent white muscle disease ( Kott et al., 1998 ). The label dose for selenium is 2.5–3 mg/45 kg of body weight. Combination products are available and can be used in goats at the sheep dose ( Smith and Sherman, 1994 ). Proper mineral balance in the diet is critical. Etiology. White muscle disease, also known as stiff lamb disease, is a nutritional muscular dystrophy caused by a deficiency of selenium or vitamin E. Clinical signs and diagnosis. Clinically two forms of the disease have been identified: cardiac and skeletal. The cardiac form occurs most commonly in neonates. In these, respiratory difficulty will be a manifestation of damage to cardiac, diaphragmatic, and intercostal muscles. Young will be able to nurse when assisted. In slightly older animals, the disease is characterized by locomotor disturbances and/or circulatory failure. Clinically, animals may display paresis, stiffness or inability to stand, rapid but weak pulse, and acute death. Mortality may reach 70% ( Jensen and Swift, 1982 ). Paresis and sudden death in neonates with associated pathological signs are frequently diagnostic. With the skeletal form, affected animals are stiff and reluctant to move, and muscles of affected animals are painful. Young will be reluctant to get up but will readily nurse when assisted. Peracute to acute myocardial degeneration may occur in the cardiac form, and animals may simply be found dead. Serum selenium levels are usually below 50 ppb (normal is 158–160 ppb) ( Nelson, 1983 ). Diagnosis may also include determination of antemortem whole blood levels of selenium and plasma levels of vitamin E. Glutathione peroxidase levels in red blood cells can be measured as an indirect test. Clinical biochemistry findings of significant elevations of aspartate aminotransferase (AST) in creatinine kinase (CK) are also supportive of the diagnosis. Epizootiology and transmission. Selenium deficiency has been associated with formulated diets deficient in selenium, forages grown on selenium-deficient soils in certain geographic regions, and forages such as alfalfa and clover that have an inability to efficiently extract available selenium from the soils. Rumen bacterial reduction of selenium compounds to unavailable elemental selenium may also contribute to the disease. Necropsy findings. Necropsy lesions include petechial hemorrhages and muscle edema. Hallmarks are pale white streaking of affected skeletal and cardiac muscle. These are due to coagulation necrosis. Pale striated muscles of the limb, diaphragm, and tongue are also seen. Pathogenesis. Selenium and vitamin E function together as antioxidants that protect lipid membranes from oxidative destruction. Selenium is a cofactor for glutathione peroxidase, which converts hydrogen peroxide to water and other nontoxic compounds. Lack of one or both results in loss of membrane integrity. Differential diagnosis. In neonatal ruminants presenting with respiratory and cardiac dysfunction, differentials include congenital cardiac anomalies. Differentials generally for weak neonates or sudden or peracute neonatal deaths should include septicemia, pneumonia, toxicity, diarrhea, and dehydration. Prevention and control. Awareness of regional selenium deficiencies is important. Control involves providing goodquality roughage, vitamin E and selenium supplementation, and parenteral injections prior to parturition and weaning. Treatment. Affected animals may be treated by administering vitamin E or selenium injections. Administering vitamin E or selenium to ewes in late pregnancy can prevent white muscle disease ( Kott et al., 1998 ). The label dose for selenium is 2.5–3 mg/45 kg of body weight. Combination products are available and can be used in goats at the sheep dose ( Smith and Sherman, 1994 ). Proper mineral balance in the diet is critical. Etiology. White muscle disease, also known as stiff lamb disease, is a nutritional muscular dystrophy caused by a deficiency of selenium or vitamin E. Clinical signs and diagnosis. Clinically two forms of the disease have been identified: cardiac and skeletal. The cardiac form occurs most commonly in neonates. In these, respiratory difficulty will be a manifestation of damage to cardiac, diaphragmatic, and intercostal muscles. Young will be able to nurse when assisted. In slightly older animals, the disease is characterized by locomotor disturbances and/or circulatory failure. Clinically, animals may display paresis, stiffness or inability to stand, rapid but weak pulse, and acute death. Mortality may reach 70% ( Jensen and Swift, 1982 ). Paresis and sudden death in neonates with associated pathological signs are frequently diagnostic. With the skeletal form, affected animals are stiff and reluctant to move, and muscles of affected animals are painful. Young will be reluctant to get up but will readily nurse when assisted. Peracute to acute myocardial degeneration may occur in the cardiac form, and animals may simply be found dead. Serum selenium levels are usually below 50 ppb (normal is 158–160 ppb) ( Nelson, 1983 ). Diagnosis may also include determination of antemortem whole blood levels of selenium and plasma levels of vitamin E. Glutathione peroxidase levels in red blood cells can be measured as an indirect test. Clinical biochemistry findings of significant elevations of aspartate aminotransferase (AST) in creatinine kinase (CK) are also supportive of the diagnosis. Epizootiology and transmission. Selenium deficiency has been associated with formulated diets deficient in selenium, forages grown on selenium-deficient soils in certain geographic regions, and forages such as alfalfa and clover that have an inability to efficiently extract available selenium from the soils. Rumen bacterial reduction of selenium compounds to unavailable elemental selenium may also contribute to the disease. Necropsy findings. Necropsy lesions include petechial hemorrhages and muscle edema. Hallmarks are pale white streaking of affected skeletal and cardiac muscle. These are due to coagulation necrosis. Pale striated muscles of the limb, diaphragm, and tongue are also seen. Pathogenesis. Selenium and vitamin E function together as antioxidants that protect lipid membranes from oxidative destruction. Selenium is a cofactor for glutathione peroxidase, which converts hydrogen peroxide to water and other nontoxic compounds. Lack of one or both results in loss of membrane integrity. Differential diagnosis. In neonatal ruminants presenting with respiratory and cardiac dysfunction, differentials include congenital cardiac anomalies. Differentials generally for weak neonates or sudden or peracute neonatal deaths should include septicemia, pneumonia, toxicity, diarrhea, and dehydration. Prevention and control. Awareness of regional selenium deficiencies is important. Control involves providing goodquality roughage, vitamin E and selenium supplementation, and parenteral injections prior to parturition and weaning. Treatment. Affected animals may be treated by administering vitamin E or selenium injections. Administering vitamin E or selenium to ewes in late pregnancy can prevent white muscle disease ( Kott et al., 1998 ). The label dose for selenium is 2.5–3 mg/45 kg of body weight. Combination products are available and can be used in goats at the sheep dose ( Smith and Sherman, 1994 ). Proper mineral balance in the diet is critical. d. Selenium Toxicity Selenium toxicity occurs most frequently as the result of excessive dosing to prevent or correct selenium deficiency or as the result of ingestion of selenium-converting plants. The main preventive measure for the former is the use of the appropriate product for the species. Secondarily, the concentration of the available product should be double-checked. In the United States, ruminants in the Midwest and western areas may be subject to selenium toxicity when pastured in areas containing selenium-converting plants. Signs of overdosing include weakness, dyspnea, bloating, and diarrhea. Shock, paresis, and death may occur. Initial clinical signs of excessive selenium intake from plants are observed in the distal limb, with cracked hoof walls and subsequent infection and irregular hoof growth. e. Thiamin Deficiency (Polioencephalomalacia) Etiology. Polioencephalomalacia (PEM) is a noninfectious, noncontagious disease characterized by neurological signs. Growing and adult ruminants on high-concentrate diets are typically affected. Animals exposed to toxic plants or moldy feed containing thiaminases, feed high in sulfates, or unusually high doses of some medications are also at risk. Clinical signs and diagnosis. An early sign may be mild diarrhea. Acute clinical signs include bruxism, hyperesthesia, involuntary muscle contractions, depression, partial or complete opisthotonus, nystagmus, dorsomedial strabismus, seizures, and death. In subacute cases of the disease, animals may appear to walk aimlessly as if blind or may display head-pressing postures. Hypersalivation may be present, but body temperatures and ocular reflexes are normal. Morbidity and mortality may be high, especially in younger animals. Diagnosis is suggestive from clinical signs and from response to intensive parental thiamine hydrochloride. Epizootiology and transmission. PEM is caused by a thiamin deficiency. The disease tends to be seen more frequently in cattle and sheep feedlots where the concentrates fed are high in fermentable carbohydrates. Pastured animals are also vulnerable if grain is feed. Thiaminase-containing plants, such as bracken fern, are often unpalatable so will less likely be a contributing factor. Recent studies have also indicated that high levels of sulfate in the diet, such as in the fermentable, low-fiber concentrates, may play an important role. Medications such as as amprolium, levamisole, and thiabendazole have thiaminantagonizing activity when given in excessive doses. Necropsy signs. Cerebral lesions characterized by softening and discoloration are grossly observed in the gray matter. Microscopically, neurons will exhibit edema, chromatolysis, and shrinkage. Gliosis and cerebral capillary proliferation may be observed. Pathogenesis. A lack of thiamin results in inappropriate carbohydrate metabolism and accumulation of pyruvate and other intermediaries that lead to cerebral edema and neuronal degeneration. Differential diagnosis. Several important differentials include acute lead poisoning, nitrofuran toxicity, hypomagnesemia, vitamin A deficiency, listeriosis, pregnancy toxemia, infectious thromboembolic meningoencephalitis, and type D clostridial enterotoxemia. Prevention and control. The disease can be prevented by monitoring the diet and by providing adequate roughage necessary to prevent overgrowth of thiaminase-producing ruminal flora and to maximize ruminal production of B vitamins. If excess sulfur is the primary factor, immediate removal of the source is critical. Treatment. Early aggressive treatment is essential to save animals. The disease is treated by frequent parenteral administration of thiamine hydrochloride, the first dose being administered intravenously. Dexamethasone, B vitamins, and diazepam may also be required. Treatment is less successful when sulfur plays a prominent role in the etiology. Research complications. This disease is preventable. Although the disease is less likely to occur in smaller groups of confined ruminants, the risks of feeding concentrates or moldy feed, for example, with minimal good-quality roughage, should be kept in mind. Etiology. Polioencephalomalacia (PEM) is a noninfectious, noncontagious disease characterized by neurological signs. Growing and adult ruminants on high-concentrate diets are typically affected. Animals exposed to toxic plants or moldy feed containing thiaminases, feed high in sulfates, or unusually high doses of some medications are also at risk. Clinical signs and diagnosis. An early sign may be mild diarrhea. Acute clinical signs include bruxism, hyperesthesia, involuntary muscle contractions, depression, partial or complete opisthotonus, nystagmus, dorsomedial strabismus, seizures, and death. In subacute cases of the disease, animals may appear to walk aimlessly as if blind or may display head-pressing postures. Hypersalivation may be present, but body temperatures and ocular reflexes are normal. Morbidity and mortality may be high, especially in younger animals. Diagnosis is suggestive from clinical signs and from response to intensive parental thiamine hydrochloride. Epizootiology and transmission. PEM is caused by a thiamin deficiency. The disease tends to be seen more frequently in cattle and sheep feedlots where the concentrates fed are high in fermentable carbohydrates. Pastured animals are also vulnerable if grain is feed. Thiaminase-containing plants, such as bracken fern, are often unpalatable so will less likely be a contributing factor. Recent studies have also indicated that high levels of sulfate in the diet, such as in the fermentable, low-fiber concentrates, may play an important role. Medications such as as amprolium, levamisole, and thiabendazole have thiaminantagonizing activity when given in excessive doses. Necropsy signs. Cerebral lesions characterized by softening and discoloration are grossly observed in the gray matter. Microscopically, neurons will exhibit edema, chromatolysis, and shrinkage. Gliosis and cerebral capillary proliferation may be observed. Pathogenesis. A lack of thiamin results in inappropriate carbohydrate metabolism and accumulation of pyruvate and other intermediaries that lead to cerebral edema and neuronal degeneration. Differential diagnosis. Several important differentials include acute lead poisoning, nitrofuran toxicity, hypomagnesemia, vitamin A deficiency, listeriosis, pregnancy toxemia, infectious thromboembolic meningoencephalitis, and type D clostridial enterotoxemia. Prevention and control. The disease can be prevented by monitoring the diet and by providing adequate roughage necessary to prevent overgrowth of thiaminase-producing ruminal flora and to maximize ruminal production of B vitamins. If excess sulfur is the primary factor, immediate removal of the source is critical. Treatment. Early aggressive treatment is essential to save animals. The disease is treated by frequent parenteral administration of thiamine hydrochloride, the first dose being administered intravenously. Dexamethasone, B vitamins, and diazepam may also be required. Treatment is less successful when sulfur plays a prominent role in the etiology. Research complications. This disease is preventable. Although the disease is less likely to occur in smaller groups of confined ruminants, the risks of feeding concentrates or moldy feed, for example, with minimal good-quality roughage, should be kept in mind. Etiology. Polioencephalomalacia (PEM) is a noninfectious, noncontagious disease characterized by neurological signs. Growing and adult ruminants on high-concentrate diets are typically affected. Animals exposed to toxic plants or moldy feed containing thiaminases, feed high in sulfates, or unusually high doses of some medications are also at risk. Clinical signs and diagnosis. An early sign may be mild diarrhea. Acute clinical signs include bruxism, hyperesthesia, involuntary muscle contractions, depression, partial or complete opisthotonus, nystagmus, dorsomedial strabismus, seizures, and death. In subacute cases of the disease, animals may appear to walk aimlessly as if blind or may display head-pressing postures. Hypersalivation may be present, but body temperatures and ocular reflexes are normal. Morbidity and mortality may be high, especially in younger animals. Diagnosis is suggestive from clinical signs and from response to intensive parental thiamine hydrochloride. Epizootiology and transmission. PEM is caused by a thiamin deficiency. The disease tends to be seen more frequently in cattle and sheep feedlots where the concentrates fed are high in fermentable carbohydrates. Pastured animals are also vulnerable if grain is feed. Thiaminase-containing plants, such as bracken fern, are often unpalatable so will less likely be a contributing factor. Recent studies have also indicated that high levels of sulfate in the diet, such as in the fermentable, low-fiber concentrates, may play an important role. Medications such as as amprolium, levamisole, and thiabendazole have thiaminantagonizing activity when given in excessive doses. Necropsy signs. Cerebral lesions characterized by softening and discoloration are grossly observed in the gray matter. Microscopically, neurons will exhibit edema, chromatolysis, and shrinkage. Gliosis and cerebral capillary proliferation may be observed. Pathogenesis. A lack of thiamin results in inappropriate carbohydrate metabolism and accumulation of pyruvate and other intermediaries that lead to cerebral edema and neuronal degeneration. Differential diagnosis. Several important differentials include acute lead poisoning, nitrofuran toxicity, hypomagnesemia, vitamin A deficiency, listeriosis, pregnancy toxemia, infectious thromboembolic meningoencephalitis, and type D clostridial enterotoxemia. Prevention and control. The disease can be prevented by monitoring the diet and by providing adequate roughage necessary to prevent overgrowth of thiaminase-producing ruminal flora and to maximize ruminal production of B vitamins. If excess sulfur is the primary factor, immediate removal of the source is critical. Treatment. Early aggressive treatment is essential to save animals. The disease is treated by frequent parenteral administration of thiamine hydrochloride, the first dose being administered intravenously. Dexamethasone, B vitamins, and diazepam may also be required. Treatment is less successful when sulfur plays a prominent role in the etiology. Research complications. This disease is preventable. Although the disease is less likely to occur in smaller groups of confined ruminants, the risks of feeding concentrates or moldy feed, for example, with minimal good-quality roughage, should be kept in mind. f. Vitamin D Toxicity Vitamin D toxicity can result either from iatrogenic overadministration or ingestion of the plant Trisetum flavescens. Serum calcium levels may be high enough that blood in EDTA tubes will clot. g. Nutritional Deficiencies In goats, nutritional deficiencies often manifest as a generalized poor coat that is dry, scaly, thin, and erectile. Zinc-responsive dermatitis has been reported in goats ( Smith and Sherman, 1994 ). Vitamin A deficiencies associated with hyperkeratosis have been reported, as well as vitamin E-responsive and selenium-responsive dermatitis. 4. Management-Related Diseases a. Failure of Passive Transfer Neonatal ruminants are born without immunoglobulins and must receive colostrum by 24 hr after birth. The morbidity and mortality associated with failure of or inadequate passive transfer, such as enteric and respiratory illnesses, can be severe. Measures to assure passive immunity for neonatal ruminants are covered in Section II,B,5, and clinical signs of illness associated with lack of immunity are addressed in the discussions of bacterial diseases (e.g., Escherichia coli infections) and, of viral diseases (e.g., diarrheas) in Section III,A,1 and III,A,2. Generally, transfer of less than 600 mg/dl of immunoglobulins in the serum is classified as failure of transfer, 600–1600 mg/dl is partial, and above 1600 mg/dl is complete transfer. Methods to determine success of transfer should be performed within a week of birth and include single radial immunodiffusion (quantitates immunogloblin classes); zinc sulfate turbidity (semiquantitative); sodium sulfite precipitation (semiquantitative); glutaraldehyde coagulation (coagulates above specific level); and, γ-glutamyltransferase (assays enzyme in high concentration in colostrum and absorbed simultaneously with colostrum). b. Laminitis Laminitis is common in ruminants and can be caused by sudden changes in diet, excess dietary energy, and grain overload (or overeating). Laminitis is also associated with mastitis and metritis. Facility conditions, such as concrete flooring, poor manure management, and inadequate resting areas may also contribute to the pathogenesis of the disease. The complete pathogenesis of laminitis is poorly understood; however, it is thought that changes in the diet cause changes in rumen microbial populations, resulting in acidosis and endotoxemia. Dramatic changes in the vascular endothelium result in chronic inflammation of the sensitive laminae of the hoof, separation of corium and hoof wall, and rotation of the third phalanx. Affected animals may be reluctant to get up or walk, will shift their weight frequently, and will grind teeth or walk on carpi. Chronically, the hoof wall takes on a "slipper" appearance. Treatment consists of identifying the underlying cause, administering antiinflammatories (phenylbutazone, flunixin meglumin), feeding good-quality forages only, and regular foot trimming. c. Nutritional Diarrhea Otherwise normal, well-managed lambs, kids, and calves can develop loose, pasty feces due to a nutritional imbalance caused by overfeeding and/or improper mixing of milk replacers. Only milk replacer formulated for the particular species should be used. Once nutritional imbalances are corrected, the feces readily return to normal. Sudden changes in diet can also result in loose feces. d. Photosensitization (Bighead) Photosensitization is an acute dermatitis associated with an interaction between photosensitive chemicals and sunlight. The photosensitive chemicals are usually ingested, but in some cases exposure may be by contact. Animals with a lack of pigment are more susceptible to the disease. Three types of photosensitization occur: primary; secondary, or hepatogenous; and aberrant. Primary photosensitization is related to uncommon plant pigments or to drugs such as phenothiazine, sulfonamides, or tetracyclines. Secondary photosensitization is more common in large animals and is specifically related to the plant pigment phylloerythrin. Phylloerythrin, a porphyrin compound, is a degradation product of chlorophyll released by rumen microbial digestion. Liver disease or injury, which prevents normal conjugation of phylloerythrin and excretion through the biliary system, predisposes to photosensitization. The only example of aberrant photosensitization is congenital porphyria of cattle (see Section III,B,1). Pathologically, the photosensitive chemical is deposited in the skin and is activated by absorbed sunlight. The activated pigments transfer their energy to local proteins and amino acids, which, in the presence of oxygen, are converted to vasoactive substances. The vasoactive substances increase the permeability of capillaries, leading to fluid and plasma protein losses and eventually to local tissue necrosis. Photosensitization can occur within hours to days after sun exposure and produces lesions of the face, vulva, and coronary bands; lesions are most likely to occur on white-haired areas. Initially, edema of the lips, corneas, eyelids, nasal planum, face, vulva, or coronary bands occurs. The facial edema, nostril constriction, and swollen lips potentially lead to difficulty in breathing. With secondary photosensitization, icterus is also common. Necrosis and gangrene may occur. Diagnosis is based on clinical lesions and exposure to the photosensitive chemicals and sunlight. Treatment is symptomatic. The prognosis for hepatogenous type may be guarded if hepatic disease is severe. e. Reproductive Prolapses (Vaginal, Uterine) Vaginal and uterine prolapses occur in ewes, does, and cows. The conditions are not common in does. Vaginal prolapses usually occur during late gestation and may be related to relaxation of the pelvic ligaments in response to hormone levels. In sheep, these are most common in overconditioned ewes that are also carrying twins or triplets. Overconsumption of roughages, which distends the rumen, and lack of exercise leading to intra-abdominal fat may predispose an animal to vaginal prolapse by increasing intra-abdominal pressure. The condition may result from excessive straining associated with dysuria from the pressure of the fetuses and/or abdominal contents on the bladder. If the prolapse obstructs subsequent urination, rupture of the bladder may occur. The vaginal prolapse can be reduced and repaired if discovered early, and techniques in small and large ruminants are comparable. The animal should be restrained, and the prolapsed tissue should be cleansed with disinfectants. Best done under epidural anesthesia, the vagina is replaced into the pelvic canal and the vulvar or vestibular opening is sutured closed (Buhner suture). Alternatively, a commercial device called a bearing retainer (or truss) can be placed into the reduced vagina and tied to the wool, thereby holding the vagina in proper orientation without interfering with subsequent lambing. Vaginal prolapses may have a hereditary basis in ewes and cows and may prolapse the following year. These animals should be culled. Vaginal prolapses may occur in nonpregnant animals that graze estrogenic plants or as a sequela to docking the tail too close to the body ( Ross, 1989 ). Uterine prolapses occur sporadically in postpartum ewes and cattle. The gravid horn invaginates after delivery and protrudes from the vulva. The cause is unknown, but excessive traction utilized to correct dystocia or retained placenta, uterine atony, hypocalcemia, and overconditioning or lack of exercise have been implicated. In cattle, the uterine prolapses usually develop within 1 week of calving, are more common in dairy cows than in beef cows, and are often associated with dystocia or hypocalcemia. Cows may also have concurrent parturient paresis. Initially, the tissue will appear normal, but edema and environmental contamination or injuries of the tissue develop quickly. Clinical signs will include increased pulse and respiratory rates, straining, restlessness, and anorexia. If identified early, the uterus can be replaced as for vaginal prolapses. Electrolyte imbalances should be corrected if present. Additional supportive therapy, including the use of antibiotics should always be considered. Tetanus prophylaxis should be included. Oxytocin should be administered to induce uterine reduction. Vaginal closures are less successful at retaining uterine prolapses. Preventive and control measures include regular exercise for breeding animals, and management of prepartum nutrition and body condition. f. Rectal Prolapse Rectal prolapse is common in growing, weaned lambs and in cattle from 6 months to 2 years old. The physical eversion of the rectum through the anal sphincter is usually secondary to other diseases or management-related circumstances. Rectal prolapses may occur secondary to gastrointestinal infection or inflammation, especially when the colon is involved. Diseases that cause tenesmus, such as coccidiosis, salmonellosis, and intestinal worms, may result in prolapse. Urolithiasis may result in prolapses as the animal strains to urinate. Any form of cystitis or urethritis, vaginal irritation, or vaginal prolapse and some forms of hepatic disease may lead to rectal prolapse. Abdominal enlargement related to advanced stages of pregnancy, excessive rumen filling or bloat, and overconditioning may cause prolapse. Finally, excessive coughing during respiratory tract infections, improper tail docking (too short), growth implants, prolonged recumbency, or overcrowded housing with animal piling may lead to prolapses. Diagnosis is based on clinical signs. Early prolapses may be corrected by holding the animal with the head down, while a colleague places a pursestring suture around the anus. The mucosa and underlying tissue of prolapses that have been present for longer periods of time will often become necrotic, dry, friable, and devitalized and will require surgical amputation or the placement of prolapse rings to remove the tissue. Rectal prolapse may also be accompanied by intestinal intussusceptions that will further complicate the treatment and increase mortality. Occasionally, acute rectal prolapse with evisceration will result in shock and prompt death of the animal. Prognosis depends on the cause and extent of the prolapse as well as the timeliness of intervention. In all cases of treatment, determination and elimination of the underlying cause are essential. g. Trichobezoars Gastrointestinal accumulations or obstructions of hair (and/or sometimes very coarse roughage, forming bezoars) occur in cattle and sheep. Cattle that are maintained on a low-roughage diet, that lick their coats frequently, that have long hair coats from outdoor housing, or that have heavy lice or mite infestations and associated pruritus will often develop bezoars. In addition, younger calves with abomasal ulcers have been found to be more likely to have abomasal trichobezoars as well. Clinical signs may be mild or severe according to size, number, and location. Ruminal trichobezoars rarely result in clinical signs. Obstruction will be accompanied by signs of pain, development of bloat, and decreased fecal production. Serum profiles will show hypochloridemia; other imbalances depend on the duration of the problem. Diagnosis is also based on abdominal auscultation, rectal palpation, and ultrasound (useful in calves and smaller ruminants). Treatment is surgical, such as paracostal laparotomy (for abomasal), paralumbar celiotomy with manual breakdown, or enterotomy. Supportive care should be administered as necessary to correct electrolyte imbalances and to prevent inflammation and sepsis. Prognosis is generally good if the condition is diagnosed and treated before dehydration and imbalances become severe and peritonitis develops. Prevention includes providing good-quality roughage and treating lice and mange infestations. a. Failure of Passive Transfer Neonatal ruminants are born without immunoglobulins and must receive colostrum by 24 hr after birth. The morbidity and mortality associated with failure of or inadequate passive transfer, such as enteric and respiratory illnesses, can be severe. Measures to assure passive immunity for neonatal ruminants are covered in Section II,B,5, and clinical signs of illness associated with lack of immunity are addressed in the discussions of bacterial diseases (e.g., Escherichia coli infections) and, of viral diseases (e.g., diarrheas) in Section III,A,1 and III,A,2. Generally, transfer of less than 600 mg/dl of immunoglobulins in the serum is classified as failure of transfer, 600–1600 mg/dl is partial, and above 1600 mg/dl is complete transfer. Methods to determine success of transfer should be performed within a week of birth and include single radial immunodiffusion (quantitates immunogloblin classes); zinc sulfate turbidity (semiquantitative); sodium sulfite precipitation (semiquantitative); glutaraldehyde coagulation (coagulates above specific level); and, γ-glutamyltransferase (assays enzyme in high concentration in colostrum and absorbed simultaneously with colostrum). b. Laminitis Laminitis is common in ruminants and can be caused by sudden changes in diet, excess dietary energy, and grain overload (or overeating). Laminitis is also associated with mastitis and metritis. Facility conditions, such as concrete flooring, poor manure management, and inadequate resting areas may also contribute to the pathogenesis of the disease. The complete pathogenesis of laminitis is poorly understood; however, it is thought that changes in the diet cause changes in rumen microbial populations, resulting in acidosis and endotoxemia. Dramatic changes in the vascular endothelium result in chronic inflammation of the sensitive laminae of the hoof, separation of corium and hoof wall, and rotation of the third phalanx. Affected animals may be reluctant to get up or walk, will shift their weight frequently, and will grind teeth or walk on carpi. Chronically, the hoof wall takes on a "slipper" appearance. Treatment consists of identifying the underlying cause, administering antiinflammatories (phenylbutazone, flunixin meglumin), feeding good-quality forages only, and regular foot trimming. c. Nutritional Diarrhea Otherwise normal, well-managed lambs, kids, and calves can develop loose, pasty feces due to a nutritional imbalance caused by overfeeding and/or improper mixing of milk replacers. Only milk replacer formulated for the particular species should be used. Once nutritional imbalances are corrected, the feces readily return to normal. Sudden changes in diet can also result in loose feces. d. Photosensitization (Bighead) Photosensitization is an acute dermatitis associated with an interaction between photosensitive chemicals and sunlight. The photosensitive chemicals are usually ingested, but in some cases exposure may be by contact. Animals with a lack of pigment are more susceptible to the disease. Three types of photosensitization occur: primary; secondary, or hepatogenous; and aberrant. Primary photosensitization is related to uncommon plant pigments or to drugs such as phenothiazine, sulfonamides, or tetracyclines. Secondary photosensitization is more common in large animals and is specifically related to the plant pigment phylloerythrin. Phylloerythrin, a porphyrin compound, is a degradation product of chlorophyll released by rumen microbial digestion. Liver disease or injury, which prevents normal conjugation of phylloerythrin and excretion through the biliary system, predisposes to photosensitization. The only example of aberrant photosensitization is congenital porphyria of cattle (see Section III,B,1). Pathologically, the photosensitive chemical is deposited in the skin and is activated by absorbed sunlight. The activated pigments transfer their energy to local proteins and amino acids, which, in the presence of oxygen, are converted to vasoactive substances. The vasoactive substances increase the permeability of capillaries, leading to fluid and plasma protein losses and eventually to local tissue necrosis. Photosensitization can occur within hours to days after sun exposure and produces lesions of the face, vulva, and coronary bands; lesions are most likely to occur on white-haired areas. Initially, edema of the lips, corneas, eyelids, nasal planum, face, vulva, or coronary bands occurs. The facial edema, nostril constriction, and swollen lips potentially lead to difficulty in breathing. With secondary photosensitization, icterus is also common. Necrosis and gangrene may occur. Diagnosis is based on clinical lesions and exposure to the photosensitive chemicals and sunlight. Treatment is symptomatic. The prognosis for hepatogenous type may be guarded if hepatic disease is severe. e. Reproductive Prolapses (Vaginal, Uterine) Vaginal and uterine prolapses occur in ewes, does, and cows. The conditions are not common in does. Vaginal prolapses usually occur during late gestation and may be related to relaxation of the pelvic ligaments in response to hormone levels. In sheep, these are most common in overconditioned ewes that are also carrying twins or triplets. Overconsumption of roughages, which distends the rumen, and lack of exercise leading to intra-abdominal fat may predispose an animal to vaginal prolapse by increasing intra-abdominal pressure. The condition may result from excessive straining associated with dysuria from the pressure of the fetuses and/or abdominal contents on the bladder. If the prolapse obstructs subsequent urination, rupture of the bladder may occur. The vaginal prolapse can be reduced and repaired if discovered early, and techniques in small and large ruminants are comparable. The animal should be restrained, and the prolapsed tissue should be cleansed with disinfectants. Best done under epidural anesthesia, the vagina is replaced into the pelvic canal and the vulvar or vestibular opening is sutured closed (Buhner suture). Alternatively, a commercial device called a bearing retainer (or truss) can be placed into the reduced vagina and tied to the wool, thereby holding the vagina in proper orientation without interfering with subsequent lambing. Vaginal prolapses may have a hereditary basis in ewes and cows and may prolapse the following year. These animals should be culled. Vaginal prolapses may occur in nonpregnant animals that graze estrogenic plants or as a sequela to docking the tail too close to the body ( Ross, 1989 ). Uterine prolapses occur sporadically in postpartum ewes and cattle. The gravid horn invaginates after delivery and protrudes from the vulva. The cause is unknown, but excessive traction utilized to correct dystocia or retained placenta, uterine atony, hypocalcemia, and overconditioning or lack of exercise have been implicated. In cattle, the uterine prolapses usually develop within 1 week of calving, are more common in dairy cows than in beef cows, and are often associated with dystocia or hypocalcemia. Cows may also have concurrent parturient paresis. Initially, the tissue will appear normal, but edema and environmental contamination or injuries of the tissue develop quickly. Clinical signs will include increased pulse and respiratory rates, straining, restlessness, and anorexia. If identified early, the uterus can be replaced as for vaginal prolapses. Electrolyte imbalances should be corrected if present. Additional supportive therapy, including the use of antibiotics should always be considered. Tetanus prophylaxis should be included. Oxytocin should be administered to induce uterine reduction. Vaginal closures are less successful at retaining uterine prolapses. Preventive and control measures include regular exercise for breeding animals, and management of prepartum nutrition and body condition. f. Rectal Prolapse Rectal prolapse is common in growing, weaned lambs and in cattle from 6 months to 2 years old. The physical eversion of the rectum through the anal sphincter is usually secondary to other diseases or management-related circumstances. Rectal prolapses may occur secondary to gastrointestinal infection or inflammation, especially when the colon is involved. Diseases that cause tenesmus, such as coccidiosis, salmonellosis, and intestinal worms, may result in prolapse. Urolithiasis may result in prolapses as the animal strains to urinate. Any form of cystitis or urethritis, vaginal irritation, or vaginal prolapse and some forms of hepatic disease may lead to rectal prolapse. Abdominal enlargement related to advanced stages of pregnancy, excessive rumen filling or bloat, and overconditioning may cause prolapse. Finally, excessive coughing during respiratory tract infections, improper tail docking (too short), growth implants, prolonged recumbency, or overcrowded housing with animal piling may lead to prolapses. Diagnosis is based on clinical signs. Early prolapses may be corrected by holding the animal with the head down, while a colleague places a pursestring suture around the anus. The mucosa and underlying tissue of prolapses that have been present for longer periods of time will often become necrotic, dry, friable, and devitalized and will require surgical amputation or the placement of prolapse rings to remove the tissue. Rectal prolapse may also be accompanied by intestinal intussusceptions that will further complicate the treatment and increase mortality. Occasionally, acute rectal prolapse with evisceration will result in shock and prompt death of the animal. Prognosis depends on the cause and extent of the prolapse as well as the timeliness of intervention. In all cases of treatment, determination and elimination of the underlying cause are essential. g. Trichobezoars Gastrointestinal accumulations or obstructions of hair (and/or sometimes very coarse roughage, forming bezoars) occur in cattle and sheep. Cattle that are maintained on a low-roughage diet, that lick their coats frequently, that have long hair coats from outdoor housing, or that have heavy lice or mite infestations and associated pruritus will often develop bezoars. In addition, younger calves with abomasal ulcers have been found to be more likely to have abomasal trichobezoars as well. Clinical signs may be mild or severe according to size, number, and location. Ruminal trichobezoars rarely result in clinical signs. Obstruction will be accompanied by signs of pain, development of bloat, and decreased fecal production. Serum profiles will show hypochloridemia; other imbalances depend on the duration of the problem. Diagnosis is also based on abdominal auscultation, rectal palpation, and ultrasound (useful in calves and smaller ruminants). Treatment is surgical, such as paracostal laparotomy (for abomasal), paralumbar celiotomy with manual breakdown, or enterotomy. Supportive care should be administered as necessary to correct electrolyte imbalances and to prevent inflammation and sepsis. Prognosis is generally good if the condition is diagnosed and treated before dehydration and imbalances become severe and peritonitis develops. Prevention includes providing good-quality roughage and treating lice and mange infestations. C. Traumatic Disorders (Wounds, Bites, and Entrapped Foreign Bodies) Wounds may be sustained from poorly constructed pens or fences, or from skirmishes among animals. Predators will usually be sources of bite wounds. Standard veterinary wound assessment and care are essential for wounds or bites. Tetanus antitoxin may be indicated. Use of approved antibiotics may be appropriate. The lesion should be cleaned with disinfectants and repaired with primary closure if it is clean and uncontaminated. Thorough cleaning, regular monitoring, and healing by second intention are recommended for older wounds. Abscesses may also occur in the soft tissues of the hooves (sole abscesses; see Section III,C,3) because of entrapped foreign bodies or hoof cracks that fill with dirt. Preventive measures include improvement of housing facilities, pens, and pastures; monitoring hierarchies among animals penned together; and implementing predator control measures, such as sound fencing, flock guard dogs, or donkeys, in pasture situations. D. Iatrogenic Diseases 1. Anaphylactic Reactions Acute anaphylatic reactions in sheep, goats, and cattle are often clinically referable to the respiratory system. Anaphylactic vaccine reactions cause acute lung edema; lungs are the primary site of lesions if collapse and death are sequelae. The animals will also be anxious and shivering and will become hyperthermic. Salivation, diarrhea, and bloat also occur. Immediate therapy must include epinephrine by intravenous infusion at (1 ml of 1:1000 per 50 kg of body weight for goats and 1:10,000 (0.1 mg/ml) or 0.01 mg/kg (about 5 ml) for adult cows.) Furosemide (5 mg/kg) may be beneficial to reduce edema. Prognosis is usually guarded. Recovery can occur within 2 hr. 2. Catheter Sites and Experimental Surgeries In a research environment, catheter sites or experimental surgeries may be sources of iatrogenic infection. Traumatic injuries to peripheral nerves can cause acute lameness. Improper administration of therapeutics can easily cause this type of lameness. Injections given in gluteals or between the semimembranosus and semitendinosus can cause irritation to the sciatic nerve and subsequent lameness. Contraction of the quadriceps results in the limb being pulled forward. Injections in the caudal thigh can damage the peroneal nerve and cause knuckling at the fetlock. Traumatic injury to the radial nerve can result in a "dropped elbow" ( Nelson, 1983 ). Husbandry procedures such as tail docking, castration, dehorning, dosing with a balling gun, and shearing may result in superficial lesions, dermal infections, or cases of tetanus. Balling-gun injuries to the pharynx may lead to cellulitis with coughing, decreased appetite, and sensitivity to palpation. Standard veterinary assessment and care are essential for these cases. Local and systemic antibiotics with supportive care may be indicated. Swelling around peripheral nerves caused by inoculations may be reduced by diuretics and anti-inflammatories. Mild cases of peripheral nerve damage may recover in 7–14 days. Personnel training, including review of relevant anatomy, preprocedure preparation, appropriate technique, careful surgical site preparation, rigorous instrument sanitation, and sterile technique will minimize the incidence of potential complications from surgical procedures. 1. Anaphylactic Reactions Acute anaphylatic reactions in sheep, goats, and cattle are often clinically referable to the respiratory system. Anaphylactic vaccine reactions cause acute lung edema; lungs are the primary site of lesions if collapse and death are sequelae. The animals will also be anxious and shivering and will become hyperthermic. Salivation, diarrhea, and bloat also occur. Immediate therapy must include epinephrine by intravenous infusion at (1 ml of 1:1000 per 50 kg of body weight for goats and 1:10,000 (0.1 mg/ml) or 0.01 mg/kg (about 5 ml) for adult cows.) Furosemide (5 mg/kg) may be beneficial to reduce edema. Prognosis is usually guarded. Recovery can occur within 2 hr. 2. Catheter Sites and Experimental Surgeries In a research environment, catheter sites or experimental surgeries may be sources of iatrogenic infection. Traumatic injuries to peripheral nerves can cause acute lameness. Improper administration of therapeutics can easily cause this type of lameness. Injections given in gluteals or between the semimembranosus and semitendinosus can cause irritation to the sciatic nerve and subsequent lameness. Contraction of the quadriceps results in the limb being pulled forward. Injections in the caudal thigh can damage the peroneal nerve and cause knuckling at the fetlock. Traumatic injury to the radial nerve can result in a "dropped elbow" ( Nelson, 1983 ). Husbandry procedures such as tail docking, castration, dehorning, dosing with a balling gun, and shearing may result in superficial lesions, dermal infections, or cases of tetanus. Balling-gun injuries to the pharynx may lead to cellulitis with coughing, decreased appetite, and sensitivity to palpation. Standard veterinary assessment and care are essential for these cases. Local and systemic antibiotics with supportive care may be indicated. Swelling around peripheral nerves caused by inoculations may be reduced by diuretics and anti-inflammatories. Mild cases of peripheral nerve damage may recover in 7–14 days. Personnel training, including review of relevant anatomy, preprocedure preparation, appropriate technique, careful surgical site preparation, rigorous instrument sanitation, and sterile technique will minimize the incidence of potential complications from surgical procedures. E. Neoplastic Diseases Neoplasia and tumors are relatively rare in ruminants. Lymphosarcoma/leukemia in sheep has been shown to result from infection by a virus related (or identical) to the bovine leukemia virus. Pulmonary carcinoma (pulmonary adenomatosis) and hepatic tumors are found in sheep. Virus-induced papillomatosis (warts), discussed in Section III,A,2,s, and squamous cell carcinomas have also been reported in sheep. In goats, thymoma is one of the two most common neoplasias reported, although no distinct clinical syndrome has been described. Cutaneous papillomas are the most common skin and udder tumor of goats, and although outbreaks involve multiple animals, no wart virus has been identified. Persistent udder papillomas may progress to squamous cell carcinoma. Lymphosarcoma is reported rarely in goats. Although adrenocortical adenomas have been reported frequently and almost exclusively in older wethers, no clinical condition has been described. Lymphosarcoma of various organ systems and "cancer eye" (bovine ocular squamous cell carcinoma, or OSCC) are the most commonly reported cancers in cattle. Lymphosarcoma is described in Section III,A,2,c. Lack of periocular pigmentation and the amount and intensity of exposure to solar ultraviolet light are considered important factors in OSCC. Genetic factors may also play a role. Many cases occur in Herefords. This is a disease of older cattle; no case has been reported in animals less than 4 years of age. The cancer metastasizes through the lymph system to major organs. Treatment in either lymphosarcoma or OSCC is recommended only as a palliative measure. The extent of ocular neoplastic involvement is a significant criterion for carcass condemnation. Papillomatosis (warts) are common in cattle (see Section III,A,2,s). F. Miscellaneous 1. Amyloidosis Amyloidosis in adult cattle is due to accumulations of amyloid protein in the kidney, liver, adrenal glands, and gastrointestinal tract. The disease has been classified as AA type, or associated with chronic inflammatory disease, although other unknown factors are believed to be involved in some cases. Clinical signs include chronic diarrhea, weight loss, decreased production, nonpainful renomegaly, and generalized edema. The loss of protein in the urine contributes to abnormal plasma albumin values and foaming urine. The proteinuria also distinguishes amyloidosis (and glomerulonephritis) from other causes of weight loss and diarrhea in cattle such as Johne's disease, parasitism, copper deficiency, salmonellosis, and bovine viral diarrhea virus infection. Prognosis is poor, and no treatment is reported. 2. Dental Wear Dental wear is seen most commonly in sheep. As sheep age, excessive dental wear may lead to an inability to properly masticate feed, manifesting as weight loss and unthriftiness. Several factors predisposing to dental wear should be considered. The diet should be properly balanced for minerals, especially calcium and phosphorus, because primary or secondary calcium deficiency during teeth development results in softening of the enamel and dentin. Dietary contamination with silica (i.e., hays and grains harvested in sandy regions) will lead to mechanical wear on the teeth. Likewise, animals grazing or being fed in sandy environments will have excessive tooth wear. Sheep older than about 5 years of age are especially prone to tooth wear and should be checked frequently, especially if signs of weight loss or malnutrition are evident. Managing the content and consistency of the diets can best prevent the disease. 3. Sole abscesses Of the ruminants, cows are the most frequently affected by subsolar absesses. Dirt becomes packed into cracks in the horny layer of the sole of the hoof, and contamination eventually extends into the sensitive areas of the hoof, with lameness and infection resulting. Animals maintained in very soiled or muddy conditions, combined with poor hoof care, are more likely affected. Fusobacterium necrophorum is often the pathogen involved. Separation of the animal, supportive care, surgical drainage, and antibiotic treatment are indicated. 1. Amyloidosis Amyloidosis in adult cattle is due to accumulations of amyloid protein in the kidney, liver, adrenal glands, and gastrointestinal tract. The disease has been classified as AA type, or associated with chronic inflammatory disease, although other unknown factors are believed to be involved in some cases. Clinical signs include chronic diarrhea, weight loss, decreased production, nonpainful renomegaly, and generalized edema. The loss of protein in the urine contributes to abnormal plasma albumin values and foaming urine. The proteinuria also distinguishes amyloidosis (and glomerulonephritis) from other causes of weight loss and diarrhea in cattle such as Johne's disease, parasitism, copper deficiency, salmonellosis, and bovine viral diarrhea virus infection. Prognosis is poor, and no treatment is reported. 2. Dental Wear Dental wear is seen most commonly in sheep. As sheep age, excessive dental wear may lead to an inability to properly masticate feed, manifesting as weight loss and unthriftiness. Several factors predisposing to dental wear should be considered. The diet should be properly balanced for minerals, especially calcium and phosphorus, because primary or secondary calcium deficiency during teeth development results in softening of the enamel and dentin. Dietary contamination with silica (i.e., hays and grains harvested in sandy regions) will lead to mechanical wear on the teeth. Likewise, animals grazing or being fed in sandy environments will have excessive tooth wear. Sheep older than about 5 years of age are especially prone to tooth wear and should be checked frequently, especially if signs of weight loss or malnutrition are evident. Managing the content and consistency of the diets can best prevent the disease. 3. Sole abscesses Of the ruminants, cows are the most frequently affected by subsolar absesses. Dirt becomes packed into cracks in the horny layer of the sole of the hoof, and contamination eventually extends into the sensitive areas of the hoof, with lameness and infection resulting. Animals maintained in very soiled or muddy conditions, combined with poor hoof care, are more likely affected. Fusobacterium necrophorum is often the pathogen involved. Separation of the animal, supportive care, surgical drainage, and antibiotic treatment are indicated.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8767654/

Modulating Antibody Functionality in Infectious Disease and Vaccination

Induction of pathogen-specific binding antibodies has long been considered a signature of protective immunity following vaccination and infection. The humoral immune response is a complex network of antibodies that target different specificities and drive different functions, collectively acting to limit and clear infection either directly, via pathogen neutralization, or indirectly, via pathogen clearance by the innate immune system. Emerging data suggest that not all antibody responses are equal, and qualitative features of antibodies may be key to defining protective immune profiles. Here, we review the most recent advances in our understanding of protective functional antibody responses in natural infection, vaccination, and monoclonal antibody therapeutics. Moreover, we highlight opportunities to augment or modulate antibody-mediated protection through enhancement of antibody functionality.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9996316/

Chemically modified aptamers for improving binding affinity to the target proteins via enhanced non-covalent bonding

Nucleic acid aptamers are ssDNA or ssRNA fragments that specifically recognize targets. However, the pharmacodynamic properties of natural aptamers consisting of 4 naturally occurring nucleosides (A, G, C, T/U) are generally restricted for inferior binding affinity than the cognate antibodies. The development of high-affinity modification strategies has attracted extensive attention in aptamer applications. Chemically modified aptamers with stable three-dimensional shapes can tightly interact with the target proteins via enhanced non-covalent bonding, possibly resulting in hundreds of affinity enhancements. This review overviewed high-affinity modification strategies used in aptamers, including nucleobase modifications, fluorine modifications (2′-fluoro nucleic acid, 2′-fluoro arabino nucleic acid, 2′,2′-difluoro nucleic acid), structural alteration modifications (locked nucleic acid, unlocked nucleic acid), phosphate modifications (phosphorothioates, phosphorodithioates), and extended alphabets. The review emphasized how these high-affinity modifications function in effect as the interactions with target proteins, thereby refining the pharmacodynamic properties of aptamers. 1 Introduction Aptamers are ssDNA or ssRNA fragments, which are typically screened from oligonucleotide pools using the systematic evolution of ligands by exponential enrichment (SELEX) technology. Because of the three-dimensional shapes, aptamers enable to recognition of abundant targets like antibodies ( Chen and Yang, 2015 ). Aptamers take many advantages over antibodies, including easier synthesis, less time and cost consumption, lower immunogenicity, higher stability, and superior re-foldability. Hence, aptamers are promising substitutes for homologous antibodies. In 2004, the United States Food and Drug Administration (FDA) approved the first commercialized aptamer drug (Macugen ® ) that binds vascular endothelial growth factor protein 165 (VEGF165) for the wet-form neovascular age-related macular degeneration (AMD) ( Ruckman et al., 1998 ; Rothlisberger and Hollenstein, 2018 ). Nowadays, several therapeutic aptamers have entered phase II-III clinical trials. In ionic environments, aptamers enable the folding of numerous 3-dimensional (3D) motifs, e.g., hairpins, bulges, loops and pseudoknots ( Strehlitz et al., 2012 ). It permits aptamers to form different shapes to interact with the ligands through non-covalent bonding, including hydrogen bonding, π–π stacking, London dispersion forces, ion-ion interactions, and dipole-dipole interactions. It is critical for aptamers to tightly interact with the target ligand (usually protein) to obtain high binding affinity ( Hu et al., 2022 ). Nevertheless, aptamers should form a 3D structure for target recognition, possibly impeding the amplification efficiency during polymerase chain reaction (PCR). It provides a mechanistic insight involved in the undesired binding affinity of the resultant aptamers screened by SELEX to targets ( Hasegawa et al., 2016 ). More importantly, natural DNA aptamers are composed of naturally occurring 4 deoxynucleotides (A, C, G, T), while natural antibodies are composed of 20 amino acids. Hence, the chemical variety of DNA aptamers is much more restricted in comparison with those of antibodies. Typically, naturally occurring aptamers show a decreased binding affinity than cognate antibodies. It indicates that the interactions between the aptamer and its ligand protein have spaces to be optimized. Researchers sought to determine whether chemical modifications could facilitate interactions between the modified aptamer and its ligand protein, with no increase in off-target effects ( Lipi et al., 2016 ) ( Figure 1 .). Totally, the interactions can be classified as non-covalent and covalent, respectively. Although covalent bonding enhances the highest affinity, the miserable off-target effects demonstrate reduced therapeutic or diagnostic applications in aptamers. In contrasts, non-covalent bonding embodies the properties of high affinity and specificity. The absence of interactions of aptamers to targets can be remedied with modifications at either the nucleobase, sugar ring or phosphate backbone. The naturally occurring aptamers have reached a plateau in the treatment of diseases, and there is an urgent sense that affinity enhancement must now come from fresh modification approaches, such as hydrophobic groups, amino acids, positive charges and phosphorothioates. Importantly, expanding aptamer epitopes, i.e. , increasing the contact area between the modified aptamer and protein, largely contributes to the affinity enhancement ( Zon, 2022 ). FIGURE 1 Chemically modified aptamers for improving binding affinity to the target protein via enhancing non-covalent bonding (the red dot represented amino acid residues on the protein, the yellow rectangle represented modified nucleotides on the aptamer, the black arrow represented non-covalent bond). In this review, we summarized an outlook of chemically modified aptamers for improving binding affinity to the target protein via enhanced non-covalent bonding. It will help guide the next-generation of chemically modified aptamers with high affinity for disease diagnosis and treatment. 2 Strategies for improving the binding affinity of the modified aptamer to the target protein via enhanced non-covalent bonding 2.1 Nucleobases modifications 2.1.1 Nucleobases with amino acid-like side chains Nucleobases can be modified with a wide variety of functional groups to improve the affinity between aptamer and protein. Given the inherent properties of nucleic acids and proteins nucleobases with amino acid-like side chains could confer further advantages to modified aptamers, because those amino acid side chains can directly participate in the interaction with protein. By modifying the C5 position of deoxyuridine triphosphate (dUTP) with some hydrophobic groups ( Figure 2 ), Gold et al. combined the conformational flexibility of nucleic acids with the functional variety of proteins, which greatly enhanced the binding affinity of modified aptamers to proteins ( Davies et al., 2012 ). dUTP modification at C5 can form additional hydrophobic interactions with hydrophobic pockets of proteins. These aptamers embodied slow off-rate to proteins, and were termed as SOMAmers (slow off-rate modified aptamers). Nebojsa et al. expanded nucleobases with amino acid-like side chains from uracil to cytosine. It was found that aptamers comprising modified uracil and cytosine exhibited higher binding affinity than that comprising modified uracil ( Gawande et al., 2017 ). FIGURE 2 The nucleobases modifications on the aptamers to enhance binding affinity. (A) SOMAmers modification; (B) Modified uridine by click chemistry; (C) Base-appended bases modification; (D) Amino acid (leu) modification. Different from the SOMAmers in which an amido linker was used between hydrophobic group and dU, Günter Mayer et al. developed triazole linker by click chemistry between alkyne-modified uridine (5-ethynyl-deoxyuridine (EdU)) instead of thymidine and hydrophobic group-azide ( Figure 2B ). Combining the compatibility of EdU to DNA polymerase in SELEX procedure and highly efficiency of click chemistry, they succeeded to screen an indole modified aptamer against cycle 3 GFP with 18.4 nM of K D value ( Tolle et al., 2015 ; Pfeiffer et al., 2018 ). Apart from hydrophobic modifications, hydrophilic modifications may facilitate forming additional hydrogen bonds and ion-ion interactions. Buyst et al. conjugated histamine to C5 of the thymine base in the aptamer by amido linkages ( Buyst et al., 2015 ). The single modified thymine was placed at four different positions in the center of the 14mer double helix, from which the interaction between imidazole and double chain and its influence on imidazole pKa were studied. A structural motif is established by unrestricted molecular dynamics and nuclear magnetic resonance, involving the formation of hydrogen bonds between imidazole and the Hoogsteen side of two adjacent GC base pairs, which has been shown to significantly enhance the DNA Thermal stability of double strands. It was reported that base modifications contribute to the thermal stability of synthetic DNA duplexes ( Verdonck et al., 2018 ). Unconstrained molecular dynamics (MD) simulation showed that attributable hydrogen bonds could be formed between imidazole and the nearby guanosine. A principal validation study showed that the imidazole thymine modification could enhance the stability of L-arginine amide conjugated aptamers, indicating a vital role in stability enhancement. Park's group demonstrated that the hydrophobic amino acids in aptamers could be regarded as a novel kind of unnatural nucleotide ( Yum et al., 2021 ). They synthesized a series of modified thrombin aptamers (TBA) to investigate their affinity and antithrombin activity. Then found that the incorporation of amine acids could improve the binding affinity of TBA (K D = 2.94 nM), and make the 3-fold of antithrombin activity enhancement. 2.1.2 Base-appended base modifications Horii and Waga's group developed the base-appended bases ( Figure 2C ) to enhance the binding affinity of modified aptamers ( Minagawa et al., 2017 ). Using modified SELEX, a high affinity aptamer could be isolated after several rounds of selection. For example, a Salivary α-amylase (sAA) aptamer (AMYm1) was selected, which had high affinity (K D 100-fold enhanced over the aptamers which only contained natural nucleotides. Hirao's group also improved the ExSELEX in a subsequent study, in which they constructed a Ds-randomized library improving the complexity of the initial version library ( Matsunaga et al., 2017 ). Using the enhanced version ExSELEX, they identified the aptamer targeting von Willebrand factor A1-domain (vWF) (K D = 75 p.m.). In addition, Hirao's group also developed the mini-hairpin DNA to modify aptamer for improving the stability of unnatural-nucleotides aptamers without declining their affinity ( Matsunaga et al., 2015 ; Kimoto et al., 2016 ). Benner's group constructed a laboratory in vitro evolution system including A, G, C, T, Z ( Figure 7B ) and P ( Figure 7C ) ( Biondi et al., 2016 ). They generated an aptamer binding protective antigen (PA) PA63 with a dissociation constant of ∼35 nM. Furthermore, by combining cell engineering technology and a laboratory in vitro evolution system, Benner and his colleagues found a series of aptamers including unnatural nucleotides targeting glypican 3 (GPC3) which was expressed on the surface of liver cells ( Zhang et al., 2016 ). Artificial nucleoside incorporation could increase the complexity of the SELEX library, and it made aptamers more like proteins, thereby allowing aptamers to bind target proteins with high affinity. Recently, the Hachimoji eight-letter DNA/RNA was reported, which might promote significantly the aptamer field in the future ( Hoshika et al., 2019 ). FIGURE 7 The artificial nucleotides incorporating into aptamer to improve binding affinity. (A) The (7- (2-thienyl) imidazo [4, 5-b] pyridine) (Ds) artificial nucleotide; (B) the 6-amino-5-nitro-3- (1′-β-D-2′-deoxyribofuranosyl)-2 (1H)-pyridone (Z) artificial nucleotide; (C) the 2-amino-8- (1′-β-D-2′-deoxy-ribofuranosyl)-imidazo-[1,2-a]-1,3,5-triazin-4 (8H)-one) (P) artificial nucleotide. 2.1 Nucleobases modifications 2.1.1 Nucleobases with amino acid-like side chains Nucleobases can be modified with a wide variety of functional groups to improve the affinity between aptamer and protein. Given the inherent properties of nucleic acids and proteins nucleobases with amino acid-like side chains could confer further advantages to modified aptamers, because those amino acid side chains can directly participate in the interaction with protein. By modifying the C5 position of deoxyuridine triphosphate (dUTP) with some hydrophobic groups ( Figure 2 ), Gold et al. combined the conformational flexibility of nucleic acids with the functional variety of proteins, which greatly enhanced the binding affinity of modified aptamers to proteins ( Davies et al., 2012 ). dUTP modification at C5 can form additional hydrophobic interactions with hydrophobic pockets of proteins. These aptamers embodied slow off-rate to proteins, and were termed as SOMAmers (slow off-rate modified aptamers). Nebojsa et al. expanded nucleobases with amino acid-like side chains from uracil to cytosine. It was found that aptamers comprising modified uracil and cytosine exhibited higher binding affinity than that comprising modified uracil ( Gawande et al., 2017 ). FIGURE 2 The nucleobases modifications on the aptamers to enhance binding affinity. (A) SOMAmers modification; (B) Modified uridine by click chemistry; (C) Base-appended bases modification; (D) Amino acid (leu) modification. Different from the SOMAmers in which an amido linker was used between hydrophobic group and dU, Günter Mayer et al. developed triazole linker by click chemistry between alkyne-modified uridine (5-ethynyl-deoxyuridine (EdU)) instead of thymidine and hydrophobic group-azide ( Figure 2B ). Combining the compatibility of EdU to DNA polymerase in SELEX procedure and highly efficiency of click chemistry, they succeeded to screen an indole modified aptamer against cycle 3 GFP with 18.4 nM of K D value ( Tolle et al., 2015 ; Pfeiffer et al., 2018 ). Apart from hydrophobic modifications, hydrophilic modifications may facilitate forming additional hydrogen bonds and ion-ion interactions. Buyst et al. conjugated histamine to C5 of the thymine base in the aptamer by amido linkages ( Buyst et al., 2015 ). The single modified thymine was placed at four different positions in the center of the 14mer double helix, from which the interaction between imidazole and double chain and its influence on imidazole pKa were studied. A structural motif is established by unrestricted molecular dynamics and nuclear magnetic resonance, involving the formation of hydrogen bonds between imidazole and the Hoogsteen side of two adjacent GC base pairs, which has been shown to significantly enhance the DNA Thermal stability of double strands. It was reported that base modifications contribute to the thermal stability of synthetic DNA duplexes ( Verdonck et al., 2018 ). Unconstrained molecular dynamics (MD) simulation showed that attributable hydrogen bonds could be formed between imidazole and the nearby guanosine. A principal validation study showed that the imidazole thymine modification could enhance the stability of L-arginine amide conjugated aptamers, indicating a vital role in stability enhancement. Park's group demonstrated that the hydrophobic amino acids in aptamers could be regarded as a novel kind of unnatural nucleotide ( Yum et al., 2021 ). They synthesized a series of modified thrombin aptamers (TBA) to investigate their affinity and antithrombin activity. Then found that the incorporation of amine acids could improve the binding affinity of TBA (K D = 2.94 nM), and make the 3-fold of antithrombin activity enhancement. 2.1.2 Base-appended base modifications Horii and Waga's group developed the base-appended bases ( Figure 2C ) to enhance the binding affinity of modified aptamers ( Minagawa et al., 2017 ). Using modified SELEX, a high affinity aptamer could be isolated after several rounds of selection. For example, a Salivary α-amylase (sAA) aptamer (AMYm1) was selected, which had high affinity (K D 100-fold enhanced over the aptamers which only contained natural nucleotides. Hirao's group also improved the ExSELEX in a subsequent study, in which they constructed a Ds-randomized library improving the complexity of the initial version library ( Matsunaga et al., 2017 ). Using the enhanced version ExSELEX, they identified the aptamer targeting von Willebrand factor A1-domain (vWF) (K D = 75 p.m.). In addition, Hirao's group also developed the mini-hairpin DNA to modify aptamer for improving the stability of unnatural-nucleotides aptamers without declining their affinity ( Matsunaga et al., 2015 ; Kimoto et al., 2016 ). Benner's group constructed a laboratory in vitro evolution system including A, G, C, T, Z ( Figure 7B ) and P ( Figure 7C ) ( Biondi et al., 2016 ). They generated an aptamer binding protective antigen (PA) PA63 with a dissociation constant of ∼35 nM. Furthermore, by combining cell engineering technology and a laboratory in vitro evolution system, Benner and his colleagues found a series of aptamers including unnatural nucleotides targeting glypican 3 (GPC3) which was expressed on the surface of liver cells ( Zhang et al., 2016 ). Artificial nucleoside incorporation could increase the complexity of the SELEX library, and it made aptamers more like proteins, thereby allowing aptamers to bind target proteins with high affinity. Recently, the Hachimoji eight-letter DNA/RNA was reported, which might promote significantly the aptamer field in the future ( Hoshika et al., 2019 ). FIGURE 7 The artificial nucleotides incorporating into aptamer to improve binding affinity. (A) The (7- (2-thienyl) imidazo [4, 5-b] pyridine) (Ds) artificial nucleotide; (B) the 6-amino-5-nitro-3- (1′-β-D-2′-deoxyribofuranosyl)-2 (1H)-pyridone (Z) artificial nucleotide; (C) the 2-amino-8- (1′-β-D-2′-deoxy-ribofuranosyl)-imidazo-[1,2-a]-1,3,5-triazin-4 (8H)-one) (P) artificial nucleotide. 3 Precision strike, new driving force for aptamer affinity improvement by chemical modification The current researches put forward higher requirements for aptamers - precision strike, in which the target of the aptamer is not no longer the whole molecule but a part of it. Therefore, high affinity and specificity is required. For this purpose, chemical modification must be the most powerful sword. Recent works from Ichiro Hirao's group succeeded to identify four dengue non-structural protein 1 (DEN-NS1) serotypes from clinical samples using expended alphabet Ds included aptamers (K D : 27–182 p.m.). The specificity of each aptamer is remarkably high, and the aptamers can recognize the subtle variants of DENNS1 with at least 96.9% amino acid sequence identity, beyond the capability of serotype identification (69–80% sequence identities ( Matsunaga et al., 2021a ; Matsunaga et al., 2021b ). The presence of several Ds bases in these aptamers significantly increased the affinities and specificities to each target. Not alone, Ge Zhang's group identified an aptamer, named aptscl56, which specifically targets the structural domain loop3 of sclerostin to promote bone formation without cardiovascular risks. Because the loop2 of plays a protective role in cardiovascular system, once the aptamer binds to it, it will lead to cardiovascular risk just like the previously reported monoclonal antibody. In their aptamer, four methoxy modifications at each 5′and 3′terminals not only provided nuclease-resistant ability, but also enhanced affinity ( Yu et al., 2022 ). Following these concepts, it will be a trend to explore subdomain binding aptamers, in which chemical modification will definitely play his role well. 4 Discussion and future perspectives The affinity of aptamers to the target protein is a key factor for their pharmacodynamics. Generally, the higher binding affinity was linked to better pharmacodynamics. The reduced dosage could contribute to less toxicity. The naturally occurring aptamer are composed of four kinds of nucleosides (A, G, C, T or U). Typically, naturally occurring aptamers show a decreased binding affinity than cognate antibodies. To enhance the affinity of aptamers, chemical modifications have great potentials to optimize the interactions between the aptamer and its ligand protein. Until now, many high affinity aptamers were generated by using chemical modifications either in pre-SELEX or post-SELEX procedures ( Table 1 ). In the pre-SELEX procedure, the chemically modified nucleotide analogs were involved early in the construction of the SELEX library through phosphoramidite chemistry. Importantly, it has reached a conclusion that the involved nucleotide analogs must be compatible with DNA/RNA polymerase during PCR procedure. The next challenge is to understand which nucleotide analogs need what kind of DNA/RNA polymerase to maximize the PCR yield for improving the success rate of aptamer screening. Just like SOMAmers, the hydrophobicity of amino acid like side chains were demonstrated to have great potential in increasing hydrophobic interactions between the aptamers and target proteins ( Vaught et al., 2010 ; Rohloff et al., 2014 ). Although chemical modifications benefit aptamers with abundant diversity and preferred nuclease resistance, there is rare evidence on which types of chemical modifications have the most impact. Accordingly, most chemical modifications hindered the integrity of SELEX libraries during their construction and the enrichment of high-affinity sequences, as well as the final sequencing procedure. In the post-SELEX procedure, the choice of chemical modifications is usually unrestricted, which significantly widens the diversity of modified aptamers. Additionally, chemical modifications in post-SELEX are site-specific. It indicates that diverse modifications can be simultaneously incorporated into aptamers to meet the criteria for optimizing properties. On contrary to pre-SELEX modification, pre-SELEX modification becomes complex and costly in the screening procedure. Not only did many sites were needed to be considered in the initial preparation of the modified aptamers, but also the screening method was limited. For example, depending on the researchers' will if we want to manipulate 2 types of modification groups on a 40-nt long DNA aptamer, a total of 3 40 possibilities needed to be considered, which cannot be completed by manpower. One of the shortcuts is to consider only one modification at one site of a known aptamer. Just as phosphorodithioate modification in an RNA aptamer, Xianbin Yang's group tested every phosphorodithioate linkage at a single site and finally obtained ∼1000-fold of affinity enhancement in a case ( Abeydeera et al., 2016 ). Additionally, structure-guided post-SELEX optimization is strongly recommended after a deep understanding of the interactions between the aptamer and its target ( Xu et al., 2019 ). Last but not the least, high-throughput screening methods, such as DNA microchips, artificial intelligence, and combinational chemistry, could be involved to increase the capacity of post-SELEX modification. The possibilities of the modification types and modification sites in aptamer are both huge. It is impossible to characterize all the interactions of the modified aptamers to the target manually. Thus, it is necessary to seek an efficient virtual prediction strategy to optimize the modification types and modification sites of the aptamers for high binding affinity. Artificial intelligence (AI) has witnessed successes in predicting the interactions between targets and ligands in drug discovery as previously reported. Using high-throughput DNA chip technology, the binding ability data of aptamers with some modifications at some sites could be obtained. Based on the above data, the researchers could further build and train AI models to predict the enormous binding affinity of aptamers with different modifications at different sites. TABLE 1 Chemically modified aptamers for improving binding affinity. Target Aptamer (length) Modification strategy K D Ref platelet-derived growth factor B SL1 (29) SOMAmer 0.02 nM Davies et al. (2012) Interleukin-8 8A-35 (35) 2′-fluoro-pyrimidine 1.72 pM Sung et al. (2014) HIV-1 reverse transcriptase FA1 (77) 2′-deoxy-2′-fluoroarabinonucleotide 4 pM Alves Ferreira-Bravo et al. (2015) Salivary α-amylase AMYm1 (75) base-appended base <1 nM Minagawa et al. (2017) vascular endothelial cell growth factor-165 AF83-7 (24) PS2 modification 1 pM Abeydeera et al. (2016) Interferon-γ IFd1-3Ds-49 (49) Artificial Nucleotide Ds 0.038 nM Kimoto et al. (2013) vascular endothelial cell growth factor-165 VGd1-2Ds-47 (47) Artificial Nucleotide Ds 0.65 pM Kimoto et al. (2013) Author contributions ZC, HL, AG, and YM wrote the manuscript; HZ, HD, and YZ helped in revising the manuscript; SY and BZ proposed constructive discussions; YM, AL, and GZ supervised the preparation of the manuscript. All authors contributed to the article and approved the submitted version. Conflict of interest Author AG was employed by the "Aptacure Therapeutics Limited". The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851142/

Clinical practice guideline development manual: A quality-driven approach for translating evidence into action

Background Guidelines translate best evidence into best practice. A well-crafted guideline promotes quality by reducing healthcare variations, improving diagnostic accuracy, promoting effective therapy, and discouraging ineffective – or potentially harmful – interventions. Despite a plethora of published guidelines, methodology is often poorly defined and varies greatly within and among organizations. Purpose This manual describes the principles and practices used successfully by the American Academy of Otolaryngology – Head and Neck Surgery to produce quality-driven, evidence-based guidelines using efficient and transparent methodology for action-ready recommendations with multi-disciplinary applicability. The development process, which allows moving from conception to completion in twelve months, emphasizes a logical sequence of key action statements supported by amplifying text, evidence profiles, and recommendation grades that link action to evidence. Conclusions As clinical practice guidelines become more prominent as a key metric of quality healthcare, organizations must develop efficient production strategies that balance rigor and pragmatism. Equally important, clinicians must become savvy in understanding what guidelines are – and are not – and how they are best utilized to improve care. The information in this manual should help clinicians and organizations achieve these goals. Background Guidelines translate best evidence into best practice. A well-crafted guideline promotes quality by reducing healthcare variations, improving diagnostic accuracy, promoting effective therapy, and discouraging ineffective – or potentially harmful – interventions. Despite a plethora of published guidelines, methodology is often poorly defined and varies greatly within and among organizations. Purpose This manual describes the principles and practices used successfully by the American Academy of Otolaryngology – Head and Neck Surgery to produce quality-driven, evidence-based guidelines using efficient and transparent methodology for action-ready recommendations with multi-disciplinary applicability. The development process, which allows moving from conception to completion in twelve months, emphasizes a logical sequence of key action statements supported by amplifying text, evidence profiles, and recommendation grades that link action to evidence. Conclusions As clinical practice guidelines become more prominent as a key metric of quality healthcare, organizations must develop efficient production strategies that balance rigor and pragmatism. Equally important, clinicians must become savvy in understanding what guidelines are – and are not – and how they are best utilized to improve care. The information in this manual should help clinicians and organizations achieve these goals.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368938/

Role of Sphingomyelinase in Infectious Diseases Caused by Bacillus cereus

Bacillus cereus ( B. cereus ) is a pathogen in opportunistic infections. Here we show that Bacillus cereus sphingomyelinase ( Bc -SMase) is a virulence factor for septicemia. Clinical isolates produced large amounts of Bc -SMase, grew in vivo, and caused death among mice, but ATCC strains isolated from soil did not. A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc- SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not. Administration of an anti- Bc -SMase antibody and immunization against Bc -SMase prevented death caused by the clinical isolates, showing that Bc -SMase plays an important role in the diseases caused by B. cereus . Treatment of mouse macrophages with Bc -SMase resulted in a reduction in the generation of H 2 O 2 and phagocytosis of macrophages induced by peptidoglycan (PGN), but no effect on the release of TNF-α and little release of LDH under our experimental conditions. Confocal laser microscopy showed that the treatment of mouse macrophages with Bc -SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that the cells treated with Bc -SMase exhibited a reduction in membrane fluidity. The results suggest that Bc -SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis. Introduction B. cereus is well-known for its role as a mediator of food-borne illness [1] , [2] , [3] , [4] . The microorganism, which forms spores, is found worldwide in dust, air, and water [5] . Therefore, B. cereus is ubiquitous in the hospital environment, indicating that contamination of dressings, intravenous catheters, and linen provides an opportunity for infection [5] . It is possible that B. cereus is a pathogen of nosocomial infections transmitted via towels, linen, and balloons to compromised patients [6] , [7] . In recent years, there has been an increasing appreciation for its potential as an opportunistic pathogen in immunocompromised hosts [5] , [6] , [8] , [9] . The microorganism secretes a wide variety of membrane-damaging toxins, phospholipases such as Bc -SMase, phosphatidylinositol-specific phospholipase C (PIPLC) and phosphatidylcholine-specific phospholipase C (PCPLC), and hemolysins such as cereolysin O, hemolysins and proteases [3] , [10] , [11] , [12] , [13] , [14] . However, there has been little research into the contributions of these enzymes and toxins to the infectious diseases caused by B . cereus . The SMase produced by the intracellular pathogen Listeria ivanovii was shown to mediate bacterial escape from the phagocytic vacuole following internalization, thereby promoting intracellular survival and propagation [15] . Helicobacter pylori -derived SMase was found to contribute toward cytotoxicity for gastric cells [16] . β-Hemolysin containing SMase activity from methicillin-resistant Staphylococcus aureus was expressed by 91% of strains in a high-toxicity group [17] . A mutant strain with deletions of β-hemolysin and catalase was significantly less virulent to mice than the wild-type Staphylococcus aureus strain [18] . We reported that Bc -SMase lysed sheep erythrocytes containing large amounts of SM in the outer lipid layer of their plasma membranes [19] . However, the enzyme is known not to be lethal or cytotoxic. Bc -SMase belongs to a family of Mg 2+ -dependent neutral SMases (nSMase) that includes SMases produced by Staphylococcus aureus , and Listeria ivanovii [20] . The members of this family share a high degree of homology in amino acid sequence [20] , [21] , [22] . However, the role of Bc -SMase in the virulence of B. cereus remains controversial. To investigate the relationship between Bc -SMase and B. cereus infections, we examined the relationship between Bc -SMase and the growth in vivo of clinical isolates of B. cereus . Results Pathogenicity of the Clinical Isolates of B. cereus To investigate if B. cereus JMU-06B-31 and JMU-06B-1, isolated from a patient with septicemia, and JMU-06B-35, isolated from a patient with endophthalmitis, grow in mice in vivo, six- to eight-week old male wild-type mice of the ICR mice were each injected intraperitoneally with 5×10 8 CFU of the clinical isolates or ATCC21928, ATCC31429, and ATCC6464 isolated from soil. Mice administered with the clinical isolates began to die after 12 h, and all mice died within 30 h of the administration ( Fig. 1A ). Mice injected with ATCC21928, ATCC31429, and ATCC6464 did not die within 100 h ( Fig. 1A ). The number of microorganisms in the blood of mice about 12 h after the administration of JMU-06B-31, JMU-06B-35, and JMU-06B-1 was 300–400 CFU/100 µL, whereas the ATCC strains were not detected in blood ( Fig. 1B ). 10.1371/journal.pone.0038054.g001 Figure 1 Lethal challenges with clinical isolates and ATCC strains of B. cereus. Mice were intraperitoneally administered with clinical isolates and ATCC strains of B. cereus (3×10 8 CFU/mouse). Clinical isolates; JMU-06B-31 (•), JMU-06B-35 (▪), and JMU-06B-1 (▴). ATCC strains ; ATCC21928 (□), ATCC31429 (○), and ATCC6464 (△). A) Mice were monitored every five hours after the injection. The duration of the experiment was set at 100 h. B) The microorganisms in the blood of mice about 12 h after the administration of various strains were cultured on Luria Broth agar plates. Values represent the mean ± SEM; n = 5 independent experiments. ND: not detected. Production of Phospholipases by the Clinical Isolates and the ATCC Strains of B. cereus Phospholipases produced by bacteria such as Staphylococcus aureus, Clostridium perfringens, and Helicobacter pylori are reported to be associated with local infections and of importance in the establishment of systemic diseases [4] , [16] , [23] , [24] . To analyze the production of phospholipases by B. cereus , we measured the amount of phospholipases produced by the clinical isolates and the ATCC strains in Luria Broth medium. These strains were cultured to an optical density at 620 nm of 0.8 in the medium. The enzyme samples fractionated from the culture supernatants were subjected to SDS-PAGE and Western blotting using anti- Bc -SMase, -PCPLC, and -PIPLC antibodies. As shown in Fig. 2A , large amounts (>5 µg/ml) of Bc -SMase, PCPLC, and PIPLC were detected in the culture supernatants of the clinical isolates, but very small amounts or undetectable levels in those of the ATCC strains. These phospholipase C genes were detected in every clinical and ATCC strain ( Figure S1 ). 10.1371/journal.pone.0038054.g002 Figure 2 Expression of phospholipases and promoter sequences of smase from clinical isolates and ATCC strains of B. cereus . A) 50% Ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) were subjected to SDS-PAGE and Western blotting using anti- Bc -SMase, -PCPLC, and -PIPLC antibodies. Lane: 1, JMU-06B-31; 2, JMU-06B-35; 3, JMU-06B-1; 4, ATCC21928; 5, ATCC31429; 6, ATCC6464. A representative result from one of three experiments is shown. B, C) The sequences of the promoter region of plc and smase from clinical isolates and ATCC strains of B. cereus were aligned by the program T-Coffee [44] . Consensus sequences of regulatory elements are indicated in bold type. Gray areas indicate nucleotide sequence differences. Next, we focused on the promoter sequence for the Bc -SMase gene ( smase ) or PLC gene ( plc ) of clinical isolates and ATCC isolates. The −35 and −10 promoter sequences of smase or plc from clinical isolates were almost the same as those of ATCC strains ( Fig. 2B and 2C ). In B. cereus, the transcriptional regulator PlcR (Phospholipase C regulator) controls most known virulence factors [25] , [26] , and activates gene expression by binding to a nucleotidic sequence called the 'PlcR box' [25] . As shown in Fig. 2B and 2C , there was no clear difference in the sequence of the PlcR box between clinical isolates and ATCC strains. In addition, the amino acid sequence of Bc -SMase was highly conserved in all strains ( Figure S2 ). Effect of Anti-phospholipases on Growth of B. cereus in Mice To provide clues regarding the growth of B. cereus in vivo, the effect of anti-phospholipases on the growth of JMU-06B-35 in mice was investigated. Mice were intraperitoneally injected with the clinical isolate (JMU-06B-35, 5×10 8 CFU) 2 h after the intraperitoneally administration of 50 µg of anti-PCPLC, -PIPLC, or -SMase antibody. The anti- Bc -SMase antibody completely inhibited the growth of JMU-06B-35 in the bloodstream ( Fig. 3A ). In addition, the mice injected with the anti- Bc -SMase antibody did not die within 100 h ( Fig. 3B ). The administration of the anti-PIPLC and -PCPLC antibodies had no effect on the growth and lethality of JMU-06B-35 in mice ( Fig. 3A and 3B ). The concentration of these antibodies was enough to neutralize the activity of the three enzymes (10 µg) in vitro (data not shown). It therefore appears that Bc -SMase plays an important role in the propagation of B. cereus in vivo in our experimental condition. 10.1371/journal.pone.0038054.g003 Figure 3 Effect of antibody and immunization against Bc -SMase, PCPLC, or PIPLC on lethality of B. cereus. Mice intraperitoneally received 50 µg of anti-SMase, -PCPLC, or -PIPLC antibodies and 2 h after the injection, were intraperitoneally administered B. cereus (JMU-06B-35). A) B. cereus in blood was cultured on Luria Broth agar plates 12 h after the intraperitoneal injection. Values represent the mean ± SEM; n = 3 independent experiments. ND: not detected. B) Mice were monitored every five hours after the injection of B. cereus . The duration of the experiment was set at 100 h. ▪, B. cereus ; ▴, anti-PIPLC antibody + B. cereus ; •, anti-PCPLC antibody + B. cereus ; ○, anti- Bc -SMase antibody + B. cereus . C) Mice subcutaneously received an emulsion of the enzyme ( Bc -SMase (○), PCPLC (•), or PIPLC (▴)) and CFA (▪) 2 times every 2 weeks. The immunized mice received B. cereus (JMU-06B-35, 3×10 8 CFU/mouse). The duration of the experiment was set at 100 h. To confirm the relationship between Bc -SMase and the growth of B. cereus in vivo, we investigated the effect of immunization of mice with Bc -SMase, PCPLC, or PIPLC on the death induced by JMU-06B-35. The BALB/c mice were immunized with 25 µg mixture of PCPLC, PIPLC, or Bc -SMase with Complete Freund's adjuvant (CFA) two times at two-week intervals. Sham-immunized mice administered the clinical isolate began to die after approximately 10 h, and all mice died within 30 h of the administration ( Fig. 3C ). The survival rate of mice immunized against Bc -SMase, PCPLC, or PIPLC was 100%, 0%, and 0% 30 h after infection, respectively ( Fig. 3C ). Effect of Bc -SMase on Infection of Mice with ATCC Strain Isolated from Soil To investigate the role of Bc -SMase in B. cereus infections, we examined the effect of Bc -SMase on B. cereus -induced death in mice. The animals were intraperitoneally injected with mixtures of ATCC21928 (5.0×10 7 CFU/mouse), which did not produce Bc -SMase in the culture supernatants, and various concentrations of Bc -SMase. As shown in Fig. 4A , the increase in the rate of death was dependent on the dose of Bc -SMase above 1.0 µg/mouse. On administration of ATCC21928 and 5.0 µg of Bc -SMase, the death rate was 100% within 30 h ( Fig. 4A ). Mice injected with ATCC21928 or Bc -SMase alone survived after 100 h under the experimental conditions ( Fig. 4A ). In addition, the number of microorganisms in blood 12 h after the administration of the mixture of ATCC21928 and 1.0 or 5.0 µg of Bc -SMase was 50–100 and 300–400 CFU/100 µl, respectively ( Fig. 4B ). On the other hand, the administration of ATCC21928 with PCPLC (5.0 µg/mouse) or PIPLC (5.0 µg/mouse) resulted in no death under the conditions (data not shown). 10.1371/journal.pone.0038054.g004 Figure 4 Effect of Bc -SMase on the infection with B. cereus or B. subtilis. Mice received various concentrations of Bc -SMase and B. cereus (ATCC21928, 5×10 7 CFU/mouse). A) Mice were monitored every five hours after the injection. The duration of the experiment was set at 100 h. ○, B. cereus ; □, 1.0 µg Bc -SMase; △, 5.0 µg Bc -SMase; ▴, 0.1 µg Bc -SMase + B. cereus ; •, 1.0 µg Bc -SMase + B. cereus ; ▪, 5.0 µg Bc -SMase + B. cereus . B) B. cereus in blood was cultured on Luria broth agar plates. Values represent the mean ± SEM; n = 5 independent experiments. Effect of Overexpression of Bc -SMase on Growth of B. cereus or B. subtilis in Mice To investigate the effect of Bc -SMase on growth of B. cereus in vivo, we transfected a vector expressing smase or the gene for E53A ( e53a ), a variant which has little enzymatic activity [27] ( Table S1 ), into ATCC21928 or Bacillus subtilis (ISW1215), which did not produce Bc -SMase in the culture supernatants. The ammonium sulfate precipitation fraction of the culture supernatant fluids of these transfected strains was subjected to SDS-PAGE and Western blotting using anti- Bc -SMase antibody. As shown in Fig. 5A , these proteins (>5.0 µg/ml) were detected in the culture supernatants of these transformants carrying smase or e53a , but not in each microorganism transformed with empty vector. When the mice intraperitoneally received ATCC21928 or ISW1215 transformants carrying the smase , the microorganisms were detected about 300–400 CFU/100 µl in bloodstream ( Fig. 5B ). However, administration of these bacteria carrying e53a had no effect on the growth of each microorganism in vivo ( Fig. 5B ). The results showed that overexpression of Bc -SMase in ATCC21928 or ISW1215 induced growth of these strains in vivo. In addition, the survival rate 100 h after administration of ATCC21928 transformants carrying the smase was approximately 50%, but that of ISW1215 was 100% ( Fig. 5C ). 10.1371/journal.pone.0038054.g005 Figure 5 Effect of overexpression of Bc -SMase on growth of B. cereus or B. subtilis in mice. A) B. cereus (ATCC21928) or B. subtilis (ISW1215) was transfected with the plasmid carrying smase or e53a . A) 50% ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) of each microorganism were subjected to SDS-PAGE and Western blotting using anti- Bc -SMase antibody. A representative result from one of three experiments is shown. B, C) Mice intraperitoneally received B. cereus or B. subtilis transformants (1×10 8 CFU/mouse) carrying empty vector (vector), smase , or e53a . The microorganisms in the blood of mice about 12 h after the administration of these strains were cultured on Luria Broth agar plates. Values represent the mean ± SEM; n = 5 independent experiments. C) Mice were monitored every five hours after the injection. The duration of the experiment was set at 100 h. •, vector ( B. cereus ); ▪, smase ( B. cereus ); ▴, e53a ( B.cereus ); ○, vector ( B. subtilis ); □, smase ( B. subtilis ); △, e53a ( B. subtilis ). Effect of Bc -SMase on Activation of Macrophages by Peptidoglycan González Zorn et al. reported that the SMase from Listeria ivanovii mediates bacterial escape from phagocytic cells [15] . The activation of macrophages is known to be related to bactericidal action in vivo. To investigate the effect of Bc -SMase on the activation of macrophages, we assessed the effect of PGN, an activator of macrophages, on macrophages treated with Bc -SMase. Bc -SMase attenuated PGN-activated H 2 O 2 generation and phagocytosis of macrophages in a dose-dependent manner ( Fig. 6A and 6B ). However, Bc -SMase had no effect on the release of TNF-α induced by PGN from macrophages and induced no release of lactate dehydrogenase (LDH) from the cells ( Fig. 6C and 6D ). It therefore is likely that Bc -SMase specifically influences H 2 O 2 generation and phagocytosis without impairing membranes of macrophages, suggesting that treatment of macrophages with Bc -SMase results in a change in function of the membranes. It was thought that the frustrated phagocytosis may be dependent on the formation of ceramide in macrophage membranes. 10.1371/journal.pone.0038054.g006 Figure 6 Effect of Bc -SMase on activation of mouse macrophages. Mouse macrophages were incubated with or without Bc -SMase at 37°C for 60 min (D), and then treated with PGN (5 µg/ml) for 60 min (A, B, C). H 2 O 2 production, phagocytosis, TNF-α release, and LDH release were measured as described in Materials and Methods . Values represent the mean ± SEM; n = 7; * P 5 µg/ml) of Bc -SMase, PCPLC, and PIPLC were detected in the culture supernatants of the clinical isolates, but very small amounts or undetectable levels in those of the ATCC strains. These phospholipase C genes were detected in every clinical and ATCC strain ( Figure S1 ). 10.1371/journal.pone.0038054.g002 Figure 2 Expression of phospholipases and promoter sequences of smase from clinical isolates and ATCC strains of B. cereus . A) 50% Ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) were subjected to SDS-PAGE and Western blotting using anti- Bc -SMase, -PCPLC, and -PIPLC antibodies. Lane: 1, JMU-06B-31; 2, JMU-06B-35; 3, JMU-06B-1; 4, ATCC21928; 5, ATCC31429; 6, ATCC6464. A representative result from one of three experiments is shown. B, C) The sequences of the promoter region of plc and smase from clinical isolates and ATCC strains of B. cereus were aligned by the program T-Coffee [44] . Consensus sequences of regulatory elements are indicated in bold type. Gray areas indicate nucleotide sequence differences. Next, we focused on the promoter sequence for the Bc -SMase gene ( smase ) or PLC gene ( plc ) of clinical isolates and ATCC isolates. The −35 and −10 promoter sequences of smase or plc from clinical isolates were almost the same as those of ATCC strains ( Fig. 2B and 2C ). In B. cereus, the transcriptional regulator PlcR (Phospholipase C regulator) controls most known virulence factors [25] , [26] , and activates gene expression by binding to a nucleotidic sequence called the 'PlcR box' [25] . As shown in Fig. 2B and 2C , there was no clear difference in the sequence of the PlcR box between clinical isolates and ATCC strains. In addition, the amino acid sequence of Bc -SMase was highly conserved in all strains ( Figure S2 ). Effect of Anti-phospholipases on Growth of B. cereus in Mice To provide clues regarding the growth of B. cereus in vivo, the effect of anti-phospholipases on the growth of JMU-06B-35 in mice was investigated. Mice were intraperitoneally injected with the clinical isolate (JMU-06B-35, 5×10 8 CFU) 2 h after the intraperitoneally administration of 50 µg of anti-PCPLC, -PIPLC, or -SMase antibody. The anti- Bc -SMase antibody completely inhibited the growth of JMU-06B-35 in the bloodstream ( Fig. 3A ). In addition, the mice injected with the anti- Bc -SMase antibody did not die within 100 h ( Fig. 3B ). The administration of the anti-PIPLC and -PCPLC antibodies had no effect on the growth and lethality of JMU-06B-35 in mice ( Fig. 3A and 3B ). The concentration of these antibodies was enough to neutralize the activity of the three enzymes (10 µg) in vitro (data not shown). It therefore appears that Bc -SMase plays an important role in the propagation of B. cereus in vivo in our experimental condition. 10.1371/journal.pone.0038054.g003 Figure 3 Effect of antibody and immunization against Bc -SMase, PCPLC, or PIPLC on lethality of B. cereus. Mice intraperitoneally received 50 µg of anti-SMase, -PCPLC, or -PIPLC antibodies and 2 h after the injection, were intraperitoneally administered B. cereus (JMU-06B-35). A) B. cereus in blood was cultured on Luria Broth agar plates 12 h after the intraperitoneal injection. Values represent the mean ± SEM; n = 3 independent experiments. ND: not detected. B) Mice were monitored every five hours after the injection of B. cereus . The duration of the experiment was set at 100 h. ▪, B. cereus ; ▴, anti-PIPLC antibody + B. cereus ; •, anti-PCPLC antibody + B. cereus ; ○, anti- Bc -SMase antibody + B. cereus . C) Mice subcutaneously received an emulsion of the enzyme ( Bc -SMase (○), PCPLC (•), or PIPLC (▴)) and CFA (▪) 2 times every 2 weeks. The immunized mice received B. cereus (JMU-06B-35, 3×10 8 CFU/mouse). The duration of the experiment was set at 100 h. To confirm the relationship between Bc -SMase and the growth of B. cereus in vivo, we investigated the effect of immunization of mice with Bc -SMase, PCPLC, or PIPLC on the death induced by JMU-06B-35. The BALB/c mice were immunized with 25 µg mixture of PCPLC, PIPLC, or Bc -SMase with Complete Freund's adjuvant (CFA) two times at two-week intervals. Sham-immunized mice administered the clinical isolate began to die after approximately 10 h, and all mice died within 30 h of the administration ( Fig. 3C ). The survival rate of mice immunized against Bc -SMase, PCPLC, or PIPLC was 100%, 0%, and 0% 30 h after infection, respectively ( Fig. 3C ). Effect of Bc -SMase on Infection of Mice with ATCC Strain Isolated from Soil To investigate the role of Bc -SMase in B. cereus infections, we examined the effect of Bc -SMase on B. cereus -induced death in mice. The animals were intraperitoneally injected with mixtures of ATCC21928 (5.0×10 7 CFU/mouse), which did not produce Bc -SMase in the culture supernatants, and various concentrations of Bc -SMase. As shown in Fig. 4A , the increase in the rate of death was dependent on the dose of Bc -SMase above 1.0 µg/mouse. On administration of ATCC21928 and 5.0 µg of Bc -SMase, the death rate was 100% within 30 h ( Fig. 4A ). Mice injected with ATCC21928 or Bc -SMase alone survived after 100 h under the experimental conditions ( Fig. 4A ). In addition, the number of microorganisms in blood 12 h after the administration of the mixture of ATCC21928 and 1.0 or 5.0 µg of Bc -SMase was 50–100 and 300–400 CFU/100 µl, respectively ( Fig. 4B ). On the other hand, the administration of ATCC21928 with PCPLC (5.0 µg/mouse) or PIPLC (5.0 µg/mouse) resulted in no death under the conditions (data not shown). 10.1371/journal.pone.0038054.g004 Figure 4 Effect of Bc -SMase on the infection with B. cereus or B. subtilis. Mice received various concentrations of Bc -SMase and B. cereus (ATCC21928, 5×10 7 CFU/mouse). A) Mice were monitored every five hours after the injection. The duration of the experiment was set at 100 h. ○, B. cereus ; □, 1.0 µg Bc -SMase; △, 5.0 µg Bc -SMase; ▴, 0.1 µg Bc -SMase + B. cereus ; •, 1.0 µg Bc -SMase + B. cereus ; ▪, 5.0 µg Bc -SMase + B. cereus . B) B. cereus in blood was cultured on Luria broth agar plates. Values represent the mean ± SEM; n = 5 independent experiments. Effect of Overexpression of Bc -SMase on Growth of B. cereus or B. subtilis in Mice To investigate the effect of Bc -SMase on growth of B. cereus in vivo, we transfected a vector expressing smase or the gene for E53A ( e53a ), a variant which has little enzymatic activity [27] ( Table S1 ), into ATCC21928 or Bacillus subtilis (ISW1215), which did not produce Bc -SMase in the culture supernatants. The ammonium sulfate precipitation fraction of the culture supernatant fluids of these transfected strains was subjected to SDS-PAGE and Western blotting using anti- Bc -SMase antibody. As shown in Fig. 5A , these proteins (>5.0 µg/ml) were detected in the culture supernatants of these transformants carrying smase or e53a , but not in each microorganism transformed with empty vector. When the mice intraperitoneally received ATCC21928 or ISW1215 transformants carrying the smase , the microorganisms were detected about 300–400 CFU/100 µl in bloodstream ( Fig. 5B ). However, administration of these bacteria carrying e53a had no effect on the growth of each microorganism in vivo ( Fig. 5B ). The results showed that overexpression of Bc -SMase in ATCC21928 or ISW1215 induced growth of these strains in vivo. In addition, the survival rate 100 h after administration of ATCC21928 transformants carrying the smase was approximately 50%, but that of ISW1215 was 100% ( Fig. 5C ). 10.1371/journal.pone.0038054.g005 Figure 5 Effect of overexpression of Bc -SMase on growth of B. cereus or B. subtilis in mice. A) B. cereus (ATCC21928) or B. subtilis (ISW1215) was transfected with the plasmid carrying smase or e53a . A) 50% ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) of each microorganism were subjected to SDS-PAGE and Western blotting using anti- Bc -SMase antibody. A representative result from one of three experiments is shown. B, C) Mice intraperitoneally received B. cereus or B. subtilis transformants (1×10 8 CFU/mouse) carrying empty vector (vector), smase , or e53a . The microorganisms in the blood of mice about 12 h after the administration of these strains were cultured on Luria Broth agar plates. Values represent the mean ± SEM; n = 5 independent experiments. C) Mice were monitored every five hours after the injection. The duration of the experiment was set at 100 h. •, vector ( B. cereus ); ▪, smase ( B. cereus ); ▴, e53a ( B.cereus ); ○, vector ( B. subtilis ); □, smase ( B. subtilis ); △, e53a ( B. subtilis ). Effect of Bc -SMase on Activation of Macrophages by Peptidoglycan González Zorn et al. reported that the SMase from Listeria ivanovii mediates bacterial escape from phagocytic cells [15] . The activation of macrophages is known to be related to bactericidal action in vivo. To investigate the effect of Bc -SMase on the activation of macrophages, we assessed the effect of PGN, an activator of macrophages, on macrophages treated with Bc -SMase. Bc -SMase attenuated PGN-activated H 2 O 2 generation and phagocytosis of macrophages in a dose-dependent manner ( Fig. 6A and 6B ). However, Bc -SMase had no effect on the release of TNF-α induced by PGN from macrophages and induced no release of lactate dehydrogenase (LDH) from the cells ( Fig. 6C and 6D ). It therefore is likely that Bc -SMase specifically influences H 2 O 2 generation and phagocytosis without impairing membranes of macrophages, suggesting that treatment of macrophages with Bc -SMase results in a change in function of the membranes. It was thought that the frustrated phagocytosis may be dependent on the formation of ceramide in macrophage membranes. 10.1371/journal.pone.0038054.g006 Figure 6 Effect of Bc -SMase on activation of mouse macrophages. Mouse macrophages were incubated with or without Bc -SMase at 37°C for 60 min (D), and then treated with PGN (5 µg/ml) for 60 min (A, B, C). H 2 O 2 production, phagocytosis, TNF-α release, and LDH release were measured as described in Materials and Methods . Values represent the mean ± SEM; n = 7; * P <0.01 compared with H 2 O 2 production or phagocytosis induced by PGN alone. Localization of Ceramide in Membranes of Macrophages Treated with Bc -SMase To determine the amount of ceramide formed in the macrophages treated with Bc -SMase, macrophages were incubated with Bc -SMase at 37°C for 30 min. The lipids extracted from the treated cells were phosphorylated by diacylglycerol kinase from Escherichia coli, and developed by reverse-phase thin layer chromatography (TLC). The level of ceramide in the cells treated with Bc -SMase increased in a dose-dependent manner ( Fig. 7A ). Using confocal microscopy, Montes et al. found that phospholipase C from P. aeruginosa caused the formation of ceramide-rich domains in biological membranes [28] . We reported that Bc -SMase induced the formation of ceramide-rich domains in membranes of sheep erythrocytes and a decrease in the fluidity of membranes, leading to destabilization under physical stimulation [19] . We investigated whether treatment of macrophages with Bc -SMase results in the local accumulation of BODIPY-ceramide formed in membranes of cells preincubated with BODIPY FL-C 12 -SM (BODIPY-SM). Fig. 7B (left) shows that the fluorescent substance in membranes of macrophages preincubated with BODIPY-SM was not localized. However, when BODIPY-SM -incubated macrophages were treated with Bc -SMase, the local accumulation of the fluorescent substance was found on membranes of the cells, as shown by the white arrows ( Fig. 7B , right). To test whether the site where the substance accumulates coincides with a ceramide-rich site, BODIPY–SM-preincubated membranes treated with Bc -SMase were analyzed using Cy3-labeled anti-ceramide antibody. As shown in Fig. 7C , the distribution of the fluorescence of the antibody was different from that of BODIPY–SM in the untreated cells. In the case of BODIPY–SM-preincubated macrophages treated with Bc -SMase, the location of the fluorescence of Cy3-anti-ceramide antibody was consistent with that of BODIPY, as shown in Fig. 7B , suggesting that BODIPY-ceramide formed from BODIPY-SM in the macrophages treated with Bc -SMase is mostly located in ceramide-rich domains. Klein et al. reported that membrane fluidity of cells was evaluated by measurement of lateral diffusion of fluorescence-labeled SM by FRAP with a confocal laser microscopy [29] . A FRAP analysis revealed that the recovery of effective diffusion for the fluorescence of BODIPY in ceramide-rich domains of the macrophages treated with Bc -SMase decreased to about 70–80%, compared with that of BODIPY-SM in the untreated cells ( Fig. 7D ). It therefore appears that the Bc -SMase-induced formation of ceramide from SM in membranes of macrophages results in a decrease in the fluidity of membranes. 10.1371/journal.pone.0038054.g007 Figure 7 Effect of ceramide on activation of mouse macrophages. A) Mouse macrophages were incubated with various concentrations of Bc -SMase at 37°C for 60 min. Ceramide was phosphorylated by 1, 2-diacylglycerol kinase and separated by reverse-phase TLC. Values represent the mean ± SEM; n = 3; * P <0.01 compared with Bc -SMase-untreated cells. (B) Macrophages pretreated with BODIPY-SM were incubated without (left) or with (right) Bc -SMase at 37°C for 60 min. (C) Mouse macrophages pretreated with BODIPY-SM were incubated with or without Bc -SMase at 37°C for 60 min. The cells were treated with paraformaldehyde, and stained with Cy3-coupled anti-ceramide anti-bodies. (D) Representative recovery curves for the diffusion of BODIPY fluorescence following Bc -SMase treatment (broken line, ceramide-rich domain) or without treatment (solid line, ceramide-poor domain) are shown. A representative result from one of ten experiments is shown. Discussion The present study showed that clinical isolates of B. cereus, which produced large amounts of Bc -SMase, from patients with sepsis and endophthalmitis were lethal to mice. The production of Bc -SMase from clinical isolates was greater than that from ATCC strains under our culture conditions. B. cereus produces several secreted toxins, the expression of which is controlled by the PlcR [25] , [26] . A difference in protein level from PlcR regulated proteins has been observed for pathogenic factors such as nonhemolytic enterotoxin and hemolysin BL [30] , [31] . In addition, variations of the InhA1, NprA, and HlyII, which regulation is independent on PlcR, between pathogenic and nonpathogenic B. cereus strains has also been observed [32] . In this study, the sequences of the PlcR box in clinical isolates were almost the same as that in ATCC strains. Therefore, it appears that various factors participate in the pathogenic expression of B. cereus and production of Bc- SMase. Bc -SMase enhanced the growth of B. cereus in the peritoneal cavity, and in addition, invaded the bloodstream in mice, causing death. Furthermore, overexpression of Bc -SMase in B. subtilis , an avirulent strain, induced growth in mice. The administration of a mixture of ATCC21928 and Bc -SMase resulted in the death of mice, but that of PIPLC or PCPLC did not. In addition, the loss of PlcR-regulated factors, which include Bc -SMase, significantly attenuated the severity of Bacillus endophthalmitis [10] . Furthermore, mice administered the anti- Bc -SMase antibody or immunized with Bc -SMase were protected from the lethality of clinical isolates of B. cereus. Callegan et al., reported that intraocular infection with wild type B. cereus or isogenic mutants specifically deficient in PIPLC or PCPLC resulted in similar degrees of destruction of the retinal architecture, and a complete loss of retinal function [33] , [34] . These results suggested that Bc -SMase plays an important role in the propagation of B. cereus in vivo. Antibody and immunization against Bc -SMase protected mice from B. cereus, suggesting Bc -SMase to be a candidate vaccine against infectious diseases caused by B. cereus. Furthermore, it has been reported that nSMase is secreted by several bacteria involved in infectious diseases [17] , [18] . Drobnik et al. found that an increase in ceramide levels mediated by nSMase in serum was associated with sepsis-related mortality [35] . Gonzalez-Zorn et al. reported that SMase from Listeria ivanovii was involved in avoiding phagocytic vacuoles in the bovine epithelial-cell line MDBK [15] . Heffernan et al. showed that the deletion of three phospholipase (PIPLC, PCPLC, and SMase) genes was required for the attenuation of virulence in a murine model of anthrax and that these enzymes play an important role in the growth of Bacillus anthracis in alveolar macrophages [36] . Lo et al. reported that a combination of β-hemolysin immunization and Christie Atkins Munch-Peterson factor neutralization cooperatively suppressed the skin lesions caused by a coinfection of Staphylococcus aureus and Propionibacterium acnes [37] . The result suggested the need for immunotherapy targeting the interaction of Staphylococcus aureus with a skin commensal. Therefore, our findings may provide significant opportunities for the development of new vaccines against infections caused by SMase-producing bacteria. Treatment of macrophages with SMase resulted in a reduction in the generation of H 2 O 2 and phagocytosis of macrophages induced by PGN, but no effect on the release of TNF-α and little release of LDH under our experimental conditions. Therefore, it appears that Bc -SMase specifically inhibits the activation of macrophages induced by PGN without harmful effects on the cells. The treatment with Bc -SMase is known to induce changes in the lipid content of plasma membranes by generating ceramide upon the cleavage of SM [38] . Ceramide is reported to promote the coalescence of rafts, which have been termed "membrane platforms" [39] . We reported that Bc -SMase induced the formation of ceramide-rich domains in membranes of sheep erythrocytes, and a decrease in the fluidity of membranes, leading to destabilization under physical stimulation and finally the disruption of erythrocytes [19] . The treatment of mouse macrophages with Bc -SMase caused the formation of ceramide, and inhibition of the production of H 2 O 2 and phagocytosis induced by PGN. Treatment of macrophages with ceramide significantly inhibited the generation of H 2 O 2 and phagocytosis of macrophages activated by PGN, suggesting that the formation of ceramide induced by Bc -SMase is closely related to a reduction in the activation of macrophages. Nakabo and Pabst have reported that C2- or C6-ceramide inhibited the release of superoxides from monocytes primed with lipopolysaccharide, and induced the secretion of small amounts of TNF-α and IL-1β [40] . The treatment of macrophages with Bc -SMase resulted in the clustering of ceramide recognized by a Cy3-anti-ceramide antibody. Furthermore, using a confocal laser scanning microscope, we found that the effective diffusion rate for BODIPY-ceramide at ceramide-rich domains in macrophages treated with Bc -SMase was reduced about 70%, compared with that for BODIPY-SM, ceramide-poor domains, in control cells. Goni and Alonso reported that the ceramide-rich domains formed a rigid phase in membranes [28] , [41] . Therefore, it appears that the formation of ceramide in macrophages treated with Bc -SMase results in functional differences of the membranes through the coalescence of ceramide-rich domains and formation of the interface between rigid and fluid domains, leading specifically to inhibition of the production of H 2 O 2 and phagocytosis induced by PGN. In conclusion, the hydrolysis of SM to form ceramide in the macrophage membrane treated with Bc -SMase induced the attenuation of membrane fluidity and the frustrated phagocytosis. Bc -SMase plays a crucial role in the evasion from immune response by macrophages during the early stages of infections of B. cereus . Materials and Methods Strains The clinical isolates of B. cereus (JMU-06B-31, JMU-06B-35, and JMU-06B-1) were isolated at Jichi Medical University. These isolates, obtained from patients diagnosed with Bacillus bacteremia or endophthalmitis according to the CDC definition, were identified and characterized as B. cereus , as described previously [6] . The ATCC strains of B. cereus from soil (ATCC21928, ATCC31429, ATCC6464) were purchased from DS Pharma Biomedical, Tokyo, Japan. The characteristics of the clinical isolates were reported previously [6] . Mice Six- to eight-week old male wild-type mice of the ICR and BALB/c strains (Nihon SLC, Japan) were used. Experimental protocols were approved by the Institute Animal Care and Use Committee at Tokushima Bunri University. The mice were housed in plastic cage under controlled environmental conditions (temperature 22°C, humidity 55%). Food and water were freely available. Detection of Genes Encoding Bc -SMase, PCPLC, and PIPLC The genomic DNA from various strains of B. cereus was extracted with the bacteria genomicPrep Mini Spin kit from GE healthcare (UK). The phospholipase C genes of the genomic DNA were confirmed by PCR using the primer sets described below. Bc -SMase primers were forward, 5′-CAAATGGCCAATCGCTGAA-3′ , reverse, 5′-GGTTCCTACGTACAGATGCTGGTG-3′ . PCPLC primers were forward, 5′-CTTTACAAAGCGTTGCATTTGCTC-3′ , reverse, 5′-CAATCGCACGGTTTACAATCCATA-3′ . PIPLC primers were forward, 5′-ACCTGATAGTATCCCGTTAGCACGA-3′ , reverse, 5′-CGAGCTCCATGGTCCATTTG-3′ . DNA Cloning and Sequencing The plc-smase region from B. cereus (JMU-06B-31, JMU-06B-35, and JMU-06B-1, ATCC21928, ATCC31429, ATCC6464) was obtained as a 2.1-kb DNA fragment by PCR using primer sets described below. A1 forward primer: 5′-GTATTCATTCATTATTCACTGTG-3′ , A2 reverse primer: 5′-CTACTTCATAGAAATAGTCGCCT-3′ . The fragment isolated from agarose gels was cloned into the vector pGEM-T (Promega, USA). Nucleotide sequencing of the cloned fragments was performed by the dideoxy chain termination technique with a BigDye terminator v1.1 cycle sequencing kit (Applied Biosystems, USA) using M13 reverse and forward primers. The genetic sequence was confirmed with an ABI3500 genetic analyzer (Applied Biosystems, USA). Site-directed Mutagenesis The transformer site-directed mutagenesis kit (LA PCR in vitro Mutagenesis Kit, Takara, Japan) was used with the primer E53A: 5′-GTTATTTTAAATGCCGTGTTTGATAATAGC-3′ to prepare the modified plasmid. The genetic sequence of Bc -SMase in each plasmid was confirmed with an ABI310 PRISM™ genetic analyzer (Life technologies, USA). Preparation of Bc -SMase and Variants Bc- SMase and E53A were overexpressed in B. subtilis ISW1214 or B. cereus ATCC21928 transformed with the plasmid vector pHY300PLK carrying smase or e53a . The expression and purification of the recombinant Bc -SMase and variants were performed as described previously [21] . Purification of PCPLC and PIPLC PCPLC and PIPLC were overexpressed in B. subtilis ISW1214 transformed with pHY300PLK carrying the gene of PCPLC or PIPLC cloned from B. cereus IAM1029. The PCPLC and PIPLC were secreted into the culture medium. The 80% (w/v) ammonium sulfate fraction of the Luria Broth was fractionated by chromatography using a Cu 2+ -column and then a DEAE-Sepharose column. The purity of samples was verified using SDS-PAGE, and staining with coomasie Brilliant Blue. The PCPLC and PIPLC were observed as a single band at 28 kDa and 34 kDa, respectively. Preparation of Antibodies Anti- Bc -SMase, -PCPLC, and -PIPLC antibodies were prepared by immunizing rabbits with 100 µg of the purified phospholipases. CFA (Difco, USA) (1.0 ml) was used for the preparation of antigens. Two hypodermic booster injections were made. Antiserum was obtained 2 weeks after the last injection. Purification of these antisera was performed using Ab-Rapid PuRe (Protenova, Japan). Immunization of Mice Inbred 6–8-week old male BALB/c mice were immunized subcutaneously two times at two-week intervals. The immunogens were used 25 µg mixture of PCPLC, PIPLC, or Bc -SMase with CFA. ELISA Procedure The purified recombinant PCPLC, PIPLC, or Bc -SMase was diluted to 5 µg/ml in a carbonate buffer (0.05, pH 9.5) and used to coat the wells of polystyrene plates (100 µl/well: Nunc-Immuno plates with a Maxisorp surface) The plates were incubated overnight at 4°C, and the next morning washed three times with PBST (PBS/0.05% Tween-20). The remaining sites of absorption were blocked by the addition of 200 µl/well in PBS containing 3% BSA for 2 h at 37°C. The plates were washed three times with PBST. Serum from each group of immunized animals was serially diluted 2-fold (1∶500 to 1∶128,000) and examined in triplicate wells (100 µl/well) of the blocked antigen-coated plates and incubated for 1 h at 37°C. The plates were then washed five times with PBST and further incubated at 37°C for 1 h with HRP-conjugated anti-mouse IgG (1∶2000). The plates were washed five times with PBST and developed with 100 µl of ortho-pheylenediamine (0.4 mg/ml) in a fleshly prepared citrate phosphate buffer (0.1 M, pH5.0) and H 2 O 2 (0.4 µg/ml). The reaction was terminated by the addition of 50 µl of 2.5 N H 2 SO 4 /well. Absorbance was read at 492 nm with a microtiter plate reader. Determination of ELISA Titer by Endpoint Dilution The serum was diluted 2-fold from 1∶500 to 1∶128,000, and an absorbance value was determined for each dilution. The cut-off value for the assay was calculated from the reference curve for the control serum. The titer of immune serum was calculated as the reciprocal of the highest dilution yielding a specific optical density above the cut-off value. A significant ( P <0.05) value of IgG antibody against recombinant PCPLC, PIPLC, and Bc -SMase was 128,000, 64,000, and 64,000, respectively, when compared with CFA-treated mouse serum. Measurement of Cytokines The concentration of TNF-α was determined with enzyme-linked absorbent assay kits (R&D systems, USA). Culture of Macrophages Mouse macrophages were isolated from cells in peritoneal exudates with 2 ml of phenol red-free RPMI1640 medium (Wako Pure Chemical Industries, Japan) supplemented with 5% fetal bovine serum (FBS) (Biowest, USA). After centrifugation at 170×g for 10 min at 4°C, the cell pellet was resuspended in phenol red-free RPMI1640 medium supplemented with 5% FBS. Adherent macrophage monolayers were obtained by plating the cells in 96- or 48-well plastic trays (Falcon, USA). Preparation of sheep erythrocytes Sheep erythrocytes were suspended in 0.02 M Tris-HCl buffer (pH 7.5) containing 0.9% NaCl (TBS), and centrifuged at 1,100× g for 3 min. The erythrocytes were washed by the centrifugation three times. The number of erythrocytes was determined with a cell counter (Celltac; Nihon Kohden, Japan). Determination of Hemolytic Activity Bc -SMase and E53A was incubated with sheep erythrocytes (12×10 10 cells/ml) in TBS at 37°C for 30 min, and the cells were chilled at 4°C. The hemolysis of the erythrocytes was measured, as described previously [42] . Hemolysis was expressed as a percentage of the amount of hemoglobin released from 0.1 ml of erythrocytes suspended in 0.4 ml of 0.4% NaCl. Preparation of Liposomes SM (Nacalai Tesque, Japan) from bovine brain and cholesterol (1∶1) in chloroform-methanol (2∶1 v/v) were dried with N 2 gas, resuspended in TBS containing 0.1 M calboxyfluoroscein (CF). The liposome suspensions were centrifuged at 22,000×g for 15 min at 4°C to remove the nonencapsulated marker, and washed three times by centrifugation. The resulting liposomes were suspended in 200 µl of TBS. The SM-liposome-disruption activity The SM-liposome-disruption activity was evaluated at the amount of released-CF in the test aliquot. The SM-liposomes in TBS containing 1 mM MgCl 2 were incubated with Bc -SMase or E53A for 30 min at 37°C. The wavelengths for excitation and measurement were 490 and 530 nm, respectively. SMase Activity Assay SMase activity was measured using an Amplex Red Sphingomyelinase assay kit (Invitogen, USA). Measurement of Intracellular H 2 O 2 Mouse macrophages (80% confluent in 48-well plates) isolated from mouse peritoneal exudates were activated with 5 µg/ml PGN (Sigma, USA) for 60 min in the presence of phenol red-free RPMI1640 medium (supplemented with 5% FBS). H 2 O 2 was measured in the supernatants using an H 2 O 2 assay kit (Oxis International, USA). Assay of Phagocytosis Phagocytic activity was determined by measuring the uptake of fluorescent microspheres (Fluoresbrite Carboxylate Microspheres, 1.75 µm in diameter, Polysciences), as described [34] . Mouse macrophages (80% confluent in 48-well plates) were stimulated by PGN in the presence of 5.0×10 5 fluorescent microspheres per ml. After 3 h incubation, cells were washed, and fluorescent intensity in the cells was determined with a fluorescence imaging analyzer (FLA-1000, Fujifilm, Japan). Measurement of LDH LDH activity was determined with LDH assay kits (Wako Pure Chemical Industries, Japan), according to the manufacturer's instructions. Determination of Ceramide Mouse macrophages were incubated with various concentrations of Bc -SMase at 37°C for 60 min in phenol red-free RPMI1640 medium supplemented with 5% FBS. The isolation and the measurement of ceramide were performed as described previously [42] , [43] . The ceramide, which is from bovine brain, used as stimulants or standard was purchased from Sigma, USA. Immunofluorescence Staining and Confocal Imaging Mouse macrophages stained with 2 µM BODIPY-SM were plated on 35-mm glass-bottomed dishes (MatTek, USA). The cells were incubated with Bc -SMase in phenol red-free RPMI 1640 medium supplemented with 5% FBS at 37°C for 60 min, and the reaction was stopped by 1.0% paraformaldehyde at room temperature. For antibody labeling, the cells were incubated in 50 mM NH 4 Cl in phosphate- buffered saline (PBS) at room temperature for 10 min. After being washed with PBS, the cells were incubated in PBS containing 4% BSA at room temperature for 60 min, followed by mouse Cy3-labeled-anti-ceramide antibody in PBS for 60 min. Fluorescence Microscopy A confocal fluorescence microscope (A1; Nikon, Japan) was used. The excitation wavelength was 488 nm for BODIPY FL C 12 -SM (Molecular probes, USA). The fluorescence signals were simultaneously collected using NIS-Elements C (Nikon software, Japan) into a channel using bandpass filters of 525/50. The objective used in all experiments was a 60×oil immersion, CFI Plan Apo VC 60×oil/1.40 objective (Nikon). The objective lens was used with a zoom factor of 2. The experiments were performed at room temperature. Fluorescence Recovery after Photobleaching Fluorescence recovery after photobleaching (FRAP), a technology used to measure the lateral mobility of membranes, was performed with a Nikon A1R confocal laser scanning microscope, according to the manual. Mouse macrophages stained with 2 µM BODIPY-SM were plated on 35-mm glass-bottomed dishes (MatTek, USA). The photobleaching was performed in a 1.5 µm, visually uniform region of the cell membranes. Bleaching was performed with 5% laser intensity for a duration of approximately 1 s (10 scans of the laser) to achieve 20% bleaching of the BODIPY fluorescence. After photobleaching, images were acquired 200 times at 1 s intervals. Statistics Results were expressed as the mean ± SEM. n equals the sample size. Statistical comparisons were performed using an unpaired t -test or one-way analysis of variance (ANOVA) with Bonferroni correction. p Values less than 0.05 were considered statistically significant. Strains The clinical isolates of B. cereus (JMU-06B-31, JMU-06B-35, and JMU-06B-1) were isolated at Jichi Medical University. These isolates, obtained from patients diagnosed with Bacillus bacteremia or endophthalmitis according to the CDC definition, were identified and characterized as B. cereus , as described previously [6] . The ATCC strains of B. cereus from soil (ATCC21928, ATCC31429, ATCC6464) were purchased from DS Pharma Biomedical, Tokyo, Japan. The characteristics of the clinical isolates were reported previously [6] . Mice Six- to eight-week old male wild-type mice of the ICR and BALB/c strains (Nihon SLC, Japan) were used. Experimental protocols were approved by the Institute Animal Care and Use Committee at Tokushima Bunri University. The mice were housed in plastic cage under controlled environmental conditions (temperature 22°C, humidity 55%). Food and water were freely available. Detection of Genes Encoding Bc -SMase, PCPLC, and PIPLC The genomic DNA from various strains of B. cereus was extracted with the bacteria genomicPrep Mini Spin kit from GE healthcare (UK). The phospholipase C genes of the genomic DNA were confirmed by PCR using the primer sets described below. Bc -SMase primers were forward, 5′-CAAATGGCCAATCGCTGAA-3′ , reverse, 5′-GGTTCCTACGTACAGATGCTGGTG-3′ . PCPLC primers were forward, 5′-CTTTACAAAGCGTTGCATTTGCTC-3′ , reverse, 5′-CAATCGCACGGTTTACAATCCATA-3′ . PIPLC primers were forward, 5′-ACCTGATAGTATCCCGTTAGCACGA-3′ , reverse, 5′-CGAGCTCCATGGTCCATTTG-3′ . DNA Cloning and Sequencing The plc-smase region from B. cereus (JMU-06B-31, JMU-06B-35, and JMU-06B-1, ATCC21928, ATCC31429, ATCC6464) was obtained as a 2.1-kb DNA fragment by PCR using primer sets described below. A1 forward primer: 5′-GTATTCATTCATTATTCACTGTG-3′ , A2 reverse primer: 5′-CTACTTCATAGAAATAGTCGCCT-3′ . The fragment isolated from agarose gels was cloned into the vector pGEM-T (Promega, USA). Nucleotide sequencing of the cloned fragments was performed by the dideoxy chain termination technique with a BigDye terminator v1.1 cycle sequencing kit (Applied Biosystems, USA) using M13 reverse and forward primers. The genetic sequence was confirmed with an ABI3500 genetic analyzer (Applied Biosystems, USA). Site-directed Mutagenesis The transformer site-directed mutagenesis kit (LA PCR in vitro Mutagenesis Kit, Takara, Japan) was used with the primer E53A: 5′-GTTATTTTAAATGCCGTGTTTGATAATAGC-3′ to prepare the modified plasmid. The genetic sequence of Bc -SMase in each plasmid was confirmed with an ABI310 PRISM™ genetic analyzer (Life technologies, USA). Preparation of Bc -SMase and Variants Bc- SMase and E53A were overexpressed in B. subtilis ISW1214 or B. cereus ATCC21928 transformed with the plasmid vector pHY300PLK carrying smase or e53a . The expression and purification of the recombinant Bc -SMase and variants were performed as described previously [21] . Purification of PCPLC and PIPLC PCPLC and PIPLC were overexpressed in B. subtilis ISW1214 transformed with pHY300PLK carrying the gene of PCPLC or PIPLC cloned from B. cereus IAM1029. The PCPLC and PIPLC were secreted into the culture medium. The 80% (w/v) ammonium sulfate fraction of the Luria Broth was fractionated by chromatography using a Cu 2+ -column and then a DEAE-Sepharose column. The purity of samples was verified using SDS-PAGE, and staining with coomasie Brilliant Blue. The PCPLC and PIPLC were observed as a single band at 28 kDa and 34 kDa, respectively. Preparation of Antibodies Anti- Bc -SMase, -PCPLC, and -PIPLC antibodies were prepared by immunizing rabbits with 100 µg of the purified phospholipases. CFA (Difco, USA) (1.0 ml) was used for the preparation of antigens. Two hypodermic booster injections were made. Antiserum was obtained 2 weeks after the last injection. Purification of these antisera was performed using Ab-Rapid PuRe (Protenova, Japan). Immunization of Mice Inbred 6–8-week old male BALB/c mice were immunized subcutaneously two times at two-week intervals. The immunogens were used 25 µg mixture of PCPLC, PIPLC, or Bc -SMase with CFA. ELISA Procedure The purified recombinant PCPLC, PIPLC, or Bc -SMase was diluted to 5 µg/ml in a carbonate buffer (0.05, pH 9.5) and used to coat the wells of polystyrene plates (100 µl/well: Nunc-Immuno plates with a Maxisorp surface) The plates were incubated overnight at 4°C, and the next morning washed three times with PBST (PBS/0.05% Tween-20). The remaining sites of absorption were blocked by the addition of 200 µl/well in PBS containing 3% BSA for 2 h at 37°C. The plates were washed three times with PBST. Serum from each group of immunized animals was serially diluted 2-fold (1∶500 to 1∶128,000) and examined in triplicate wells (100 µl/well) of the blocked antigen-coated plates and incubated for 1 h at 37°C. The plates were then washed five times with PBST and further incubated at 37°C for 1 h with HRP-conjugated anti-mouse IgG (1∶2000). The plates were washed five times with PBST and developed with 100 µl of ortho-pheylenediamine (0.4 mg/ml) in a fleshly prepared citrate phosphate buffer (0.1 M, pH5.0) and H 2 O 2 (0.4 µg/ml). The reaction was terminated by the addition of 50 µl of 2.5 N H 2 SO 4 /well. Absorbance was read at 492 nm with a microtiter plate reader. Determination of ELISA Titer by Endpoint Dilution The serum was diluted 2-fold from 1∶500 to 1∶128,000, and an absorbance value was determined for each dilution. The cut-off value for the assay was calculated from the reference curve for the control serum. The titer of immune serum was calculated as the reciprocal of the highest dilution yielding a specific optical density above the cut-off value. A significant ( P <0.05) value of IgG antibody against recombinant PCPLC, PIPLC, and Bc -SMase was 128,000, 64,000, and 64,000, respectively, when compared with CFA-treated mouse serum. Measurement of Cytokines The concentration of TNF-α was determined with enzyme-linked absorbent assay kits (R&D systems, USA). Culture of Macrophages Mouse macrophages were isolated from cells in peritoneal exudates with 2 ml of phenol red-free RPMI1640 medium (Wako Pure Chemical Industries, Japan) supplemented with 5% fetal bovine serum (FBS) (Biowest, USA). After centrifugation at 170×g for 10 min at 4°C, the cell pellet was resuspended in phenol red-free RPMI1640 medium supplemented with 5% FBS. Adherent macrophage monolayers were obtained by plating the cells in 96- or 48-well plastic trays (Falcon, USA). Preparation of sheep erythrocytes Sheep erythrocytes were suspended in 0.02 M Tris-HCl buffer (pH 7.5) containing 0.9% NaCl (TBS), and centrifuged at 1,100× g for 3 min. The erythrocytes were washed by the centrifugation three times. The number of erythrocytes was determined with a cell counter (Celltac; Nihon Kohden, Japan). Preparation of sheep erythrocytes Sheep erythrocytes were suspended in 0.02 M Tris-HCl buffer (pH 7.5) containing 0.9% NaCl (TBS), and centrifuged at 1,100× g for 3 min. The erythrocytes were washed by the centrifugation three times. The number of erythrocytes was determined with a cell counter (Celltac; Nihon Kohden, Japan). Determination of Hemolytic Activity Bc -SMase and E53A was incubated with sheep erythrocytes (12×10 10 cells/ml) in TBS at 37°C for 30 min, and the cells were chilled at 4°C. The hemolysis of the erythrocytes was measured, as described previously [42] . Hemolysis was expressed as a percentage of the amount of hemoglobin released from 0.1 ml of erythrocytes suspended in 0.4 ml of 0.4% NaCl. Preparation of Liposomes SM (Nacalai Tesque, Japan) from bovine brain and cholesterol (1∶1) in chloroform-methanol (2∶1 v/v) were dried with N 2 gas, resuspended in TBS containing 0.1 M calboxyfluoroscein (CF). The liposome suspensions were centrifuged at 22,000×g for 15 min at 4°C to remove the nonencapsulated marker, and washed three times by centrifugation. The resulting liposomes were suspended in 200 µl of TBS. The SM-liposome-disruption activity The SM-liposome-disruption activity was evaluated at the amount of released-CF in the test aliquot. The SM-liposomes in TBS containing 1 mM MgCl 2 were incubated with Bc -SMase or E53A for 30 min at 37°C. The wavelengths for excitation and measurement were 490 and 530 nm, respectively. The SM-liposome-disruption activity The SM-liposome-disruption activity was evaluated at the amount of released-CF in the test aliquot. The SM-liposomes in TBS containing 1 mM MgCl 2 were incubated with Bc -SMase or E53A for 30 min at 37°C. The wavelengths for excitation and measurement were 490 and 530 nm, respectively. SMase Activity Assay SMase activity was measured using an Amplex Red Sphingomyelinase assay kit (Invitogen, USA). Measurement of Intracellular H 2 O 2 Mouse macrophages (80% confluent in 48-well plates) isolated from mouse peritoneal exudates were activated with 5 µg/ml PGN (Sigma, USA) for 60 min in the presence of phenol red-free RPMI1640 medium (supplemented with 5% FBS). H 2 O 2 was measured in the supernatants using an H 2 O 2 assay kit (Oxis International, USA). Assay of Phagocytosis Phagocytic activity was determined by measuring the uptake of fluorescent microspheres (Fluoresbrite Carboxylate Microspheres, 1.75 µm in diameter, Polysciences), as described [34] . Mouse macrophages (80% confluent in 48-well plates) were stimulated by PGN in the presence of 5.0×10 5 fluorescent microspheres per ml. After 3 h incubation, cells were washed, and fluorescent intensity in the cells was determined with a fluorescence imaging analyzer (FLA-1000, Fujifilm, Japan). Measurement of LDH LDH activity was determined with LDH assay kits (Wako Pure Chemical Industries, Japan), according to the manufacturer's instructions. Determination of Ceramide Mouse macrophages were incubated with various concentrations of Bc -SMase at 37°C for 60 min in phenol red-free RPMI1640 medium supplemented with 5% FBS. The isolation and the measurement of ceramide were performed as described previously [42] , [43] . The ceramide, which is from bovine brain, used as stimulants or standard was purchased from Sigma, USA. Immunofluorescence Staining and Confocal Imaging Mouse macrophages stained with 2 µM BODIPY-SM were plated on 35-mm glass-bottomed dishes (MatTek, USA). The cells were incubated with Bc -SMase in phenol red-free RPMI 1640 medium supplemented with 5% FBS at 37°C for 60 min, and the reaction was stopped by 1.0% paraformaldehyde at room temperature. For antibody labeling, the cells were incubated in 50 mM NH 4 Cl in phosphate- buffered saline (PBS) at room temperature for 10 min. After being washed with PBS, the cells were incubated in PBS containing 4% BSA at room temperature for 60 min, followed by mouse Cy3-labeled-anti-ceramide antibody in PBS for 60 min. Fluorescence Microscopy A confocal fluorescence microscope (A1; Nikon, Japan) was used. The excitation wavelength was 488 nm for BODIPY FL C 12 -SM (Molecular probes, USA). The fluorescence signals were simultaneously collected using NIS-Elements C (Nikon software, Japan) into a channel using bandpass filters of 525/50. The objective used in all experiments was a 60×oil immersion, CFI Plan Apo VC 60×oil/1.40 objective (Nikon). The objective lens was used with a zoom factor of 2. The experiments were performed at room temperature. Fluorescence Recovery after Photobleaching Fluorescence recovery after photobleaching (FRAP), a technology used to measure the lateral mobility of membranes, was performed with a Nikon A1R confocal laser scanning microscope, according to the manual. Mouse macrophages stained with 2 µM BODIPY-SM were plated on 35-mm glass-bottomed dishes (MatTek, USA). The photobleaching was performed in a 1.5 µm, visually uniform region of the cell membranes. Bleaching was performed with 5% laser intensity for a duration of approximately 1 s (10 scans of the laser) to achieve 20% bleaching of the BODIPY fluorescence. After photobleaching, images were acquired 200 times at 1 s intervals. Statistics Results were expressed as the mean ± SEM. n equals the sample size. Statistical comparisons were performed using an unpaired t -test or one-way analysis of variance (ANOVA) with Bonferroni correction. p Values less than 0.05 were considered statistically significant. Supporting Information Figure S1 Detection of genes encoding Bc -SMase, PCPLC, and PIPLC. The various strains of B. cereus were determined for mRNA of Bc -SMase, PCPLC, and PIPLC. (TIF) Click here for additional data file. Figure S2 The amino acid sequence alignment of Bc -SMase from clinical isolates or ATCC strains. The amino acid sequences of Bc -SMase from clinical isolates (JMU-06B-1, 31, 35) or ATCC strains (ATCC21928, 31429, 6464) were aligned by the program T-Coffee. The sequences of signal peptide are indicated in bold type. Gray areas indicate amino acid sequence differences. The amino acid residues participating in the enzymatic activity are shown by black circles. (TIF) Click here for additional data file. Table S1 Biological activities of E53A. Activity (%) was expressed as the percentage of each activity in the wild-type enzyme. Each value is the mean of five experiments. (DOCX) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2601556/

A Combination of Flt3 Ligand cDNA and CpG ODN as Nasal Adjuvant Elicits NALT Dendritic Cells for Prolonged Mucosal Immunity

Summary We explore cellular and molecular mechanisms of nasal adjuvant of a combination of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG oligodeoxynucleotides (CpG ODN). The double DNA adjuvant given with OVA maintained prolonged OVA-specific secretory IgA (S-IgA) Ab responses in external secretions for more than twenty-five weeks after the final immunization. Further, both Th1- and Th2-type cytokine responses were induced by this combined adjuvant regimen. The frequencies of plasmacytoid DCs (pDCs) and CD8 + DCs were significantly increased in nasopharyngeal-associated lymphoreticular tissue (NALT) of mice given the combined adjuvant. Importantly, when we examined adjuvanticity of pFL plus CpG ODN in 2-year-old mice, significant levels of mucosal IgA Ab responses were also induced. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for the development of an effective mucosal vaccine for the elderly.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9476378/

Famous traditional Mongolian medicine Xieriga-4 (Turmeric-4) decoction: A review

Xieriga-4 Decoction, composed of dried rhizomes of Curcumae longae , barks of Phellodendron chinense or Phellodendron amurense , fruits of Cardenia jasminoides , and fruits of Tribulus terrestris , is a famous prescription of traditional Mongolian medicine for the treatment of urinary system diseases such as frequent urination, urgent urination, urine occlusion, hematuria, bladder irritation and pain. This paper reviewed Xieriga-4 Decoction from the aspects of historical description, prescription principle, chemical components, pharmacology, clinical application and quality control. 1 Introduction Mongolian medicine is an empirical medicine gradually accumulated and inherited by Mongolians during their struggle with nature and disease over the long course of history. In the long-term medication practice, a systematic and complete theoretical system has been gradually formed during its development through unique drug resources, processing technique, administration method, continuous absorption, and theoretical development. It is not only a valuable cultural heritage of Mongolia, but also an indispensable part of traditional Chinese medicine. Xieriga-4 Decoction, also known as Mongolian transliteration, from classics Tongwagajid , and refers to a prescription composed of four drugs such as turmeric. In the Mongolian Medicine Volume of the Drug Standard of Ministry of Public Health of the Peoples Republic of China , it is prepared from four drugs, including rhizomes of Curcuma longa L., ripe fruits of Tribulus terrestris L., ripe fruits of Gardenia jasminoides Ellis, and barks of Phellodendron chinense Schneid. or Phellodendron amurense Rupr . by extraction, concentration and drying. With functions of dieresis and clearing away damp-heat, the medicine is mainly used to treat urine occlusion, frequent urination, urgent urination, hematuria and bladder irritation and pain. It is the primary choice in treating the heat syndrome of the bladder. Recent studies on the historical origin, prescription analysis, chemical composition, pharmacological action, clinical application, and quality control of Xieriga-4 Decoction are reviewed to provide a theoretical basis for its further clinical application. Let it be added here that Xieriga-4 (Yongwa-4) has two different prescriptions in Mongolian medicine. In addition to Xieriga-4 from the Tongwagajid reviewed in this paper, there is also a prescription contained in the Encyclopedia of Chinese Medicine – Mongolian Medicine (first) (1986 Edition) edited by Qingyun Bai and the Clinical Research of Mongolian Medicine (Mongolian Edition, Volume II) edited by Professor Surongzab. It has the same name as Xieriga-4 Decoction. The prescription also takes turmeric as the main drug, but the other components are different (croton-dried ripe fruits of Croton tiglium L., Rabiagar-sulphide mineral- Realgar , grasshopper – the head of Locusta migratoria L.) as pills. The efficacy (detoxification, killing viscosity, anti severe diarrhea, and detumescence) and main diseases (diphtheria, anthrax, rabies, carbuncle caused by infectious diseases, etc.) are also different from the decoction in this paper ( Bai, 1986 , Surongzab, 1999 ). 2 Historical description Xieriga-4 decoction was first recorded in the classic Medical Canon in Four Sections . It is an authoritative reference book of Tibetan medicine, known as the encyclopedia of Tibetan medicine with the most systematic, complete and fundamental theoretical system of Tibetan medicine. It was completed in the second half of the 8th century CE and written by the famous Tibetan medical expert Yutok Yonden Gonbu, including 156 chapters in four parts. According to the encyclopedia, the medicine is mainly used to treat frequent urination and is composed of Curcumae Longae Rhizoma (turmeric), Berberidis Cortex , Phyllanthi Fructus , Tribuli Fructus ( Gonbu, 8th century ). However, the formula recorded in this book is slightly different from that in Mongolian medicine. Four Nectar Treatises (Chapter 30 of Volume II) recorded the treatment of frequent urination with Yongwa-4, Xieriga-4's another name, which was the first time for its name to appear in historical records ( 'Byor, 18th century ). Obidasen Dalai (Chapter 90) included this prescription in the treatment of frequent urination: Xieriga-4 Decoction (Yongwa Decoction)-5 qian (a traditional unit of weight in China, 1 qian = 5 g) of Curcumae Longae Rhizoma (turmeric), Pnellodendri Chinensis Cortex and Gardeniae Fructus , respectively, 7 qian of Tribuli Fructus , make decoction and take several times. The book first recorded the dosage of the ingredients in the prescription ( Zhanbula, 1829 ). Tongwagajid (Part 3, Chapter 56) also recorded the use of Yongwa-4 decoction for the treatment of frequent urination, but with different dosage of each component, i.e., 5 qian of Curcumae Longae Rhizoma (turmeric), 3 qian of Phellodendri Chinensis Cortex , 4 qian of Gardeniae Fructus , and 5 qian of Tribuli Fructus (Part 5) ( Jigmud, 1888 ). So far, the composition of Xieriga-4 that we have used is recorded in this book, so we generally believe that Xieriga-4 comes from the Tongwagajid . The above prescription enhanced clinical efficacy for a long time, especially in treating urinary system diseases such as frequent urination with damp-heat (gonorrhea) and urinary tract infection, boosting its application since ancient times. It was explored in depth in the 20th century, as it attracted the attention of many scholars, and was included in Jilin Province Drug Standard ( Jilin Provincial Health Bureau, 1977 ). Standards for Mongolian Patent Medicine in Inner Mongolia ( Standards for Mongolian Patent Medicine in Inner Mongolia , 1984). Mongolian Medicine Volume of the Drug Standard of Ministry of Public Health of the Peoples Republic of China ( Commission, 1998 ) becoming a national registered standard preparation ( Fig.1 ). Fig. 1 Historical description books of Xieriga-4. Medical Canon in Four Sections (A): It is an authoritative reference book of Tibetan medicine. It was written by the famous Tibetan medical expert Yutok Yonden Gonbu in the second half of the 8th century A. D. Four Nectar Treatises (B): It is four medical works by the Mongolian physician Ye Shes Dpal 'Byor in the 18th century during his more than 50 years of medical practice and research career. Obidasen Dalai (C): It was written by Zhanbula, a famous Mongolian pharmacist from the 18th to 19th centuries. It is a complete and systematic monograph on Mongolian pharmacy. Tongwagjid (D): Tongwagajid is the Tibetan name of this book, which is also known as Traditional Prescription of Mongolian Medicine and Joy of the Viewer . Works on clinical prescriptions of Mongolian medicine. At the beginning of the 20th century, it was written in Tibetan by Jigmud Danjin Zamsu, a pharmacist in East Sunit banner, Xilingol League, Inner Mongolia. 3 Prescription principles Urinary tract infection is urinary tract inflammation caused by pathogens invading urinary tract mucosa. In Mongolian medicine, it is called "Xijing" (equivalent to urinary tract infections in modern medicine). It is believed that the main cause of the disease is "Muo Qisu" (You can think of it as blood with disease), excessive "Xierin Halun" (Mongolian medicine has a unique theoretical system, the core of which is the theory of Heyi, Xieri, and Badgan. Being referred to as "three roots or three essence", they run through the whole process of Mongolian medicine pathophysiology, etiology and pathogenesis, syndrome differentiation and treatment, and protecting against evil and keeping healthy. Therefore, they are the essence of Mongolian medicine theory. We can understand that Heyi is the gaseous substance of human body, Xieri is a flammable substance, and Badgan refers to the viscous substance of human body, respectively, and thus Xierin Halun can be seen as the heat of the body.), and local redness by "sticky poison", swelling, and pain, resulting in the functional disorder of "Down cleared-Hey" (There are five types of Heyi, and Down cleared-Heyi is responsible for the body's excretory function) in charge of the urinary tract. The symptoms include urethral tingling, dripping discomfort, frequent urination and other symptoms. Xieriga-4 is mainly made of Curcumae Longae Rhizoma (turmeric, rhizomes of C. longa ), Mongolian medicine believed it can kill stickiness, prevent rot, detoxify, and these correspond to the anti-inflammatory, detoxification and anti-infection effects of modern medicine. Xieriga-4 is supplemented by Tribuli Fructus (ripe fruits of T. terrestris ) with diuretic and detumescence effects and supplemented by Phellodendri Chinensis Cortex (barks of P. chinense or P. amurense ) and Gardeniae Fructus (ripe fruits of G. jasminoides ). The combination of all drugs has the effects of killing viscosity, removing heat and diuresis, which are equivalent to anti-inflammatory, diuretic and analgesic in western medicine. Miraculously, the main component of the prescription removes the main cause of "Muo Qisu", "Xierin Halun", and "Nieyi" (similar to bacteria and viruses in modern medicine) of the disease; the auxiliary drug of Gardeniae Fructus heals the disease symptoms. The auxiliary component can improve the prognosis of the disease, removes the root of the disease and achieves the effect of preventing disease recurrence. As mentioned above, although the composition of the prescription is simplified, the components are perfectly matched, and the therapeutic purpose is accurately achieved for the etiology, symptoms and prognosis of the disease. It is in line with not only the law of drug compatibility in Mongolian medicine theory, but also the precision medical policy. In short, it is a drug worthy of in-depth study. 4 Chemical components 4.1 Curcumae Longae Rhizoma (Turmeric) Curcumae Longae Rhizoma (turmeric) also known as "Xieriga" in Mongolian, is the main component of Xieriga-4 decoction. It is the rhizomes of C. longa mainly containing phenolic pigments (curcumin, demethoxycurcumin, bisdemethoxycurcumin, dihydrocurcumin, etc. ( Zhang, Oldenqimuge, Unierjirigala, & Alatenqimuge, 2021 ), volatile oil including α -turmerone, β -turmerone, α -pinene, β -pinene, thujone, ar -turmerone, curdione, limonene, isoborneol, 2-octanol, terpinen-4-ol, etc., carbohydrate such as stigmasterol, β -sitosterol, fatty acids, monoenoic acids, dienoic acid, p -coumaroylferuloylmethane, di- p -doumaroylmethane, etc., and others ( Lu & Mou, 2017 , Yan et al., 2021 ). Curcumin in turmeric is one of the characteristic active components known to dominate the drug action of this prescription and is categorized as a polyphenolic compound ( Moghadamtousi et al., 2014 ). Curcumin ( Fig. 2 ) has significant antibacterial and antiviral functions. It is also the main pharmacodynamic effect of Xieriga-4 decoction ( Fig. 3 ). Fig. 2 Chemical structures of main chemical components in each component. Fig. 3 Pictures of each component in Xieriga-4. Curcumae Longae Rhizoma (turmeric) (from China medical information platform, https://www.dayi.org.cn ). (A): dried rhizomes of C. longa ; Gardeniae Fructus (B): dried ripe fruit of G. jasminoides ; Tribuli Fructus (C): dried fruit of T. terrestris . Phellodendri Chinensis Cortex (D): dried barks of P. chinense or P. amurense. 4.2 Tribuli Fructus Tribuli Fructus also known as ImanJangu in Mongolian, is an adjuvant of Xieriga-4 Decoction, it is the ripe fruits of T. terrestris . Tribuli Fructus contains steroidal saponins, β -sitosterol, stigmasterol, flavonoids which are more abundant in the leaves and rhizomes of T. terrestris than in its fruits, alkaloids such as harman, harmine, harmol, β -carboline, norharmane, N - p -hydroxyacetophenyl-3-methoxy-4-hydroxy substituted cinnamamide, N - trans -feruloyltyramine, N - trans - p -coumaroyltyramine, terrestriamide, tribulusamides A and B, etc., volatile oil, fatty oil, tannin resin, inorganic salt, T. terrestris polysaccharides that are less in processed fruits, amino acids, etc ( Ren, Zhou, & Wang, 2019 ). The steroidal saponins, the main components of T. terrestris , are mainly of furostanol and spirostanol types ( Fig. 2 ). These compounds have significant anti-inflammatory and antibacterial effects ( Stefanescu, Tero-Vescan, Negroiu, Aurica, & Vari, 2020 ). 4.3 Gardeniae Fructus Gardeniae Fructus , also known as Zhurura in Mongolian, is an adjuvant of Xierigar-4 Decoction. It is ripe fruits of G. jasminoides . It contains iridoid glycosides, diterpenes, triterpenoids including crocin, crocetin, and its homologues, organic acids including chlorogenic acid, 3,4-di- O -caffeoylquinic acids, 3- O -caffeoyl-4- O -sina poylqouinic acid, etc., pigments such as water-soluble carotenoids, volatile components, saponins, lignans, glycoproteins, etc. ( Zhou et al., 2017 ). Geniposide is the index and main effective component of G. jasminoides ( Fig. 2 ), which is categorized as iridoid glycosides. Moreover, it has strong anti-inflammatory effect ( Shan et al., 2017 ). 4.4 Phellodendri Chinensis Cortex Phellodendri Chinensis Cortex , also known as Xier Modun Dors in Mongolian, is an adjuvant of Xieriga-4 powder, and it is bark of P. chinense or P. amurense. It contains alkaloids such as berberine, jatrorrhizine, magnoflorine, phellodendrine, candicine, palmatine, and dauricine, flavonoids, terpenoids most of which are triterpenoid compounds including obacunone, obaculactone, obacunonic acid, which are also important components of P. chinense and P. amurense sterols such as 7-dehydrostigmasterol, β -sitosterol, campesterol, etc., fatty acids, volatile, amides and other compounds ( Tian, 2020 ). Berberine is one of the characteristic active components known to dominate the drug action of this prescription ( Fig. 2 ). Besides, other alkaloids can effectively inhibit bacteria and inflammation. They all have inhibitory effects on urinary tract infection ( Sun, Lenon, & Yang, 2019 ). Besides, there are limonoid triterpenoids including obacunone, obaculactone, obacunonic acid, sterols including 7-dehydrostigmasterol, β -sitosterol, campesterol, etc. 4.1 Curcumae Longae Rhizoma (Turmeric) Curcumae Longae Rhizoma (turmeric) also known as "Xieriga" in Mongolian, is the main component of Xieriga-4 decoction. It is the rhizomes of C. longa mainly containing phenolic pigments (curcumin, demethoxycurcumin, bisdemethoxycurcumin, dihydrocurcumin, etc. ( Zhang, Oldenqimuge, Unierjirigala, & Alatenqimuge, 2021 ), volatile oil including α -turmerone, β -turmerone, α -pinene, β -pinene, thujone, ar -turmerone, curdione, limonene, isoborneol, 2-octanol, terpinen-4-ol, etc., carbohydrate such as stigmasterol, β -sitosterol, fatty acids, monoenoic acids, dienoic acid, p -coumaroylferuloylmethane, di- p -doumaroylmethane, etc., and others ( Lu & Mou, 2017 , Yan et al., 2021 ). Curcumin in turmeric is one of the characteristic active components known to dominate the drug action of this prescription and is categorized as a polyphenolic compound ( Moghadamtousi et al., 2014 ). Curcumin ( Fig. 2 ) has significant antibacterial and antiviral functions. It is also the main pharmacodynamic effect of Xieriga-4 decoction ( Fig. 3 ). Fig. 2 Chemical structures of main chemical components in each component. Fig. 3 Pictures of each component in Xieriga-4. Curcumae Longae Rhizoma (turmeric) (from China medical information platform, https://www.dayi.org.cn ). (A): dried rhizomes of C. longa ; Gardeniae Fructus (B): dried ripe fruit of G. jasminoides ; Tribuli Fructus (C): dried fruit of T. terrestris . Phellodendri Chinensis Cortex (D): dried barks of P. chinense or P. amurense. 4.2 Tribuli Fructus Tribuli Fructus also known as ImanJangu in Mongolian, is an adjuvant of Xieriga-4 Decoction, it is the ripe fruits of T. terrestris . Tribuli Fructus contains steroidal saponins, β -sitosterol, stigmasterol, flavonoids which are more abundant in the leaves and rhizomes of T. terrestris than in its fruits, alkaloids such as harman, harmine, harmol, β -carboline, norharmane, N - p -hydroxyacetophenyl-3-methoxy-4-hydroxy substituted cinnamamide, N - trans -feruloyltyramine, N - trans - p -coumaroyltyramine, terrestriamide, tribulusamides A and B, etc., volatile oil, fatty oil, tannin resin, inorganic salt, T. terrestris polysaccharides that are less in processed fruits, amino acids, etc ( Ren, Zhou, & Wang, 2019 ). The steroidal saponins, the main components of T. terrestris , are mainly of furostanol and spirostanol types ( Fig. 2 ). These compounds have significant anti-inflammatory and antibacterial effects ( Stefanescu, Tero-Vescan, Negroiu, Aurica, & Vari, 2020 ). 4.3 Gardeniae Fructus Gardeniae Fructus , also known as Zhurura in Mongolian, is an adjuvant of Xierigar-4 Decoction. It is ripe fruits of G. jasminoides . It contains iridoid glycosides, diterpenes, triterpenoids including crocin, crocetin, and its homologues, organic acids including chlorogenic acid, 3,4-di- O -caffeoylquinic acids, 3- O -caffeoyl-4- O -sina poylqouinic acid, etc., pigments such as water-soluble carotenoids, volatile components, saponins, lignans, glycoproteins, etc. ( Zhou et al., 2017 ). Geniposide is the index and main effective component of G. jasminoides ( Fig. 2 ), which is categorized as iridoid glycosides. Moreover, it has strong anti-inflammatory effect ( Shan et al., 2017 ). 4.4 Phellodendri Chinensis Cortex Phellodendri Chinensis Cortex , also known as Xier Modun Dors in Mongolian, is an adjuvant of Xieriga-4 powder, and it is bark of P. chinense or P. amurense. It contains alkaloids such as berberine, jatrorrhizine, magnoflorine, phellodendrine, candicine, palmatine, and dauricine, flavonoids, terpenoids most of which are triterpenoid compounds including obacunone, obaculactone, obacunonic acid, which are also important components of P. chinense and P. amurense sterols such as 7-dehydrostigmasterol, β -sitosterol, campesterol, etc., fatty acids, volatile, amides and other compounds ( Tian, 2020 ). Berberine is one of the characteristic active components known to dominate the drug action of this prescription ( Fig. 2 ). Besides, other alkaloids can effectively inhibit bacteria and inflammation. They all have inhibitory effects on urinary tract infection ( Sun, Lenon, & Yang, 2019 ). Besides, there are limonoid triterpenoids including obacunone, obaculactone, obacunonic acid, sterols including 7-dehydrostigmasterol, β -sitosterol, campesterol, etc. 5 Pharmacology 5.1 Diuresis and detumescence Gao et al. studied the effect of Xieriga-4 on the urine volume and the number of urination (by filter paper weighing method) in rats and mice, and the results showed that the total urine volume at 4 h in the Xieriga group was significantly increased compared with that in the hydrochlorothiazide and Bilinqing capsule groups. The experiment also found that its diuretic effect of Xieriga group is quick (1–2 h) and long-lasting ( Gao, Xing, & Dong, 2015 ). Studies collected the urine by the bell-jar process for testing and measured the swelling degree by carrageenan-induced paw swelling method in experimental rats intervened by Xieriga-4 Decoction powder, tested the urine volume by filter paper weighing method, and measured the swelling degree by xylene-induced ear swelling method in experimental mice. The experimental results showed that the urine volume of rats in the Xieriga-4 Decoction powder group saw a significant increase after 1, 2, 3 and 4 h of administration (most significant at 2–3 h). Xieriga-4 Decoction powder had a similar diuretic effect with the control group (Relinqing granule), with a later effect and longer duration. The results of the ear swelling test showed that the anti-inflammation and detumescence effects of Xieriga-4 Decoction were significantly better than that of the control group. The results of the paw swelling test showed that Xieriga-4 Decoction had a significantly increased detumescence effect 4 h after the intervention ( Qing, 2013 , Qing and Bagenna, 2009 , Shuang, Wei, Qing, & Han, 2015 ). 5.2 Renal protection After continuous administration of Xieriga-4 Decoction for 14 days for the gentamicin-induced renal injury model, Li et al. (2019) found that, compared with the model group, Xieriga-4 Decoction could effectively reduce kidney index (KI) and prostate index (PI) levels, serum creatinine (Scr) and blood urea nitrogen (BUN) levels in the serum, and urinary protein (UP), N -acetyl-beta- D -glucoaminidase (NAG), and kidney injury molecule-1 (KIM-1) expression levels in the urine of rats. Pathological sections showed that glomerular mesangial cell proliferation improved significantly and tubular epithelial cell shedding significantly inhibited in the Xieriga-4 Decoction group. Researchers applied Xieriga-4 Decoction of four drugs combined with Sugmel-10 and Narenmandula-11 to diagnose and treat diabetic nephropathy (DN) based on syndrome differentiation. They found that the combined administration of the three prescriptions for one day could effectively inhibit renal injury. The study also revealed that its renal protective mechanism might be associated with pathways such as Matrix metalloproteinase-2 (MMP-2) and Transforming growth factor-β (TGF-β) ( Wang, Li, Liu, Wang, & Wei, 2015 ). 5.3 Anti-inflammation and labor pain Zhang (2019) found that the levels of tumor necrosis factor-α (TNF-α), prostate specific antigen (PSA), interleukin-4 (IL-4), and prostaglandin E2 (PGE2) factors in the serum of experimental rats were significantly reduced in the Xieriga-4 group compared with the model group, and HE staining of prostate tissues revealed that the number of inflammatory cells was significantly decreased and inflammatory characteristics alleviated considerably in the Xieriga-4 group compared with the model group. Qu et al. found that Xieriga granule effectively inhibited not only acetic acid-induced abdominal and hot plate irritation and pain, but also xylene-induced inflammation by writhing and hot plate test and xylene-induced inflammation test in mice ( Qu, Altanqimeg, & Odunqimeg, 2018 ). 5.4 Bacteriostasis Qu also found that Xieriga-4 could significantly inhibit Staphylococcus aureus and Escherichia coli in vitro , but with no significant protective effect on intraperitoneal inoculation of E. coli in mice through bacteriostasis test in vivo and in vitro in mice ( Qu et al., 2018 ). Durina and Wurina (2013) performed bacterial and fungal inhibition experiments on Xieriga lotion by the test tube and cup-plate methods. They found that the lotion could effectively inhibit such fungi as Microsporum lauosum , M. gypseum , M. canis , Trichophyton rubrum , Candida albicans , and Malassezia . The results of inhibition of bacteria showed that for the inhibition of G- bacteria: the lotion could effectively inhibit Pseudomonas aeruginosa (with a stronger effect on gentamicin); it was ineffective against Bacterium vulgare and resistant E. coli (20% ethanol applied in the control group also had no inhibitory effect on the above two strains). For the inhibition of G + bacteria: the lotion could significantly inhibit Staphylococcus aureus , Bacillus anthracis , Staphylococcus albus , etc. (with a stronger effect on penicillin, while 20% ethanol applied in the control group had no inhibitory effect on the above strains). Wurina et al. studied Xieriga lotion by conducting in vitro fungal inhibition tests in culture media and found that the lotion could effectively inhibit fungi: M. gypseum (with an effective rate of 100%), M. lauosum (94%), Candida albicans (100%), and Trichophyton rubrum (93%) ( Wurina & Gao, 2013 ). In addition, an article on pharmacology of various component networks of Xieriga-4 reported 12 targets such as estrogen receptor, Bcl-2, COX-2, S5AR, ALR2, AR, PAP, IL-6, IL-8, aromatase, etc. associated with prostate hyperplasia and prostate cancer ( Bai, Li, Dong, & Zhang, 2018 ). They also found that Xieriga-4 may regulate the expression of prostate CA-related target genes through Wnt/β-catenin and AR signaling pathways. It is precisely because Xieriga-4 Decoction has the above-mentioned pharmacological effects corresponding to nephritis, cystitis, prostatic hyperplasia, urinary tract infection and other urinary infectious disease, for example, diuresis and detumescence, anti-inflammation and labor pain, bacteriostasis, renal protection, as well as its advantages of high safety and small toxic and side effects, which shows an excellent therapeutic effect in urinary diseases, especially infectious urinary diseases. 5.1 Diuresis and detumescence Gao et al. studied the effect of Xieriga-4 on the urine volume and the number of urination (by filter paper weighing method) in rats and mice, and the results showed that the total urine volume at 4 h in the Xieriga group was significantly increased compared with that in the hydrochlorothiazide and Bilinqing capsule groups. The experiment also found that its diuretic effect of Xieriga group is quick (1–2 h) and long-lasting ( Gao, Xing, & Dong, 2015 ). Studies collected the urine by the bell-jar process for testing and measured the swelling degree by carrageenan-induced paw swelling method in experimental rats intervened by Xieriga-4 Decoction powder, tested the urine volume by filter paper weighing method, and measured the swelling degree by xylene-induced ear swelling method in experimental mice. The experimental results showed that the urine volume of rats in the Xieriga-4 Decoction powder group saw a significant increase after 1, 2, 3 and 4 h of administration (most significant at 2–3 h). Xieriga-4 Decoction powder had a similar diuretic effect with the control group (Relinqing granule), with a later effect and longer duration. The results of the ear swelling test showed that the anti-inflammation and detumescence effects of Xieriga-4 Decoction were significantly better than that of the control group. The results of the paw swelling test showed that Xieriga-4 Decoction had a significantly increased detumescence effect 4 h after the intervention ( Qing, 2013 , Qing and Bagenna, 2009 , Shuang, Wei, Qing, & Han, 2015 ). 5.2 Renal protection After continuous administration of Xieriga-4 Decoction for 14 days for the gentamicin-induced renal injury model, Li et al. (2019) found that, compared with the model group, Xieriga-4 Decoction could effectively reduce kidney index (KI) and prostate index (PI) levels, serum creatinine (Scr) and blood urea nitrogen (BUN) levels in the serum, and urinary protein (UP), N -acetyl-beta- D -glucoaminidase (NAG), and kidney injury molecule-1 (KIM-1) expression levels in the urine of rats. Pathological sections showed that glomerular mesangial cell proliferation improved significantly and tubular epithelial cell shedding significantly inhibited in the Xieriga-4 Decoction group. Researchers applied Xieriga-4 Decoction of four drugs combined with Sugmel-10 and Narenmandula-11 to diagnose and treat diabetic nephropathy (DN) based on syndrome differentiation. They found that the combined administration of the three prescriptions for one day could effectively inhibit renal injury. The study also revealed that its renal protective mechanism might be associated with pathways such as Matrix metalloproteinase-2 (MMP-2) and Transforming growth factor-β (TGF-β) ( Wang, Li, Liu, Wang, & Wei, 2015 ). 5.3 Anti-inflammation and labor pain Zhang (2019) found that the levels of tumor necrosis factor-α (TNF-α), prostate specific antigen (PSA), interleukin-4 (IL-4), and prostaglandin E2 (PGE2) factors in the serum of experimental rats were significantly reduced in the Xieriga-4 group compared with the model group, and HE staining of prostate tissues revealed that the number of inflammatory cells was significantly decreased and inflammatory characteristics alleviated considerably in the Xieriga-4 group compared with the model group. Qu et al. found that Xieriga granule effectively inhibited not only acetic acid-induced abdominal and hot plate irritation and pain, but also xylene-induced inflammation by writhing and hot plate test and xylene-induced inflammation test in mice ( Qu, Altanqimeg, & Odunqimeg, 2018 ). 5.4 Bacteriostasis Qu also found that Xieriga-4 could significantly inhibit Staphylococcus aureus and Escherichia coli in vitro , but with no significant protective effect on intraperitoneal inoculation of E. coli in mice through bacteriostasis test in vivo and in vitro in mice ( Qu et al., 2018 ). Durina and Wurina (2013) performed bacterial and fungal inhibition experiments on Xieriga lotion by the test tube and cup-plate methods. They found that the lotion could effectively inhibit such fungi as Microsporum lauosum , M. gypseum , M. canis , Trichophyton rubrum , Candida albicans , and Malassezia . The results of inhibition of bacteria showed that for the inhibition of G- bacteria: the lotion could effectively inhibit Pseudomonas aeruginosa (with a stronger effect on gentamicin); it was ineffective against Bacterium vulgare and resistant E. coli (20% ethanol applied in the control group also had no inhibitory effect on the above two strains). For the inhibition of G + bacteria: the lotion could significantly inhibit Staphylococcus aureus , Bacillus anthracis , Staphylococcus albus , etc. (with a stronger effect on penicillin, while 20% ethanol applied in the control group had no inhibitory effect on the above strains). Wurina et al. studied Xieriga lotion by conducting in vitro fungal inhibition tests in culture media and found that the lotion could effectively inhibit fungi: M. gypseum (with an effective rate of 100%), M. lauosum (94%), Candida albicans (100%), and Trichophyton rubrum (93%) ( Wurina & Gao, 2013 ). In addition, an article on pharmacology of various component networks of Xieriga-4 reported 12 targets such as estrogen receptor, Bcl-2, COX-2, S5AR, ALR2, AR, PAP, IL-6, IL-8, aromatase, etc. associated with prostate hyperplasia and prostate cancer ( Bai, Li, Dong, & Zhang, 2018 ). They also found that Xieriga-4 may regulate the expression of prostate CA-related target genes through Wnt/β-catenin and AR signaling pathways. It is precisely because Xieriga-4 Decoction has the above-mentioned pharmacological effects corresponding to nephritis, cystitis, prostatic hyperplasia, urinary tract infection and other urinary infectious disease, for example, diuresis and detumescence, anti-inflammation and labor pain, bacteriostasis, renal protection, as well as its advantages of high safety and small toxic and side effects, which shows an excellent therapeutic effect in urinary diseases, especially infectious urinary diseases. 6 Clinical applications 6.1 Urinary infectious diseases Xieriga-4 Decoction is widely adopted in Mongolian medicine clinically for the treatment of urinary infectious diseases such as nephritis, cystitis, benign prostatic hyperplasia, and urinary tract infection. Yan & Zhang, 2020 divided 75 patients with benign prostatic hyperplasia into two groups. The Mongolian medicine group was given Xieriga-4 Decoction combined with Sugmel-10, while the Western medicine group was given finasteride tablets and tamsulosin hydrochloride sustained-release capsules (Harnal). Both groups were treated for three months. The symptoms, signs, and ultrasonic inspections were observed before and after treatment to measure prostate parameters, and the results showed that the Mongolian medicine group had a significant effect, with high safety, as well as less toxic and side effects. Shi & Lu, 2015 applied an Xieriga capsule of four drugs combined with levofloxacin to treat 30 lower urinary tract infection patients. After 7 to 10 d, it was found that the total effective rate in this group hit 93.3% (73.3% in the Western medicine alone group), demonstrating the ideal clinical efficacy of Xieriga capsule of four drugs combined with Western medicine in the treatment of lower urinary tract infection. This regimen had witnessed a significantly improved efficacy compared with levofloxacin alone, with no significant adverse reactions. A clinical observation observed 60 elderly female patients with urinary tract infection, with Xieriga decoction of four drugs orally administrated in the morning as the main drug and Xieriga-4 lotion sitz bath before bedtime for 4–6 weeks, and the indicators such as symptoms, urinalysis showed that the total effective rate for 60 patients with urinary tract infection hit 98.3%. With complete eradication effect, this treatment regimen was also found to have better long-term efficacy and low recurrence rate at the follow-up visit after twelve months ( Xiu, Qi, & Zhang, 2018 ). Another clinical observation randomly divided 72 patients with urinary tract infection into the Xieriga-4 capsule combined with gatifloxacin group and the gatifloxacin alone group. After treatment for 7–10 d, the total effective rate hit 91.9% and 74.2%, respectively, and the negative conversion ratio of bacteria hit 91.4% and 78.1%, respectively, both with significant differences. The incidence rate of adverse reactions was similar between the two groups, demonstrating the better clinical efficacy of Xieriga-4 capsule combined with Western medicine in the treatment of urinary tract infection, with no significant adverse reactions ( Wang & Wang, 2014 ). Moreover, in recent years, many scholars have also obtained better efficacy in treating other diseases using this prescription. 6.2 Diabetes treatment Many studies and clinical applications have demonstrated that Xieriga decoction of four drugs has a hypoglycemic effect. For example, studies demonstrated that Xieriga decoction exhibited significant hypoglycemic and anti-inflammatory effects, with less probability of complications and low price, etc., and was expected to serve as an ideal Mongolian medicine for the intervention of diabetic nephropathy. This research group randomly divided 80 DN patients into the Western medicine group (repaglinide tablets combined with metformin hydrochloride sustained-release tablets) and Mongolian and Western medicine treatment group (Western medicine + Sali-Gardi + Xieriga decoction of four drugs). After treatment for eight weeks, the clinical symptoms and 24 h urine microalbumin changes before and after treatment in each group were observed. The results showed that the total effective rate in the Mongolian medicine combined with Western medicine group hit 90%, higher than 77.5% in the Western medicine group; the 24 h urine microalbumin excretion rate in the former group was significantly lower than that in the latter. Compared with the Western medicine group, symptoms such as turbid urine, frequent urination with profuse urine, soreness and weakness of waist and knees, and tiredness and weakness were significantly improved in the Mongolian medicine and Western medicine group, with no significant difference in the effect of improving dry mouth and desire to drink, and feverishness in palms and soles between the groups. There was no adverse reaction in all patients before and after treatment including examinations of liver and kidney function, blood, urine and stool routine, and ECG ( Fang and Liu, 2020 , Liu, Zhang, & Wu, 2013 ). Shi et al. applied Xieriga decoction of four drugs and Sali Garidi combined with Western medicine (repaglinide tablets and metformin hydrochloride sustained-release tablets) to treat 50 patients with early type 2 DN based on syndrome differentiation. The results showed that the total effective rate of urine microalbumin excretion rate, symptoms and signs, UAER, and recovery of renal function hit 96% in the Mongolian medicine and Western medicine treatment group, and 82% in the 50 patients in the Western medicine alone group (repaglinide tablets and metformin hydrochloride sustained-release tablets), demonstrating the effectiveness and safety of the combination in treating early DN ( Shi, Zhang, & Li, 2020 ). Bao (2019) found through clinical studies that after two weeks of treatment with Sali-Garidi combined with Xieriga decoction of four drugs in 50 DN patients, the 24 h urinary protein and urinary albumin excretion rate of patients were significantly reduced in the treatment group, with no significant changes in liver and kidney function, blood routine and other examinations of all patients before and after treatment. It demonstrated that Xieriga-4 Decoction combined with Sali Gardi was safe and effective in treating DN. Zhu (2021) randomly divided 120 DN patients into two groups. The Western medicine group was given conventional drugs for diabetes (insulin for patients with type 1 diabetes and repaglinide tablets and metformin hydrochloride sustained-release tablets for patients with type 2 diabetes). The Xieriga-4 group was given Xieriga-4 Decoction combined with the above hypoglycemic drug regimen. After 8 weeks of treatment, it was found that the proportion of patients with improved symptoms such as soreness and weakness of waist and knees, turbid urine, frequent urination with profuse urine in the Xieriga-4 group was significantly higher than that in the Western medicine group; the symptoms including tiredness, feverishness in palms and soles, and dry mouth and desire to drink were also relieved to vary degrees in each group. Fasting blood glucose, postprandial blood glucose and glycosylated hemoglobin levels, Scr, BUN, and 24 h urine microalbumin excretion rate were significantly decreased in each group, and the above blood glucose levels and kidney function levels were significantly lower in the Xieriga-4 group compared with the Western medicine group. The Scr level was significantly increased in each group and was significantly higher in the Xieriga-4 group compared with the Western medicine alone group. 6.3 Acne A study applied Xieriga capsule of four drugs to treat 67 patients with acne with the accumulation of damp-heat in the body, while another 49 patients were being treated with minocyline (minocycline) as the control group. After six weeks, the total effective rate in the Xieriga capsule of four drugs group hit 85.1% (65.3% in the control group). Through observation, it was found that Xieriga capsule of four drugs could effectively prevent pigmentation and scars and reduce the pigmentation caused by acne, with high safety ( Wu, 2009 ). 6.1 Urinary infectious diseases Xieriga-4 Decoction is widely adopted in Mongolian medicine clinically for the treatment of urinary infectious diseases such as nephritis, cystitis, benign prostatic hyperplasia, and urinary tract infection. Yan & Zhang, 2020 divided 75 patients with benign prostatic hyperplasia into two groups. The Mongolian medicine group was given Xieriga-4 Decoction combined with Sugmel-10, while the Western medicine group was given finasteride tablets and tamsulosin hydrochloride sustained-release capsules (Harnal). Both groups were treated for three months. The symptoms, signs, and ultrasonic inspections were observed before and after treatment to measure prostate parameters, and the results showed that the Mongolian medicine group had a significant effect, with high safety, as well as less toxic and side effects. Shi & Lu, 2015 applied an Xieriga capsule of four drugs combined with levofloxacin to treat 30 lower urinary tract infection patients. After 7 to 10 d, it was found that the total effective rate in this group hit 93.3% (73.3% in the Western medicine alone group), demonstrating the ideal clinical efficacy of Xieriga capsule of four drugs combined with Western medicine in the treatment of lower urinary tract infection. This regimen had witnessed a significantly improved efficacy compared with levofloxacin alone, with no significant adverse reactions. A clinical observation observed 60 elderly female patients with urinary tract infection, with Xieriga decoction of four drugs orally administrated in the morning as the main drug and Xieriga-4 lotion sitz bath before bedtime for 4–6 weeks, and the indicators such as symptoms, urinalysis showed that the total effective rate for 60 patients with urinary tract infection hit 98.3%. With complete eradication effect, this treatment regimen was also found to have better long-term efficacy and low recurrence rate at the follow-up visit after twelve months ( Xiu, Qi, & Zhang, 2018 ). Another clinical observation randomly divided 72 patients with urinary tract infection into the Xieriga-4 capsule combined with gatifloxacin group and the gatifloxacin alone group. After treatment for 7–10 d, the total effective rate hit 91.9% and 74.2%, respectively, and the negative conversion ratio of bacteria hit 91.4% and 78.1%, respectively, both with significant differences. The incidence rate of adverse reactions was similar between the two groups, demonstrating the better clinical efficacy of Xieriga-4 capsule combined with Western medicine in the treatment of urinary tract infection, with no significant adverse reactions ( Wang & Wang, 2014 ). Moreover, in recent years, many scholars have also obtained better efficacy in treating other diseases using this prescription. 6.2 Diabetes treatment Many studies and clinical applications have demonstrated that Xieriga decoction of four drugs has a hypoglycemic effect. For example, studies demonstrated that Xieriga decoction exhibited significant hypoglycemic and anti-inflammatory effects, with less probability of complications and low price, etc., and was expected to serve as an ideal Mongolian medicine for the intervention of diabetic nephropathy. This research group randomly divided 80 DN patients into the Western medicine group (repaglinide tablets combined with metformin hydrochloride sustained-release tablets) and Mongolian and Western medicine treatment group (Western medicine + Sali-Gardi + Xieriga decoction of four drugs). After treatment for eight weeks, the clinical symptoms and 24 h urine microalbumin changes before and after treatment in each group were observed. The results showed that the total effective rate in the Mongolian medicine combined with Western medicine group hit 90%, higher than 77.5% in the Western medicine group; the 24 h urine microalbumin excretion rate in the former group was significantly lower than that in the latter. Compared with the Western medicine group, symptoms such as turbid urine, frequent urination with profuse urine, soreness and weakness of waist and knees, and tiredness and weakness were significantly improved in the Mongolian medicine and Western medicine group, with no significant difference in the effect of improving dry mouth and desire to drink, and feverishness in palms and soles between the groups. There was no adverse reaction in all patients before and after treatment including examinations of liver and kidney function, blood, urine and stool routine, and ECG ( Fang and Liu, 2020 , Liu, Zhang, & Wu, 2013 ). Shi et al. applied Xieriga decoction of four drugs and Sali Garidi combined with Western medicine (repaglinide tablets and metformin hydrochloride sustained-release tablets) to treat 50 patients with early type 2 DN based on syndrome differentiation. The results showed that the total effective rate of urine microalbumin excretion rate, symptoms and signs, UAER, and recovery of renal function hit 96% in the Mongolian medicine and Western medicine treatment group, and 82% in the 50 patients in the Western medicine alone group (repaglinide tablets and metformin hydrochloride sustained-release tablets), demonstrating the effectiveness and safety of the combination in treating early DN ( Shi, Zhang, & Li, 2020 ). Bao (2019) found through clinical studies that after two weeks of treatment with Sali-Garidi combined with Xieriga decoction of four drugs in 50 DN patients, the 24 h urinary protein and urinary albumin excretion rate of patients were significantly reduced in the treatment group, with no significant changes in liver and kidney function, blood routine and other examinations of all patients before and after treatment. It demonstrated that Xieriga-4 Decoction combined with Sali Gardi was safe and effective in treating DN. Zhu (2021) randomly divided 120 DN patients into two groups. The Western medicine group was given conventional drugs for diabetes (insulin for patients with type 1 diabetes and repaglinide tablets and metformin hydrochloride sustained-release tablets for patients with type 2 diabetes). The Xieriga-4 group was given Xieriga-4 Decoction combined with the above hypoglycemic drug regimen. After 8 weeks of treatment, it was found that the proportion of patients with improved symptoms such as soreness and weakness of waist and knees, turbid urine, frequent urination with profuse urine in the Xieriga-4 group was significantly higher than that in the Western medicine group; the symptoms including tiredness, feverishness in palms and soles, and dry mouth and desire to drink were also relieved to vary degrees in each group. Fasting blood glucose, postprandial blood glucose and glycosylated hemoglobin levels, Scr, BUN, and 24 h urine microalbumin excretion rate were significantly decreased in each group, and the above blood glucose levels and kidney function levels were significantly lower in the Xieriga-4 group compared with the Western medicine group. The Scr level was significantly increased in each group and was significantly higher in the Xieriga-4 group compared with the Western medicine alone group. 6.3 Acne A study applied Xieriga capsule of four drugs to treat 67 patients with acne with the accumulation of damp-heat in the body, while another 49 patients were being treated with minocyline (minocycline) as the control group. After six weeks, the total effective rate in the Xieriga capsule of four drugs group hit 85.1% (65.3% in the control group). Through observation, it was found that Xieriga capsule of four drugs could effectively prevent pigmentation and scars and reduce the pigmentation caused by acne, with high safety ( Wu, 2009 ). 7 Quality control Li et al. determined the contents of baicalin, bisdemethoxycurcumin, demethoxycurcumin, and curcumin in Xieriga decoction of four drugs by high-performance liquid chromatography. The results showed a good linear relationship ( r > 0.9998) between the concentration of the determined components and their peak areas within the linear range. The average recoveries were 97.2%, 97.1%, 96.4%, and 96.8%, the RSDs of 1.1%, 1.4%, 0.86%, and 1.0%, and the average contents were 0.0080%, 0.0377%, 0.0292%, and 0.0631%, respectively ( Li, Bai, Zhao, & Dong, 2018 ). Li et al. determined the contents of jasminoidin, curcumin, and berberine hydrochloride in Xieriga decoction of four drugs by HPLC. The results showed that the optimum conditions for effective separation, good reproducibility, linearity and precision as well as rapid, simple and accurate determination were Elite C 18 (4.6 mm × 250 mm, 5 μm) chromatographic column, acetonitrile (A)-0.4% potassium dihydrogen phosphate aqueous solution (B) as the mobile phase, a chromatographic system established at 30 °C, detection wavelength of 238 nm for geniposide and 346 nm for curcumin and berberine hydrochloride, as well as the flow rate of 1.0 mL/min ( Li, Yang, & Dong, 2014 ). Bagenna et al. performed fluorescence scanning by TLC with n -butanol: glacial acetic acid: water (7:1:2) and an excitation wavelength of 365 nm, and detected Curcumae Longae Rhizoma (turmeric), Phellodendri Chinensis Cortex , and Gardeniae Fructus , respectively in Xieriga Decoction of four drugs. The results showed a linear relationship between berberine hydrochloride and its corresponding integrated value of fluorescence intensity within the range of 0.0196–0.1568 μg, with r = 0.998; an average recovery of 100.14%, and RSD of 1.87% ( n = 6) ( Bagenna, Qiqigma, & Li, 2007 ). 8 Conclusion and prospects To sum up, the prescription is cool in nature. With killing stickiness, antiseptic and detoxifying, Curcumae Longae Rhizoma (turmeric) as the main drug, supplemented by Tribuli Fructus for diuretic and detumescence, Gardeniae Fructus for blood heat eliminating and Phellodendri Chinensis Cortex for heat clearing, the organic combination has become a prescription with killing stickiness, heat clearing and diuretic effects. It is mainly used for the treatment of frequent urination, urgent urination, urine occlusion, hematuria, and bladder irritation and pain. Xieriga decoction of four drugs has served as a special prescription for urinary system diseases for a long time and has excellent clinical feedback. With the great progress in modern scientific research in recent years, many scientific and clinical studies have demonstrated that Xieriga-4 Decoction enjoys significant pharmacological actions such as diuresis, inflammation elimination, bacteriostasis, pain relief, and immune regulation, with higher safety. Moreover, the components of this prescription are simple and easy to obtain, but the components are perfectly matched, and the therapeutic purpose is accurately achieved for the etiology, symptoms and prognosis of the disease. It is in line with not only the law of drug compatibility in Mongolian medicine theory, but also the precision medical policy. At present, in addition to the original dosage form of decoction, there are capsule, tablet, granule, spray, lotion, and other improved dosage forms. It is a group of safe, effective preparations with Mongolian characteristics. However, the basic research on this preparation is not deep enough, which is a real problem faced by many excellent Mongolian medicines. Efforts need to be made to carry out more in-depth pharmacological analysis on the basis of existing research, or even to analyze the quality of its chemical components using advanced techniques such as molecular biology and cell biology, to elucidate the material basis of its medicinal effects and to clarify its mechanism of action and targets. By doing so, a stable and reliable specification can be established to ensure the safety of clinical application, thus promoting the overall development of Xieriga-4 Decoction and its wider clinical application. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152148/

Safety precautions

INFECTIOUS DISEASE: THE BAD NEWS The incidence of infectious diseases, particularly those that are incurable or difficult to treat, is rising. In a study of patients undergoing major surgery in New York, 1 5.2% were HCV positive, 1.4% HBV positive, and 1.6% HIV positive (or 6.7% with one or more of these viruses). Often, the presence of infection is unknown or is not reported to the pathology department. Healthcare workers are at risk for contracting these diseases when working with patients ( Box 8-1 ). The risk is lower for pathology personnel, but exposure can occur by aerosolization of tissues, needlestick injury, scalpel wounds, and mucocutaneous exposure during the processing of pathology specimens. Box 8-1 Diseases that have been transmitted to healthcare workers • Hepatitis B, C, and A • Tuberculosis (including strains resistant to multiple drugs) • HIV • Syphilis • Creutzfeldt–Jakob disease • Coccidioides immitis (the risk arises primarily from cultures of the fungus in microbiology laboratories); if this infection is suspected, all specimens must be labeled appropriately • Parvovirus and H. pylori infection, cryptosporidiosis, scabies, pertussis Other infectious agents (e.g., other types of bacteria or fungi, Pneumocystis carinii , other viral agents) are also potential dangers, particularly to immunocompromised healthcare workers, but transmission is very rare and has not yet been reported. Diseases that have been transmitted to healthcare workers • Hepatitis B, C, and A • Tuberculosis (including strains resistant to multiple drugs) • HIV • Syphilis • Creutzfeldt–Jakob disease • Coccidioides immitis (the risk arises primarily from cultures of the fungus in microbiology laboratories); if this infection is suspected, all specimens must be labeled appropriately • Parvovirus and H. pylori infection, cryptosporidiosis, scabies, pertussis INFECTIOUS DISEASE: THE GOOD NEWS The actual incidence of transmission of infectious agents from unfixed surgical specimens to pathology department personnel is extremely low. There are only three reported cases, all involving conversion to positive tuberculin skin tests after use of an aerosolized gas coolant to freeze a tissue block during an intraoperative consultation. 2, 3 However, transmission of other types of infectious disease is theoretically possible: transmission of HBV, HIV, and TB has occurred during the performance of autopsies. The good news is that pathology personnel can take action to protect themselves by educating themselves about risks, taking physical precautions to protect themselves and others, avoiding the use of hollow-bore needles, and making sure they are vaccinated for HBV ( Table 8-1 ). Personnel who are immunocompromised must be especially vigilant. Table 8-1 Risk of exposure to common infectious agents AGENT PERCENTAGE OF HOSPITAL PATIENTS RISK OF INFECTION AFTER PERCUTANEOUS INJURY a RISK AFTER MUCOCUTANEOUS EXPOSURE RISK OF ENVIRONMENTAL EXPOSURE POSTEXPOSURE PROPHYLAXIS AVAILABLE HIV ∼0.2-14% 0.3% 0.09% Possible, but very rare Yes, effective HCV ∼2-5% 1.8% Rare Yes, but rapidly degrades No, not shown to be effective HBV ∼2% 30% Yes, probably high Occurs, can be found in dried blood ∼1 week Yes, effective TB ∼10% Yes, risk not quantified Yes, risk not quantified Yes No, treatment initiated only if skin test converts a Percutaneous injury: needlestick injury (majority) or other penetrating injury with a sharp object (e.g., scalpel, broken glass). Hepatitis B virus 4, 5 The CDC estimated that 18,000 healthcare workers whose jobs entailed exposure to blood became infected with HBV each year prior to widespread vaccination. Of these, 200 to 300 died of complications of HBV infection. Prior to widespread vaccination, 25% to 30% of pathologists were positive for HBV, their exposure likely being due to the performance of autopsies. However, the incidence of HBV infection has sharply declined with vaccination. All pathology department workers who come into contact with tissue should be vaccinated. OSHA bloodborne standards require that employers offer the vaccine at no cost to all employees at risk. ( www.osha.gov ). After a needlestick injury, the seroconversion rate is 30% from HBeAg-positive blood and <6% from HBeAg-negative blood in non-vaccinated individuals. Mucocutaneous exposure can also occur. Postexposure prophylaxis with HBV hyperimmune globulin and vaccine is suggested for non-vaccinated individuals or vaccinated persons with low antibody titers. Treatment provides approximately 75% protection from infection if instituted within a week. Hepatitis C virus 4 , 5 , 6 The seroprevalence of HCV in healthcare workers has ranged from 0% to 1.7% in multiple studies. Occupational infections in pathology personnel have not been reported. Eighty percent to 90% of infections will become chronic with risk for the development of chronic hepatitis, cirrhosis (3% to 20% of patients), and hepatocellular carcinoma. HCV has also been linked to cryoglobulinemia and many other immune system related diseases. The risk is approximately 1-8% for HCV transmission after a needlestick injury. The risk after skin or mucous membrane exposure is likely to be very low. Postexposure treatment has not been shown to be effective. If there has been a potential exposure, the person should be monitored for infection in order to start treatment as early as possible. Human immunodeficiency virus 5 , 7 , 8 , 9 , 10 As of 2001, 57 healthcare workers had developed HIV infection following documented occupational exposure; an additional 138 workers were considered possible cases. Most exposures (88%) were percutaneous and involved hollow-bore needles, scalpels, and broken vials: 20% of exposures occurred during the disposal of sharp objects. Mucous membrane and skin exposure were responsible in about 10% of cases. The source in almost all cases was infected blood (86%). The risk is increased with the volume of blood, the depth of the injury, and the viral titer of the patient (with an increased risk with patients close to death). A pathologist was infected by HIV after a scalpel wound to the hand during an autopsy. 11 Surgical specimens containing blood could also potentially transmit the virus, if an injury occurs. HIV can be cultured from cadavers hours to days after death. 12 The effect of fixation has not been studied but would presumably lower or eliminate risk. Approximately 0.3% of persons will seroconvert after a needlestick exposure to HIV, 0.1% after mucocutaneous exposure, and <0.1% after skin exposure. Postexposure treatment with antiviral agents can decrease the risk of seroconversion by 81%. Treatment should be started as soon as possible, as it may be less effective after 2 to 3 days. Additional agents used in combination for prophylaxis may be more effective, as the source patients for occupational cases have a high prevalence of drug-resistant HIV. 13 There have been 21 cases of healthcare personnel becoming infected with HIV despite postexposure prophylaxis. Tuberculosis The risk of transmission of TB to autopsy personnel during the performance of necroscopies is well documented. TB can be transmitted not only as an aerosol but also percutaneously. 14 It must be kept in mind that many cases of TB are first diagnosed after death. Multiple individuals had conversion to a positive skin test after the autopsy of an infected person. 15 Exposure can be diminished by wearing special respiratory protection. Healthcare workers also have a significant risk of contracting multiple-drug-resistant tuberculosis. Although healthcare workers have been infected by drug-resistant TB, no fatal case has yet been reported in the absence of an underlying immunodeficiency disorder. There are no definitive studies on the survival of mycobacteria in fixed surgical specimens. 16, 17 Formalin probably kills mycobacteria, but the time required for it to do so is unknown. Special respiratory protective devices are recommended for personnel who may be exposed to tuberculosis. If an exposed person does not develop a positive skin test, no treatment is necessary. Converters and persons who are immunocompromised should be treated. Hospital workers are required to undergo yearly TB testing. Severe acute respiratory syndrome 18 Severe acute respiratory syndrome (SARS) was first identified in China in late 2002. It is caused by SARS-associated coronavirus (SARS-CoV) and spreads via respiratory droplets contacting the mucous membranes of a second person. Occupationally acquired cases have occurred among healthcare workers. The risk to surgical pathology personnel is likely to be low, as most patients will not undergo surgical procedures. However, autopsies may be performed. There are no reported cases of SARS being transferred via the handling of pathology specimens. However, as there is little experience with this virus, all cases from patients with known or suspected SARS may best be handled as for cases of HBV. All tissue should be promptly fixed and the cryostat decontaminated if necessary. Creutzfeldt–Jakob disease 19, 20 The only cases of infection in laboratory personnel from fixed tissue are due to Creutzfeldt–Jakob disease (CJD). As of 1995, 24 healthcare workers had developed CJD, including two histotechnologists and one pathologist. Infectious units are present in fixed and paraffin-embedded tissue for years. Any adult patient with a rapidly progressive dementia, myoclonus, and nonspecific neurologic findings should be considered as potentially having the disease. All tissues from affected patients can potentially cause infection. The virus is not inactivated by standard formalin fixation or boiling water. Tissues should be fixed in formalin for 24 hours, then in 95% formic acid for one hour followed by formalin fixation for one day. Hepatitis B virus 4, 5 The CDC estimated that 18,000 healthcare workers whose jobs entailed exposure to blood became infected with HBV each year prior to widespread vaccination. Of these, 200 to 300 died of complications of HBV infection. Prior to widespread vaccination, 25% to 30% of pathologists were positive for HBV, their exposure likely being due to the performance of autopsies. However, the incidence of HBV infection has sharply declined with vaccination. All pathology department workers who come into contact with tissue should be vaccinated. OSHA bloodborne standards require that employers offer the vaccine at no cost to all employees at risk. ( www.osha.gov ). After a needlestick injury, the seroconversion rate is 30% from HBeAg-positive blood and <6% from HBeAg-negative blood in non-vaccinated individuals. Mucocutaneous exposure can also occur. Postexposure prophylaxis with HBV hyperimmune globulin and vaccine is suggested for non-vaccinated individuals or vaccinated persons with low antibody titers. Treatment provides approximately 75% protection from infection if instituted within a week. Hepatitis C virus 4 , 5 , 6 The seroprevalence of HCV in healthcare workers has ranged from 0% to 1.7% in multiple studies. Occupational infections in pathology personnel have not been reported. Eighty percent to 90% of infections will become chronic with risk for the development of chronic hepatitis, cirrhosis (3% to 20% of patients), and hepatocellular carcinoma. HCV has also been linked to cryoglobulinemia and many other immune system related diseases. The risk is approximately 1-8% for HCV transmission after a needlestick injury. The risk after skin or mucous membrane exposure is likely to be very low. Postexposure treatment has not been shown to be effective. If there has been a potential exposure, the person should be monitored for infection in order to start treatment as early as possible. Human immunodeficiency virus 5 , 7 , 8 , 9 , 10 As of 2001, 57 healthcare workers had developed HIV infection following documented occupational exposure; an additional 138 workers were considered possible cases. Most exposures (88%) were percutaneous and involved hollow-bore needles, scalpels, and broken vials: 20% of exposures occurred during the disposal of sharp objects. Mucous membrane and skin exposure were responsible in about 10% of cases. The source in almost all cases was infected blood (86%). The risk is increased with the volume of blood, the depth of the injury, and the viral titer of the patient (with an increased risk with patients close to death). A pathologist was infected by HIV after a scalpel wound to the hand during an autopsy. 11 Surgical specimens containing blood could also potentially transmit the virus, if an injury occurs. HIV can be cultured from cadavers hours to days after death. 12 The effect of fixation has not been studied but would presumably lower or eliminate risk. Approximately 0.3% of persons will seroconvert after a needlestick exposure to HIV, 0.1% after mucocutaneous exposure, and <0.1% after skin exposure. Postexposure treatment with antiviral agents can decrease the risk of seroconversion by 81%. Treatment should be started as soon as possible, as it may be less effective after 2 to 3 days. Additional agents used in combination for prophylaxis may be more effective, as the source patients for occupational cases have a high prevalence of drug-resistant HIV. 13 There have been 21 cases of healthcare personnel becoming infected with HIV despite postexposure prophylaxis. Tuberculosis The risk of transmission of TB to autopsy personnel during the performance of necroscopies is well documented. TB can be transmitted not only as an aerosol but also percutaneously. 14 It must be kept in mind that many cases of TB are first diagnosed after death. Multiple individuals had conversion to a positive skin test after the autopsy of an infected person. 15 Exposure can be diminished by wearing special respiratory protection. Healthcare workers also have a significant risk of contracting multiple-drug-resistant tuberculosis. Although healthcare workers have been infected by drug-resistant TB, no fatal case has yet been reported in the absence of an underlying immunodeficiency disorder. There are no definitive studies on the survival of mycobacteria in fixed surgical specimens. 16, 17 Formalin probably kills mycobacteria, but the time required for it to do so is unknown. Special respiratory protective devices are recommended for personnel who may be exposed to tuberculosis. If an exposed person does not develop a positive skin test, no treatment is necessary. Converters and persons who are immunocompromised should be treated. Hospital workers are required to undergo yearly TB testing. Severe acute respiratory syndrome 18 Severe acute respiratory syndrome (SARS) was first identified in China in late 2002. It is caused by SARS-associated coronavirus (SARS-CoV) and spreads via respiratory droplets contacting the mucous membranes of a second person. Occupationally acquired cases have occurred among healthcare workers. The risk to surgical pathology personnel is likely to be low, as most patients will not undergo surgical procedures. However, autopsies may be performed. There are no reported cases of SARS being transferred via the handling of pathology specimens. However, as there is little experience with this virus, all cases from patients with known or suspected SARS may best be handled as for cases of HBV. All tissue should be promptly fixed and the cryostat decontaminated if necessary. Creutzfeldt–Jakob disease 19, 20 The only cases of infection in laboratory personnel from fixed tissue are due to Creutzfeldt–Jakob disease (CJD). As of 1995, 24 healthcare workers had developed CJD, including two histotechnologists and one pathologist. Infectious units are present in fixed and paraffin-embedded tissue for years. Any adult patient with a rapidly progressive dementia, myoclonus, and nonspecific neurologic findings should be considered as potentially having the disease. All tissues from affected patients can potentially cause infection. The virus is not inactivated by standard formalin fixation or boiling water. Tissues should be fixed in formalin for 24 hours, then in 95% formic acid for one hour followed by formalin fixation for one day. BIOLOGIC TERRORISM 21 , 22 , 23 , 24 , 25 , 26 , 27 ( Table 8-2 ) It is to be hoped that pathologists will not receive specimens from acts of biologic terrorism, but, should such an event occur, pathologists can aid in recognizing the disease and the likely method of infection. The first anthrax case in 2001 was suspected when typical organisms were seen on a Gram stain of CSF. The autopsy determined that the mode of exposure was inhalation and this finding helped direct investigators to search for possible sources of airborne spores. In the event of an actual or threatened bioterrorist attack, local health and law enforcement agencies should be contacted. Additional information can be found at www.bt.cdc.gov/emcontact/index.asp or the CDC Emergency Response Hotline 770-488-7100. The CDC recommends saving tissue from autopsies (and other specimens) from possible victims of biologic terrorism: • Fixed tissue: Histologic examination for patterns of tissue damage and special stains for identification of organisms. IHC and DFA assays are available at the CDC and most can be performed on fixed tissue. • Blood, CSF, tissue samples or swabs for bacterial and viral culture. Mucosal swabs for cases of possible botulinum toxin inhalation. • Serum for biologic and serologic assays. • Frozen tissue for PCR. • Fixed tissue (glutaraldehyde) for EM to identify viral particles. The Laboratory Response Network The Laboratory Response Network (LRN) is a partnership of local, state, and federal public health laboratories, and veterinary, food, and environmental laboratories, the CDC, the Food and Drug Administration, the Environmental Protection Agency, the US Army Medical Research Institute of Infectious Diseases, and other Department of Defense laboratories. The network functions to channel specimens from sentinel laboratories to advanced laboratories for confirmation and final identification of pathogens. Specimens from cases suspected to be related to biologic terrorism can be submitted to the state public health laboratory. Contact information for all state laboratories is included in the CDC guidebook listed in the resources. If the suspected agent is smallpox, the state laboratory should be notified as such specimens may be transported directly to the CDC. Risks to pathology personnel All of the infectious agents listed in Table 8-2 could potentially be transmitted to personnel during the performance of an autopsy or by handling fresh tissue, except for botulinum toxin. Smallpox, tularemia, and viral hemorrhagic fevers have been transmitted to persons performing autopsies. Biologic terrorism raises an additional risk of surface contamination by the agent (e.g., powders used to transmit anthrax or botulinum toxin). Because of the incubation period, it is likely victims will have changed clothes and bathed so that such contamination, in most cases, will likely be minimal. Standard universal safety precautions should be used in all cases and should be protective. Table 8-2 Agents most likely to be used for biologic terrorism (category A agents) AGENT MODE OF TRANSMISSION CLINICAL SYNDROME PATHOLOGIC FINDINGS APPEARANCE OF ORGANISM/AVAILABLE TESTS a TREATMENT/PROPHYLAXIS Smallpox virus (variola major) Inhalation: aerosols Direct contact with lesions or contaminated surfaces Person to person spread Diffuse rash (including palms and soles): deep-seated, firm/hard, round well-circumscribed vesicles or pustules, all in same stage of development Hemorrhage into skin and Gl tract Early vesicles are multilocular (but coalesce in later stages), ballooning degeneration of epithelial cells (not multinucleated), eosinophilic intracytoplasmic viral inclusions (Guarnieri bodies) Viral inclusions present in cytoplasm IHC EM: fluid from vesicles can be used to detect viral particles PCR: viral DNA Vaccine available. b Routine vaccination in the US ended in 1972. Persons with remote vaccination probably have some, but not complete, immunity Bacillus anthracis (anthrax) Direct contact with spores (skin or ingestion) Inhalation of spores No person to person spread Cutaneous: eschar with hemorrhage, edema, necrosis, perivascular infiltrate, vasculitis Gastrointestinal: hemorrhagic enteritis, hemorrhagic lymphadenitis, mucosal ulcers with necrosis in the terminal ileum and cecum, peritonitis Inhalational: hemorrhagic mediastinitis, hemorrhagic lymphadenitis, hemorrhagic pleural effusion CNS: hemorrhagic meningitis Skin: edema, focal necrosis, vasculitis, acute inflammation, ulceration Organisms only rarely seen by H&E Lymph nodes: hemorrhage, necrosis After antibiotic treatment, organisms may only be visible by silver stains and IHC Gram, silver stains: large, broad (3×5 μm) encapsulated Gram-positive bacilli with flattened ends in short chains India ink: shows capsule in blood and CSF IHC: sensitive and specific DFA (but cannot be used on formalin-fixed tissue) PCR: formalin or fresh tissue Vaccine available Antibiotic prophylaxis available Yersinia pestis (plague) Flea bites Inhalation: aerosols Person to person spread Bubonic: acute lymphadenitis with surrounding edema (a bubo is a local painful swelling) Pneumonic: severe, hemorrhagic bronchopneumonia, often with fibrinous pleuritis, diffuse alveolar damage (ARDS), sepsis with DIC CNS: meningitis Lung: Severe, confluent, hemorrhagic, necrotizing bronchopneumonia, often with fibrinous pleuritis Lymph nodes: Necrosis—preferred for histologic examination and culture Gram, silver, Giemsa stains: short, fat Gram-negative bacilli IHC DFA Vaccine available (but does not protect against pneumonia) Antibiotic prophylaxis available Clostridium botulinum toxin (botulism) Ingestion or inhalation of preformed neurotoxin No person to person spread CNS: hyperemia and microthrombosis of small vessels associated with symmetrical, descending pattern of weakness and paralysis of cranial nerves, limbs, and trunk No specific findings for cases due to ingestion or inhalation of preformed toxin Swabs of mucosal surfaces or serum may be used for the botulinum toxin mouse bioassay Samples should be taken prior to the use of antitoxin Gram-positive bacteria -however organisms unlikely to be present in a terror attack Antitoxin available Francisella tularensis (tularemia) Tick bite Direct contact with infected fluids or tissues Ingestion of infected meat No person to person spread Ulceroglandular: skin ulcer with associated suppurative lymphadenitis Glandular: suppurative necrotizing lymphadenitis without associated skin ulcer Oculoglandular: eyelid edema, acute conjunctivitis and edema, small conjunctival ulcers, regional lymphadenitis Pharyngeal: exudative pharyngitis or tonsillitis with ulceration, pharyngeal membrane formation, regional lymphadenitis Typhoidal: systemic involvement, DIC, focal necrosis of major organs Pneumonic: acute inflammation, diffuse alveolar damage Ulcer with a nonspecific inflammatory infiltrate and a granulomatous reaction. In some cases, large necrotizing granulomas with giant cells may be present Lymph nodes: extensive necrosis, irregular microabscesses and multiple granulomas with caseous necrosis Lung: necrotizing pneumonia with abundant fibrin, acute inflammation Small encapsulated Gram-negative coccobacilli—difficult to see with histochemical stains IHC DFA Antibiotic prophylaxis available Hemorrhagic fever viruses, including filoviruses (including Ebola and Marburg viruses) and arenaviruses (e.g., Lassa fever) Close personal contact with infected person, blood, tissue, or body fluids Diffuse rash, massive hepatocellular necrosis, extensive necrosis in other major organs, diffuse alveolar damage Massive hepatic necrosis with filamentous viral inclusions in hepatocytes, extensive necrosis of other organs Viral inclusions in hepatocytes IHC EM: viral inclusions PCR No specific treatment ARDS, acute respiratory distress syndrome; DFA, direct fluorescent assay; DIC, disseminated intravascular coagulopathy; IHC, immunohistochemistry a IHC and DFA tests for each of these organisms are available at the CDC. Consult the CDC website to determine how to decide if a specimen is appropriate for testing and how to send such a sample: call the CDC at 404-639-3 1 33 or fax the CDC at 404-639-3043 for more information. b Vaccination is not currently recommended for individuals without a known exposure. Vaccination for smallpox may be considered for selected personnel who would be first responders for the examination of the remains or specimens from patients dying of smallpox. Cadavers of patients dying of B. anthracis, Y. pestis , or botulinum toxin are unlikely to pose a threat to non-autopsy personnel (e.g., funeral home workers). However, smallpox virus and hemorrhagic fever viruses could be transmitted and should only be handled with safety precautions. In general, such bodies should not be embalmed as this might impose increased risk. Sending specimens to reference laboratories Detailed instructions for the packaging and shipping of specimens to reference laboratories are available at the CDC website ( www.cdc.gov ). In general, such specimens must have three levels of containment and must be marked with an "Infectious Substance" label. The laboratory director of the state health department should be contacted before a specimen with a suspected biologic agent is shipped. The Laboratory Response Network The Laboratory Response Network (LRN) is a partnership of local, state, and federal public health laboratories, and veterinary, food, and environmental laboratories, the CDC, the Food and Drug Administration, the Environmental Protection Agency, the US Army Medical Research Institute of Infectious Diseases, and other Department of Defense laboratories. The network functions to channel specimens from sentinel laboratories to advanced laboratories for confirmation and final identification of pathogens. Specimens from cases suspected to be related to biologic terrorism can be submitted to the state public health laboratory. Contact information for all state laboratories is included in the CDC guidebook listed in the resources. If the suspected agent is smallpox, the state laboratory should be notified as such specimens may be transported directly to the CDC. Risks to pathology personnel All of the infectious agents listed in Table 8-2 could potentially be transmitted to personnel during the performance of an autopsy or by handling fresh tissue, except for botulinum toxin. Smallpox, tularemia, and viral hemorrhagic fevers have been transmitted to persons performing autopsies. Biologic terrorism raises an additional risk of surface contamination by the agent (e.g., powders used to transmit anthrax or botulinum toxin). Because of the incubation period, it is likely victims will have changed clothes and bathed so that such contamination, in most cases, will likely be minimal. Standard universal safety precautions should be used in all cases and should be protective. Table 8-2 Agents most likely to be used for biologic terrorism (category A agents) AGENT MODE OF TRANSMISSION CLINICAL SYNDROME PATHOLOGIC FINDINGS APPEARANCE OF ORGANISM/AVAILABLE TESTS a TREATMENT/PROPHYLAXIS Smallpox virus (variola major) Inhalation: aerosols Direct contact with lesions or contaminated surfaces Person to person spread Diffuse rash (including palms and soles): deep-seated, firm/hard, round well-circumscribed vesicles or pustules, all in same stage of development Hemorrhage into skin and Gl tract Early vesicles are multilocular (but coalesce in later stages), ballooning degeneration of epithelial cells (not multinucleated), eosinophilic intracytoplasmic viral inclusions (Guarnieri bodies) Viral inclusions present in cytoplasm IHC EM: fluid from vesicles can be used to detect viral particles PCR: viral DNA Vaccine available. b Routine vaccination in the US ended in 1972. Persons with remote vaccination probably have some, but not complete, immunity Bacillus anthracis (anthrax) Direct contact with spores (skin or ingestion) Inhalation of spores No person to person spread Cutaneous: eschar with hemorrhage, edema, necrosis, perivascular infiltrate, vasculitis Gastrointestinal: hemorrhagic enteritis, hemorrhagic lymphadenitis, mucosal ulcers with necrosis in the terminal ileum and cecum, peritonitis Inhalational: hemorrhagic mediastinitis, hemorrhagic lymphadenitis, hemorrhagic pleural effusion CNS: hemorrhagic meningitis Skin: edema, focal necrosis, vasculitis, acute inflammation, ulceration Organisms only rarely seen by H&E Lymph nodes: hemorrhage, necrosis After antibiotic treatment, organisms may only be visible by silver stains and IHC Gram, silver stains: large, broad (3×5 μm) encapsulated Gram-positive bacilli with flattened ends in short chains India ink: shows capsule in blood and CSF IHC: sensitive and specific DFA (but cannot be used on formalin-fixed tissue) PCR: formalin or fresh tissue Vaccine available Antibiotic prophylaxis available Yersinia pestis (plague) Flea bites Inhalation: aerosols Person to person spread Bubonic: acute lymphadenitis with surrounding edema (a bubo is a local painful swelling) Pneumonic: severe, hemorrhagic bronchopneumonia, often with fibrinous pleuritis, diffuse alveolar damage (ARDS), sepsis with DIC CNS: meningitis Lung: Severe, confluent, hemorrhagic, necrotizing bronchopneumonia, often with fibrinous pleuritis Lymph nodes: Necrosis—preferred for histologic examination and culture Gram, silver, Giemsa stains: short, fat Gram-negative bacilli IHC DFA Vaccine available (but does not protect against pneumonia) Antibiotic prophylaxis available Clostridium botulinum toxin (botulism) Ingestion or inhalation of preformed neurotoxin No person to person spread CNS: hyperemia and microthrombosis of small vessels associated with symmetrical, descending pattern of weakness and paralysis of cranial nerves, limbs, and trunk No specific findings for cases due to ingestion or inhalation of preformed toxin Swabs of mucosal surfaces or serum may be used for the botulinum toxin mouse bioassay Samples should be taken prior to the use of antitoxin Gram-positive bacteria -however organisms unlikely to be present in a terror attack Antitoxin available Francisella tularensis (tularemia) Tick bite Direct contact with infected fluids or tissues Ingestion of infected meat No person to person spread Ulceroglandular: skin ulcer with associated suppurative lymphadenitis Glandular: suppurative necrotizing lymphadenitis without associated skin ulcer Oculoglandular: eyelid edema, acute conjunctivitis and edema, small conjunctival ulcers, regional lymphadenitis Pharyngeal: exudative pharyngitis or tonsillitis with ulceration, pharyngeal membrane formation, regional lymphadenitis Typhoidal: systemic involvement, DIC, focal necrosis of major organs Pneumonic: acute inflammation, diffuse alveolar damage Ulcer with a nonspecific inflammatory infiltrate and a granulomatous reaction. In some cases, large necrotizing granulomas with giant cells may be present Lymph nodes: extensive necrosis, irregular microabscesses and multiple granulomas with caseous necrosis Lung: necrotizing pneumonia with abundant fibrin, acute inflammation Small encapsulated Gram-negative coccobacilli—difficult to see with histochemical stains IHC DFA Antibiotic prophylaxis available Hemorrhagic fever viruses, including filoviruses (including Ebola and Marburg viruses) and arenaviruses (e.g., Lassa fever) Close personal contact with infected person, blood, tissue, or body fluids Diffuse rash, massive hepatocellular necrosis, extensive necrosis in other major organs, diffuse alveolar damage Massive hepatic necrosis with filamentous viral inclusions in hepatocytes, extensive necrosis of other organs Viral inclusions in hepatocytes IHC EM: viral inclusions PCR No specific treatment ARDS, acute respiratory distress syndrome; DFA, direct fluorescent assay; DIC, disseminated intravascular coagulopathy; IHC, immunohistochemistry a IHC and DFA tests for each of these organisms are available at the CDC. Consult the CDC website to determine how to decide if a specimen is appropriate for testing and how to send such a sample: call the CDC at 404-639-3 1 33 or fax the CDC at 404-639-3043 for more information. b Vaccination is not currently recommended for individuals without a known exposure. Vaccination for smallpox may be considered for selected personnel who would be first responders for the examination of the remains or specimens from patients dying of smallpox. Cadavers of patients dying of B. anthracis, Y. pestis , or botulinum toxin are unlikely to pose a threat to non-autopsy personnel (e.g., funeral home workers). However, smallpox virus and hemorrhagic fever viruses could be transmitted and should only be handled with safety precautions. In general, such bodies should not be embalmed as this might impose increased risk. Sending specimens to reference laboratories Detailed instructions for the packaging and shipping of specimens to reference laboratories are available at the CDC website ( www.cdc.gov ). In general, such specimens must have three levels of containment and must be marked with an "Infectious Substance" label. The laboratory director of the state health department should be contacted before a specimen with a suspected biologic agent is shipped. TRANSMISSION OF TUMORS In general, malignant tumors do not pose a risk to any person other than the patient. However, malignancies can be transferred from a graft to an organ transplant recipient. 28 There has been one case of a sarcoma transferred to the hand of a non-immunocompromised surgeon after a scalpel injury. 29 Thus, although the risk is extremely small, tumors (and all human tissue) must be handled with appropriate safety precautions. GUIDELINES FOR PROCESSING SPECIMENS WITH KNOWN OR PROBABLE INFECTIOUS DISEASE • Specimens from patients with infections not posing a risk to immunocompetent individuals (e.g., routine bacterial and fungal infections, opportunistic pathogens) can be processed as for other pathology specimens using universal precautions. Specimens from patients with infections (or suspected infections) posing a greater risk to pathology personnel (TB, HBV, HCV, HIV, CJD) must be handled with special precautions. All specimens must be fixed as soon as possible and stored in rigid leak-proof containers. Gloves must always be worn when handling specimens. • Fresh tissues are potentially infective and all specimens are placed in fixative as soon as possible. Formalin effectively inactivates viruses (including HIV and HBV) and reduces the infectivity of mycobacteria. Procedures that could aerosolize an infectious agent (e.g., cutting a specimen with a bone saw) should not be performed. Tissue from a CJD patient requires special procedures for handling it safely (see "Creutzfeldt–Jakob disease," above). • Small specimens (e.g., colon biopsies and open lung biopsies) are usually of immediate diagnostic importance and can be processed as usual as long as the specimens are fixed in formalin for at least 4 to 6 hours. • Larger specimens , if of no immediate diagnostic importance (e.g., a placenta from a normal delivery or a colon resection for trauma), can be sectioned thinly and placed in an adequate volume of fixative (1:10 specimen/formalin fixative ratio) for 72 hours before submitting for histologic processing. Placentas and products of conception must be fixed for 7 days before processing. If such a specimen is of diagnostic importance, small sections can be cut for blocks and fixed as above before processing. • Potentially infectious cases are not photographed in the fresh state. If it is an especially interesting case, pictures may be taken after fixation if special precautions are used in order not to contaminate surfaces or the camera. • Frozen sections on potentially infectious cases may be performed but should be avoided if cytologic preparations can be used or an intraoperative diagnosis is not necessary. Freezing does not inactivate infectious agents. If an infectious case is cut in a cryostat, the cryostat should be decontaminated. Pressurized sprays should not be used as this can aerosolize infectious agents. Air-dried slides should be considered potentially infectious and are not saved or submitted to the histology laboratory. Any smears submitted for special stains must be fixed in methanol. Prevention of injuries and exposures Prevention of injuries and exposures is the goal of all pathology personnel. Most injuries and exposure to blood and other body fluids can be prevented if the following guidelines are followed: Gloves • All fresh and fixed tissues must be handled with gloves. The use of two pairs of gloves is recommended as small tears in gloves are common. Metal mesh and Kevlar cloth type gloves are available and should be worn if puncture injuries are possible. • Latex gloves protect against biohazards but not against fixatives. Nitrile gloves provide protection from fixatives. Some individuals (5% to 10%) have or develop allergic reactions (usually dermatitis but sometimes asthma or anaphylaxis) to latex antigens. • Do not touch objects in general use (door handles, telephone, computer, etc.) with contaminated gloves. Hands must always be washed after handling specimens and after leaving a specimen handling area because gloves are not completely leak-proof. Protective clothing • Scrub suits or disposable jumpsuits are recommended if large, bloody specimens need to be processed. • Aprons must be worn when handling many specimens (e.g., at a cutting bench) or handling large specimens. • Protective clothing, including gloves, must be removed and disposed of properly before leaving the surgical cutting or OR consultation rooms. Sharps • Any person using a scalpel blade, razor blade, or syringe needle is responsible for disposing of it properly. Scalpel blades are removed from the handle with extreme caution after gross blood and tissue have been removed. OCT blocks are not removed from the chuck with a razor blade. Holding the stem for a few seconds will melt the OCT sufficiently for removal with a fingertip. Syringe needles are never recapped. All blades, needles, and disposable scissors must be discarded into impervious labeled sharps containers. Broken glass slides and coverslips must also be disposed of into designated containers. • The most common site of an injury is the nondominant hand. • Reusable but contaminated equipment should be decontaminated with bleach. Tissue fixation • All tissues are fixed as soon as possible. Unfixed specimens must be kept in leak-proof containers and stored in an appropriate biohazard refrigerator or freezer. • Always dispose of all blood and tissue fragments before leaving a worksite. All tissues, or non-reusable material contaminated by any body fluid or tissue, must be disposed of in labeled hazardous waste containers (containers with red bags and biohazard symbols). Urine, blood, and feces may be disposed of directly into the municipal sewerage system. Eye protection • Areas contaminated after handling a known infectious case should be immediately cleaned with dilute bleach. • Eye protection should be worn when cutting into large specimens. Cysts may feel deceptively solid when filled with fluid. Such fluid may be under pressure and can travel several feet when the cyst is opened (this has been documented by many pathologists!). Place the specimen near a sink on a surgical drape or blue barrier and make a small nick near the bottom in order to let fluid slowly drain out of the cyst. Food • Food or beverages must not be consumed, or brought into, the cutting room or the OR consultation room. Foods cannot be stored in refrigerators used to store specimens. Food or food containers (e.g., an empty coffee cup) cannot be disposed of into containers in these areas as this may be used as evidence that food consumption is occurring in these areas. Evidence of food consumption is monitored by OSHA and can be grounds for penalties or closure. Prevention of injuries and exposures Prevention of injuries and exposures is the goal of all pathology personnel. Most injuries and exposure to blood and other body fluids can be prevented if the following guidelines are followed: Gloves • All fresh and fixed tissues must be handled with gloves. The use of two pairs of gloves is recommended as small tears in gloves are common. Metal mesh and Kevlar cloth type gloves are available and should be worn if puncture injuries are possible. • Latex gloves protect against biohazards but not against fixatives. Nitrile gloves provide protection from fixatives. Some individuals (5% to 10%) have or develop allergic reactions (usually dermatitis but sometimes asthma or anaphylaxis) to latex antigens. • Do not touch objects in general use (door handles, telephone, computer, etc.) with contaminated gloves. Hands must always be washed after handling specimens and after leaving a specimen handling area because gloves are not completely leak-proof. Protective clothing • Scrub suits or disposable jumpsuits are recommended if large, bloody specimens need to be processed. • Aprons must be worn when handling many specimens (e.g., at a cutting bench) or handling large specimens. • Protective clothing, including gloves, must be removed and disposed of properly before leaving the surgical cutting or OR consultation rooms. Sharps • Any person using a scalpel blade, razor blade, or syringe needle is responsible for disposing of it properly. Scalpel blades are removed from the handle with extreme caution after gross blood and tissue have been removed. OCT blocks are not removed from the chuck with a razor blade. Holding the stem for a few seconds will melt the OCT sufficiently for removal with a fingertip. Syringe needles are never recapped. All blades, needles, and disposable scissors must be discarded into impervious labeled sharps containers. Broken glass slides and coverslips must also be disposed of into designated containers. • The most common site of an injury is the nondominant hand. • Reusable but contaminated equipment should be decontaminated with bleach. Tissue fixation • All tissues are fixed as soon as possible. Unfixed specimens must be kept in leak-proof containers and stored in an appropriate biohazard refrigerator or freezer. • Always dispose of all blood and tissue fragments before leaving a worksite. All tissues, or non-reusable material contaminated by any body fluid or tissue, must be disposed of in labeled hazardous waste containers (containers with red bags and biohazard symbols). Urine, blood, and feces may be disposed of directly into the municipal sewerage system. Eye protection • Areas contaminated after handling a known infectious case should be immediately cleaned with dilute bleach. • Eye protection should be worn when cutting into large specimens. Cysts may feel deceptively solid when filled with fluid. Such fluid may be under pressure and can travel several feet when the cyst is opened (this has been documented by many pathologists!). Place the specimen near a sink on a surgical drape or blue barrier and make a small nick near the bottom in order to let fluid slowly drain out of the cyst. Food • Food or beverages must not be consumed, or brought into, the cutting room or the OR consultation room. Foods cannot be stored in refrigerators used to store specimens. Food or food containers (e.g., an empty coffee cup) cannot be disposed of into containers in these areas as this may be used as evidence that food consumption is occurring in these areas. Evidence of food consumption is monitored by OSHA and can be grounds for penalties or closure. Gloves • All fresh and fixed tissues must be handled with gloves. The use of two pairs of gloves is recommended as small tears in gloves are common. Metal mesh and Kevlar cloth type gloves are available and should be worn if puncture injuries are possible. • Latex gloves protect against biohazards but not against fixatives. Nitrile gloves provide protection from fixatives. Some individuals (5% to 10%) have or develop allergic reactions (usually dermatitis but sometimes asthma or anaphylaxis) to latex antigens. • Do not touch objects in general use (door handles, telephone, computer, etc.) with contaminated gloves. Hands must always be washed after handling specimens and after leaving a specimen handling area because gloves are not completely leak-proof. Protective clothing • Scrub suits or disposable jumpsuits are recommended if large, bloody specimens need to be processed. • Aprons must be worn when handling many specimens (e.g., at a cutting bench) or handling large specimens. • Protective clothing, including gloves, must be removed and disposed of properly before leaving the surgical cutting or OR consultation rooms. Sharps • Any person using a scalpel blade, razor blade, or syringe needle is responsible for disposing of it properly. Scalpel blades are removed from the handle with extreme caution after gross blood and tissue have been removed. OCT blocks are not removed from the chuck with a razor blade. Holding the stem for a few seconds will melt the OCT sufficiently for removal with a fingertip. Syringe needles are never recapped. All blades, needles, and disposable scissors must be discarded into impervious labeled sharps containers. Broken glass slides and coverslips must also be disposed of into designated containers. • The most common site of an injury is the nondominant hand. • Reusable but contaminated equipment should be decontaminated with bleach. Tissue fixation • All tissues are fixed as soon as possible. Unfixed specimens must be kept in leak-proof containers and stored in an appropriate biohazard refrigerator or freezer. • Always dispose of all blood and tissue fragments before leaving a worksite. All tissues, or non-reusable material contaminated by any body fluid or tissue, must be disposed of in labeled hazardous waste containers (containers with red bags and biohazard symbols). Urine, blood, and feces may be disposed of directly into the municipal sewerage system. Eye protection • Areas contaminated after handling a known infectious case should be immediately cleaned with dilute bleach. • Eye protection should be worn when cutting into large specimens. Cysts may feel deceptively solid when filled with fluid. Such fluid may be under pressure and can travel several feet when the cyst is opened (this has been documented by many pathologists!). Place the specimen near a sink on a surgical drape or blue barrier and make a small nick near the bottom in order to let fluid slowly drain out of the cyst. Food • Food or beverages must not be consumed, or brought into, the cutting room or the OR consultation room. Foods cannot be stored in refrigerators used to store specimens. Food or food containers (e.g., an empty coffee cup) cannot be disposed of into containers in these areas as this may be used as evidence that food consumption is occurring in these areas. Evidence of food consumption is monitored by OSHA and can be grounds for penalties or closure. Gloves • All fresh and fixed tissues must be handled with gloves. The use of two pairs of gloves is recommended as small tears in gloves are common. Metal mesh and Kevlar cloth type gloves are available and should be worn if puncture injuries are possible. • Latex gloves protect against biohazards but not against fixatives. Nitrile gloves provide protection from fixatives. Some individuals (5% to 10%) have or develop allergic reactions (usually dermatitis but sometimes asthma or anaphylaxis) to latex antigens. • Do not touch objects in general use (door handles, telephone, computer, etc.) with contaminated gloves. Hands must always be washed after handling specimens and after leaving a specimen handling area because gloves are not completely leak-proof. Protective clothing • Scrub suits or disposable jumpsuits are recommended if large, bloody specimens need to be processed. • Aprons must be worn when handling many specimens (e.g., at a cutting bench) or handling large specimens. • Protective clothing, including gloves, must be removed and disposed of properly before leaving the surgical cutting or OR consultation rooms. Sharps • Any person using a scalpel blade, razor blade, or syringe needle is responsible for disposing of it properly. Scalpel blades are removed from the handle with extreme caution after gross blood and tissue have been removed. OCT blocks are not removed from the chuck with a razor blade. Holding the stem for a few seconds will melt the OCT sufficiently for removal with a fingertip. Syringe needles are never recapped. All blades, needles, and disposable scissors must be discarded into impervious labeled sharps containers. Broken glass slides and coverslips must also be disposed of into designated containers. • The most common site of an injury is the nondominant hand. • Reusable but contaminated equipment should be decontaminated with bleach. Tissue fixation • All tissues are fixed as soon as possible. Unfixed specimens must be kept in leak-proof containers and stored in an appropriate biohazard refrigerator or freezer. • Always dispose of all blood and tissue fragments before leaving a worksite. All tissues, or non-reusable material contaminated by any body fluid or tissue, must be disposed of in labeled hazardous waste containers (containers with red bags and biohazard symbols). Urine, blood, and feces may be disposed of directly into the municipal sewerage system. Eye protection • Areas contaminated after handling a known infectious case should be immediately cleaned with dilute bleach. • Eye protection should be worn when cutting into large specimens. Cysts may feel deceptively solid when filled with fluid. Such fluid may be under pressure and can travel several feet when the cyst is opened (this has been documented by many pathologists!). Place the specimen near a sink on a surgical drape or blue barrier and make a small nick near the bottom in order to let fluid slowly drain out of the cyst. Food • Food or beverages must not be consumed, or brought into, the cutting room or the OR consultation room. Foods cannot be stored in refrigerators used to store specimens. Food or food containers (e.g., an empty coffee cup) cannot be disposed of into containers in these areas as this may be used as evidence that food consumption is occurring in these areas. Evidence of food consumption is monitored by OSHA and can be grounds for penalties or closure. Gloves • All fresh and fixed tissues must be handled with gloves. The use of two pairs of gloves is recommended as small tears in gloves are common. Metal mesh and Kevlar cloth type gloves are available and should be worn if puncture injuries are possible. • Latex gloves protect against biohazards but not against fixatives. Nitrile gloves provide protection from fixatives. Some individuals (5% to 10%) have or develop allergic reactions (usually dermatitis but sometimes asthma or anaphylaxis) to latex antigens. • Do not touch objects in general use (door handles, telephone, computer, etc.) with contaminated gloves. Hands must always be washed after handling specimens and after leaving a specimen handling area because gloves are not completely leak-proof. Protective clothing • Scrub suits or disposable jumpsuits are recommended if large, bloody specimens need to be processed. • Aprons must be worn when handling many specimens (e.g., at a cutting bench) or handling large specimens. • Protective clothing, including gloves, must be removed and disposed of properly before leaving the surgical cutting or OR consultation rooms. Sharps • Any person using a scalpel blade, razor blade, or syringe needle is responsible for disposing of it properly. Scalpel blades are removed from the handle with extreme caution after gross blood and tissue have been removed. OCT blocks are not removed from the chuck with a razor blade. Holding the stem for a few seconds will melt the OCT sufficiently for removal with a fingertip. Syringe needles are never recapped. All blades, needles, and disposable scissors must be discarded into impervious labeled sharps containers. Broken glass slides and coverslips must also be disposed of into designated containers. • The most common site of an injury is the nondominant hand. • Reusable but contaminated equipment should be decontaminated with bleach. Tissue fixation • All tissues are fixed as soon as possible. Unfixed specimens must be kept in leak-proof containers and stored in an appropriate biohazard refrigerator or freezer. • Always dispose of all blood and tissue fragments before leaving a worksite. All tissues, or non-reusable material contaminated by any body fluid or tissue, must be disposed of in labeled hazardous waste containers (containers with red bags and biohazard symbols). Urine, blood, and feces may be disposed of directly into the municipal sewerage system. Eye protection • Areas contaminated after handling a known infectious case should be immediately cleaned with dilute bleach. • Eye protection should be worn when cutting into large specimens. Cysts may feel deceptively solid when filled with fluid. Such fluid may be under pressure and can travel several feet when the cyst is opened (this has been documented by many pathologists!). Place the specimen near a sink on a surgical drape or blue barrier and make a small nick near the bottom in order to let fluid slowly drain out of the cyst. Food • Food or beverages must not be consumed, or brought into, the cutting room or the OR consultation room. Foods cannot be stored in refrigerators used to store specimens. Food or food containers (e.g., an empty coffee cup) cannot be disposed of into containers in these areas as this may be used as evidence that food consumption is occurring in these areas. Evidence of food consumption is monitored by OSHA and can be grounds for penalties or closure. Gloves • All fresh and fixed tissues must be handled with gloves. The use of two pairs of gloves is recommended as small tears in gloves are common. Metal mesh and Kevlar cloth type gloves are available and should be worn if puncture injuries are possible. • Latex gloves protect against biohazards but not against fixatives. Nitrile gloves provide protection from fixatives. Some individuals (5% to 10%) have or develop allergic reactions (usually dermatitis but sometimes asthma or anaphylaxis) to latex antigens. • Do not touch objects in general use (door handles, telephone, computer, etc.) with contaminated gloves. Hands must always be washed after handling specimens and after leaving a specimen handling area because gloves are not completely leak-proof. Protective clothing • Scrub suits or disposable jumpsuits are recommended if large, bloody specimens need to be processed. • Aprons must be worn when handling many specimens (e.g., at a cutting bench) or handling large specimens. • Protective clothing, including gloves, must be removed and disposed of properly before leaving the surgical cutting or OR consultation rooms. Sharps • Any person using a scalpel blade, razor blade, or syringe needle is responsible for disposing of it properly. Scalpel blades are removed from the handle with extreme caution after gross blood and tissue have been removed. OCT blocks are not removed from the chuck with a razor blade. Holding the stem for a few seconds will melt the OCT sufficiently for removal with a fingertip. Syringe needles are never recapped. All blades, needles, and disposable scissors must be discarded into impervious labeled sharps containers. Broken glass slides and coverslips must also be disposed of into designated containers. • The most common site of an injury is the nondominant hand. • Reusable but contaminated equipment should be decontaminated with bleach. Tissue fixation • All tissues are fixed as soon as possible. Unfixed specimens must be kept in leak-proof containers and stored in an appropriate biohazard refrigerator or freezer. • Always dispose of all blood and tissue fragments before leaving a worksite. All tissues, or non-reusable material contaminated by any body fluid or tissue, must be disposed of in labeled hazardous waste containers (containers with red bags and biohazard symbols). Urine, blood, and feces may be disposed of directly into the municipal sewerage system. Eye protection • Areas contaminated after handling a known infectious case should be immediately cleaned with dilute bleach. • Eye protection should be worn when cutting into large specimens. Cysts may feel deceptively solid when filled with fluid. Such fluid may be under pressure and can travel several feet when the cyst is opened (this has been documented by many pathologists!). Place the specimen near a sink on a surgical drape or blue barrier and make a small nick near the bottom in order to let fluid slowly drain out of the cyst. Food • Food or beverages must not be consumed, or brought into, the cutting room or the OR consultation room. Foods cannot be stored in refrigerators used to store specimens. Food or food containers (e.g., an empty coffee cup) cannot be disposed of into containers in these areas as this may be used as evidence that food consumption is occurring in these areas. Evidence of food consumption is monitored by OSHA and can be grounds for penalties or closure. Gloves • All fresh and fixed tissues must be handled with gloves. The use of two pairs of gloves is recommended as small tears in gloves are common. Metal mesh and Kevlar cloth type gloves are available and should be worn if puncture injuries are possible. • Latex gloves protect against biohazards but not against fixatives. Nitrile gloves provide protection from fixatives. Some individuals (5% to 10%) have or develop allergic reactions (usually dermatitis but sometimes asthma or anaphylaxis) to latex antigens. • Do not touch objects in general use (door handles, telephone, computer, etc.) with contaminated gloves. Hands must always be washed after handling specimens and after leaving a specimen handling area because gloves are not completely leak-proof. Protective clothing • Scrub suits or disposable jumpsuits are recommended if large, bloody specimens need to be processed. • Aprons must be worn when handling many specimens (e.g., at a cutting bench) or handling large specimens. • Protective clothing, including gloves, must be removed and disposed of properly before leaving the surgical cutting or OR consultation rooms. Sharps • Any person using a scalpel blade, razor blade, or syringe needle is responsible for disposing of it properly. Scalpel blades are removed from the handle with extreme caution after gross blood and tissue have been removed. OCT blocks are not removed from the chuck with a razor blade. Holding the stem for a few seconds will melt the OCT sufficiently for removal with a fingertip. Syringe needles are never recapped. All blades, needles, and disposable scissors must be discarded into impervious labeled sharps containers. Broken glass slides and coverslips must also be disposed of into designated containers. • The most common site of an injury is the nondominant hand. • Reusable but contaminated equipment should be decontaminated with bleach. Tissue fixation • All tissues are fixed as soon as possible. Unfixed specimens must be kept in leak-proof containers and stored in an appropriate biohazard refrigerator or freezer. • Always dispose of all blood and tissue fragments before leaving a worksite. All tissues, or non-reusable material contaminated by any body fluid or tissue, must be disposed of in labeled hazardous waste containers (containers with red bags and biohazard symbols). Urine, blood, and feces may be disposed of directly into the municipal sewerage system. Eye protection • Areas contaminated after handling a known infectious case should be immediately cleaned with dilute bleach. • Eye protection should be worn when cutting into large specimens. Cysts may feel deceptively solid when filled with fluid. Such fluid may be under pressure and can travel several feet when the cyst is opened (this has been documented by many pathologists!). Place the specimen near a sink on a surgical drape or blue barrier and make a small nick near the bottom in order to let fluid slowly drain out of the cyst. Food • Food or beverages must not be consumed, or brought into, the cutting room or the OR consultation room. Foods cannot be stored in refrigerators used to store specimens. Food or food containers (e.g., an empty coffee cup) cannot be disposed of into containers in these areas as this may be used as evidence that food consumption is occurring in these areas. Evidence of food consumption is monitored by OSHA and can be grounds for penalties or closure. RECOMMENDATIONS FOR POSTEXPOSURE TREATMENT AND INCIDENT REPORTING Unfortunately, accidents will occasionally occur. First aid is administered at the site. Bleeding injuries are allowed to bleed liberally. The site should be cleaned with soap and water. If there has been an eye or mucous membrane exposure, these sites are liberally flushed with water. All exposures involving percutaneous inoculation or contact with an open wound, non-intact skin (e.g., chapped, abraded, weeping, or dermatitic), or mucous membranes by blood or tissue should be seen by a physician. The exposed person should record the name of the patient and the surgical specimen number and file an incident report. The exposed individual should be informed of current recommendations for postexposure prophylaxis (HIV and HBV). The exposed individual should be counseled on the relative risks and benefits of this treatment, if available. The blood of the source individual can only be tested for HIV after appropriate consent is obtained. The results of such a test may be made available to the exposed individual after he or she is made aware of applicable laws and regulations concerning disclosure of the identity and infectious status of a source individual and after signing a confidentiality statement. RADIATION Radioactive substances are widely used in the evaluation of patients and may be present in tissues submitted to pathology departments. In some cases patients have been injected with radioactive agents for the purpose of localizing and surgically removing a lesion (e.g., sentinel nodes, octreotide-positive lesions). Little published information is available about the incidence of such specimens and the risk to pathology personnel. 30 In general, patients are injected with small amounts (<5 millicuries) and typical half-lives are short. Specimens should have minimal residual radioactivity and can be generally handled and disposed of without special precautions. However, radiation safety personnel should be consulted for unusual cases or unusual isotopes.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8756770/

StarD13 negatively regulates invadopodia formation and invasion in high-grade serous (HGS) ovarian adenocarcinoma cells by inhibiting Cdc42

Metastasis remains the main challenge to overcome for treating ovarian cancers. In this study, we investigate the potential role of the Cdc42 GAP StarD13 in the modulation of cell motility, invasion in ovarian cancer cells. StarD13 depletion does not affect the 2D motility of ovarian cancer cells. More importantly, StarD13 inhibits matrix degradation, invadopodia formation and cell invasion through the inhibition of Cdc42. StarD13 does not localize to mature TKS4-labeled invadopodia that possess matrix degradation ability, while a Cdc42 FRET biosensor, detects Cdc42 activation in these invadopodia. In fact, StarD13 localization and Cdc42 activation appear mutually exclusive in invadopodial structures. Finally, for the first time we uncover a potential role of Cdc42 in the direct recruitment of TKS4 to invadopodia. This study emphasizes the specific role of StarD13 as a narrow spatial regulator of Cdc42, inhibiting invasion, suggesting the suitability of StarD13 for targeted therapy. 1. Introduction Ovarian cancer is the second most common cancer of the female reproductive system and the primary cause of death of patients with female reproductive system malignancies ( Hunn and Rodriguez, 2012 ). The American Cancer Society estimates that there will be around 21,750 new cases of ovarian cancer and 13,940 deaths from ovarian cancer in the United States in 2020 ( Hunn and Rodriguez, 2012 ). Tumor metastasis from the ovaries to a secondary site remains the main cause of death in patients with ovarian cancer ( Yeung et al., 2015 ). Hence, there is a need to understand the mechanisms regulating ovarian cancer cell motility and invasion as well as identifying the key molecular players involved in these processes for effective treatment of ovarian cancer. Metastasis is a result of the dysregulation of molecules that regulate actin polymerization and cell migration, leading to the acquired ability of cancer cells to metastasize and invade ( Giese et al., 2003 ; Nakada et al., 2007 ). Initially, the cell protrudes towards the direction of the chemoattractant through de novo actin polymerization ( Bailly et al., 1998 ). The cell adheres to the substratum, thereby committing to movement ( Gupton and Waterman-Storer, 2006 ), moves itself forward through actomyosin contractility and finally detaches its tail in order to propel itself forward ( Condeelis et al., 2001 ). Following initial migration, cancer cells form 3D invasive structures called invadopodia, which possess matrix metalloproteinase activity. This enables cancer cells to degrade the extracellular matrix and to invade the surrounding tissues ( Artym et al., 2006 ; Gimona and Buccione, 2006 ; Yamaguchi et al., 2005 ). Invadopodia are F-actin-rich vertical protrusions, that contain regulators of the actin cytoskeleton, such as cortactin, cofilin, Arp2/3 and matrix metalloproteinases ( Artym et al., 2006 ; Gimona and Buccione, 2006 ; Oser and Condeelis, 2009 ; Oser et al., 2009 ; Yamaguchi et al., 2005 ), as well as members of the Rho family of small guanosine triphosphatases (GTPases) ( Sakurai-Yageta et al., 2008 ). Rho GTPases are small monomeric G proteins that belong to the Ras superfamily ( Takai et al., 2001 ). These are molecular switches that regulate all signal transduction pathways involving the reorganization of the actin cytoskeleton ( Ridley and Hall, 1992 ). Almost all processes and events of tumor cell proliferation, motility and invasion including cellular polarity, cytoskeletal re-organization, and signal transduction pathways are controlled through the interplay between the different Rho-GTPases ( El Atat et al., 2019 ; Hanna and El-Sibai, 2013 ; Sahai and Marshall, 2002 ; Tang et al., 2008 ). Rho GTPases are found in two forms, a GDP-bound inactive and a GTP-bound active form ( Bourne et al., 1990 ). Rho GTPases are regulated by three classes of proteins, Guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). GAPs negatively regulate Rho GTPases by stimulating the intrinsic GTPase activity of Rho GTPases and promoting the formation of the inactive GDP-bound form ( Moon and Zheng, 2003 ). Steroidogenic acute regulatory protein-related lipid transfer domain-containing protein 13 (StarD13) is a GAP for RhoA and Cdc42 with potential tumor suppressor functions ( Ching et al., 2003 ; El-Sitt and El-Sibai, 2013 ; El-Sitt et al., 2012 ; Ullmannova and Popescu, 2006 ). Indeed, several studies have shown that StarD13 is under expressed in many types of tumors, and that the overexpression of StarD3 inhibits cancer cell growth and proliferation ( Al Haddad et al., 2020 ; Ching et al., 2003 ; El-Sitt et al., 2012 ; Gao et al., 2018 ; Yang et al., 2019a ). In contrast with its tumor suppressor function however, we have demonstrated that StarD13 positively regulates cell migration of different cancer types including astrocytoma, breast, lung, and colorectal cancer ( Al Haddad et al., 2020 ; Hanna et al., 2014a ; Khalil et al., 2014 ; Nasrallah et al., 2014 ). However, the expression of StarD13 in ovarian cancer tissues versus normal ovarian tissues as well as the role of StarD13 in ovarian cancer cell proliferation, motility and invasion remains unknown. Therefore, this study aims at comparing the expression of StarD13 in normal and cancerous tissues, analyzing the effects of StarD13 on the migration and invasion of different ovarian cancer cell lines, as well as uncovering the mechanisms and downstream targets mediating the role of StarD13 in ovarian cancer. The results obtained support the tumor suppressor role of StarD13 and that StarD13 is a GAP for Cdc42 in ovarian cancer cell lines, as was previously shown in other tumor types ( Ching et al., 2003 ). Moreover, we show that StarD13 inhibits cell adhesion, cell protrusions, cell invasion, matrix degradation and the formation of invadopodia of ovarian cancer cells while having no effect on their 2D motility. Furthermore, we dissect, for the first time, the role of StarD13 in the suppression of invadopodia formation, through the suppression of Cdc42 activation. Finally, the data show that RhoA is required for matrix degradation and invasion of ovarian cancer cells independently from StarD13. 2. Materials and methods 2.1. Antibodies and reagents Cdc42 and RhoA biosensors were kind gifts from Dr. Louis Hodgson (Albert Einstein College of Medicine). RhoA/Rac1/Cdc42 Activation Assay Combo Kit was purchased from Cell BioLabs (Sand Diego, CA, USA). QCM Gelatin Invadopodia Assay was obtained from Millipore (Massachusetts, USA). Primary antibodies against Cdc42, StarD13, actin, vinculin, Arp2, cortactin (Mouse and Rabbit) and TKS5 antibodies were obtained from Abcam (Cambridge, UK). Primary mouse anti-TKS4 was obtained from Merck Millipore. Primary mouse anti-StarD13 and rabbit anti-Cdc42 were obtained from Santa Cruz Biotech. HRP-conjugated secondary antibodies were obtained from Promega (Wisconsin, USA). Fluorescent secondary antibodies Alexa Fluor 488-green and Alexa Fluor 594-red as well as Rhodamine-phalloidin stain were purchased from Invitrogen (Massachusetts, USA). DAPI stain, and cell proliferation reagent were acquired from Roche Diagnostics (Roche Ltd, Mannheim, Germany). Hiperfect transfection reagent, luciferase and human Flexi Tubes siRNA for luciferase, StarD13, Cdc42 and RhoA were obtained from Qiagen (Hilden, Germany). Lipofectamine LTX was from Waltham (Massachusetts, USA) and crystal violet was from SCP Science (Quebec, Canada). 2.2. Cell culture Metastatic SKOV-3 (Human ovarian adenocarcinoma derived from ovaries and from metastasis in ascites), Caov-3 (Human ovarian adenocarcinoma) and PA-1 (Human ovarian teratocarcinoma derived from metastasis in ascites) cell lines were purchased from ATCC (American Type Culture Collection). All cell lines were culture in DMEM medium supplemented with 10% fetal bovine serum and 100U penicillin/streptomycin at 37 °C and 5% CO 2 . 2.3. Expression analysis To determine the expression of StarD13 in human ovarian tumors, we mined the publicly available Repository Oncoming gene expression microarray database (National Cancer Institute, https://www.oncomine.org/resource/login.html ). Data was plotted using the normal versus ovarian cancer data sets and parameters and the threshold was set at p-value of 0.001. The analysis reflects the expression of StarD13 in normal ovarian tissue as well as different groups of ovarian cancers (NT: None tumorigenic, OCCA: ovarian clear cell adenocarcinoma, OEA: ovarian endometrioid adenocarcinoma, OMA: ovarian mucinous adenocarcinoma, OSA: ovarian serous adenocarcinoma). StarD13 gene expression from two different microarray data sets was plotted. 2.4. Immunostaining Ovarian cancer cells were plated on glass coverslips and transfected with siRNA or biosensors as indicated. The media was removed and the cells were washed with PBS1X before fixation with 4% paraformaldehyde for 10 min. Following, the cells were permeabilized for 15 min with 0.5% Triton-X100, and then blocked with 1% BSA blocking solution for 1 h. All cells were incubated with the different primary antibodies overnight at 4 °C and with the appropriate fluorescent secondary alexa fluor antibody for 1 h the day after. Finally, all coverslips were washed and stained with Rhodamine-phalloidin for 30 min before mounting using a mounting solution and sealing the slides. Fluorescent cell images were taken using the 63X objective lens of the fluorescent Zeiss Observer Z1 microscope operated by the Zen software (Oberkochen, Germany). 2.5. Immunohistochemistry Normal ovarian and ovarian adenocarcinoma paraffin-embedded tissues were purchased from OriGene technologies (Maryland, USA). Paraffin was removed by soaking the slides in xylol before rehydrating the tissues with PBS for 20 min at room temperature. Next, 200–300 ml of pre-heated antigen retrieval buffer (1 mM EDTA pH=8) were added to the slides before heating twice for 5 min in the microwave (700 W). Following, the tissues were delimited using a clear nail polish and blocked using a 4% BSA blocking solution before incubation with the primary StarD13 antibody for 2 h. After washing the slides with ice-cold PBS1X, the tissues were incubated with the appropriate secondary antibody coupled to Alexa fluor-488 fluorophore for 30 min before mounted using a mounting solution mixed with DAPI. Fluorescent cell images were taken using the 20X objective lens of the fluorescent Zeiss Observer Z1 microscope operated by the Zen software (Oberkochen, Germany). 2.6. Cell transfection with plasmids and small interfering RNA SKOV3, Caov-3 and PA-1 ovarian cancer cells were transfected with 10 nM of control Luciferase siRNA or RhoA siRNA, Cdc42 siRNA, Rac1 siRNA and StarD13 siRNA, alone or in combination, using the Hiperfect transfection reagent. Where indicated, 48 h after transfection with the siRNA, the cells were co-transfected with 5 μg of the RhoA or Cdc42 biosensors or with 5 μg of the constitutively active RhoA construct. All assays were performed 72 h after transfection with the siRNA. 2.7. Western blot Proteins extracted from ovarian cancer cells by scraping with Laemmeli sample buffer, were run by SDS-PAGE under standard conditions as previously described ( Al Haddad et al., 2020 ; Al Hassan et al., 2018 ; Nicolas et al., 2019 ). After transfer onto a PVDF membrane, the proteins were blocked with 5% bovine serum albumin solution for 1 h, before incubating with the different primary antibodies overnight at 4 °C. Following, the membranes were washed and immunoblotted with the appropriate secondary antibodies for 1 h at room temperature. The bands were visualized with an enhanced chemiluminescent reagent and the images were captured with the Chemidoc imaging system. Densitometry analysis of the expression levels of the different proteins was performed in ImageJ software (National Institute of Health, Massachusetts, USA). 2.8. Pull down assay Proteins were extracted from ovarian cancer cells using the cell lysis buffer provided with the RhoA/Rac1/Cdc42 Activation Assay Combo Kit. The cell lysate was divided in two parts: the first part (500 μl) was incubated with GST-RBD beads or GST-PBD-PAK1 beads, respectively. After incubation on a shaker for 1 h at 4 °C, the samples were centrifuged, and the pellet was washed several times before resuspension in Laemelli sample buffer as described previously ( Al Haddad et al., 2020 ; El Atat et al., 2019 ; Khalil et al., 2014 ). The second part, which was not incubated with the beads, was mixed with Laemelli buffer and used as a loading control for RhoA (total RhoA), Rac1 (total Rac1) or Cdc42 (total Cdc42). All proteins, namely, total RhoA, total Rac1, total Cdc42 as well as GTP-RhoA, GTP-Rac1 and GTP-Cdc2 were boiled 5 min at 100 °C before separation by SDS-PAGE as described earlier. 2.9. Cell proliferation assay Ovarian cancer cell Proliferation was determined by adding 10 μl of the WST-1 reagent to control and transfected ovarian cancer cells which were plated in the different wells of a 96 well plate (dilution of WST-1 to media 1:10). The cells were then incubated with WST-1 for 2 h in the incubator (37 °C and 5% CO 2 ). Cell proliferation was finally quantified at 450 nm using the Varioskan microplate reader from ThermoFisher scientific (Massachusetts, USA). 2.10. Wound healing assay Control and transfected ovarian cancer cells were grown to confluency before making a wound in the cell monolayer using a sterile pipette tip. The media was then discarded and replaced with serum-free fresh media. Wound healing in control and transfected ovarian cancer cells was monitored by capturing phase-contrast images of the same wound area at t = 0 and t = 48 h post-wounding, using the 10X objective of the Leica inverted microscope. The rate of wound closure (μm/h) presented in the figures was obtained in ImageJ by measuring the distance between the cells at 11 different points of the wound region, and calculating and averaging the speed of wound closure by dividing the distance over time (48 h). 2.11. Random cell motility Random cell motility assay was performed as previously described ( Al-Dimassi et al., 2016 ; Al Haddad et al., 2020 ; Al Hassan et al., 2018 ; Hanna et al., 2014a ; Khalil et al., 2014 ; Nasrallah et al., 2014 ). Briefly, control and transfected ovarian cancer cells plated in 3.5 cm plates were monitored randomly moving in a controlled environment (37 °C and 5% CO 2 ). Phase-contrast images of the randomly moving cells were collected every 60 s for 120 min using the 20X objective of the Zeiss Observer Z1 microscope. The total distance traveled by at least 50 randomly moving cells was determined using the ROI tracker in ImageJ. To obtain the average speed of migration in μm/min, the distance was divided over time. 2.12. Adhesion assay Cell adhesion was measured as previously described. Briefly, the wells of a 96-well plate were coated with collagen type I overnight, before washing with washing buffer (0.1% BSA in DMEM media) and blocking in 0.5% BSA blocking solution in the incubator for 1 h. After that, 50 μl of control or transfected ovarian cancer cells suspension containing 4 × 10 5 cells/ml were added to the wells before placing the plates back in the incubator 30 min. Non-adherent cells were then removed by washing the wells 3 times. Adherent cells in the wells were fixed with 4% paraformaldehyde solution for 10 min, before staining with crystal violet (5 mg/ml in 2% ethanol) for 10 min. Finally, the plates were thoroughly washed and dried before solubilizing crystal violet in DMSO for 30 min. Cell adhesion was quantified at 550 nm using the Varioskan microplate reader from ThermoFisher scientific (Massachusetts, USA). 2.13. Invasion assay Invasion assay was performed as previously described using the collagen-based invasion assay kit from Millipore (Burlington, MA) ( Al-Koussa et al., 2020b ; Al Haddad et al., 2020 ; Al Hassan et al., 2018 ). Briefly, control and transfected ovarian cancer cells were starved for 24 h before resuspension in serum-free quenching medium and plating onto the hydrated inserts. The cells were then placed in wells containing complete medium (10% FBS) and incubated for 24 h. Following, the cells at the bottom surface of the inserts were stained with 400 μl of cell stain for 20 min at room temperature. After extracting the stain with the extraction buffer, 100 μl of the extracted stain were transferred to the wells of a 96-well plate. Finally, the optical density of each sample was measured at 560 nm using the Varioskan microplate reader from ThermoFisher scientific (Massachusetts, USA). 2.14. Invadopodia assay The invasion of control and transfected ovarian cancer cells was examined using the QCM Gelatin Invadopodia Assay kit from Millipore (Massachusetts, USA). Briefly, ovarian cancer cells were plated on fluorescently labeled gelatin matrix for 24 h before fixing the cells with 4% paraformaldehyde solution for 10 min at 37°C, and permeabilizing them with 0.5% Triton-X 100 for 15 min on ice. All cells were blocked in 1% BSA solution for 1 h, before incubation with cortactin or TKS4 primary antibodies overnight at 4°C, and with the appropriate fluorophore-conjugated secondary antibodies for 1 h. The cells were imaged using a 63X objective lens on Zeiss Observer Z1 fluorescent microscope and the degradation of the matrix was measured upon quantification of dark areas lacking cortactin or TKS4 signaling using ImageJ. 2.15. FRET imaging and analysis FRET analysis was performed as previously described ( Al Haddad et al., 2020 ). Briefly, ovarian cancer cells were transfected with 2.5 μg of RhoA fluorescence resonance energy transfer 2 (FRET)-based biosensor ( Pertz et al., 2006 ) or Cdc42-based FRET biosensor ( Hanna et al., 2014b ) using lipofectamine. After 24 h, the cells were imaged in CFP, YFP, FRET and DIC channels using the 63X objective lens of the Zeiss Observer Z1 fluorescent microscope (Oberkochen, Germany). YFP exciter and emitter were S500/20 and S535/30 (YFP/acceptor image), respectively. CFP exciter and emitter were S430/25 and S470/30 (CFP/donor image) or S535/30 for FRET image ( Al Haddad et al., 2020 ). For FRET analysis, the background was flatfield corrected and subtracted from the signal and a threshold was applied to the YFP images to obtain a binary mask with values of 1 for the area inside the cell and 0 for the background. Next, the background was removed from the ratio calculations by multiplying the CFP and FRET images by the mask. RhoA and Cdc42 activation were calculated by dividing the FRET image over the donor image. Finally, FRET signals were quantified by averaging the mean FRET ratio in all the cell area, normalizing the values to control cells (untreated) and expressing the difference as fold change as described previously ( Al Haddad et al., 2020 ; Hodgson et al., 2010 ). All single cell analysis experiments are performed as three independent experiments and images of 15 cells from every experiment collected (data is average from 45 cells). 2.16. Quantification of focal adhesions The area and number of focal adhesion were quantified in ImageJ using the CLAHE and Log3D plugins as described previously ( Al-Koussa et al., 2019 ; Al Haddad et al., 2020 ; Horzum et al., 2014 ). CLAHE enhances the local contrast of the image and Log3D filters the image based on predefined parameters for focal adhesions detection and analysis ( Al Haddad et al., 2020 ; Horzum et al., 2014 ). The area of focal adhesions observed upon staining with vinculin was expressed in arbitrary unit (a. u.). The number of focal adhesion was presented as absolute values of the means for each condition. 2.17. Statistical analysis The results reported represent average values from three independent experiments. The error estimates are given as ± SEM. The p -values were calculated by two way ANOVA or t -test to check if the changes observed in the results were significant. 2.1. Antibodies and reagents Cdc42 and RhoA biosensors were kind gifts from Dr. Louis Hodgson (Albert Einstein College of Medicine). RhoA/Rac1/Cdc42 Activation Assay Combo Kit was purchased from Cell BioLabs (Sand Diego, CA, USA). QCM Gelatin Invadopodia Assay was obtained from Millipore (Massachusetts, USA). Primary antibodies against Cdc42, StarD13, actin, vinculin, Arp2, cortactin (Mouse and Rabbit) and TKS5 antibodies were obtained from Abcam (Cambridge, UK). Primary mouse anti-TKS4 was obtained from Merck Millipore. Primary mouse anti-StarD13 and rabbit anti-Cdc42 were obtained from Santa Cruz Biotech. HRP-conjugated secondary antibodies were obtained from Promega (Wisconsin, USA). Fluorescent secondary antibodies Alexa Fluor 488-green and Alexa Fluor 594-red as well as Rhodamine-phalloidin stain were purchased from Invitrogen (Massachusetts, USA). DAPI stain, and cell proliferation reagent were acquired from Roche Diagnostics (Roche Ltd, Mannheim, Germany). Hiperfect transfection reagent, luciferase and human Flexi Tubes siRNA for luciferase, StarD13, Cdc42 and RhoA were obtained from Qiagen (Hilden, Germany). Lipofectamine LTX was from Waltham (Massachusetts, USA) and crystal violet was from SCP Science (Quebec, Canada). 2.2. Cell culture Metastatic SKOV-3 (Human ovarian adenocarcinoma derived from ovaries and from metastasis in ascites), Caov-3 (Human ovarian adenocarcinoma) and PA-1 (Human ovarian teratocarcinoma derived from metastasis in ascites) cell lines were purchased from ATCC (American Type Culture Collection). All cell lines were culture in DMEM medium supplemented with 10% fetal bovine serum and 100U penicillin/streptomycin at 37 °C and 5% CO 2 . 2.3. Expression analysis To determine the expression of StarD13 in human ovarian tumors, we mined the publicly available Repository Oncoming gene expression microarray database (National Cancer Institute, https://www.oncomine.org/resource/login.html ). Data was plotted using the normal versus ovarian cancer data sets and parameters and the threshold was set at p-value of 0.001. The analysis reflects the expression of StarD13 in normal ovarian tissue as well as different groups of ovarian cancers (NT: None tumorigenic, OCCA: ovarian clear cell adenocarcinoma, OEA: ovarian endometrioid adenocarcinoma, OMA: ovarian mucinous adenocarcinoma, OSA: ovarian serous adenocarcinoma). StarD13 gene expression from two different microarray data sets was plotted. 2.4. Immunostaining Ovarian cancer cells were plated on glass coverslips and transfected with siRNA or biosensors as indicated. The media was removed and the cells were washed with PBS1X before fixation with 4% paraformaldehyde for 10 min. Following, the cells were permeabilized for 15 min with 0.5% Triton-X100, and then blocked with 1% BSA blocking solution for 1 h. All cells were incubated with the different primary antibodies overnight at 4 °C and with the appropriate fluorescent secondary alexa fluor antibody for 1 h the day after. Finally, all coverslips were washed and stained with Rhodamine-phalloidin for 30 min before mounting using a mounting solution and sealing the slides. Fluorescent cell images were taken using the 63X objective lens of the fluorescent Zeiss Observer Z1 microscope operated by the Zen software (Oberkochen, Germany). 2.5. Immunohistochemistry Normal ovarian and ovarian adenocarcinoma paraffin-embedded tissues were purchased from OriGene technologies (Maryland, USA). Paraffin was removed by soaking the slides in xylol before rehydrating the tissues with PBS for 20 min at room temperature. Next, 200–300 ml of pre-heated antigen retrieval buffer (1 mM EDTA pH=8) were added to the slides before heating twice for 5 min in the microwave (700 W). Following, the tissues were delimited using a clear nail polish and blocked using a 4% BSA blocking solution before incubation with the primary StarD13 antibody for 2 h. After washing the slides with ice-cold PBS1X, the tissues were incubated with the appropriate secondary antibody coupled to Alexa fluor-488 fluorophore for 30 min before mounted using a mounting solution mixed with DAPI. Fluorescent cell images were taken using the 20X objective lens of the fluorescent Zeiss Observer Z1 microscope operated by the Zen software (Oberkochen, Germany). 2.6. Cell transfection with plasmids and small interfering RNA SKOV3, Caov-3 and PA-1 ovarian cancer cells were transfected with 10 nM of control Luciferase siRNA or RhoA siRNA, Cdc42 siRNA, Rac1 siRNA and StarD13 siRNA, alone or in combination, using the Hiperfect transfection reagent. Where indicated, 48 h after transfection with the siRNA, the cells were co-transfected with 5 μg of the RhoA or Cdc42 biosensors or with 5 μg of the constitutively active RhoA construct. All assays were performed 72 h after transfection with the siRNA. 2.7. Western blot Proteins extracted from ovarian cancer cells by scraping with Laemmeli sample buffer, were run by SDS-PAGE under standard conditions as previously described ( Al Haddad et al., 2020 ; Al Hassan et al., 2018 ; Nicolas et al., 2019 ). After transfer onto a PVDF membrane, the proteins were blocked with 5% bovine serum albumin solution for 1 h, before incubating with the different primary antibodies overnight at 4 °C. Following, the membranes were washed and immunoblotted with the appropriate secondary antibodies for 1 h at room temperature. The bands were visualized with an enhanced chemiluminescent reagent and the images were captured with the Chemidoc imaging system. Densitometry analysis of the expression levels of the different proteins was performed in ImageJ software (National Institute of Health, Massachusetts, USA). 2.8. Pull down assay Proteins were extracted from ovarian cancer cells using the cell lysis buffer provided with the RhoA/Rac1/Cdc42 Activation Assay Combo Kit. The cell lysate was divided in two parts: the first part (500 μl) was incubated with GST-RBD beads or GST-PBD-PAK1 beads, respectively. After incubation on a shaker for 1 h at 4 °C, the samples were centrifuged, and the pellet was washed several times before resuspension in Laemelli sample buffer as described previously ( Al Haddad et al., 2020 ; El Atat et al., 2019 ; Khalil et al., 2014 ). The second part, which was not incubated with the beads, was mixed with Laemelli buffer and used as a loading control for RhoA (total RhoA), Rac1 (total Rac1) or Cdc42 (total Cdc42). All proteins, namely, total RhoA, total Rac1, total Cdc42 as well as GTP-RhoA, GTP-Rac1 and GTP-Cdc2 were boiled 5 min at 100 °C before separation by SDS-PAGE as described earlier. 2.9. Cell proliferation assay Ovarian cancer cell Proliferation was determined by adding 10 μl of the WST-1 reagent to control and transfected ovarian cancer cells which were plated in the different wells of a 96 well plate (dilution of WST-1 to media 1:10). The cells were then incubated with WST-1 for 2 h in the incubator (37 °C and 5% CO 2 ). Cell proliferation was finally quantified at 450 nm using the Varioskan microplate reader from ThermoFisher scientific (Massachusetts, USA). 2.10. Wound healing assay Control and transfected ovarian cancer cells were grown to confluency before making a wound in the cell monolayer using a sterile pipette tip. The media was then discarded and replaced with serum-free fresh media. Wound healing in control and transfected ovarian cancer cells was monitored by capturing phase-contrast images of the same wound area at t = 0 and t = 48 h post-wounding, using the 10X objective of the Leica inverted microscope. The rate of wound closure (μm/h) presented in the figures was obtained in ImageJ by measuring the distance between the cells at 11 different points of the wound region, and calculating and averaging the speed of wound closure by dividing the distance over time (48 h). 2.11. Random cell motility Random cell motility assay was performed as previously described ( Al-Dimassi et al., 2016 ; Al Haddad et al., 2020 ; Al Hassan et al., 2018 ; Hanna et al., 2014a ; Khalil et al., 2014 ; Nasrallah et al., 2014 ). Briefly, control and transfected ovarian cancer cells plated in 3.5 cm plates were monitored randomly moving in a controlled environment (37 °C and 5% CO 2 ). Phase-contrast images of the randomly moving cells were collected every 60 s for 120 min using the 20X objective of the Zeiss Observer Z1 microscope. The total distance traveled by at least 50 randomly moving cells was determined using the ROI tracker in ImageJ. To obtain the average speed of migration in μm/min, the distance was divided over time. 2.12. Adhesion assay Cell adhesion was measured as previously described. Briefly, the wells of a 96-well plate were coated with collagen type I overnight, before washing with washing buffer (0.1% BSA in DMEM media) and blocking in 0.5% BSA blocking solution in the incubator for 1 h. After that, 50 μl of control or transfected ovarian cancer cells suspension containing 4 × 10 5 cells/ml were added to the wells before placing the plates back in the incubator 30 min. Non-adherent cells were then removed by washing the wells 3 times. Adherent cells in the wells were fixed with 4% paraformaldehyde solution for 10 min, before staining with crystal violet (5 mg/ml in 2% ethanol) for 10 min. Finally, the plates were thoroughly washed and dried before solubilizing crystal violet in DMSO for 30 min. Cell adhesion was quantified at 550 nm using the Varioskan microplate reader from ThermoFisher scientific (Massachusetts, USA). 2.13. Invasion assay Invasion assay was performed as previously described using the collagen-based invasion assay kit from Millipore (Burlington, MA) ( Al-Koussa et al., 2020b ; Al Haddad et al., 2020 ; Al Hassan et al., 2018 ). Briefly, control and transfected ovarian cancer cells were starved for 24 h before resuspension in serum-free quenching medium and plating onto the hydrated inserts. The cells were then placed in wells containing complete medium (10% FBS) and incubated for 24 h. Following, the cells at the bottom surface of the inserts were stained with 400 μl of cell stain for 20 min at room temperature. After extracting the stain with the extraction buffer, 100 μl of the extracted stain were transferred to the wells of a 96-well plate. Finally, the optical density of each sample was measured at 560 nm using the Varioskan microplate reader from ThermoFisher scientific (Massachusetts, USA). 2.14. Invadopodia assay The invasion of control and transfected ovarian cancer cells was examined using the QCM Gelatin Invadopodia Assay kit from Millipore (Massachusetts, USA). Briefly, ovarian cancer cells were plated on fluorescently labeled gelatin matrix for 24 h before fixing the cells with 4% paraformaldehyde solution for 10 min at 37°C, and permeabilizing them with 0.5% Triton-X 100 for 15 min on ice. All cells were blocked in 1% BSA solution for 1 h, before incubation with cortactin or TKS4 primary antibodies overnight at 4°C, and with the appropriate fluorophore-conjugated secondary antibodies for 1 h. The cells were imaged using a 63X objective lens on Zeiss Observer Z1 fluorescent microscope and the degradation of the matrix was measured upon quantification of dark areas lacking cortactin or TKS4 signaling using ImageJ. 2.15. FRET imaging and analysis FRET analysis was performed as previously described ( Al Haddad et al., 2020 ). Briefly, ovarian cancer cells were transfected with 2.5 μg of RhoA fluorescence resonance energy transfer 2 (FRET)-based biosensor ( Pertz et al., 2006 ) or Cdc42-based FRET biosensor ( Hanna et al., 2014b ) using lipofectamine. After 24 h, the cells were imaged in CFP, YFP, FRET and DIC channels using the 63X objective lens of the Zeiss Observer Z1 fluorescent microscope (Oberkochen, Germany). YFP exciter and emitter were S500/20 and S535/30 (YFP/acceptor image), respectively. CFP exciter and emitter were S430/25 and S470/30 (CFP/donor image) or S535/30 for FRET image ( Al Haddad et al., 2020 ). For FRET analysis, the background was flatfield corrected and subtracted from the signal and a threshold was applied to the YFP images to obtain a binary mask with values of 1 for the area inside the cell and 0 for the background. Next, the background was removed from the ratio calculations by multiplying the CFP and FRET images by the mask. RhoA and Cdc42 activation were calculated by dividing the FRET image over the donor image. Finally, FRET signals were quantified by averaging the mean FRET ratio in all the cell area, normalizing the values to control cells (untreated) and expressing the difference as fold change as described previously ( Al Haddad et al., 2020 ; Hodgson et al., 2010 ). All single cell analysis experiments are performed as three independent experiments and images of 15 cells from every experiment collected (data is average from 45 cells). 2.16. Quantification of focal adhesions The area and number of focal adhesion were quantified in ImageJ using the CLAHE and Log3D plugins as described previously ( Al-Koussa et al., 2019 ; Al Haddad et al., 2020 ; Horzum et al., 2014 ). CLAHE enhances the local contrast of the image and Log3D filters the image based on predefined parameters for focal adhesions detection and analysis ( Al Haddad et al., 2020 ; Horzum et al., 2014 ). The area of focal adhesions observed upon staining with vinculin was expressed in arbitrary unit (a. u.). The number of focal adhesion was presented as absolute values of the means for each condition. 2.17. Statistical analysis The results reported represent average values from three independent experiments. The error estimates are given as ± SEM. The p -values were calculated by two way ANOVA or t -test to check if the changes observed in the results were significant. 3. Results 3.1. StarD13 is a potential tumor suppressor in ovarian cancer To understand the role of StarD13 in ovarian cancer cells, we determined the expression profile of StarD13 in normal ovarian tissues as well as in tissues obtained from patients with ovarian adenocarcinoma ( Fig. 1A ). Immunohistochemistry analysis showed a significant 2-fold decrease in the expression levels of StarD13 in ovarian adenocarcinoma biopsies as compared to normal ones ( Fig. 1B ). This was further confirmed by mining the Oncomine database for microarray analysis of StarD13 expression in different ovarian cancer types from two datasets. StarD13 expression levels were significantly lower in almost all ovarian cancer types investigated relative to the normal tissues, thus suggesting a potential tumor suppressor role for StarD13 in ovarian cancers ( Fig. 1C ). To further test this hypothesis, we first knocked down StarD13 in three different ovarian cancer cell lines: SKOV-3 ( Supplemental Fig. S1A ), Caov-3 ( Supplemental Fig. SIB ) and PA-1 ( Supplemental Fig. SIC ) then examined the impact of this knock down on cell proliferation. This was achieved by transfecting cells with two different StarD13 specific siRNA oligos (oligo 1 and oligo 2). Supplemental Fig. S1 demonstrates that both oligos efficiently decreased StarD13 expression levels as compared to controls. Specifically, StarD13 expression was reduced by around 80% in SKOV-3 and PA-1 cell lines and around 50% in Caov-3 cell line as compared to the luciferase control ( Supplemental Fig. S1D ). Proliferation of Caov-3 cells depleted of StarD13 increased by around 25%, compared to controls, while proliferation of SKOV-3 and PA-1 cells depleted of StarD13 increased by around 50% compared to controls ( Fig. 1D ), further demonstrating a potential tumor suppressor role played by StarD13 in ovarian cancer cells as previously observed in other tumor types ( Leung et al., 2005 ). 3.2. StarD13 depletion does not affect the 2D migration of ovarian cancer cells After having established the anti-proliferative potential of StarD13 in ovarian cancer cells, we investigated its ability to modulate cell motility using two approaches: wound healing and time-lapse assays. The results show similar wound closure areas in control and StarD13-depleted SKOV-3, Caov-3 and PA-1 cells ( Fig. 2A ). Quantitatively, StarD13 knock down did not affect the rate of wound closure in any of these cell lines either ( Fig. 2B ). The time lapse assays performed on control and StarD13-depleted SKOV-3 and Caov-3 also support these conclusions whereby both control and SKOV-3 depleted cells exhibit similar cell speed as the luciferase control ( Table 1 ). This data potentially exclude a role for StarD13 in regulating the migration of ovarian cancer cells in 2D. This was surprising since previous reports from studies performed in astrocytoma, breast cancer, colon cancer and lung cancer, showed that StarD13 depletion inhibited cancer cell migration due to the dysregulation of RhoA activation ( Al Haddad et al., 2020 ; Hanna et al., 2014a ; Khalil et al., 2014 ; Nasrallah et al., 2014 ). Indeed in these cells, the dysregulation of RhoA led to a decrease in cell migration ( Supplemental Fig. S2 ). When RhoA was depleted in SKOV-3 and Caov-3 (The western blot results presented in Supplemental Fig. S2 show significant reduction of RhoA expression in both SKOV-3 and Caov-3 cells) upon transfection with the RhoA siRNA or overexpressed in the constitutively active form (cells transfected with RhoA-CA), both cells showed a decrease in cell motility ( Supplemental Fig. S2B and S2D and Supplemental movie S1 ). This shows that the regulation of activation/inactivation cycling of RhoA is also needed in ovarian cancer cells for effective cell migration, however, this regulation seems to be StarD13-independent. Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . 3.3. StarD13 is a GAP for Cdc42 in ovarian cancer cells To confirm the GAP function of StarD13, we performed a pull-down assay to investigate the differences in GTP loading of RhoA and Cdc42 in SKOV-3 and Caov-3 cancer cells in the presence or absence of StarD13. Western blot analysis revealed an approximate 3-fold increase in GTP-Cdc42 levels in both SKOV-3 and Caov-3 cells upon StarD13 depletion, compared to controls ( Fig. 3B ). This strongly suggests that StarD13 is a GAP for Cdc42 in ovarian cancer as it was shown in other tumor types ( Ching et al., 2003 ; Kawai et al., 2007 ; Wong et al., 2003 ). On the other hand, Fig. 3A reveals that GTP-RhoA levels were not affected by the depletion of StarD13 in both cell lines. This could explain why RhoA affects cell migration independently of StraD13, as described above, in these cells. 3.4. StarD13 attenuates ovarian cancer cells adhesion through the inhibition of Cdc42/Rac1 Previous studies from our laboratory and others reported an effect of StarD13 on cell adhesion and a potential localization of StarD13 to focal adhesions ( Al Haddad et al., 2020 ; Hanna et al., 2014a ; Holeiter et al., 2008 ; Kawai et al., 2007 , 2009 ; Khalil et al., 2014 ). Here, we investigate the effects of StarD13 knock down on the adhesion of SKOV-3 and Caov-3 cells to the extra cellular matrix. Fig. 4A reveals a 40% and 20% increase in cellular adhesion to collagen of SKOV-3 and Caov-3 cells transfected with StarD13 siRNA, respectively, as compared to controls. Immunostaining for focal adhesion using anti-vinculin in control and StarD13-depleted SKOV-3 cells further confirmed the role of StarD13 in ovarian cancer cell adhesion to the matrix. Indeed, the silencing of StarD13 triggered a 60% increase in the number of focal adhesions in StarD13 siRNA transfected cells as compared to the luciferase control ( Fig. 4B ). Previously, we had also established that the effect of StarD13 depletion on adhesion to be mediated through a lack of RhoA inactivation ( Al Haddad et al., 2020 ; Hanna et al., 2014a ; Khalil et al., 2014 ). Having established in ovarian cancer that StarD13 has only Cdc42 as a downstream target, we tested the possibility that the increase in adhesion in the Stard13-depleted cells is mediated through the absence of Cdc42 inactivation, which leads to an increase in Rac1 activation. We knocked down Cdc42 in SKOV-3 and Caov-3 cells using sequence specific siRNA targeted against Cdc42 (Cdc42 siRNA oligo 1 and oligo 2). Supplemental Fig. S3A shows that Cdc42 siRNA oligo 1 and oligo 2 efficiently decreased the expression level of Cdc42 in SKOV-3 and Caov-3 cancer cells by 80% and 60%, respectively as compared to the control. As expected, Cdc42 depletion led to a decrease in levels of GTP-Rac1 in SKOV-3 cells ( Fig. 4C ). In addition, StarD13 depletion led to an increase in GTP-Rac1 levels which was reversed in the Cdc42 double knock down but not in the RhoA double knock down ( Fig. 4C ). This potentially suggests that Cdc42 is upstream of Rac. Hence, the depletion of StarD13 might relieve the Cdc42 inhibition, potentially leading to GTP-Rac1 accumulation. Having established the increase in GTP-Rac1 in response to StarD13 depletion, we then directly tested the involvement of the Rac1 Rho GTPase in mediating the increase in cellular adhesion upon StarD13 depletion. To this aim, we effectively knocked down Rac1 (by approximately 80%) ( Supplemental Fig. S3B ) and assessed the effects of StarD13, Rac1, Cdc42, StarD13 + Cdc42 or StarD13 + Rac1 depletion on SKOV-3 cell adhesion. In contrast to StarD13 depletion, which increased cell adhesion by 40% as compared to the control, both Cdc42 and Rac1 depletions decreased SKOV-3 cell adhesion to collagen by 60% and 80%, respectively. Knocking down StarD13 with either Cdc42 or Rac1 countered StarD13 depletion effects and decreased cell adhesion as compared to the control ( Fig. 4D ). Collectively, this suggests that StarD13 attenuates adhesion through the inhibition of Cdc42, which in turn leads to the inhibition of Rac1. 3.5. Cdc42 mediates inhibition of ovarian cancer cell protrusions by StarD13 Knowing that Cdc42 is the main effector of StarD13 in these cells, we then looked at the effect of StarD13 depletion on cell protrusion, a main event during cancer migration regulated by Cdc42 ( El-Sibai et al., 2007 ). In order to stimulate pronounced protrusions in these cells, we stimulated the cells with the epidermal growth factor (EGF), a known chemoattractant of ovarian cancer cells ( Alper et al., 2001 ; Hudson et al., 2009 ; Moss et al., 2009 ). SKOV-3 cells were transfected with Luciferase siRNA, StarD13 siRNA, Cdc42 siRNA, or StarD13 siRNA in combination with Cdc42 siRNA. The cells were then starved and stimulated with EGF before staining with rhodamine-phalloidin and Arp2 to mark actin rich protrusions and subsequently quantify the area of membrane ruffles/protrusions. Starved and EGF stimulated SKOV-3 cells depleted of StarD13 exhibited an increase in dorsal membrane ruffles and protrusions as well as a loss of directionality of these structures as compared to the control ( Fig. 5A and Supplemental movie S2 ). Moreover, StarD13-depleted cells showed a higher increase in protrusion and ruffles in response to EGF stimulation. Depletion of Cdc42 in combination with StarD13 eliminated the phenotype observed in both starved and EGF-stimulated cells ( Fig. 5A and Supplemental movie S2 ). Interestingly, Fig. 5B shows that even though Cdc42 depleted SKOV-3 cells lack protrusion and membrane ruffles, the cells show "pockets" of Arp2 and actin accumulation. This suggests that in the absence of Cdc42, the actin nucleator Arp2/3 does not distribute properly for the protrusions to mature and branch. Quantitatively, the protrusion/ruffle area of EGF-stimulated SKOV-3 cells depleted of StarD13 increased by 30% as compared to the control ( Fig. 5C ). The results also show that Cdc42 depleted SKOV3 cells fold change in ruffle area was reduced by 15% as compared to the control and by 20% for StarD13 and Cdc42 depleted SKOV3 cells ( Fig. 5C ). Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . 3.6. Cdc42 mediates StarD13 inhibition of ovarian cancer cell invasion Cdc42 is directly implicated in the regulation of cancer cell invasion, therefore we investigated the role of StarD13 in regulating the invasion of ovarian cancer cells ( Al-Koussa et al., 2020a ; Al Haddad et al., 2020 ; Evers et al., 2000 ; Fortin Ensign et al., 2013 ; Keely et al., 1997 ; Kwiatkowska et al., 2012 ; Yang et al., 2019b ). Using transwell inserts, we tested the ability of control, StarD13 siRNA or StarD13 siRNA + Cdc42 siRNA transfected SKOV-3 or Caov-3 cells to invade in vitro. The micrographs presented in Fig. 6A reveal an increased number of SKOV-3 invaded cells in response to the depletion of StarD13. However, fewer invading cells were observed when Cdc42 is depleted in combination with StarD13 ( Fig. 6A ). Quantitatively, SKOV3 and Caov-3 cells depleted of StarD13 exhibited 80% and 30% increase in invasion, respectively as compared to the control ( Figs. 6B and 6C ). However, Cdc42 depletion was able to reverse the effect of StarD13 depletion in SKOV-3 and Caov-3 cancer cells and reduce the number of invaded cells by around 50% as compared to the StarD13 depleted cells. 3.7. StarD13 inhibits matrix degradation in ovarian cancer cells by inhibiting Cdc42 Matrix degradation is a critical step in cancer invasion and metastasis. We therefore next tested the ability of StarD13 to modulate ovarian cancer cell matrix degradation using the gelatin invadopodia assay. StarD13 and Cdc42 were silenced alone and in combination cells before plating SKOV-3 and Caov-3 cells on fluorescently labeled gelatin matrix for 24 h and subsequent staining with cortactin to the mark invadopodia. Fluorescent micrographs show that StarD13 depletion increases the matrix degraded area in SKOV-3 and Caov-3 cells ( Figs. 6D and 6F ). Cdc42 depletion alone or in combination with StarD13 decreases matrix degradation in both cell lines as compared to the control. Quantitatively, StarD13 depletion increased the degraded area by around 70% and 50% in SKOV-3 and Caov-3, respectively as compared to the control ( Figs. 6E and 6G ). Cdc42 depletion alone or in combination with StarD13 reduces matrix degradation by around 80% and 60% respectively, in SKOV-3 and Caov-3 cells as compared to the control ( Figs. 6E and 6G ). 3.8. StarD13 inhibits potential invadopodia formation in ovarian cancer cells by inhibiting Cdc42 In a recent study, we showed that Cdc42 localizes to TKS4-labeled invadopodia and to the sites of matrix degradation in lung cancer cells. We also showed that StarD13 depletion leads to an increase in these potential invadopodial structures and that this is mediated through an increase in GTP-Cdc42 levels ( Al Haddad et al., 2020 ). In addition, Cdc42 is a known component and regulator of invadopodia ( Murphy and Courtneidge, 2011 ). To further understand the mechanism of StarD13 regulation of ovarian cancer invasion, we investigated the effects of StarD13 and Cdc42 inhibition on invadopodia formation. In order to correctly detect invadopodia, the cells were immunostained against TKS4, a key and exclusive component of invadopodia ( Murphy and Courtneidge, 2011 ). Fig. 7A shows that Cdc42 co-localizes with TKS4-labeled (mature) invadopodia and the area of matrix degradation. Staining the cells with cortactin (and rhodamine-phalloidin) revealed an increase in the collective number of actin-rich dots in StarD13-depleted SKOV-3 cells ( Fig. 7B ). In contrast, SKOV-3 cells depleted of Cdc42 (alone or in combination with StarD13) did not display cortactin-stained invadopodia ( Fig. 7B ). These phenotypes were further confirmed upon staining the cells with either cortactin or WASP and quantitation of the total number of actin-rich dots per cell ( Figs. 7C and 7D ). The data support that StarD13 knock down increases the potential number of invadopodia as compared to the luciferase control by up to two folds, and that Cdc42 depletion either alone or in combination with StarD13 decreases the potential number of invadopodia by around 1–1.5 fold ( Figs. 7C and 7D ). After establishing that Cdc42 localizes to TKS5-positive invadopodia, we looked at the localization of GTP-Cdc42 with FRET analysis of control and StarD13-depleted SKOV-3 cells transfected with a Cdc42 activation biosensor ( Figs. 7E and 7F and Supplemental movies S3 - S6 ) ( Hanna et al., 2014b ). StarD13-depleted SKOV-3 cells exhibited a significant 1.5-fold increase in GTP-Cdc42-positive dots (potential invadopodia with Cdc42 activation), as compared to the luciferase control ( Figs. 7E and 7F ). The data also reveals that all invadopodia in StarD13-depleted SKOV-3 have GTP-Cdc42 ( Fig. 7E and Supplemental movies S6 - S6 ). This is possibly due to the exaggerated activation of Cdc42 in response to StarD13 knock down and the persistence of GTP-Cdc42 in invadopodia ( Supplemental movie S6 ). Conversely, the YFP channel in luciferase control SKOV-3 cells transfected with the Cdc42 biosensor display more dots than those seen in the Cdc42 FRET ratio image ( Fig. 7E ). This would indicate the localization of the Cdc42 biosensor (seen in YFP) but lack of Cdc42 activation (potentially due to the presence of StarD13). Indeed, Fig. 7E and Supplemental movies S3 and S6 further indicate that potential Cdc42 activation signal is lost in invadopodia of Luciferase siRNA control cells after around 1 min, while the invadopodia of SKOV-3 cells depleted of StarD13 persisted as revealed through the Cdc42 FRET signal ( Fig. 7E and Supplemental movies S3 - S6 ). Interestingly, the first frames in StarD13-depleted cells show that, at an earlier stage, invadopodia lack GTP-Cdc42 as evidenced by the lowest FRET ratio pointed out with arrows in Fig. 7E , while the following ones reveal dot like spheres with GTP-Cdc42 which is also evidenced by the highest FRET ratio pointed out with the arrows in Fig. 7E ( Supplemental movies S3 - S6 ). This is an opportune visualization of Cdc42 localization to the invadopodia (seen as a low FRET signal since YFP signal is high reflecting localization while the biosensor has not activated yet) followed shortly by its activation as the FRET signal increased. Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . 3.9. RhoA is required for matrix degradation and invasion independently from StarD13 RhoA has been previously shown to regulate matrix degradation through a mechanism that involves delivery of matrix metalloproteinases to the invadopodia ( Sakurai-Yageta et al., 2008 ). While transfecting SKOV-3 cells with a RhoA FRET biosensor ( Pertz et al., 2006 ), we were able to detect a concentration of GTP-RhoA in dots (potential invadopodia) ( Supplemental Fig. S4A ). Supplemental Fig. S4A and Supplemental movies S7 and S8 illustrate an increase in the number of invadopodia in StarD13-depleted cells as compared to the control (in line with our previous findings) and a localization of GTP-RhoA to the invadopodia in both control and StarD13-depleted cells. This was evidenced by the FRET signal measured in all cell area and per invadopodia where no significant change in levels of GTP-RhoA was detected upon the depletion of StarD13 ( Supplemental Fig. S4B and C and Supplemental movies S7 and S8 ). The movies revealed that RhoA activation potentially persists along with invadopodia presence. RhoA depletion also inhibited gelatin degradation and invasion by 70% and 40% respectively ( Supplemental Fig. S4D and E ). This data proved that RhoA is required for SKOV-3 cancer cells invasion. Having established no significant effect of StarD13 depletion on levels of GTP-RhoA in these cells, this suggests that RhoA plays a role in invadopodial function and in matrix degradation and invasion independently of Stard13 in ovarian cancer cells. Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . 3.10. StarD13 depletion activates Cdc42 and triggers the maturation of TKS-rich dots Having isolated a subset of invadopodia which lacked the accumulation of GTP-Cdc42 ( Fig. 7E ), we sought to further characterize these invadopodia structures by immunostaining SKOV-3 for different markers that make different stages of invadopodia, mainly TKS5 and TKS4. TKS5 and TKS4 are both required for invadopodia formation and the invasive property for multiple human cancer cell lines, with TKS5 playing an early role in the assembly of invadopodia and TKS4 leading to the activation ECM degradation in mature invadopodia ( Buschman et al., 2009 ; Courtneidge et al., 2005 ; Murphy and Courtneidge, 2011 ; Saykali and El-Sibai, 2014 ; Seals et al., 2005 ). Fig. 8A and B reveal two different populations of cortactin-labeled invadopodia in SKOV-3 cells: Cortactin/TKS5 double stained and Cortactin/TKS4 double stained invadopodia. Co-staining the cells with both TKS4 and TKS5 further proves that these two populations are, for the most part, distinct ( Fig. 8C ), whereby most invadopodia have either TKS4 or TKS5 staining and few have both ( Supplemental Fig. S5 ). This could indicate the invadopodia in two stages of formation, where TKS5 localizes to the early stage and TKS4 to the late maturation stage ( Fig. 9 ). In addition, the results indicate that some TKS5 positive invadopodia co-localize with StarD13 ( Fig. 8D ) and lack matrix degradation ability, but none of the TKS4 positive invadopodia (mature/with matrix degradation ability) express StarD13. This might indicate that, at a later stage of invadopodia formation (TKS4-positive invadopodia) StarD13 has to leave the site of invadopodia, in order for Cdc42 to activate ( Fig. 8D and 8E ). Interestingly, TKS5 invadopodia positive for StarD13 lack Cdc42 FRET activation whereas those which are negative for StarD13 exhibit Cdc42 FRET signal ( Fig. 8F ). Similarly, TKS4-positive invadopodia negative for StarD13 also display Cdc42 FRET signal ( Fig. 8G ). Fig. 8H and 8I also highlight that StarD13 never coincides with potentially active Cdc42 in invadopodia, thus suggesting an inhibitory role of StarD13 on Cdc42 in invadopodia. Finally, we show that StarD13 depletion has no effect on the number of TKS5 positive invadopodia but that it significantly increases the number of TKS4 positive invadopodia as compared to the control ( Fig. 8J ). The opposite is observed following Cdc42 depletion, specifically, Cdc42 depletion has no effect on the number of TKS5 positive invadopodia but it significantly decreases the number of TKS4 positive invadopodia as compared to the control ( Fig. 8J ). This suggests a role of Cdc42 in the maturation of invadopodia and suggests that StarD13 suppresses the formation of mature invadopodia through the inhibition of Cdc42. 3.1. StarD13 is a potential tumor suppressor in ovarian cancer To understand the role of StarD13 in ovarian cancer cells, we determined the expression profile of StarD13 in normal ovarian tissues as well as in tissues obtained from patients with ovarian adenocarcinoma ( Fig. 1A ). Immunohistochemistry analysis showed a significant 2-fold decrease in the expression levels of StarD13 in ovarian adenocarcinoma biopsies as compared to normal ones ( Fig. 1B ). This was further confirmed by mining the Oncomine database for microarray analysis of StarD13 expression in different ovarian cancer types from two datasets. StarD13 expression levels were significantly lower in almost all ovarian cancer types investigated relative to the normal tissues, thus suggesting a potential tumor suppressor role for StarD13 in ovarian cancers ( Fig. 1C ). To further test this hypothesis, we first knocked down StarD13 in three different ovarian cancer cell lines: SKOV-3 ( Supplemental Fig. S1A ), Caov-3 ( Supplemental Fig. SIB ) and PA-1 ( Supplemental Fig. SIC ) then examined the impact of this knock down on cell proliferation. This was achieved by transfecting cells with two different StarD13 specific siRNA oligos (oligo 1 and oligo 2). Supplemental Fig. S1 demonstrates that both oligos efficiently decreased StarD13 expression levels as compared to controls. Specifically, StarD13 expression was reduced by around 80% in SKOV-3 and PA-1 cell lines and around 50% in Caov-3 cell line as compared to the luciferase control ( Supplemental Fig. S1D ). Proliferation of Caov-3 cells depleted of StarD13 increased by around 25%, compared to controls, while proliferation of SKOV-3 and PA-1 cells depleted of StarD13 increased by around 50% compared to controls ( Fig. 1D ), further demonstrating a potential tumor suppressor role played by StarD13 in ovarian cancer cells as previously observed in other tumor types ( Leung et al., 2005 ). 3.2. StarD13 depletion does not affect the 2D migration of ovarian cancer cells After having established the anti-proliferative potential of StarD13 in ovarian cancer cells, we investigated its ability to modulate cell motility using two approaches: wound healing and time-lapse assays. The results show similar wound closure areas in control and StarD13-depleted SKOV-3, Caov-3 and PA-1 cells ( Fig. 2A ). Quantitatively, StarD13 knock down did not affect the rate of wound closure in any of these cell lines either ( Fig. 2B ). The time lapse assays performed on control and StarD13-depleted SKOV-3 and Caov-3 also support these conclusions whereby both control and SKOV-3 depleted cells exhibit similar cell speed as the luciferase control ( Table 1 ). This data potentially exclude a role for StarD13 in regulating the migration of ovarian cancer cells in 2D. This was surprising since previous reports from studies performed in astrocytoma, breast cancer, colon cancer and lung cancer, showed that StarD13 depletion inhibited cancer cell migration due to the dysregulation of RhoA activation ( Al Haddad et al., 2020 ; Hanna et al., 2014a ; Khalil et al., 2014 ; Nasrallah et al., 2014 ). Indeed in these cells, the dysregulation of RhoA led to a decrease in cell migration ( Supplemental Fig. S2 ). When RhoA was depleted in SKOV-3 and Caov-3 (The western blot results presented in Supplemental Fig. S2 show significant reduction of RhoA expression in both SKOV-3 and Caov-3 cells) upon transfection with the RhoA siRNA or overexpressed in the constitutively active form (cells transfected with RhoA-CA), both cells showed a decrease in cell motility ( Supplemental Fig. S2B and S2D and Supplemental movie S1 ). This shows that the regulation of activation/inactivation cycling of RhoA is also needed in ovarian cancer cells for effective cell migration, however, this regulation seems to be StarD13-independent. Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . 3.3. StarD13 is a GAP for Cdc42 in ovarian cancer cells To confirm the GAP function of StarD13, we performed a pull-down assay to investigate the differences in GTP loading of RhoA and Cdc42 in SKOV-3 and Caov-3 cancer cells in the presence or absence of StarD13. Western blot analysis revealed an approximate 3-fold increase in GTP-Cdc42 levels in both SKOV-3 and Caov-3 cells upon StarD13 depletion, compared to controls ( Fig. 3B ). This strongly suggests that StarD13 is a GAP for Cdc42 in ovarian cancer as it was shown in other tumor types ( Ching et al., 2003 ; Kawai et al., 2007 ; Wong et al., 2003 ). On the other hand, Fig. 3A reveals that GTP-RhoA levels were not affected by the depletion of StarD13 in both cell lines. This could explain why RhoA affects cell migration independently of StraD13, as described above, in these cells. 3.4. StarD13 attenuates ovarian cancer cells adhesion through the inhibition of Cdc42/Rac1 Previous studies from our laboratory and others reported an effect of StarD13 on cell adhesion and a potential localization of StarD13 to focal adhesions ( Al Haddad et al., 2020 ; Hanna et al., 2014a ; Holeiter et al., 2008 ; Kawai et al., 2007 , 2009 ; Khalil et al., 2014 ). Here, we investigate the effects of StarD13 knock down on the adhesion of SKOV-3 and Caov-3 cells to the extra cellular matrix. Fig. 4A reveals a 40% and 20% increase in cellular adhesion to collagen of SKOV-3 and Caov-3 cells transfected with StarD13 siRNA, respectively, as compared to controls. Immunostaining for focal adhesion using anti-vinculin in control and StarD13-depleted SKOV-3 cells further confirmed the role of StarD13 in ovarian cancer cell adhesion to the matrix. Indeed, the silencing of StarD13 triggered a 60% increase in the number of focal adhesions in StarD13 siRNA transfected cells as compared to the luciferase control ( Fig. 4B ). Previously, we had also established that the effect of StarD13 depletion on adhesion to be mediated through a lack of RhoA inactivation ( Al Haddad et al., 2020 ; Hanna et al., 2014a ; Khalil et al., 2014 ). Having established in ovarian cancer that StarD13 has only Cdc42 as a downstream target, we tested the possibility that the increase in adhesion in the Stard13-depleted cells is mediated through the absence of Cdc42 inactivation, which leads to an increase in Rac1 activation. We knocked down Cdc42 in SKOV-3 and Caov-3 cells using sequence specific siRNA targeted against Cdc42 (Cdc42 siRNA oligo 1 and oligo 2). Supplemental Fig. S3A shows that Cdc42 siRNA oligo 1 and oligo 2 efficiently decreased the expression level of Cdc42 in SKOV-3 and Caov-3 cancer cells by 80% and 60%, respectively as compared to the control. As expected, Cdc42 depletion led to a decrease in levels of GTP-Rac1 in SKOV-3 cells ( Fig. 4C ). In addition, StarD13 depletion led to an increase in GTP-Rac1 levels which was reversed in the Cdc42 double knock down but not in the RhoA double knock down ( Fig. 4C ). This potentially suggests that Cdc42 is upstream of Rac. Hence, the depletion of StarD13 might relieve the Cdc42 inhibition, potentially leading to GTP-Rac1 accumulation. Having established the increase in GTP-Rac1 in response to StarD13 depletion, we then directly tested the involvement of the Rac1 Rho GTPase in mediating the increase in cellular adhesion upon StarD13 depletion. To this aim, we effectively knocked down Rac1 (by approximately 80%) ( Supplemental Fig. S3B ) and assessed the effects of StarD13, Rac1, Cdc42, StarD13 + Cdc42 or StarD13 + Rac1 depletion on SKOV-3 cell adhesion. In contrast to StarD13 depletion, which increased cell adhesion by 40% as compared to the control, both Cdc42 and Rac1 depletions decreased SKOV-3 cell adhesion to collagen by 60% and 80%, respectively. Knocking down StarD13 with either Cdc42 or Rac1 countered StarD13 depletion effects and decreased cell adhesion as compared to the control ( Fig. 4D ). Collectively, this suggests that StarD13 attenuates adhesion through the inhibition of Cdc42, which in turn leads to the inhibition of Rac1. 3.5. Cdc42 mediates inhibition of ovarian cancer cell protrusions by StarD13 Knowing that Cdc42 is the main effector of StarD13 in these cells, we then looked at the effect of StarD13 depletion on cell protrusion, a main event during cancer migration regulated by Cdc42 ( El-Sibai et al., 2007 ). In order to stimulate pronounced protrusions in these cells, we stimulated the cells with the epidermal growth factor (EGF), a known chemoattractant of ovarian cancer cells ( Alper et al., 2001 ; Hudson et al., 2009 ; Moss et al., 2009 ). SKOV-3 cells were transfected with Luciferase siRNA, StarD13 siRNA, Cdc42 siRNA, or StarD13 siRNA in combination with Cdc42 siRNA. The cells were then starved and stimulated with EGF before staining with rhodamine-phalloidin and Arp2 to mark actin rich protrusions and subsequently quantify the area of membrane ruffles/protrusions. Starved and EGF stimulated SKOV-3 cells depleted of StarD13 exhibited an increase in dorsal membrane ruffles and protrusions as well as a loss of directionality of these structures as compared to the control ( Fig. 5A and Supplemental movie S2 ). Moreover, StarD13-depleted cells showed a higher increase in protrusion and ruffles in response to EGF stimulation. Depletion of Cdc42 in combination with StarD13 eliminated the phenotype observed in both starved and EGF-stimulated cells ( Fig. 5A and Supplemental movie S2 ). Interestingly, Fig. 5B shows that even though Cdc42 depleted SKOV-3 cells lack protrusion and membrane ruffles, the cells show "pockets" of Arp2 and actin accumulation. This suggests that in the absence of Cdc42, the actin nucleator Arp2/3 does not distribute properly for the protrusions to mature and branch. Quantitatively, the protrusion/ruffle area of EGF-stimulated SKOV-3 cells depleted of StarD13 increased by 30% as compared to the control ( Fig. 5C ). The results also show that Cdc42 depleted SKOV3 cells fold change in ruffle area was reduced by 15% as compared to the control and by 20% for StarD13 and Cdc42 depleted SKOV3 cells ( Fig. 5C ). Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . 3.6. Cdc42 mediates StarD13 inhibition of ovarian cancer cell invasion Cdc42 is directly implicated in the regulation of cancer cell invasion, therefore we investigated the role of StarD13 in regulating the invasion of ovarian cancer cells ( Al-Koussa et al., 2020a ; Al Haddad et al., 2020 ; Evers et al., 2000 ; Fortin Ensign et al., 2013 ; Keely et al., 1997 ; Kwiatkowska et al., 2012 ; Yang et al., 2019b ). Using transwell inserts, we tested the ability of control, StarD13 siRNA or StarD13 siRNA + Cdc42 siRNA transfected SKOV-3 or Caov-3 cells to invade in vitro. The micrographs presented in Fig. 6A reveal an increased number of SKOV-3 invaded cells in response to the depletion of StarD13. However, fewer invading cells were observed when Cdc42 is depleted in combination with StarD13 ( Fig. 6A ). Quantitatively, SKOV3 and Caov-3 cells depleted of StarD13 exhibited 80% and 30% increase in invasion, respectively as compared to the control ( Figs. 6B and 6C ). However, Cdc42 depletion was able to reverse the effect of StarD13 depletion in SKOV-3 and Caov-3 cancer cells and reduce the number of invaded cells by around 50% as compared to the StarD13 depleted cells. 3.7. StarD13 inhibits matrix degradation in ovarian cancer cells by inhibiting Cdc42 Matrix degradation is a critical step in cancer invasion and metastasis. We therefore next tested the ability of StarD13 to modulate ovarian cancer cell matrix degradation using the gelatin invadopodia assay. StarD13 and Cdc42 were silenced alone and in combination cells before plating SKOV-3 and Caov-3 cells on fluorescently labeled gelatin matrix for 24 h and subsequent staining with cortactin to the mark invadopodia. Fluorescent micrographs show that StarD13 depletion increases the matrix degraded area in SKOV-3 and Caov-3 cells ( Figs. 6D and 6F ). Cdc42 depletion alone or in combination with StarD13 decreases matrix degradation in both cell lines as compared to the control. Quantitatively, StarD13 depletion increased the degraded area by around 70% and 50% in SKOV-3 and Caov-3, respectively as compared to the control ( Figs. 6E and 6G ). Cdc42 depletion alone or in combination with StarD13 reduces matrix degradation by around 80% and 60% respectively, in SKOV-3 and Caov-3 cells as compared to the control ( Figs. 6E and 6G ). 3.8. StarD13 inhibits potential invadopodia formation in ovarian cancer cells by inhibiting Cdc42 In a recent study, we showed that Cdc42 localizes to TKS4-labeled invadopodia and to the sites of matrix degradation in lung cancer cells. We also showed that StarD13 depletion leads to an increase in these potential invadopodial structures and that this is mediated through an increase in GTP-Cdc42 levels ( Al Haddad et al., 2020 ). In addition, Cdc42 is a known component and regulator of invadopodia ( Murphy and Courtneidge, 2011 ). To further understand the mechanism of StarD13 regulation of ovarian cancer invasion, we investigated the effects of StarD13 and Cdc42 inhibition on invadopodia formation. In order to correctly detect invadopodia, the cells were immunostained against TKS4, a key and exclusive component of invadopodia ( Murphy and Courtneidge, 2011 ). Fig. 7A shows that Cdc42 co-localizes with TKS4-labeled (mature) invadopodia and the area of matrix degradation. Staining the cells with cortactin (and rhodamine-phalloidin) revealed an increase in the collective number of actin-rich dots in StarD13-depleted SKOV-3 cells ( Fig. 7B ). In contrast, SKOV-3 cells depleted of Cdc42 (alone or in combination with StarD13) did not display cortactin-stained invadopodia ( Fig. 7B ). These phenotypes were further confirmed upon staining the cells with either cortactin or WASP and quantitation of the total number of actin-rich dots per cell ( Figs. 7C and 7D ). The data support that StarD13 knock down increases the potential number of invadopodia as compared to the luciferase control by up to two folds, and that Cdc42 depletion either alone or in combination with StarD13 decreases the potential number of invadopodia by around 1–1.5 fold ( Figs. 7C and 7D ). After establishing that Cdc42 localizes to TKS5-positive invadopodia, we looked at the localization of GTP-Cdc42 with FRET analysis of control and StarD13-depleted SKOV-3 cells transfected with a Cdc42 activation biosensor ( Figs. 7E and 7F and Supplemental movies S3 - S6 ) ( Hanna et al., 2014b ). StarD13-depleted SKOV-3 cells exhibited a significant 1.5-fold increase in GTP-Cdc42-positive dots (potential invadopodia with Cdc42 activation), as compared to the luciferase control ( Figs. 7E and 7F ). The data also reveals that all invadopodia in StarD13-depleted SKOV-3 have GTP-Cdc42 ( Fig. 7E and Supplemental movies S6 - S6 ). This is possibly due to the exaggerated activation of Cdc42 in response to StarD13 knock down and the persistence of GTP-Cdc42 in invadopodia ( Supplemental movie S6 ). Conversely, the YFP channel in luciferase control SKOV-3 cells transfected with the Cdc42 biosensor display more dots than those seen in the Cdc42 FRET ratio image ( Fig. 7E ). This would indicate the localization of the Cdc42 biosensor (seen in YFP) but lack of Cdc42 activation (potentially due to the presence of StarD13). Indeed, Fig. 7E and Supplemental movies S3 and S6 further indicate that potential Cdc42 activation signal is lost in invadopodia of Luciferase siRNA control cells after around 1 min, while the invadopodia of SKOV-3 cells depleted of StarD13 persisted as revealed through the Cdc42 FRET signal ( Fig. 7E and Supplemental movies S3 - S6 ). Interestingly, the first frames in StarD13-depleted cells show that, at an earlier stage, invadopodia lack GTP-Cdc42 as evidenced by the lowest FRET ratio pointed out with arrows in Fig. 7E , while the following ones reveal dot like spheres with GTP-Cdc42 which is also evidenced by the highest FRET ratio pointed out with the arrows in Fig. 7E ( Supplemental movies S3 - S6 ). This is an opportune visualization of Cdc42 localization to the invadopodia (seen as a low FRET signal since YFP signal is high reflecting localization while the biosensor has not activated yet) followed shortly by its activation as the FRET signal increased. Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . 3.9. RhoA is required for matrix degradation and invasion independently from StarD13 RhoA has been previously shown to regulate matrix degradation through a mechanism that involves delivery of matrix metalloproteinases to the invadopodia ( Sakurai-Yageta et al., 2008 ). While transfecting SKOV-3 cells with a RhoA FRET biosensor ( Pertz et al., 2006 ), we were able to detect a concentration of GTP-RhoA in dots (potential invadopodia) ( Supplemental Fig. S4A ). Supplemental Fig. S4A and Supplemental movies S7 and S8 illustrate an increase in the number of invadopodia in StarD13-depleted cells as compared to the control (in line with our previous findings) and a localization of GTP-RhoA to the invadopodia in both control and StarD13-depleted cells. This was evidenced by the FRET signal measured in all cell area and per invadopodia where no significant change in levels of GTP-RhoA was detected upon the depletion of StarD13 ( Supplemental Fig. S4B and C and Supplemental movies S7 and S8 ). The movies revealed that RhoA activation potentially persists along with invadopodia presence. RhoA depletion also inhibited gelatin degradation and invasion by 70% and 40% respectively ( Supplemental Fig. S4D and E ). This data proved that RhoA is required for SKOV-3 cancer cells invasion. Having established no significant effect of StarD13 depletion on levels of GTP-RhoA in these cells, this suggests that RhoA plays a role in invadopodial function and in matrix degradation and invasion independently of Stard13 in ovarian cancer cells. Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197 . 3.10. StarD13 depletion activates Cdc42 and triggers the maturation of TKS-rich dots Having isolated a subset of invadopodia which lacked the accumulation of GTP-Cdc42 ( Fig. 7E ), we sought to further characterize these invadopodia structures by immunostaining SKOV-3 for different markers that make different stages of invadopodia, mainly TKS5 and TKS4. TKS5 and TKS4 are both required for invadopodia formation and the invasive property for multiple human cancer cell lines, with TKS5 playing an early role in the assembly of invadopodia and TKS4 leading to the activation ECM degradation in mature invadopodia ( Buschman et al., 2009 ; Courtneidge et al., 2005 ; Murphy and Courtneidge, 2011 ; Saykali and El-Sibai, 2014 ; Seals et al., 2005 ). Fig. 8A and B reveal two different populations of cortactin-labeled invadopodia in SKOV-3 cells: Cortactin/TKS5 double stained and Cortactin/TKS4 double stained invadopodia. Co-staining the cells with both TKS4 and TKS5 further proves that these two populations are, for the most part, distinct ( Fig. 8C ), whereby most invadopodia have either TKS4 or TKS5 staining and few have both ( Supplemental Fig. S5 ). This could indicate the invadopodia in two stages of formation, where TKS5 localizes to the early stage and TKS4 to the late maturation stage ( Fig. 9 ). In addition, the results indicate that some TKS5 positive invadopodia co-localize with StarD13 ( Fig. 8D ) and lack matrix degradation ability, but none of the TKS4 positive invadopodia (mature/with matrix degradation ability) express StarD13. This might indicate that, at a later stage of invadopodia formation (TKS4-positive invadopodia) StarD13 has to leave the site of invadopodia, in order for Cdc42 to activate ( Fig. 8D and 8E ). Interestingly, TKS5 invadopodia positive for StarD13 lack Cdc42 FRET activation whereas those which are negative for StarD13 exhibit Cdc42 FRET signal ( Fig. 8F ). Similarly, TKS4-positive invadopodia negative for StarD13 also display Cdc42 FRET signal ( Fig. 8G ). Fig. 8H and 8I also highlight that StarD13 never coincides with potentially active Cdc42 in invadopodia, thus suggesting an inhibitory role of StarD13 on Cdc42 in invadopodia. Finally, we show that StarD13 depletion has no effect on the number of TKS5 positive invadopodia but that it significantly increases the number of TKS4 positive invadopodia as compared to the control ( Fig. 8J ). The opposite is observed following Cdc42 depletion, specifically, Cdc42 depletion has no effect on the number of TKS5 positive invadopodia but it significantly decreases the number of TKS4 positive invadopodia as compared to the control ( Fig. 8J ). This suggests a role of Cdc42 in the maturation of invadopodia and suggests that StarD13 suppresses the formation of mature invadopodia through the inhibition of Cdc42. 4. Discussion This study provides an in-depth understanding of the role of StarD13 in ovarian cancer cells, as well as uncovers the key targets mediating StarD13 effects on cell proliferation, motility, invadopodia formation and invasion. Most, importantly, the data provide a model for the recruitment of an ordered set of molecules and signaling events required for the formation and the maturation of invadopodia in ovarian cancer cells. This suggests StarD13 as a main regulator of invadopodia and reveals, for the first time, its clear role in the spatial regulation of Cdc42 activation. Through the use of different markers of invadopodia, this study distinguishes stages of formation of invadopodia and the potential role of StarD13 and Cdc42 during these stages. Immunohistochemistry and data microarray analysis revealed a lower expression of StarD13 in ovarian cancer tissues as compared to the normal ovarian tissues, which is similar to the results observed in lung and hepatocellular cancers ( Al Haddad et al., 2020 ; Ching et al., 2003 ). StarD13 inhibition of cell proliferation is also in line with a potential tumor suppressor function of this protein ( Al Haddad et al., 2020 ; El-Sitt et al., 2012 ; Hanna et al., 2014a ; Khalil et al., 2014 ; Nasrallah et al., 2014 ; Yang et al., 2019b , 2019a ; Zhang et al., 2017 ). However, unlike in previous studies performed on breast, lung, astrocytoma and colorectal cancers ( Al Haddad et al., 2020 ; Basak et al., 2018 ; El-Sitt et al., 2012 ; Hanna et al., 2014a ; Khalil et al., 2014 ; Nasrallah et al., 2014 ), where StarD13 was found to be necessary for cell migration, it had no effect on the 2D motility in ovarian cancer cells. This is also in contradiction to reports where StarD13 was found to negatively regulate cell migration ( Fan et al., 2012 ; Holeiter et al., 2008 ; Leung et al., 2005 ; Lin et al., 2010 ; Yang et al., 2019b ). This could be due to the lack of effect on RhoA activity in ovarian cancer cells depleted of StarD13. In contrast, StarD13 depletion led to a notable and significant increase in GTP-RhoA in other tumors studied ( Al Haddad et al., 2020 ; Ching et al., 2003 ; El-Sitt and El-Sibai, 2013 ; Jaafar et al., 2020 ; Khalil et al., 2014 ). This could be accounted for by a potential higher expression of Deleted in liver cancer-1 (DLC1), the StarD13 homolog, in ovarian cancer. DLC1 is a GAP for RhoA, which is often deleted in hepatocellular carcinoma and inactive in various types of human cancers including colon cancer ( Liu et al., 2017 ). DLC1 is not deleted in ovarian carcinoma tissues, albeit expressed at a lower level than normal ovarian tissues ( Ren et al., 2013 ), which could compensate for the GAP activity of StarD13. This could indicate that, while in other tumor types StarD13 is the main GAP for RhoA due to the absence of DLC1, in ovarian tumors DLC1 mainly drives this inhibition. Knock down of StarD13 however, showed an increase in GTP-Cdc42, strongly suggesting that StarD13 is a GAP for this Rho GTPase in ovarian cancer cells, similarly to findings in other tumor types ( El-Sitt et al., 2012 ; Hanna et al., 2014a ; Khalil et al., 2014 ; Nasrallah et al., 2014 ). In addition, a slight increase in the adhesive ability of ovarian cancer cells was observed after StarD13 depletion. This is in agreement with previously reported decrease in adhesion/cell rounding in HepG2 hepatoma cells overexpressing DLC2 ( Leung et al., 2005 ). Another main regulator of adhesion in cells is the Rho GTPase, Rac1. The depletion of StarD13 led to an increase in GTP-Rac1, which was due to a potential lack of Cdc42 inhibition (through the GAP activity). This is consistent with previous reports placing Cdc42 upstream from Rac1 in other cell types ( El Atat et al., 2019 ; Yip et al., 2007 ). This also accounts for the increase in adhesion in StarD13-depleted cells, independently of RhoA, since knocking down Rac1 in StarD13-depleted cells reversed its effect on adhesion. While in other tumor types, an increase in adhesion and the disruption of adhesion dynamics inhibited cell motility ( Al Haddad et al., 2020 ; Ching et al., 2003 ; El-Sitt and El-Sibai, 2013 ; Jaafar et al., 2020 ; Khalil et al., 2014 ), it did not affect the migratory ability of ovarian cancer cells. This discrepancy could be due to the distinct role adhesion turnover might play in migration in different cell types. Rac1 mainly drives the formation of point contacts. These are small punctate structures, found behind the front of the lamellipodium ( Kaverina et al., 2002 ) and at the edge of cells. On their own, point contacts do not confer enough contractility the cells to move ( Kaverina et al., 2002 ). Point contacts however, are precursors of focal adhesions, which are mediated by RhoA and provide the necessary mechanical strength for the cell bodies to contract and move forward ( Gupton and Waterman-Storer, 2006 ). In these cells, the depletion of StarD13 leads to an increase in Rac1 activation and potentially an increase in the number of point contacts. Since RhoA activation is unchanged, however, the point contacts would remain in excess and, while would appear higher in number, would still be bottlenecked by the maturation rate-limiting step. Consequently, the detected increase in the number of adhesion structures is mostly reflecting structures that would not paralyze the cells through over-attachment to the matrix and would not decrease cell migration, as observed. StarD13-depleted cells also showed a dramatic increase in ruffles and in protrusion (in quiesced cells as well as cells stimulated with EGF), which is mediated by Cdc42. This is consistent with previous reports highlighting the role of Cdc42 in actin polymerization and cell protrusion in cancer cells ( El-Sibai et al., 2007 ). As mentioned above however, this did not affect the directionality of the cell undergoing migration. This could be due to additional spatial suppressive factors that concentrate the protrusion at the leading edge in migrating cells ( Vega et al., 2011 ), whereas cells undergoing bath stimulation lose their directionality and a defined leading edge. Interestingly, in cells where Cdc42 was depleted, we observed concentrated ring-like structures rich in Arp2 and actin. This could be indicative of a lack of proper distribution of the Arp2/3 protein, due to the lack of activation of its organizer N-WASP ( Rohatgi et al., 1999 ). RhoA was also required for matrix degradation and invasion, independently of StarD13. In addition to the effect on actin-rich protrusion, StarD13 silencing increased matrix degradation and ovarian cancer cell invasion. This was previously observed in other tumor types ( Al Haddad et al., 2020 ; Hanna et al., 2014a ). More importantly, this was mediated by Cdc42. Indeed, silencing of Cdc42 reversed the increase observed in the StarD13-depleted cells. In addition, Cdc42 depletion completed abolished matrix degradation in control ovarian cancer cells. This is in line with the role of Cdc42 in promoting the production of metalloproteinases that degrade the extracellular matrix components facilitating the process of invasion ( Stengel and Zheng, 2011 ; Yamaguchi et al., 2005 ). In a recent study, we showed that StarD13 depletion increases invadopodia formation through Cdc42 in normal lung cells and in lung cancer cells ( Al Haddad et al., 2020 ). That corroborated the potential tumor suppressive function of StarD13 since, once depleted, normal lung cells recapitulated the tumor phenotype and formed invasive structures. In ovarian cancer cells, StarD13 depletion also led to an increase in cortactin-labeled and WASP-labeled potential invadopodia, while Cdc42 depletion completed abolished any of these structures. Cdc42 co-localized with the specific invadopodia marker TKS4. Cdc42 activation, observed through the Cdc42 FRET biosensor, also localized with TKS4 and TKS5 staining. Live imaging of Cdc42 activation through FRET, showed that StarD13 depletion leads to a higher and a more prolonged activation of Cdc42 in dots (potential invadopodia). This suggests a spatial (in invadopodia) and a temporal inhibition of Cdc42 by StarD13. This is reminiscent of the role of RhoC in the spatial regulation of cofilin in invadopodia ( Bravo-Cordero et al., 2011 ). However, while RhoC depletion did not affect the number of invadopodia but inhibited their effectiveness, StarD13 depletion increased the number of invadopodia. In addition, the increase in the number of invadopodia in ovarian cancer cells could also potentially account for the slight increase in adhesion described earlier, because these structures are in essence podosome-type adhesion clusters ( Block et al., 2008 ). Interestingly, monitoring the dynamics of StarD13, Cdc42, TKS4 and TKS5 expression in cells and in invadopodia uncovered a set of organized events required for the formation and maturation of invadopodia ( Fig. 9 ). The model proposed suggests that Cdc42 is recruited early to invadopodia but that it is not activated until StarD13 is evacuated. Cdc42 plays a direct role in the early recruitment of cortactin and WASP to the point of actin polymerization in invadopodia (as evidenced by the Cdc42 knock down in Fig. 7B ). It is worth noting that RhoA activation in invadopodia is independent of StarD13 expression. Potentially, RhoA is activated in invadopodia to recruit MMPs and lead to matrix degradation as previously demonstrated ( Chiang et al., 2016 ; Hoshino et al., 2009 ; Varon et al., 2006 ; Yu et al., 2013 ). In an earlier study, we reported StarD13 to always be absent in invadopodial structures ( Al Haddad et al., 2020 ). In that study, however, invadopodial structures were identified with TKS4 staining. In this study, we were able to identify two pools of invadopodia as labeled by TKS5 and TKS4, which co-localize with cortactin and mark early and late invadopodia, respectively ( Fig. 9 ), as previously established in the literature ( Murphy and Courtneidge, 2011 ). We also identified some TKS4/5 double-labeled invadopodia, which might be transitioning into maturation ( Supplemental Fig. S5 ). StarD13 seemed to coincide with TKS5 labeling in some of these structures, which is indicative of early invadopodia where StarD13 has not yet evacuated. Indeed, Fig. 8F shows that, while a subset of the TKS5-labeled invadopodia show Cdc42 activation through the FRET signal, others do not. The TKS5 positive invadopodia where Cdc42 has not activated yet must be short lived since, as mentioned earlier, Cdc42 plays an early role in the recruitment of cortactin and WASP ( Fig. 7B ). Indeed, the kinetics of Cdc42 activation to invadopodia-like structures in Fig. 7E showed a very quick activation of Cdc42 (reflected by the high FRET ratio) following its localization (reflected by the low FRET ratio) through the duration of the invadopodia lifetime. In summary, both RhoA and Cdc42 activation persist in invadopodia while StarD13 is only expressed in early invadopodia along with TKS5 and never coincides with TKS4 ( Fig. 8D and E ). Interestingly, when StarD13 is knocked down we observe the same number of TKS5 positive invadopodia but more TKS4 positive invadopodia, which is due to the increase in GTP-Cdc42 and which coincides with the increase in matrix degradation. In contrast, Cdc42 knock down abrogates the formation of TKS4 positive invadopodia. Collectively, this suggests that Cdc42 might be required for the recruitment of TKS4 to invadopodia to mature. Coincidentally, TKS4 has been previously found to be necessary for the recruitment of MT1-MMP to invadopodia ( Murphy and Courtneidge, 2011 ). At the same time, this was found to be regulated by Cdc42 ( Noritake et al., 2005 ; Sakurai-Yageta et al., 2008 ). Here we suspect a novel involvement of Cdc42 in matrix degradation in invadopodia through the recruitment of TKS4, in addition to its early role in WASP activation and actin polymerization. Altogether this study showed that StarD13 is a GAP for Cdc42 and emphasized the importance of StarD13 and Cdc42 in regulating the formation of invadopodia in a temporal and functional manner. Supplementary Material Supplemental Movie S5 Supplemental Movie S7 Supplemental Movie S6 Supplemental Movie S8 Supplemental Movie S3 Supplemental Movie S2 Supplemental Movie S1 Supplemental Movie S4 Supplemental Figure S5 Supplemental Figure S1 Supplemental Figure S4 Supplemental Figure S2 Supplemental Figure S3 Funding This work was supported by the Natural Science Department at the Lebanese American University, Beirut, Lebanon and by The National Institute of Health (NIH), USA R35GM136226 to Louis Hodgson.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3023272/

Current progress in the development of a prophylactic vaccine for HIV-1

Since its discovery and characterization in the early 1980s as a virus that attacks the immune system, there has been some success for the treatment of human immunodeficiency virus-1 (HIV-1) infection. However, due to the overwhelming public health impact of this virus, a vaccine is needed urgently. Despite the tireless efforts of scientist and clinicians, there is still no safe and effective vaccine that provides sterilizing immunity. A vaccine that provides sterilizing immunity against HIV infection remains elusive in part due to the following reasons: 1) degree of diversity of the virus, 2) ability of the virus to evade the hosts' immunity, and 3) lack of appropriate animal models in which to test vaccine candidates. There have been several attempts to stimulate the immune system to provide protection against HIV-infection. Here, we will discuss attempts that have been made to induce sterilizing immunity, including traditional vaccination attempts, induction of broadly neutralizing antibody production, DNA vaccines, and use of viral vectors. Some of these attempts show promise pending continued research efforts. Introduction Since its discovery and characterization in the early 1980s as a virus that attacks the immune system, leaving patients unable to fight off opportunistic infections, there has been an ebb and flow of effective treatments and hope as scientists continue to search for ways to eradicate human immunodeficiency virus-1 (HIV-1) from the human population similar to what has been accomplished in the case of smallpox. The majority of the effort and nearly all of the success has come in the area of patient treatment rather than inhibition of contraction or spread of the virus. A class of treatments, antiretroviral therapies (ARTs) and later highly active antiretroviral therapies (HAARTs), has been the mainstay of disease control during the last 15 years. Notwithstanding the increased life span of patients, increased time to full-blown AIDS, and decreased contraction of opportunistic infections and AIDS-related diseases (ie, non-Hodgkin's lymphoma, Kaposi's sarcoma, etc) by patients treated with HAART, there are several reasons why development of an HIV-1 vaccine is still warranted. Five of these reasons are as follows: 1) nearly two-thirds of the patients who contract HIV-1 live in underdeveloped countries and cannot afford the expensive HAART regimen, 1 2) both the ART and HAART regimen are complex and are disruptive to patients' lives and diets, making long-term compliance an issue, 2 3) the potential side effects of ART/HAART treatments negatively affect the long-term health of patients and include diabetes, cardiovascular disease, fractures, etc, 3 – 5 4) development of HAART drug resistance, and 5) the presence of latent HIV-1 reservoirs harboring viral strains that were produced through mutation throughout the duration of the infection of the host also play a role in the failure of HAART. 6 These reasons, as well as many others, underscore the need for a prophylactic HIV-1 vaccine. Possibly, the strongest argument for development of a prophylactic vaccine may be the need for control of the virus spread worldwide. Every day, 7500 patients worldwide are infected with HIV-1. 1 Production of a vaccine that could inhibit infection, reduce spread, or both would aid in the reduction of the burden of AIDS and AIDS-related diseases. The expenses incurred by the AIDS epidemic can hardly be calculated. They range from tens of thousands of dollars per patient for the HAART regimen, to millions of dollars required for building of orphanages by governments for children whose parents have succumbed to the disease, to the unknown cost of educational materials and condoms in the effort to prevent further spread of the disease. This public health challenge has not gone unnoticed and has been addressed by scientists' ongoing efforts to develop a safe and effective HIV-1 vaccine. Prophylactic vs therapeutic vaccines A prophylactic HIV-1 vaccine would offer sterilizing immunity to patients, preventing infection upon presentation of the virus. A prophylactic vaccine must also be effective at all possible portals of HIV-1 entry, especially the mucosa. 7 For this to occur, the vaccine must offer broad and durable immunity. Several consortia have worked diligently to produce a vaccine that will induce broadly reactive neutralizing antibodies (Nabs). These consortia include major international efforts as well as efforts of individual countries, regions, and institutions including, but not limited to: the International AIDS Vaccine Initiative Neutralizing Antibody Consortium, 8 the Center for HIV-AIDS Vaccine Immunology, the HIV Vaccine Trials Network, US Military HIV Research Program, the Collaboration for AIDS Vaccine Discovery, and the Vaccine Research Center at the National Institutes of Allergy and Infectious Diseases of the National Institutes of Health. To date, however, no HIV-1 candidate vaccine has induced broadly reactive Nabs. 8 In the absence of a vaccine that can prevent infection of HIV-1, there are still many benefits to be realized from production of a therapeutic vaccine. A therapeutic vaccine would be supremely valuable if it were able to increase the titer of virus necessary for infection, increase the time to clinical manifestation of virus, control viral load after infection, and reduce secondary transmission. 9 – 13 A vaccine that could induce this type of response would invariably decrease contagiousness, decrease the need for costly and potentially dangerous ART/HAART, and decrease the number of opportunistic infections of patients. While the effect of controlling the normal HIV-1 pathology with therapeutic vaccines will be favorable for the individual patient as well as society, the effect of preventing HIV-1 infections in humans with a prophylactic vaccine is also broadly appealing. This potential for eradicating the HIV-1 virus from human hosts drives scientists to continue to find ways to circumvent the challenges presented by this unique virus in order to induce production of the Nabs that are critical for sterilizing immunity. This review, therefore, will focus on the specific challenges presented by HIV-1 and strides that have been made toward creating a prophylactic vaccine, including past efforts that have failed and lessons that have been learned from those failures. We will also discuss novel vaccine options and some of the promising trials that are currently underway. Current challenges to creating an HIV-1 vaccine While several problems face scientists who are attempting to create an HIV-1 vaccine, three problems in particular have posed extremely daunting challenges. These three problems are 1) degree of diversity of the virus, 2) ability of the virus to evade the hosts' immunity, and 3) lack of appropriate animal models in which to test vaccine candidates. These three major problems will be discussed in more detail below. Degree of diversity Traditionally, prophylactic vaccines have been made by exposing some part of a pathogen's structure as an antigen to the host's immune system, and eliciting an immune response, resulting in the production of long-term memory lymphocytes that are capable of mounting a strong immune response upon later infection with the pathogen. The premise upon which this manipulation of the immune system is based is the ability of the immune system to make long-lasting antibodies to conserved structures on exposed proteins that are native to the pathogen. Ideally, both humoral and cell-mediated immunity would be induced creating long-lasting immunity. Traditional attempts to recreate this process using live attenuated simian immunodeficiency virus-1 (SIV-1) viruses in an effort to vaccinate macaques against SIV-1 have been proven safe and effective in macaques that were subsequently challenged with SIV-1. 14 , 15 However, an incidental study of the effect of live-attenuated HIV-1 (containing deletions of the nef gene and the long terminal repeat) was proven pathogenic in humans when three out of six treated patients developed late-onset immunosuppression. 16 – 18 Killed viruses have also been tested as a potential vaccine approach, but safety concerns have halted their use. These safety concerns include incomplete inactivation of the virus leading to potential residual infectivity during the vaccine preparation. 19 Due to the ineffectiveness of traditional vaccine approaches to date, scientists have attempted to use recombinant HIV-1 proteins to stimulate the production of Nabs. These attempts failed due to their inability to induce a lasting, broad range of Nabs that would inhibit infection in humans. 20 – 23 Perhaps these failures are a result of the inherent diversity of HIV-1. This diversity has presented a major roadblock to development of a prophylactic vaccine. There are three main groups of HIV-1 (M, O, and N) 24 as well as a recently discovered group, P. 25 Each group consists of several subtypes, clades. The various clades display biological differences with respect to transmission, 26 replication, 27 and disease progression. 28 , 29 These differences result in an inability to produce a generalizable vaccine that would induce the breadth of Nabs necessary to counter an infection by a wide range of HIV-1 clades that may be encountered in a natural setting. 30 The degree of diversity seen in HIV-1 is greater than that of any other virus observed. 31 , 32 This problem is being addressed by development of multiclade (multiple env and/or subtype B gag, pol, nef ) 33 , 34 and mosaic vaccines which incorporate sets of 10 immunogenic proteins from 4 different clades or bivalent proteins from clades B and C. 35 – 37 There are proof of principle studies that illustrate immunological protection against HIV-1 in nonhuman primates that were passively treated with broadly reactive Nabs. 38 – 40 These studies show that protection against infection with HIV-1 can be conferred by the presence of broadly reactive Nabs. The next step toward production of a prophylactic vaccine would involve induction of production of these or similar broadly reactive Nabs by the host's immune system. Immune evasion The rate at which the HIV-1 virus mutates, due to the nature of the reverse transcriptase enzyme responsible for transcribing its RNA, ensures that nearly every daughter virion will have a different genome than its parent. 41 When these changes occur in the HIV-1 Env protein that is needed for antibody recognition, they inhibit the immune system's ability to mount a sufficient response. One attempt to circumvent this problem has been to induce the production of Nabs to the conserved regions of HIV-1 proteins. A major problem with this approach is that the conserved regions of HIV-1 proteins are often shielded from exposure to Nabs within the HIV-1 envelope. The native structure of the envelope protein, reportedly the only HIV-1 protein susceptible to Nabs, 31 shields it from the immune system as a glycosylated trimer of heterodimers. The glycosylation of the envelope protein allows for the carbohydrates to masquerade as 'self ' thereby forming an immunologically silent face and protects neighboring epitopes via an 'evolving glycan shield'. 42 – 44 Additionally, the gp41 coreceptor binding site, another conserved site, is not presented until primary binding to CD4 + has occurred. 45 An attempt to create antibodies to the CD4-binding region of the gp120 protein was made in rhesus macaques in 2007 and results indicated that vaccinated hosts were able to withstand challenge with SHIV. 46 Other attempts to create an HIV-1 vaccine have focused on overcoming the ability of HIV-1 to escape immune surveillance through use of antibodies that are able to neutralize diverse isolates of HIV-1. These antibodies include PG9, PG16, 47 2F5, 2G12, 4E10, b12, 48 – 51 and most recently sCD4-17b 52 and others. 53 Identification of these antibodies gives hope that their induction or the induction of other such broadly reactive Nabs may provide the basis for a prophylactic vaccine in the future. Lack of appropriate animal models The use of animal models for development of therapeutics offers the benefit of thorough testing and validation prior to introduction of a vaccine in humans. In the past, vaccines were made by observing and then mimicking the immune response mounted by individuals who had recovered from a particular disease. To date, however, there are no known cases of individuals who have recovered from HIV-1 infection. However, data can be gathered from long-term nonprogressors – patients who have been infected with HIV-1 for at least 7 years and do not display any HIV-1-related symptoms. 54 , 55 Another option that may be critical to the development of a prophylactic vaccine is the use of relevant animal models. Such models will allow for analysis of the effect of a potential vaccine on an intact host prior to use in humans. One particular challenge with the use of animal models for development of a prophylactic HIV-1 vaccine is that there are very few naturally occurring disease models of HIV-1. Only a few nonhuman primates are susceptible to infection with HIV-1 and infected animals do not progress to AIDS. 56 Therefore, it is important to use other disease models that mimic the HIV-AIDS pathologic progression. 57 One such potential model is feline immunodeficiency virus (FIV). FIV was discovered in 1986 and is known to cause an AIDS-like disease in domestic cats and mimics HIV-related dementia in humans. 58 A vaccine for FIV was approved by the FDA in 2002. 59 While the FIV model is potentially informative, its use is not sufficient as a basis for development of a prophylactic HIV-1 vaccine. An ideal animal model would display a pathological response to infection with HIV-1 that is very similar to the one that occurs in humans. Unfortunately, HIV-1 does not cause pathology leading to the development of AIDS in any host other than humans. 60 – 63 However, animal models have been developed and used that allow partial understanding of the pathology of HIV-1, the natural immunological response to infection, and the response of the host to novel therapeutics. One of these models involves the simian immunodeficiency virus MAC (SIV MAC ) that replicates and causes an AIDS-like disease in baboons, cynomolgus, and pigtailed macaques. While the similarities of SIV MAC to HIV-1 have allowed for insight into pathology, transmission, and immunological response of the infected host to the virus, the differences between SIV MAC and HIV-1 are still too great to be able to draw conclusions regarding potential human responses to an HIV-1 prophylactic vaccine. 63 Therefore, to broaden the scope of animal model usage, a chimeric SHIV virus was engineered to incorporate both SIV and HIV-1 proteins or genes. 64 While macaques infected with SHIV do go on to develop AIDS, the time to progression is much different from the time to progression to AIDS of HIV-1-infected humans. Infection of macaques with SIV mac 251 strain mimics HIV-1 infection in humans by leading to chronic, slow disease progression. Route and dose required for infection, viral tropism, replicative capacity of the viruses, and pathology of SIV/SHIV-infected monkeys are all very different than these parameters in humans. 65 , 66 This distinction has been well characterized by the recent Phase IIb STEP trial, which involved 3000 healthy, uninfected volunteers. The result of this trial was termination at its first scheduled efficacy assessment due to its failure to suppress viral load in subsequently infected individuals and then-suspected increased HIV-1 infection due to interaction of the immune system with vaccine components. 67 The vaccine, a recombinant adenovirus serotype 5 (Ad5) virus incorporating the gag , pol , and nef genes from HIV-1, had been previously tested in an SHIV model in macaques and the results of that experiment were not suggestive of the results of the human trial. 68 This disparity underscores the need for animal models that more closely reflect the pathology seen in human infection with HIV-1 as well as identification of immunological correlates of protection that reflect control of HIV-1 viral load in human subjects. Therefore, the search for an appropriate animal model or the appropriate use of current animal models in the search for a prophylactic HIV-1 vaccine continues. Until a model can be derived that will allow for observation of each stage of infection, progression of disease, and response of the immune system in a way that is comparable to this process in humans, we will not be able to logically predict which vaccine candidates should be moved forward to clinical trials. Several attempts to stimulate the immune system to provide protection against HIV infection have been attempted so far ( Table 1 ). Hope for creating a prophylactic vaccine lies in the ability of the scientific community to identify and induce a broad neutralizing antibody response that would offer sterilizing immunity to vaccinated patients. To this end, several novel approaches are being studied. Degree of diversity Traditionally, prophylactic vaccines have been made by exposing some part of a pathogen's structure as an antigen to the host's immune system, and eliciting an immune response, resulting in the production of long-term memory lymphocytes that are capable of mounting a strong immune response upon later infection with the pathogen. The premise upon which this manipulation of the immune system is based is the ability of the immune system to make long-lasting antibodies to conserved structures on exposed proteins that are native to the pathogen. Ideally, both humoral and cell-mediated immunity would be induced creating long-lasting immunity. Traditional attempts to recreate this process using live attenuated simian immunodeficiency virus-1 (SIV-1) viruses in an effort to vaccinate macaques against SIV-1 have been proven safe and effective in macaques that were subsequently challenged with SIV-1. 14 , 15 However, an incidental study of the effect of live-attenuated HIV-1 (containing deletions of the nef gene and the long terminal repeat) was proven pathogenic in humans when three out of six treated patients developed late-onset immunosuppression. 16 – 18 Killed viruses have also been tested as a potential vaccine approach, but safety concerns have halted their use. These safety concerns include incomplete inactivation of the virus leading to potential residual infectivity during the vaccine preparation. 19 Due to the ineffectiveness of traditional vaccine approaches to date, scientists have attempted to use recombinant HIV-1 proteins to stimulate the production of Nabs. These attempts failed due to their inability to induce a lasting, broad range of Nabs that would inhibit infection in humans. 20 – 23 Perhaps these failures are a result of the inherent diversity of HIV-1. This diversity has presented a major roadblock to development of a prophylactic vaccine. There are three main groups of HIV-1 (M, O, and N) 24 as well as a recently discovered group, P. 25 Each group consists of several subtypes, clades. The various clades display biological differences with respect to transmission, 26 replication, 27 and disease progression. 28 , 29 These differences result in an inability to produce a generalizable vaccine that would induce the breadth of Nabs necessary to counter an infection by a wide range of HIV-1 clades that may be encountered in a natural setting. 30 The degree of diversity seen in HIV-1 is greater than that of any other virus observed. 31 , 32 This problem is being addressed by development of multiclade (multiple env and/or subtype B gag, pol, nef ) 33 , 34 and mosaic vaccines which incorporate sets of 10 immunogenic proteins from 4 different clades or bivalent proteins from clades B and C. 35 – 37 There are proof of principle studies that illustrate immunological protection against HIV-1 in nonhuman primates that were passively treated with broadly reactive Nabs. 38 – 40 These studies show that protection against infection with HIV-1 can be conferred by the presence of broadly reactive Nabs. The next step toward production of a prophylactic vaccine would involve induction of production of these or similar broadly reactive Nabs by the host's immune system. Immune evasion The rate at which the HIV-1 virus mutates, due to the nature of the reverse transcriptase enzyme responsible for transcribing its RNA, ensures that nearly every daughter virion will have a different genome than its parent. 41 When these changes occur in the HIV-1 Env protein that is needed for antibody recognition, they inhibit the immune system's ability to mount a sufficient response. One attempt to circumvent this problem has been to induce the production of Nabs to the conserved regions of HIV-1 proteins. A major problem with this approach is that the conserved regions of HIV-1 proteins are often shielded from exposure to Nabs within the HIV-1 envelope. The native structure of the envelope protein, reportedly the only HIV-1 protein susceptible to Nabs, 31 shields it from the immune system as a glycosylated trimer of heterodimers. The glycosylation of the envelope protein allows for the carbohydrates to masquerade as 'self ' thereby forming an immunologically silent face and protects neighboring epitopes via an 'evolving glycan shield'. 42 – 44 Additionally, the gp41 coreceptor binding site, another conserved site, is not presented until primary binding to CD4 + has occurred. 45 An attempt to create antibodies to the CD4-binding region of the gp120 protein was made in rhesus macaques in 2007 and results indicated that vaccinated hosts were able to withstand challenge with SHIV. 46 Other attempts to create an HIV-1 vaccine have focused on overcoming the ability of HIV-1 to escape immune surveillance through use of antibodies that are able to neutralize diverse isolates of HIV-1. These antibodies include PG9, PG16, 47 2F5, 2G12, 4E10, b12, 48 – 51 and most recently sCD4-17b 52 and others. 53 Identification of these antibodies gives hope that their induction or the induction of other such broadly reactive Nabs may provide the basis for a prophylactic vaccine in the future. Lack of appropriate animal models The use of animal models for development of therapeutics offers the benefit of thorough testing and validation prior to introduction of a vaccine in humans. In the past, vaccines were made by observing and then mimicking the immune response mounted by individuals who had recovered from a particular disease. To date, however, there are no known cases of individuals who have recovered from HIV-1 infection. However, data can be gathered from long-term nonprogressors – patients who have been infected with HIV-1 for at least 7 years and do not display any HIV-1-related symptoms. 54 , 55 Another option that may be critical to the development of a prophylactic vaccine is the use of relevant animal models. Such models will allow for analysis of the effect of a potential vaccine on an intact host prior to use in humans. One particular challenge with the use of animal models for development of a prophylactic HIV-1 vaccine is that there are very few naturally occurring disease models of HIV-1. Only a few nonhuman primates are susceptible to infection with HIV-1 and infected animals do not progress to AIDS. 56 Therefore, it is important to use other disease models that mimic the HIV-AIDS pathologic progression. 57 One such potential model is feline immunodeficiency virus (FIV). FIV was discovered in 1986 and is known to cause an AIDS-like disease in domestic cats and mimics HIV-related dementia in humans. 58 A vaccine for FIV was approved by the FDA in 2002. 59 While the FIV model is potentially informative, its use is not sufficient as a basis for development of a prophylactic HIV-1 vaccine. An ideal animal model would display a pathological response to infection with HIV-1 that is very similar to the one that occurs in humans. Unfortunately, HIV-1 does not cause pathology leading to the development of AIDS in any host other than humans. 60 – 63 However, animal models have been developed and used that allow partial understanding of the pathology of HIV-1, the natural immunological response to infection, and the response of the host to novel therapeutics. One of these models involves the simian immunodeficiency virus MAC (SIV MAC ) that replicates and causes an AIDS-like disease in baboons, cynomolgus, and pigtailed macaques. While the similarities of SIV MAC to HIV-1 have allowed for insight into pathology, transmission, and immunological response of the infected host to the virus, the differences between SIV MAC and HIV-1 are still too great to be able to draw conclusions regarding potential human responses to an HIV-1 prophylactic vaccine. 63 Therefore, to broaden the scope of animal model usage, a chimeric SHIV virus was engineered to incorporate both SIV and HIV-1 proteins or genes. 64 While macaques infected with SHIV do go on to develop AIDS, the time to progression is much different from the time to progression to AIDS of HIV-1-infected humans. Infection of macaques with SIV mac 251 strain mimics HIV-1 infection in humans by leading to chronic, slow disease progression. Route and dose required for infection, viral tropism, replicative capacity of the viruses, and pathology of SIV/SHIV-infected monkeys are all very different than these parameters in humans. 65 , 66 This distinction has been well characterized by the recent Phase IIb STEP trial, which involved 3000 healthy, uninfected volunteers. The result of this trial was termination at its first scheduled efficacy assessment due to its failure to suppress viral load in subsequently infected individuals and then-suspected increased HIV-1 infection due to interaction of the immune system with vaccine components. 67 The vaccine, a recombinant adenovirus serotype 5 (Ad5) virus incorporating the gag , pol , and nef genes from HIV-1, had been previously tested in an SHIV model in macaques and the results of that experiment were not suggestive of the results of the human trial. 68 This disparity underscores the need for animal models that more closely reflect the pathology seen in human infection with HIV-1 as well as identification of immunological correlates of protection that reflect control of HIV-1 viral load in human subjects. Therefore, the search for an appropriate animal model or the appropriate use of current animal models in the search for a prophylactic HIV-1 vaccine continues. Until a model can be derived that will allow for observation of each stage of infection, progression of disease, and response of the immune system in a way that is comparable to this process in humans, we will not be able to logically predict which vaccine candidates should be moved forward to clinical trials. Several attempts to stimulate the immune system to provide protection against HIV infection have been attempted so far ( Table 1 ). Hope for creating a prophylactic vaccine lies in the ability of the scientific community to identify and induce a broad neutralizing antibody response that would offer sterilizing immunity to vaccinated patients. To this end, several novel approaches are being studied. Novel vaccine options As mentioned in the previous section, there are several daunting problems facing scientists who are attempting to create an HIV-1 vaccine. In hopes of creating a vaccine which elicits sterilizing immunity to HIV-1, researchers have focused their efforts on (1) the use of plasmid DNA vaccines, (2) live recombinant vectors for vaccine development (expressing or presenting HIV antigens), and (3) mucosal immunity. These critical topics will be discussed in more detail below. Plasmid DNA vaccines Vaccines should elicit a robust immune response that is long lasting and is able to provide protection against various strains of a pathogen. Plasmid DNA vaccinations can induce a strong humoral and T-cell response. DNA-based vaccination has been used as a powerful tool to fight against parasitic, fungal, bacterial, and viral infections. 115 – 119 There are multiple advantages for using plasmid DNA for vaccination: they are generally safe, nontoxic, and through the delivery of a gene encoding important immunogenic epitopes, the DNAbased vaccine exploits biosynthetic machinery of the host cell. One such example was in 1990, whereby Wolff and colleagues illustrated protein expression after intramuscular (IM) injection of plasmid DNA into myocytes. 120 Despite these promising results, there had been speculation regarding DNA vaccination strategies. For example, it was shown that protein production in response to DNA plasmids that contained HIV inserts elicited substantial cellular response in mice and nonhuman primates. However, these products were poorly immunogenic in humans. One strategy to improve immune response of the plasmid DNA vaccine strategy is by coadministration of DNA plasmids coding for cytokines (eg, INF-g, IL-2, IL-12, IL-18, and IL-15). 121 – 124 A second strategy which has been utilized to improve plasmid DNA vaccination has been the administration of plasmid DNA with adjuvants (eg, CpG oligodeoxynucleotides), or the use of DNA-delivery systems (eg, microparticles, cochleates, and linear polyenimines). 125 – 128 A third strategy to improve vaccine efficacy involves the coadministration of plasmid DNA in combination with viral vectors. For instance, research performed by Harari and colleagues in 2008 demonstrated that vaccination by means of an HIV-1 clade C DNA prime in combination with a pox vector (NYVAC) boost induces a reliable polyfunctional and long-lasting anti-HIV T-cell response in human participants. 129 Along these same lines, work recently published by Jaoko and group demonstrated safety and immunogenicity of a multiclade HIV-1 Ad-based vaccine alone or in combination with a multiclade HIV-1 DNA vaccine in Africa. These results also demonstrated that DNA priming increased the frequency and magnitude of cellular and humoral responses; however, there was no effect of recombinant Ad5 dosage on immunogenicity endpoints. 130 The previously mentioned DNA-delivery strategies have been used in combination with viral vectors or alone by means of a variety of immunization routes (eg, IM, intravenous [IV], intradermal [ID], intranasal [IN], oral, rectal, or vaginal). In the majority of reported studies, DNA vaccines have been administered by the IM and/or ID routes. However, as it relates to HIV vaccination, mucosal immunity could potentially be an important factor to consider, with mucosal immunity being achieved optimally by IN or oral routes of administration. The topic of mucosal immunity will be discussed in more detail in a later section within this review. After immunization, it is assumed that the DNA vaccination immunogen is produced in the skeletal muscles, dendritic cells, and macrophages at the site of immunization. However, in adults, the skeletal muscles are not involved in a high level of protein synthesis as compared to the liver. Therefore, the delivery of DNA to cells, which are capable of high protein synthesis, such as hepatocytes, epithelia cells of the intestines, or salivary pancreas, may result in high levels of protein expression. The hepatocytes express enzymes involved in the formation of intrachain and interchain disulfide bonds required for proper folding and assembly of proteins. In addition, the liver expresses glycosyltranferases, which are essential for synthesis of both N- and O- linked glycan side chains; this may not be the case for other cell types, 131 , 132 the significance of this point being the fact that broadly crossclade Nabs such as 2G12 recognize glycan moieties on the heavily glycosylated HIV-1 envelope antigens. 44 , 133 , 134 Another advantage of protein expression within the liver is that significantly lower amounts of DNA are needed for protein expression of a particular antigen in the hepatocytes vs another cell type. For the immunization of humans, milligram quantities of DNA are necessary to achieve adequate levels of immune response. 119 Any method whereby there would be a reduction in DNA quantity needed to vaccinate humans would provide significant economic advantages. Based on the previously mentioned reasons, it is not a surprise that the liver has been exploited extensively as a site for gene delivery due to its ability to produce proteins and glycoproteins. 135 – 138 Hydrodynamic delivery is the application of controlled hydrodynamic pressure in capillaries to enhance endothelial and parenchymal cell permeability; this methodology had its inception in the late 1990s with investigations into intravascular injection of plasmid DNA solution for gene delivery in whole animals. 139 – 142 Hydrodynamic plasmid DNA delivery is well tolerated in mice. In 2008, Raska and colleagues demonstrated in mice that IV hydrodynamic vaccination with HIV-1 envelope DNA injections resulted in high levels of expression of HIV antigen in the liver. In mice, immunological data illustrated that hydrodynamic administration of HIV-1 plasmid DNA was superior to vaccination with DNA by IN, ID, IM, and intrasplenic routes. Further results illustrated that after boosting, hydrodynamic vaccination yielded levels of HIV-1-specific antibodies that were 40-fold higher than those elicited by other routes tested. 132 However, this delivery scheme is not feasible in large animals and humans. As an alternative, receptor-mediated DNA binding to hepatocytes could be a viable approach. Molecules with terminal galactose residues covalently linked to DNA are recognized by the hepatocyte-expressed galacto-sespecific asialoglycoprotein 143 receptor for internalization. 144 This alternative would avoid delivery through the hepatic system and the need for expansion of the blood volume. In addition, galactose-linked DNA packaged in delivery vehicles such as liposomes, choleates, or microspheres can be given by oral administration, which would be absorbed by the intestine and ultimately delivered to the hepatic vein. As an additional alternative to hydrodynamic delivery in humans, it might be possible to express HIV antigens in the liver by means of plasmid DNA delivery via viral vectors such as the Ad. Ads have been shown to transduce the liver efficiently in vivo by means of the hexon proteins. 145 , 146 In this regard, production of translation of HIV-1 proteins primarily in the liver might allow for the production of heavily glycosylated HIV-1 envelope antigens and thus the production of Nabs. Live recombinant vectors for vaccine development Viral vectors are potent inducers of cellular and humoral response. Viral vectors can express proteins from bacteria or viral pathogens to vaccinate against infectious diseases. There are several viral vaccine vectors that have been used successfully in models for vaccination. These vectors include alphaviruses, human rhinoviruses (HRVs), Ads, picornaviruses, poxviruses, measles viruses, influenza, and vaccinia viruses. 30 , 129 , 147 – 156 Each of these vectors has its respective disadvantages and advantages with respect to vaccine development. Some advantages of a few of these vectors include their ability to naturally infect a wide variety of cell types and tissues of interest. 157 – 162 Each respective vector has its own set of disadvantages. For instance, one disadvantage of using the poliovirus or the HRV as a vaccine vector is the insert size limit restriction of these vectors as compared to the large insert size (~8 kb) accommodation of Ad vectors. The most common disadvantage of the majority of viral vaccine vectors is reduced vaccine efficacy due to vector preexisting immunity (PEI). 163 – 167 Various strategies have been employed to circumvent the problems associated with vector PEI. Specifically, as it relates to Ad vectors, PEI is a tremendous problem. Of the identified serotypes of Ad vectors, human serotypes 5 (Ad5) and 2 (Ad2) have been the most extensively used for gene therapy protocols. Ad5 has been used for HIV-1 vaccination protocols, most recently in the STEP study. As it relates to Ad2 and Ad5, PEI to these vectors may be found in up to 50% of the American population and up to 95% of the population of other countries. This Ad PEI can limit the effectiveness of Ad-based vaccinations. 168 – 170 To circumvent Ad2 or Ad5 PEI, researchers have employed the use of vector chimeras, 166 , 171 use of alternative serotypes, 172 – 178 and the use of nonhuman Ads, 151 such as chimpanzee Ad. The chimpanzee Ad virus was demonstrated to not be significantly neutralized by human sera, which gives chimpanzee Ad an advantage for human vaccine development. 179 – 181 Other strategies have been used to reduce the immune response against Ad vectors such as the use of helperdependent Ad (HD-Ads) vectors, 182 – 187 the use of Ad delivery in combination with biochemical modifications such as PEGylation, 188 – 194 and the use of vector delivery by means of cell vehicles. 195 , 196 With respect to the HD-Ads, these vectors were produced to further increase the safety and cloning capacity of first-generation Ad vectors. HD-Ads lack Ad genes and contain only the packaging signals and end terminal repeats. These vectors were designed to avoid cellular immunity and diminish liver toxicity, thus promoting long-term transgene expression. 197 – 200 The reduced immune response against HD-Ads has allowed for transgene expression in mice and baboons for years. 182 , 183 , 185 , 200 This long-term transgene expression could be helpful for antigen production for an HIV vaccine, thus producing an opportunity to have increased protection against HIV, with reduced frequency of vaccinations. Although Nabs to Ad5 may reduce the immunogenicity of Ad5-based vectors in animal model systems, their effect on the immunity in subjects with previous Ad5 exposure is still largely unknown. As previously mentioned, the STEP trial, which tested a Merck recombinant Ad5 (rAd5) vaccine (encoding HIV-1 gag , pol , and ne1 genes), failed to yield protection, either by lowering viral load or by decreasing acquisition of infection. 13 Analysis of data from this study aroused speculation that subjects with pre-existing Nabs from wild-type Ad5 infection had an increased risk of HIV infection after vaccination. One recent study has shown that there was no causative role for Ad5-specific CD4 + T cells in increasing HIV-1 susceptibility in the Merck trial. 201 In this regard, there are multiple studies ongoing to elucidate a concrete finding with respect to the role of Ad5 PEI and increased activation of CD4 + T cells in the mucosal milieu. 202 , 203 Recently, there was a report by Cheng and colleagues that attempted to characterize the specificity of rAd5 Nabs in Ad5- immune subjects and determine the impact of Ad exposure on immune responses elicited by Ad5-based vaccinations. Cheng and colleagues reported that rAd5 Nabs were directed toward different components of the Ad virion, depending on whether the Ad5 infection was natural or from Ad-based HIV vaccine trials. For example, Ad Nabs generated by natural infection are directed primarily to fiber components, while vector exposure elicits responses primarily to capsid proteins other than fiber. Nabs elicited by natural infection significantly reduced the CD8 + and CD4 + cell responses to HIV Gag after DNA/rAd5 vaccination. This report concluded that Ad5 Nabs differ based on the route of exposure and that previous Ad5 exposure compromises Ad5 vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. 204 These results have a tremendous impact on HIV-1 vaccine trials and the design of next generation viral vaccine vectors. Viral vectors such as Ad, influenza, and polio have been used as vaccine vectors for many reasons. One important advantage of these vectors, which makes them attractive, is that they can provide mucosal immunity because they can easily infect the mucosal surfaces as well as act to induce cytokine and chemokine production at the mucosal entry sites. Ad, influenza, and polio also have the advantage of being able to be delivered orally, without the use of needles. This is an important fact in developing countries where needle cost is prohibitive to vaccine administration. As it relates to HIV vaccine development, mucosal immunity is a debatable factor to consider. Mucosal HIV immunity When deciding upon a vaccine agent, the importance of considering if the ultimate goal is to induce systemic immunity, mucosal immunity, or both is worth careful consideration. 205 – 207 It is believed that 80% of HIV-1 infection will occur from heterosexual viral transmission and most of the rest will occur from homosexual or perinatal transmission. 152 Although the biology of sexual transmission is poorly understood, it is clear that the essential first step in the infection pathway is the transfer of infectious virus or HIV-infected cells through the mucosal surfaces. After HIV has entered a new host, the HIV or HIV-infected cells will soon encounter susceptible host target cells at the mucosal point of entry where the virus replicates and then invades local lymphatic tissues, initiating systemic HIV infection. On this basis, strong immunity is required to provide a protective immunological barrier at the most common point of entry, the mucosal surfaces of the reproductive tract. Due to the compartmentalization of the secretory and systemic immune systems, parenterally administered antigens do not consistently stimulate mucosal immunity. 152 Therefore, it is important to consider a vaccine regime that induces mucosal immunity. Since CD4 + CCR5 + memory T cells are the primary target of HIV infection in the gut and mucosa and rapid depletion of this subset occurs early after infection, 208 , 209 several studies have investigated the role of HIV mucosal immunity. Previous studies have demonstrated the importance of a mucosal SIV/HIV vaccine producing both strong mucosal antibody and CD8 + response capable of blocking the escape of virus from the intestinal mucosa into systemic lymphoid organs. 207 , 210 – 214 However, in other instances, the necessity of exclusive mucosal HIV immunity will be further debated based on the promising results found in a heterologous prime/boost regimen using DNA/89.6-expressing SIV and HIV-1 transcripts 215 , 216 and modified vaccinia virus Ankara (MVA/89.6)-expressing SIV and HIV-1 transcripts under the control of vaccinia virus early/late promoter. In this case, either ID or IM DNA/MVA vaccination was able to provide protection against a intrarectal SHIV-89.6 challenge. 153 Along these same lines, recently, promising results were found by Hessell and colleagues in 2010. Hessell and colleagues demonstrated that after an IV administration of monoclonal antibodies 2F5 or 4E10 to six monkeys followed by a SHIV ba-L challenge, five out of six monkeys from either group showed complete protection and sterilizing immunity. A low level of viral replication could not be ruled out for the six monkeys in either group. 217 Replicative Ad yields a robust immune response at the mucosal sites partly because Ad is known to infect and replicate in epithelial cells. 218 – 221 Various strategies have been used to achieve mucosal immunity via the oral route. One such strategy embodies the development of replication-defective recombinant Ad serotype 41 (Ad41) vector. 222 Serotype 41 vectors are being currently used because Ad41 has a natural tropism for the gut and causes no pathological disease outside of the gastrointestinal tract. 223 Ad41 vectors are likely to have a preferential tropism for the gut because Ad41 appears to have a resistance to acidic pH 224 and the capsid configuration of long and short fibers allows the Ad41 virus to preferentially infect the gut. 177 , 225 Live recombinant vectors for vaccine development engineered to express/present HIV-1 antigens As previously mentioned, viral vectors are potent inducers of cellular and humoral responses. Of note, viral vectors have been practically used for human applications and have progressed treating a variety of disease contexts such as cancer and infectious diseases. 226 – 229 Traditional viral vector immunization embodies the concept that the vector uses the host cell machinery to express antigens, which are encoded as transgenes within the viral vector. Cellular and humoral immune responses are generated against these antigens. Over the last 20 years, several viral vectors have been derived to express HIV-1 antigens for vaccine purposes. Some researchers have taken an alternative approach to conventional transgene expression of antigens by means of viral vectors; this alternative approach embodies the capsid incorporation of antigens. This innovative paradigm is based upon the vector presenting the antigen as a component of the capsid rather than an encoded transgene. Incorporation of immunogenic peptides into the vector capsid offers potential advantages. In this regard, the processing of the capsid-incorporated antigen via the exogenous pathway should result in a strong humoral response similar to the response provoked by native Ad capsid proteins. In this arrangement, potentially, HIV peptide antigens accrue the potent immunostimulatory effects of the native Ad vector capsid proteins, which effectively perform an adjuvant function. On this basis, the immune response directed against vector capsid proteins with repetitive vector administration should achieve a booster effect against the incorporated antigen. 230 Most importantly, as it relates to HIV infection, this strategy yields the potential of generating antibodies to HIV proteins. Recent crystallographic, cryo-electron tomography, and molecular modeling studies have provided valuable insight to molecular surfaces recognized by antibodies as well as assisted in rationale vaccine design of immunogens. 231 – 235 These structural technologies can also potentially improve the abilities of scientists to advance the antigen capsidincorporation strategy. If the antigen capsid incorporation is effective, it can provide a way forward with respect to inducing sterilizing immunity. 68 , 236 , 237 The antigen capsid-incorporation strategy has been used for Ad-based vaccines in the context of many diseases. 230 , 238 – 242 One of the first examples where the antigen capsid-incorporation strategy was used was with research performed by Crompton in 1994. 242 Crompton and colleagues inserted an eight-amino acid sequence of the VP1 capsid protein of poliovirus type 3 into two regions of the Ad2 serotype hexon. One of the chimeric vectors produced grew well in tissue culture. In addition, antiserum raised against the Ad with the polio insert specifically recognized the VP1 capsid of polio type 3. As it relates to Ad5 serotype, Wu and group demonstrated that His 6 epitopes could be incorporated into Ad hexon hypervariable regions (HVRs) 1–7 (now reclassified as 1–9) without perturbing viral viability and any major biological characteristics such as replication, thermostability, or native infectivity. This study by Wu and colleagues demonstrated that His 6 appeared to be surface exposed at these regions. 243 With respect to peptide incorporation within Ad5 hexon, HVR2 and HVR5 appear to be the most promising locales for peptide/antigen incorporation based on X-ray and peptide analyses along with molecular studies. 244 Our laboratory and others have focused on incorporations at HVR5 or single-site incorporations (such as fiber and pIX). 230 , 238 – 241 –, 243 , 245 , 246 However, we recognized that the ability to place antigen within multiple sites of the Ad capsid protein would hold important potential for presenting multiple epitopes/antigens or several copies of the same epitope within a single Ad vector-based vaccine. In an effort to create multivalent HIV vaccine vectors, our 2008 study explored the use of Ad5 HVR2 and HVR5 in hopes of creating vectors which contained HIV antigenic epitopes at both locales. To compare the flexibility and capacities of Ad5 HVR2 and HVR5, we genetically incorporated identical epitopes of incrementally increasing size within HVR2 or HVR5 of Ad5 hexon. We incorporated identical epitopes ranging from 33 to 83 amino acids within the Ad5 hexon HVR2 or HVR5 region. Viable viruses were produced with incorporations of 33 amino acids plus a 12-amino acid linker at HVR2 or HVR5. In addition, viable viruses were produced with incorporations of up to 53 amino acids plus a 12-amino acid linker at HVR5. With respect to identical antigen incorporations at Ad5 HVR2 or HVR5, HVR5 was more permissive allowing an epitope incorporation of 65 amino acids in total. These model antigens were surface exposed via ELISA analysis. In vivo immunization with these vectors illustrated an antigen-specific immune response. 240 Along these same lines, Abe and colleagues evaluated the ability of Ad5-based vectors expressing an HIV transgene to induce antigen-specific immune responses under Ad5 preimmune conditions. To overcome limitations that are generally experienced as a result of PEI to Ad5, they constructed vectors that have a modification in the HVR5. Their study characterized various immunological parameters generated by these vectors such as vector neutralization, acquisition of adaptive immune response, and comparison of protective immunity. First, in order to evaluate the utility of the modified Ad vector, they measured the neutralizing activity of sera by a modified Ad vector. They administered Ad-Luc (luciferase protein expressed as a transgene in the Ad E1 region), Ad-HisLuc (His 6 epitope presented in HVR5 region and luciferase protein expressed as a transgene), or Ad-END/AAALuc vector (containing three amino acid mutations in HVR5 and expressing luciferase protein) to mice IM. After administration of these vectors, neutralizing activity against Ad5 was observed for 0–8 weeks. The hexon-modified vector (Ad-HisLuc) generated the lowest Ad5-specific neutralizing activity, which was significantly lower than what was generated by Ad-Luc at weeks 6 and 8, and by Ad-End/AAALuc vector at week 8. The individual neutralizing activity of Ad-HisLuc immunization was significantly lower than that of Ad-Luc immunization. Additional studies performed by Abe and colleagues support the concept that modified hexon thwarts Ad5 Nabs and promotes cellular immune responses. 247 Studies performed by this research group indicate that a change in the immunogenic epitope is necessary to avoid neutralization by pre-existing Nabs. Our recently published work exploits the antigen capsidincorporation strategy for HIV vaccination. Our novel vectors were constructed in hopes of moving toward the goal of creating vectors that will provide cellular and humoral HIV immunity. Our study is the first of its kind to genetically incorporate an HIV antigen within the Ad5 hexon HVR2 alone or in combination with genomic incorporation of a Gag transgene (Ad5/HVR2-MPER-L15(Gag)). In this study, we successfully incorporated a 24-amino acid epitope of HIV-1 within HVR2. The HIV-1 region selected for HVR2 incorporation was the membrane proximal ectodomain region (MPER) derived from HIV-1 glycoprotein 41 (gp41). Our rationale for choosing a portion of the MPER (EKNEKELLELDKWASLWNWFDITN) derived from gp41 was based on the fact that the gp41 envelope protein ectodomain is a target of three broadly neutralizing anti-HIV-1 antibodies. 248 When the MPER was incorporated into HVR2 in combination with transgene expression, we observed growth kinetics and thermostability changes similar to those of other capsid-incorporated vectors generated in other studies, 249 , 250 indicating that incorporation of the MPER epitope within HVR2 was not dramatically detrimental to virological characteristics. 250 , 251 In addition, we demonstrated that the MPER epitope is surface exposed within HVR2. Most importantly, we observed a humoral anti-HIV response in mice vaccinated with the hexon-modified vector. The MPER-modified vector allows boosting compared to AdCMVGag, possibly because the Ad5/HVR2-MPER-L15(Gag) Ad elicits less anti-Ad5 immune response. It is possible that the MPER epitope reduced the immunogenicity of the Ad5 vector. This finding is noteworthy because HVR2 has not been fully explored for antigen capsid-incorporation strategies. 252 These vectors are currently being analyzed by cryo-electron microscopic analysis to determine the critical correlates related to antigen placement/configuration and immune response. In addition, with respect to HIV-1 vaccination, the antigen capsid-incorporation strategy has been evaluated within the context of HRV. Research groups have constructed human rhinovirus:HIV-1 chimeras in an effort to stimulate immunity against HIV-1. 148 , 253 In an effort to develop HIV-1 vaccines, researchers within this same group generated combinatorial libraries of HRV capsid-incorporated HIV-1 gp41 epitope. Their results indicated that they were successful in eliciting antibodies whose activity can mimic the Nab effect. 149 Commercial and clinical Ad development of HIV-1 vaccines have progressed preferentially more than vector systems such as HRV because the flexibility of Ad generally exceeds current rhinovirus systems. For example, because HRV is a relatively small RNA virus, the HRV platform can display an array limited to 60 copies of a single HIV-1 epitope. 148 , 253 In contrast, the Ad vector platform allows incorporation of the HIV-1 MPER epitope into three structurally distinct locales, including HVR2, HVR5, 247 and protein IX (our unpublished data). In comparison, the Ad MPER antigen capsid-incorporation display platform could present an array of 720 HIV-1 epitope copies within Ad hexon and 240 HIV-1 epitope copies within pIX. If a multivalent Ad vector is generated with HIV-1 epitopes within the hexon and the pIX locales, this would represent 960 HIV epitopes within one Ad vector. Another significant difference between the Ad and HRV platforms is in the number of locales that have been successfully used for heterologous epitope insertion. Finally, in contrast to the rhinovirus that lacks this capacity, the Ad platform has sufficient coding capacity allowing for HIV-1 transgene expression in combination with presenting the same or a different antigen on the viral capsid surface. This latter finding is important because it provides the basis for constructing vectors that will provide cellular and humoral HIV-1 immunity. Vectors which provide both cellular and humoral immunity may be the way forward with respect to prophylactic HIV vaccine development. Plasmid DNA vaccines Vaccines should elicit a robust immune response that is long lasting and is able to provide protection against various strains of a pathogen. Plasmid DNA vaccinations can induce a strong humoral and T-cell response. DNA-based vaccination has been used as a powerful tool to fight against parasitic, fungal, bacterial, and viral infections. 115 – 119 There are multiple advantages for using plasmid DNA for vaccination: they are generally safe, nontoxic, and through the delivery of a gene encoding important immunogenic epitopes, the DNAbased vaccine exploits biosynthetic machinery of the host cell. One such example was in 1990, whereby Wolff and colleagues illustrated protein expression after intramuscular (IM) injection of plasmid DNA into myocytes. 120 Despite these promising results, there had been speculation regarding DNA vaccination strategies. For example, it was shown that protein production in response to DNA plasmids that contained HIV inserts elicited substantial cellular response in mice and nonhuman primates. However, these products were poorly immunogenic in humans. One strategy to improve immune response of the plasmid DNA vaccine strategy is by coadministration of DNA plasmids coding for cytokines (eg, INF-g, IL-2, IL-12, IL-18, and IL-15). 121 – 124 A second strategy which has been utilized to improve plasmid DNA vaccination has been the administration of plasmid DNA with adjuvants (eg, CpG oligodeoxynucleotides), or the use of DNA-delivery systems (eg, microparticles, cochleates, and linear polyenimines). 125 – 128 A third strategy to improve vaccine efficacy involves the coadministration of plasmid DNA in combination with viral vectors. For instance, research performed by Harari and colleagues in 2008 demonstrated that vaccination by means of an HIV-1 clade C DNA prime in combination with a pox vector (NYVAC) boost induces a reliable polyfunctional and long-lasting anti-HIV T-cell response in human participants. 129 Along these same lines, work recently published by Jaoko and group demonstrated safety and immunogenicity of a multiclade HIV-1 Ad-based vaccine alone or in combination with a multiclade HIV-1 DNA vaccine in Africa. These results also demonstrated that DNA priming increased the frequency and magnitude of cellular and humoral responses; however, there was no effect of recombinant Ad5 dosage on immunogenicity endpoints. 130 The previously mentioned DNA-delivery strategies have been used in combination with viral vectors or alone by means of a variety of immunization routes (eg, IM, intravenous [IV], intradermal [ID], intranasal [IN], oral, rectal, or vaginal). In the majority of reported studies, DNA vaccines have been administered by the IM and/or ID routes. However, as it relates to HIV vaccination, mucosal immunity could potentially be an important factor to consider, with mucosal immunity being achieved optimally by IN or oral routes of administration. The topic of mucosal immunity will be discussed in more detail in a later section within this review. After immunization, it is assumed that the DNA vaccination immunogen is produced in the skeletal muscles, dendritic cells, and macrophages at the site of immunization. However, in adults, the skeletal muscles are not involved in a high level of protein synthesis as compared to the liver. Therefore, the delivery of DNA to cells, which are capable of high protein synthesis, such as hepatocytes, epithelia cells of the intestines, or salivary pancreas, may result in high levels of protein expression. The hepatocytes express enzymes involved in the formation of intrachain and interchain disulfide bonds required for proper folding and assembly of proteins. In addition, the liver expresses glycosyltranferases, which are essential for synthesis of both N- and O- linked glycan side chains; this may not be the case for other cell types, 131 , 132 the significance of this point being the fact that broadly crossclade Nabs such as 2G12 recognize glycan moieties on the heavily glycosylated HIV-1 envelope antigens. 44 , 133 , 134 Another advantage of protein expression within the liver is that significantly lower amounts of DNA are needed for protein expression of a particular antigen in the hepatocytes vs another cell type. For the immunization of humans, milligram quantities of DNA are necessary to achieve adequate levels of immune response. 119 Any method whereby there would be a reduction in DNA quantity needed to vaccinate humans would provide significant economic advantages. Based on the previously mentioned reasons, it is not a surprise that the liver has been exploited extensively as a site for gene delivery due to its ability to produce proteins and glycoproteins. 135 – 138 Hydrodynamic delivery is the application of controlled hydrodynamic pressure in capillaries to enhance endothelial and parenchymal cell permeability; this methodology had its inception in the late 1990s with investigations into intravascular injection of plasmid DNA solution for gene delivery in whole animals. 139 – 142 Hydrodynamic plasmid DNA delivery is well tolerated in mice. In 2008, Raska and colleagues demonstrated in mice that IV hydrodynamic vaccination with HIV-1 envelope DNA injections resulted in high levels of expression of HIV antigen in the liver. In mice, immunological data illustrated that hydrodynamic administration of HIV-1 plasmid DNA was superior to vaccination with DNA by IN, ID, IM, and intrasplenic routes. Further results illustrated that after boosting, hydrodynamic vaccination yielded levels of HIV-1-specific antibodies that were 40-fold higher than those elicited by other routes tested. 132 However, this delivery scheme is not feasible in large animals and humans. As an alternative, receptor-mediated DNA binding to hepatocytes could be a viable approach. Molecules with terminal galactose residues covalently linked to DNA are recognized by the hepatocyte-expressed galacto-sespecific asialoglycoprotein 143 receptor for internalization. 144 This alternative would avoid delivery through the hepatic system and the need for expansion of the blood volume. In addition, galactose-linked DNA packaged in delivery vehicles such as liposomes, choleates, or microspheres can be given by oral administration, which would be absorbed by the intestine and ultimately delivered to the hepatic vein. As an additional alternative to hydrodynamic delivery in humans, it might be possible to express HIV antigens in the liver by means of plasmid DNA delivery via viral vectors such as the Ad. Ads have been shown to transduce the liver efficiently in vivo by means of the hexon proteins. 145 , 146 In this regard, production of translation of HIV-1 proteins primarily in the liver might allow for the production of heavily glycosylated HIV-1 envelope antigens and thus the production of Nabs. Live recombinant vectors for vaccine development Viral vectors are potent inducers of cellular and humoral response. Viral vectors can express proteins from bacteria or viral pathogens to vaccinate against infectious diseases. There are several viral vaccine vectors that have been used successfully in models for vaccination. These vectors include alphaviruses, human rhinoviruses (HRVs), Ads, picornaviruses, poxviruses, measles viruses, influenza, and vaccinia viruses. 30 , 129 , 147 – 156 Each of these vectors has its respective disadvantages and advantages with respect to vaccine development. Some advantages of a few of these vectors include their ability to naturally infect a wide variety of cell types and tissues of interest. 157 – 162 Each respective vector has its own set of disadvantages. For instance, one disadvantage of using the poliovirus or the HRV as a vaccine vector is the insert size limit restriction of these vectors as compared to the large insert size (~8 kb) accommodation of Ad vectors. The most common disadvantage of the majority of viral vaccine vectors is reduced vaccine efficacy due to vector preexisting immunity (PEI). 163 – 167 Various strategies have been employed to circumvent the problems associated with vector PEI. Specifically, as it relates to Ad vectors, PEI is a tremendous problem. Of the identified serotypes of Ad vectors, human serotypes 5 (Ad5) and 2 (Ad2) have been the most extensively used for gene therapy protocols. Ad5 has been used for HIV-1 vaccination protocols, most recently in the STEP study. As it relates to Ad2 and Ad5, PEI to these vectors may be found in up to 50% of the American population and up to 95% of the population of other countries. This Ad PEI can limit the effectiveness of Ad-based vaccinations. 168 – 170 To circumvent Ad2 or Ad5 PEI, researchers have employed the use of vector chimeras, 166 , 171 use of alternative serotypes, 172 – 178 and the use of nonhuman Ads, 151 such as chimpanzee Ad. The chimpanzee Ad virus was demonstrated to not be significantly neutralized by human sera, which gives chimpanzee Ad an advantage for human vaccine development. 179 – 181 Other strategies have been used to reduce the immune response against Ad vectors such as the use of helperdependent Ad (HD-Ads) vectors, 182 – 187 the use of Ad delivery in combination with biochemical modifications such as PEGylation, 188 – 194 and the use of vector delivery by means of cell vehicles. 195 , 196 With respect to the HD-Ads, these vectors were produced to further increase the safety and cloning capacity of first-generation Ad vectors. HD-Ads lack Ad genes and contain only the packaging signals and end terminal repeats. These vectors were designed to avoid cellular immunity and diminish liver toxicity, thus promoting long-term transgene expression. 197 – 200 The reduced immune response against HD-Ads has allowed for transgene expression in mice and baboons for years. 182 , 183 , 185 , 200 This long-term transgene expression could be helpful for antigen production for an HIV vaccine, thus producing an opportunity to have increased protection against HIV, with reduced frequency of vaccinations. Although Nabs to Ad5 may reduce the immunogenicity of Ad5-based vectors in animal model systems, their effect on the immunity in subjects with previous Ad5 exposure is still largely unknown. As previously mentioned, the STEP trial, which tested a Merck recombinant Ad5 (rAd5) vaccine (encoding HIV-1 gag , pol , and ne1 genes), failed to yield protection, either by lowering viral load or by decreasing acquisition of infection. 13 Analysis of data from this study aroused speculation that subjects with pre-existing Nabs from wild-type Ad5 infection had an increased risk of HIV infection after vaccination. One recent study has shown that there was no causative role for Ad5-specific CD4 + T cells in increasing HIV-1 susceptibility in the Merck trial. 201 In this regard, there are multiple studies ongoing to elucidate a concrete finding with respect to the role of Ad5 PEI and increased activation of CD4 + T cells in the mucosal milieu. 202 , 203 Recently, there was a report by Cheng and colleagues that attempted to characterize the specificity of rAd5 Nabs in Ad5- immune subjects and determine the impact of Ad exposure on immune responses elicited by Ad5-based vaccinations. Cheng and colleagues reported that rAd5 Nabs were directed toward different components of the Ad virion, depending on whether the Ad5 infection was natural or from Ad-based HIV vaccine trials. For example, Ad Nabs generated by natural infection are directed primarily to fiber components, while vector exposure elicits responses primarily to capsid proteins other than fiber. Nabs elicited by natural infection significantly reduced the CD8 + and CD4 + cell responses to HIV Gag after DNA/rAd5 vaccination. This report concluded that Ad5 Nabs differ based on the route of exposure and that previous Ad5 exposure compromises Ad5 vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. 204 These results have a tremendous impact on HIV-1 vaccine trials and the design of next generation viral vaccine vectors. Viral vectors such as Ad, influenza, and polio have been used as vaccine vectors for many reasons. One important advantage of these vectors, which makes them attractive, is that they can provide mucosal immunity because they can easily infect the mucosal surfaces as well as act to induce cytokine and chemokine production at the mucosal entry sites. Ad, influenza, and polio also have the advantage of being able to be delivered orally, without the use of needles. This is an important fact in developing countries where needle cost is prohibitive to vaccine administration. As it relates to HIV vaccine development, mucosal immunity is a debatable factor to consider. Mucosal HIV immunity When deciding upon a vaccine agent, the importance of considering if the ultimate goal is to induce systemic immunity, mucosal immunity, or both is worth careful consideration. 205 – 207 It is believed that 80% of HIV-1 infection will occur from heterosexual viral transmission and most of the rest will occur from homosexual or perinatal transmission. 152 Although the biology of sexual transmission is poorly understood, it is clear that the essential first step in the infection pathway is the transfer of infectious virus or HIV-infected cells through the mucosal surfaces. After HIV has entered a new host, the HIV or HIV-infected cells will soon encounter susceptible host target cells at the mucosal point of entry where the virus replicates and then invades local lymphatic tissues, initiating systemic HIV infection. On this basis, strong immunity is required to provide a protective immunological barrier at the most common point of entry, the mucosal surfaces of the reproductive tract. Due to the compartmentalization of the secretory and systemic immune systems, parenterally administered antigens do not consistently stimulate mucosal immunity. 152 Therefore, it is important to consider a vaccine regime that induces mucosal immunity. Since CD4 + CCR5 + memory T cells are the primary target of HIV infection in the gut and mucosa and rapid depletion of this subset occurs early after infection, 208 , 209 several studies have investigated the role of HIV mucosal immunity. Previous studies have demonstrated the importance of a mucosal SIV/HIV vaccine producing both strong mucosal antibody and CD8 + response capable of blocking the escape of virus from the intestinal mucosa into systemic lymphoid organs. 207 , 210 – 214 However, in other instances, the necessity of exclusive mucosal HIV immunity will be further debated based on the promising results found in a heterologous prime/boost regimen using DNA/89.6-expressing SIV and HIV-1 transcripts 215 , 216 and modified vaccinia virus Ankara (MVA/89.6)-expressing SIV and HIV-1 transcripts under the control of vaccinia virus early/late promoter. In this case, either ID or IM DNA/MVA vaccination was able to provide protection against a intrarectal SHIV-89.6 challenge. 153 Along these same lines, recently, promising results were found by Hessell and colleagues in 2010. Hessell and colleagues demonstrated that after an IV administration of monoclonal antibodies 2F5 or 4E10 to six monkeys followed by a SHIV ba-L challenge, five out of six monkeys from either group showed complete protection and sterilizing immunity. A low level of viral replication could not be ruled out for the six monkeys in either group. 217 Replicative Ad yields a robust immune response at the mucosal sites partly because Ad is known to infect and replicate in epithelial cells. 218 – 221 Various strategies have been used to achieve mucosal immunity via the oral route. One such strategy embodies the development of replication-defective recombinant Ad serotype 41 (Ad41) vector. 222 Serotype 41 vectors are being currently used because Ad41 has a natural tropism for the gut and causes no pathological disease outside of the gastrointestinal tract. 223 Ad41 vectors are likely to have a preferential tropism for the gut because Ad41 appears to have a resistance to acidic pH 224 and the capsid configuration of long and short fibers allows the Ad41 virus to preferentially infect the gut. 177 , 225 Live recombinant vectors for vaccine development engineered to express/present HIV-1 antigens As previously mentioned, viral vectors are potent inducers of cellular and humoral responses. Of note, viral vectors have been practically used for human applications and have progressed treating a variety of disease contexts such as cancer and infectious diseases. 226 – 229 Traditional viral vector immunization embodies the concept that the vector uses the host cell machinery to express antigens, which are encoded as transgenes within the viral vector. Cellular and humoral immune responses are generated against these antigens. Over the last 20 years, several viral vectors have been derived to express HIV-1 antigens for vaccine purposes. Some researchers have taken an alternative approach to conventional transgene expression of antigens by means of viral vectors; this alternative approach embodies the capsid incorporation of antigens. This innovative paradigm is based upon the vector presenting the antigen as a component of the capsid rather than an encoded transgene. Incorporation of immunogenic peptides into the vector capsid offers potential advantages. In this regard, the processing of the capsid-incorporated antigen via the exogenous pathway should result in a strong humoral response similar to the response provoked by native Ad capsid proteins. In this arrangement, potentially, HIV peptide antigens accrue the potent immunostimulatory effects of the native Ad vector capsid proteins, which effectively perform an adjuvant function. On this basis, the immune response directed against vector capsid proteins with repetitive vector administration should achieve a booster effect against the incorporated antigen. 230 Most importantly, as it relates to HIV infection, this strategy yields the potential of generating antibodies to HIV proteins. Recent crystallographic, cryo-electron tomography, and molecular modeling studies have provided valuable insight to molecular surfaces recognized by antibodies as well as assisted in rationale vaccine design of immunogens. 231 – 235 These structural technologies can also potentially improve the abilities of scientists to advance the antigen capsidincorporation strategy. If the antigen capsid incorporation is effective, it can provide a way forward with respect to inducing sterilizing immunity. 68 , 236 , 237 The antigen capsid-incorporation strategy has been used for Ad-based vaccines in the context of many diseases. 230 , 238 – 242 One of the first examples where the antigen capsid-incorporation strategy was used was with research performed by Crompton in 1994. 242 Crompton and colleagues inserted an eight-amino acid sequence of the VP1 capsid protein of poliovirus type 3 into two regions of the Ad2 serotype hexon. One of the chimeric vectors produced grew well in tissue culture. In addition, antiserum raised against the Ad with the polio insert specifically recognized the VP1 capsid of polio type 3. As it relates to Ad5 serotype, Wu and group demonstrated that His 6 epitopes could be incorporated into Ad hexon hypervariable regions (HVRs) 1–7 (now reclassified as 1–9) without perturbing viral viability and any major biological characteristics such as replication, thermostability, or native infectivity. This study by Wu and colleagues demonstrated that His 6 appeared to be surface exposed at these regions. 243 With respect to peptide incorporation within Ad5 hexon, HVR2 and HVR5 appear to be the most promising locales for peptide/antigen incorporation based on X-ray and peptide analyses along with molecular studies. 244 Our laboratory and others have focused on incorporations at HVR5 or single-site incorporations (such as fiber and pIX). 230 , 238 – 241 –, 243 , 245 , 246 However, we recognized that the ability to place antigen within multiple sites of the Ad capsid protein would hold important potential for presenting multiple epitopes/antigens or several copies of the same epitope within a single Ad vector-based vaccine. In an effort to create multivalent HIV vaccine vectors, our 2008 study explored the use of Ad5 HVR2 and HVR5 in hopes of creating vectors which contained HIV antigenic epitopes at both locales. To compare the flexibility and capacities of Ad5 HVR2 and HVR5, we genetically incorporated identical epitopes of incrementally increasing size within HVR2 or HVR5 of Ad5 hexon. We incorporated identical epitopes ranging from 33 to 83 amino acids within the Ad5 hexon HVR2 or HVR5 region. Viable viruses were produced with incorporations of 33 amino acids plus a 12-amino acid linker at HVR2 or HVR5. In addition, viable viruses were produced with incorporations of up to 53 amino acids plus a 12-amino acid linker at HVR5. With respect to identical antigen incorporations at Ad5 HVR2 or HVR5, HVR5 was more permissive allowing an epitope incorporation of 65 amino acids in total. These model antigens were surface exposed via ELISA analysis. In vivo immunization with these vectors illustrated an antigen-specific immune response. 240 Along these same lines, Abe and colleagues evaluated the ability of Ad5-based vectors expressing an HIV transgene to induce antigen-specific immune responses under Ad5 preimmune conditions. To overcome limitations that are generally experienced as a result of PEI to Ad5, they constructed vectors that have a modification in the HVR5. Their study characterized various immunological parameters generated by these vectors such as vector neutralization, acquisition of adaptive immune response, and comparison of protective immunity. First, in order to evaluate the utility of the modified Ad vector, they measured the neutralizing activity of sera by a modified Ad vector. They administered Ad-Luc (luciferase protein expressed as a transgene in the Ad E1 region), Ad-HisLuc (His 6 epitope presented in HVR5 region and luciferase protein expressed as a transgene), or Ad-END/AAALuc vector (containing three amino acid mutations in HVR5 and expressing luciferase protein) to mice IM. After administration of these vectors, neutralizing activity against Ad5 was observed for 0–8 weeks. The hexon-modified vector (Ad-HisLuc) generated the lowest Ad5-specific neutralizing activity, which was significantly lower than what was generated by Ad-Luc at weeks 6 and 8, and by Ad-End/AAALuc vector at week 8. The individual neutralizing activity of Ad-HisLuc immunization was significantly lower than that of Ad-Luc immunization. Additional studies performed by Abe and colleagues support the concept that modified hexon thwarts Ad5 Nabs and promotes cellular immune responses. 247 Studies performed by this research group indicate that a change in the immunogenic epitope is necessary to avoid neutralization by pre-existing Nabs. Our recently published work exploits the antigen capsidincorporation strategy for HIV vaccination. Our novel vectors were constructed in hopes of moving toward the goal of creating vectors that will provide cellular and humoral HIV immunity. Our study is the first of its kind to genetically incorporate an HIV antigen within the Ad5 hexon HVR2 alone or in combination with genomic incorporation of a Gag transgene (Ad5/HVR2-MPER-L15(Gag)). In this study, we successfully incorporated a 24-amino acid epitope of HIV-1 within HVR2. The HIV-1 region selected for HVR2 incorporation was the membrane proximal ectodomain region (MPER) derived from HIV-1 glycoprotein 41 (gp41). Our rationale for choosing a portion of the MPER (EKNEKELLELDKWASLWNWFDITN) derived from gp41 was based on the fact that the gp41 envelope protein ectodomain is a target of three broadly neutralizing anti-HIV-1 antibodies. 248 When the MPER was incorporated into HVR2 in combination with transgene expression, we observed growth kinetics and thermostability changes similar to those of other capsid-incorporated vectors generated in other studies, 249 , 250 indicating that incorporation of the MPER epitope within HVR2 was not dramatically detrimental to virological characteristics. 250 , 251 In addition, we demonstrated that the MPER epitope is surface exposed within HVR2. Most importantly, we observed a humoral anti-HIV response in mice vaccinated with the hexon-modified vector. The MPER-modified vector allows boosting compared to AdCMVGag, possibly because the Ad5/HVR2-MPER-L15(Gag) Ad elicits less anti-Ad5 immune response. It is possible that the MPER epitope reduced the immunogenicity of the Ad5 vector. This finding is noteworthy because HVR2 has not been fully explored for antigen capsid-incorporation strategies. 252 These vectors are currently being analyzed by cryo-electron microscopic analysis to determine the critical correlates related to antigen placement/configuration and immune response. In addition, with respect to HIV-1 vaccination, the antigen capsid-incorporation strategy has been evaluated within the context of HRV. Research groups have constructed human rhinovirus:HIV-1 chimeras in an effort to stimulate immunity against HIV-1. 148 , 253 In an effort to develop HIV-1 vaccines, researchers within this same group generated combinatorial libraries of HRV capsid-incorporated HIV-1 gp41 epitope. Their results indicated that they were successful in eliciting antibodies whose activity can mimic the Nab effect. 149 Commercial and clinical Ad development of HIV-1 vaccines have progressed preferentially more than vector systems such as HRV because the flexibility of Ad generally exceeds current rhinovirus systems. For example, because HRV is a relatively small RNA virus, the HRV platform can display an array limited to 60 copies of a single HIV-1 epitope. 148 , 253 In contrast, the Ad vector platform allows incorporation of the HIV-1 MPER epitope into three structurally distinct locales, including HVR2, HVR5, 247 and protein IX (our unpublished data). In comparison, the Ad MPER antigen capsid-incorporation display platform could present an array of 720 HIV-1 epitope copies within Ad hexon and 240 HIV-1 epitope copies within pIX. If a multivalent Ad vector is generated with HIV-1 epitopes within the hexon and the pIX locales, this would represent 960 HIV epitopes within one Ad vector. Another significant difference between the Ad and HRV platforms is in the number of locales that have been successfully used for heterologous epitope insertion. Finally, in contrast to the rhinovirus that lacks this capacity, the Ad platform has sufficient coding capacity allowing for HIV-1 transgene expression in combination with presenting the same or a different antigen on the viral capsid surface. This latter finding is important because it provides the basis for constructing vectors that will provide cellular and humoral HIV-1 immunity. Vectors which provide both cellular and humoral immunity may be the way forward with respect to prophylactic HIV vaccine development. Promising results in an effort to produce an HIV vaccine Recently, there have been encouraging developments regarding HIV vaccination. In the 1980s, in Thailand, there was a substantial increase in the prevalence of infection with HIV-1. 254 – 256 By first observation, these groups consisted of intravenous-drug users and commercial sex workers; this infected group then expanded to the general population. 101 By the mid 1990s, the overall seroprevalence of HIV-1 reached a peak of 3.7% among members of the Royal Thai Army and of 12.5% among people from Northern Thailand. 255 , 257 The Thai Ministry of Public Health acted by starting an effective HIV-prevention campaign. With this effort, the number of new HIV-1 infections per year decreased from an estimated 143,000 in 1990 to 14,000 in 2007. 255 , 258 – 260 Although this decrease was promising, there was still a desire to do more to prevent HIV infection. To achieve this goal, an HIV Phase III study was begun. The Thai Phase III HIV vaccine study, also known as RV144, opened in the fall of 2003. The placebo-controlled trial tested the safety and effectiveness of a prime-boost regimen of two vaccines: ALVAC-HIV vaccine (the prime), a modified canarypox vaccine, and AIDSVAX B/E vaccine (booster), a gp 120 vaccine. The vaccines were based on the subtype E and B HIV-1 strains that commonly circulate in Thailand. The subtype B HIV-1 strain is the most commonly found strain in the United States. The trial, conducted in the Chonburi and Rayong provinces of Thailand, enrolled 16,402 women and men aged 18–30 years at various levels of risk for HIV infection. Study participants received the placebo or ALVAC HIV vaccine at enrollment and again after 1, 3, and 6 months. The placebo or AIDSVAX B/E vaccine was given to participants at 3 and 6 months. Participants were tested for HIV-1 infection every 6 months for 3 years. During each clinic visit, study participants were counseled on how to prevent HIV-1 infection. The results showed that 74 of 8198 placebo recipients became infected with HIV-1 compared with 51 of 8197 participants who received the vaccine. This level of effectiveness in preventing HIV-1 infection was found to be statistically significant. The vaccine strategy had no effect, however, on the amount of virus in the blood of volunteers who acquired HIV-1 infection during the study. Based on the final analysis of the study, the surgeon general of the US Army, the trial sponsor, announced that the prime-boost investigational vaccine regimen was safe and 31% effective in preventing HIV-1 infection. With respect to an HIV-1 vaccine that can provide sterilizing HIV immunity, this is the best result in humans to date. However, the modest protection effect appeared limited to low-risk individuals, and there were data which suggest that this effect was confined to the first year following administration of the vaccine. Efforts must continue to focus on evaluating the immune response induced by the vaccine to establish potential correlates of protection. Conclusion Over the last three decades, the world has been faced with the emergence and subsequent epidemic of HIV/AIDS. There has been much progress with respect to diagnosis and prevention. On the treatment front, there have been several significant advances with respect to drug development (ie, ART/HAART). However, there is a desperate need for an effective and safe vaccine. There has been tremendous difficulty with regard to developing a vaccine that provides sterilizing immunity. This has been the case due to some of the factors mentioned in this review such as HIV diversity, immune evasion, and lack of appropriate animal models. Due to these obstacles, many researchers assumed that the control of HIV-1 viremia by vaccination would be a more realistic goal than the development of sterilizing immunity. The road to a safe and effective HIV-1 vaccine received a serious setback in the fall of 2007 with the premature termination of the Merck-HIV-1 Vaccine STEP trial due to the lack of efficacy and early speculation that the vaccine might have increased the risk of HIV infection in some populations of vaccinees. In late 2009, promising results came in from Thailand in response to their efforts to create a safe and effective vaccine against HIV-1. A community-based, randomized, multicenter, double-blinded, placebo-controlled efficacy trial using a prime-boost combination showed 31% effectiveness in preventing HIV-1 infection. These results lend promise to the hope of producing an HIV-1 vaccine vector that yields sterilizing HIV-1 immunity. In the future, research scientists must work together to increase HIV-1 vaccine effectiveness beyond 31%. Realization of this goal may be accomplished by some of the techniques mentioned in this review, such as acquisition of HIV mucosal immunity, development of effective prime-boost strategies, development of better animal models, better molecular antigen modeling and presentation, avoidance of PEI (by the means of using novel vector serotypes in combination with PEGylation), and/or induction of Nabs (by means of capsid incorporation of HIV antigens within viral vectors). These are just a few considerations that scientists and clinicians must consider with respect to the development of an effective and safe HIV-1 vaccine. Scientists and clinicians must also consider that one vector or scheme may not be sufficient with respect to providing effective HIV-1 immunity and some combination of the above-mentioned potential strategies may offer the most promising method of producing an effective HIV-1 prophylactic vaccine.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727399/

A large multi-ethnic genome-wide association study identifies novel genetic loci for intraocular pressure

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma, a leading cause of blindness. IOP heritability has been estimated to up to 67%, and to date only 11 IOP loci have been reported, accounting for 1.5% of IOP variability. Here, we conduct a genome-wide association study of IOP in 69,756 untreated individuals of European, Latino, Asian, and African ancestry. Multiple longitudinal IOP measurements were collected through electronic health records and, in total, 356,987 measurements were included. We identify 47 genome-wide significant IOP-associated loci ( P 60 mmHg), measurements taken on a single eye, and 116,980 IOP measurements from 3,632 participants that were taken after prescription of IOP lowering medications to exclude values influenced by treatment. When there was more than one IOP measurement on a single day, if different methods were used, we chose the highest quality measurement based on the method used (Goldmann applanation tonometer >iCare rebound tonometer >non-contact tonometer >Tono-Pen XL >other/pneumotonometer) 16 . If the same method was used for multiple measurements on the same day, we took the mean of all measurements. Finally, individual's mean IOP from both eyes for each visit was assessed, and the individual's median of the mean across all the visits was used for analysis (Fig. 1 ). In total, 356,987 IOP measurements were included in this study. Glaucoma cases and controls Among the 69,756 participants included in the current IOP study, 2,338 have been diagnosed with "glaucoma" clinically (Table 1 ). We defined "glaucoma" as having at least: two diagnoses of POAG, or two diagnoses of normal tension glaucoma, or one diagnosis of POAG and one diagnosis of normal tension glaucoma. In all cases, at least one of the diagnoses was made by a Kaiser Permanente ophthalmologist. For the control group, participants who had one or more diagnosis of any type of glaucoma (e.g., pseudoexfoliation, pigmentary, or PACG) were excluded. The final control sample included 58,172 participants. Genotyping and imputation DNA samples from GERA individuals were extracted from Oragene kits (DNA Genotek Inc., Ottawa, ON, Canada) at KPNC and genotyped at the Genomics Core Facility of the University of California, San Francisco (UCSF). DNA samples were genotyped at over 665,000 SNPs on four race/ethnicity-specific Affymetrix Axiom arrays (Affymetrix, Santa Clara, CA, USA) optimized for individuals of European, Latino, East Asian, and African-American ancestry 56 , 57 . Genotype QC (quality control) procedures for the GERA samples were performed on an array-wise basis 55 . SNPs with initial genotyping call rate ≥97%, allele frequency difference (≤0.15) between males and females for autosomal markers, and genotype concordance rate (>0.75) across duplicate samples were included. About 94% of samples and more than 98% of genetic markers assayed passed QC procedures. In addition to those QC criteria, SNPs with genotype call rates 60 mmHg), measurements taken on a single eye, and 116,980 IOP measurements from 3,632 participants that were taken after prescription of IOP lowering medications to exclude values influenced by treatment. When there was more than one IOP measurement on a single day, if different methods were used, we chose the highest quality measurement based on the method used (Goldmann applanation tonometer >iCare rebound tonometer >non-contact tonometer >Tono-Pen XL >other/pneumotonometer) 16 . If the same method was used for multiple measurements on the same day, we took the mean of all measurements. Finally, individual's mean IOP from both eyes for each visit was assessed, and the individual's median of the mean across all the visits was used for analysis (Fig. 1 ). In total, 356,987 IOP measurements were included in this study. Glaucoma cases and controls Among the 69,756 participants included in the current IOP study, 2,338 have been diagnosed with "glaucoma" clinically (Table 1 ). We defined "glaucoma" as having at least: two diagnoses of POAG, or two diagnoses of normal tension glaucoma, or one diagnosis of POAG and one diagnosis of normal tension glaucoma. In all cases, at least one of the diagnoses was made by a Kaiser Permanente ophthalmologist. For the control group, participants who had one or more diagnosis of any type of glaucoma (e.g., pseudoexfoliation, pigmentary, or PACG) were excluded. The final control sample included 58,172 participants. Genotyping and imputation DNA samples from GERA individuals were extracted from Oragene kits (DNA Genotek Inc., Ottawa, ON, Canada) at KPNC and genotyped at the Genomics Core Facility of the University of California, San Francisco (UCSF). DNA samples were genotyped at over 665,000 SNPs on four race/ethnicity-specific Affymetrix Axiom arrays (Affymetrix, Santa Clara, CA, USA) optimized for individuals of European, Latino, East Asian, and African-American ancestry 56 , 57 . Genotype QC (quality control) procedures for the GERA samples were performed on an array-wise basis 55 . SNPs with initial genotyping call rate ≥97%, allele frequency difference (≤0.15) between males and females for autosomal markers, and genotype concordance rate (>0.75) across duplicate samples were included. About 94% of samples and more than 98% of genetic markers assayed passed QC procedures. In addition to those QC criteria, SNPs with genotype call rates <90% were removed, as well as SNPs with a minor allele frequency <1%. Imputation was also conducted on an array-wise basis. Following the pre-phasing of genotypes with Shape-IT v2.r72719 58 , variants were imputed from the cosmopolitan 1000 Genomes Project reference panel (phase I integrated release; http://1000genomes.org ) using IMPUTE2 v2.3.0 59 – 61 . As a QC metric, we used the info r 2 from IMPUTE2, which is an estimate of the correlation of the imputed genotype to the true genotype 62 . We excluded variants with an imputation r 2 < 0.3, and restricted to SNPs that had a minor allele count ≥20. GWAS analysis and covariate adjustment We first analyzed each of the four race/ethnicity groups (non-Hispanic whites, Hispanic/Latinos, East Asians, and African-Americans) separately. We performed a linear regression of each individual's median of the mean IOP with the following covariates: age at the median measurement, sex, and ancestry principal components (PCs) (Supplementary Table 12 ). We then performed a linear regression of the residuals on each SNP using PLINK 63 v1.9 ( www.cog-genomics.org/plink/1.9/ ) to assess genetic associations. Data from each SNP were modeled using additive dosages to account for the uncertainty of imputation 64 . Eigenstrat 65 v4.2 was used to calculate the PCs on each of the four race/ethnicity groups 54 . The top 10 ancestry PCs were included as covariates for the non-Hispanic whites, while the top six ancestry PCs were included for the three other race/ethnicity groups. The percentage of Ashkenazi ancestry was also used as a covariate for the non-Hispanic whites to adjust for genetic ancestry, as described previously 54 . Second, we undertook a GERA meta-analysis of IOP to combine the results of the four race/ethnicity groups using the R 66 ( https://www.R-project.org ) package "meta". We calculated fixed effects summary estimates under an additive model, and we assessed heterogeneity index, I 2 (0–100%) among groups as well as Cochran's Q heterogeneity statistic. At each locus, the lead SNP was defined as the most significant SNP within a 2 Mb window. New loci were defined as those that were located more than 1 Mb apart from any previously described locus. Finally, to identify additional independent SNPs at each locus, we conducted association analyses by including all the 47 lead SNPs identified in the GERA trans-ethnic meta-analysis as covariates in the regression model. We assessed whether any additional SNPs within a 2 Mb window ( ± 1.0 Mb with respect to the original lead SNP) reached genome-wide significance. We report associations that replicate at a Bonferroni-corrected significance threshold of 0.05/500 = 0.0001 (corresponding to an estimate of ~500 independent variants per locus for 2 Mb interval surrounding each of our original signals), as previously used 67 . An epistasis analysis of all pairs of lead SNPs was also conducted in the four GERA race/ethnicity groups (non-Hispanic whites, Hispanic/Latinos, East Asians and African/Americans). For this analysis, we applied a Bonferroni-corrected significance threshold of 0.05/4,324 = 1.2 × 10 –5 (accounting for the number of SNP-pairs tested (47*46)/2, and for the four race/ethnicity groups). Glaucoma case–control analysis We evaluated the associations of the 47 lead IOP-associated SNPs with glaucoma susceptibility by logistic regression under an additive model, and adjusting for age, sex, and ancestry PCs. Replication of novel SNPs in an independent external cohort To test the 40 novel GERA genome-wide significant SNPs for replication, we evaluated associations in an independent external study. GWAS summary statistics from the study of Springelkamp et al. 11 , consisting of 37,930 individuals of European and Asian descent from 19 studies, were publicly accessible. We also combined the results for the 40 novel identified SNPs using a meta-analysis of GERA and the study of Springelkamp et al. 11 . We report fixed effects results, and associations that replicate at a strict Bonferroni threshold ( P < 0.00125, to account for a total of 40 SNPs tested). Replication analysis of previously reported SNPs in GERA To determine how many of the 11 previously reported IOP loci from genetic studies replicated in the GERA cohort, we tested 13 statistically independent lead SNPs previously reported to be associated at a genome-wide level of significance 6 – 9 , 11 , 13 , 22 . We used a nominal significance level of 0.05, and a more stringent multiple testing correction accounting for the number of SNPs tested (Bonferroni-corrected alpha level of 0.0038 ( = 0.05/13)). GWAS heritability estimates and variance explained SNP-based heritability estimates were obtained for IOP using the GCTA software 39 , which computes the phenotypic variance explained by all analyzed SNPs in the genome by restricted maximum likelihood achieved using expectation maximization. We restricted the analysis to autosomal SNPs, and a genetic relationship matrix cutoff of 0.025 was applied. For statistical power purposes, we conducted the analysis in the largest group of individuals from GERA, which is the non-Hispanic white. We also estimated the variance explained by (1) the 47 lead SNPs identified in the current study; and (2) the 13 SNPs previously identified, using a linear regression on the IOP residuals, and including either the 47 lead SNPs or the 13 previously reported SNPs as covariates in the model. To assess the impact of multiple IOP measurements on the proportion of variance explained, we also estimated the variance explained by the 47 lead SNPs using a single, randomly selected IOP measurement for each individual. In silico analyses To produce the most thorough list of candidate genes within the 47 identified loci, we used a Bayesian approach (CAVIARBF) 68 , publicly available at https://bitbucket.org/Wenan/caviarbf . Briefly, for each of the 47 signals, we computed each variant's ability to explain the observed signal within a 2 Mb window (±1.0 Mb with respect to the original lead SNP) and derived, the smallest set of variants that included the causal variant with 95% probability (95% credible set). Previous studies 69 , 70 have used similar approaches to prioritize variants near index SNPs for follow-up. These 47 credible sets included a total of 12,614 variants in 59 annotated genes (Supplementary Data 1 ). Expression of the genes that contained associated 95% credible set variants was assessed in human ocular tissues using two publicly available databases: the OTDB 34 , and EyeSAGE 35 , 36 publicly available at https://genome.uiowa.edu/otdb/ , and http://neibank.nei.nih.gov/EyeSAGE/index.shtml , respectively. The OTDB contains gene expression data for 10 eye tissues from 20 normal human donors, and the gene expression is reported as Affymetrix Probe Logarithmic Intensity Error normalized value, as previously described 34 . We then prioritized potentially causal genes for the 47 associations identified in the GERA GWAS using DEPICT 38 , a previously described bioinformatics tool that is not driven by phenotype-specific hypotheses. All the 47 lead SNPs that achieved P < 5 × 10 –8 in the GERA GWAS served as input, and information on prioritized genes was extracted. Genes that reached a nominal significance level of 0.05 in DEPICT were subsequently prioritized. Finally, enriched gene set/pathways and tissues/cell types were highlighted using DEPICT 38 and the same 47 lead SNPs input. Data availability Genotype data of GERA participants are available from the dbGaP (database of Genotypes and Phenotypes) under accession phs000674.v2.p2. This includes individuals who consented to having their data shared with dbGaP. The complete GERA data are available upon application to the KP Research Bank ( https://researchbank.kaiserpermanente.org/ ). The summary statistics generated in this study are available from the corresponding authors upon reasonable request. The GWAS summary statistics for the replication study 11 are available from ( https://www.dropbox.com/sh/3j2h9qdbzjwvaj1/AABFD1eyNetiF63I5bQooYura?dl ¼0). Electronic supplementary material Supplementary Information Description of Additional Supplementary Files Supplementary Data 1 Peer Review File Supplementary Information Description of Additional Supplementary Files Supplementary Data 1 Peer Review File Competing interests The authors declare no competing financial interests.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9854718/

Chain-Engineering-Based De Novo Drug Design against MPXVgp169 Virulent Protein of Monkeypox Virus: A Molecular Modification Approach

The unexpected appearance of the monkeypox virus and the extensive geographic dispersal of cases have prompted researchers to concentrate on potential therapeutic approaches. In addition to its vaccine build techniques, there should be some multiple integrated antiviral active compounds because of the MPV (monkeypox virus) outbreak in 2022. This study offers a computational engineering-based de novo drug discovery mediated by random antiviral active compounds that were screened against the virulent protein MPXVgp169, as one of the key players directing the pathogenesis of the virus. The screening of these candidates was supported by the use of 72 antiviral active compounds. The top candidate with the lowest binding affinity was selected for the engineering of chains or atoms. Literature assisted to identify toxic chains or atoms that were impeding the stability and effectiveness of antiviral compounds to modify them for enhanced efficacy. With a binding affinity of −9.4 Kcal/mol after chain, the lipophilicity of 0.41, the water solubility of 2.51 as soluble, and synthetic accessibility of 6.6, chain-engineered dolutegravir was one of the best active compounds, as proved by the computational engineering analysis. This study will revolutionize the era of drug engineering as a potential therapeutic strategy for monkeypox infection. 1. Introduction In the middle of the coronavirus disease 2019 (COVID-19) pandemic reaching its endemic stage, a unique global monkeypox epidemic has begun to alarm the world [ 1 ]. The MPXV virus has an enclosed double-stranded DNA genome that is around 190 kb in size. It is a member of the family Poxviridae and the genus Orthopoxvirus. Several human-infecting species of the genus Orthopoxvirus include the variola virus, MPXV, vaccinia virus, and cowpox virus [ 2 ]. The monkeypox virus (MPXV) is the causative agent of the zoonotic disease monkeypox (MPX). Although MPX is a prevalent disease in parts of the west and central Africa, its recent occurrence in several non-endemic areas outside of Africa has raised serious concerns [ 3 ]. In 2003, the United States of America reported the first monkeypox epidemic outside of Africa, which was connected to contact with infected pet prairie dogs [ 4 ]. Although in-stances may have been spreading in Europe for some months, the first verified case of the 2022 worldwide pandemic was discovered on 6 May 2022, in an adult with travel connections to Nigeria. More than 30,000 cases had been reported globally as of the first week of August 2022 [ 5 ]. Depending on the lineage of the MPV strain causing the infection and the accessibility to modern healthcare, the fatality rate ranges from 1 to 10%. Most individuals recover without treatment since the symptoms of monkeypox sickness are often minor. According to CDC (Centers for Disease Control and Prevention) recommendations, infections with the monkeypox virus do not yet have a particular therapy [ 6 ]. In vitro and preclinical investigations have shown that the antiviral drug cidofovir (Vis-tide) is effective against poxviruses by inhibiting viral DNA polymerase [ 7 ]. Tecovirimat and brincidofovir medications may be administered to very unwell monkeypox patients, although the clinical results are yet uncertain [ 8 ]. Thus, no explicit drug has been designed and approved for monkeypox specifically, which has raised the dire need to provide the necessity of monkeypox virus drugs for instantaneous and effective treatment. Direct tests on live beings have grown considerably more challenging due to the costs involved with experimenting and current ethical regulations. In this case, in silico approaches have proved effective and have developed into potent instruments for the pursuit of illness cures [ 9 ]. Since conventional drug discovery is both expensive and time-consuming, computer-aided drug design (CADD) methodologies provide a way to increase drug development efficiency while minimizing both time and expense [ 10 , 11 ]. In this study, we have proposed a chain-engineering-based drug design for monkeypox virus using homology modeling, Screening of antiviral active compounds, interaction analysis, ADMET profiling, and application of Lipinski's rule for drug safety have been performed to evaluate the best active compound for monkeypox infection. This study will be proved as a certain therapeutic approach in the treatment procedures of the monkeypox virus infection that is causing an epidemic and may lead to a pandemic in the future, similar to COVID-19. However, in vitro and in vivo experiments are still required to be considered for maximum safety and efficacy of the designed drug. 2. Materials and Methods 2.1. Identification and Preparation of Virulent Protein The targeted protein of the monkeypox virus addressed in this study is MPXVgp169 with accession number UTG40865.1, which was retrieved from the NCBI (National Centre for Biotechnology Information). The primary structure of the protein was converted into the tertiary structure by the utilization of the trRosetta ( https://yanglab.nankai.edu.cn/trRosetta/ , accessed on 17 October 2022). The tertiary structure of the selected protein was visualized by the Discovery Studio Visualizer [ 12 ]. 2.2. Prediction of Binding Pockets It is significant to identify or predict the binding sites present in the protein for better interaction analysis. For this purpose, COACH was used ( https://zhanggroup.org/COACH/ , accessed on 17 October 2022), which is a meta-server method for the recognition of active sites of the protein. It works on 2 comparative approaches: TM-SITE and S-SITE. These sites identify the ligand-binding templates from the BioLiP protein function database having sequence and substructure profiling. This step revealed the possible binding pockets lying in the protein. The PDB structure of the protein was given as the input and the COACH analysis was accomplished. 2.3. Validation of Tertiary Structure of Virulent Protein The predicted tertiary structure was validated for structural quality by PROCHECK ( https://saves.mbi.ucla.edu , accessed on 18 October 2022) and the Ramachandran Plot was constructed. The ERRAT server was utilized for the estimation of structural quality score of the constructed protein. Therefore, the structural quality was assessed by the presence of Rama-favored regions in the computed RC plot by PROCHECK. The predicted tertiary structure was submitted as input to the PROCHECK server. 2.4. Identification of Compounds Different antiviral drug components and phytochemicals were identified by the literature review. A total of 72 antiviral active components including synthetic, and phytochemicals were selected for the screening purpose. The 3D structures of these compounds were retrieved from PubChem ( https://pubchem.ncbi.nlm.nih.gov/ , accessed on 18 October 2022) and the structures were retrieved in SDF format and saved as PDB format for further utilization (Protein Databank) [ 13 ]. 2.5. Screening of Compounds All the retrieved antiviral active components were screened through multiple ligands docking by PyRx. This is a virtual screening software for computational drug designing and discovery, which screens out libraries of candidates against the drug targets. The compound with the highest binding affinity was selected for further optimizations and the removal of toxic molecules. An Excel file in dsv format was retrieved containing the interaction parameters and binding energy. 2.6. Chain Optimization of the Best-Screened Active Compounds Chain optimization is the most recent and advanced breakthrough in drug design and discovery. The unstable and toxic chains or atoms can be substituted through new chains or atoms which have the ability to stabilize the structure of the drug candidate or to enhance the pharmacophore efficiency of a drug by removing its destabilizing or toxic elements [ 14 ]. The Swiss Bioisostere from the Swiss Drug Design tool kit ( https://www.expasy.org/resources/swissdrugdesign , accessed on 18 October 2022) and it was exploited for the optimization of chains from the 4 C-terminals, 2 Carbons on benzene ring and OH-terminals of the selected component. CH 2 and Cl chains were added to stabilize the structure and improve the efficacy of the antiviral active compounds as reported in the literature. 2.7. Interaction Analysis Autodock Vina 1 revealed the docking analysis of selected best-screened active compounds [ 15 ]. This is a collection of automated docking tools that are integrated for the prediction of the interaction of small molecules with the protein. Preparation of protein and ligand was conducted, followed by the identification of active sites and grid box setting (by default). Docking was performed between the targeted protein and the chain-optimized active compound based on the lowest binding energies. The bond lengths and the types of interactions were predicted by visualizing the docked complex on PyMol. 2.8. Interpretation of Docking Results Discovery Studio Visualizer was accessed for the interpretation of the docking results. It gave the type of atoms, interactions, forces, and length of bonds present in the complex. The docked complex of protein and chain-optimized best active compound was loaded onto the PLIP server and analyzed for the presence of bond types, bond angles, and other energy parameters. 2.9. Exploitation of Host–Pathogen Interaction Network The network of host–pathogen interactions between the human host and the monkeypox virus was created using the PHIST [ 16 ]. It is a Phage–Host interaction search tool that employs accurate genome matches between the viral and host genomes to determine the prokaryotic hosts of viruses. In comparison to current alignment-based tools, alignment-free tools, and CRISPR-based tools, it increases host prediction accuracy at the species level (on average by 3 percentage points) (by 14–20 percentage points) [ 17 , 18 , 19 ]. PHIST is suited for metagenomics studies because it is also two orders of magnitude faster than alignment-based methods. 2.10. Molecular Dynamic Simulation Molecular dynamic simulation of the docked complexes was facilitated by the IMODs server ( http://imods.chaconlab.org/ , accessed on 19 October 2022) [ 20 ]. It gives information about the validation of the interaction analysis and predicts the behavior of proteins when the molecule interacts with it, so that it can be simulated in the body of the host. For this analysis, the docked complex was given as input in PDB format, and the simulations were explored for further results. 2.11. Pre-Clinical Testing SwissADME is an online platform from where the drug candidate can be tested pre-clinically ( http://www.swissadme.ch/ , accessed on 19 October 2022) [ 21 ]. It predicts important features such as absorption, distribution, metabolism, excretion, and toxicity of the drug candidate. [ 19 ] The PDB structure of the chain-optimized best active compound was given in the input and the drug likeliness properties of the drug were calculated. 2.12. Validation of Lipinski's Rule of Five Using the Molinspiration tool, the drug-like characteristics of the chain-optimized best active compound were examined [ 22 ]. The canonical smiles of the compounds were submitted as input. Using the Molinspiration service, the molecular characteristics and bioactivity of the drugs with high affinities were predicted. Molinspiration computes the following parameters such as logP, polar surface area, mass, range of atoms, range of O or N, range of OH, range of rotatable bonds, volume, ion channel modulator, enzymes, and nuclear receptors, as well as a range of Lipinski's rule violations [ 23 ]. 2.1. Identification and Preparation of Virulent Protein The targeted protein of the monkeypox virus addressed in this study is MPXVgp169 with accession number UTG40865.1, which was retrieved from the NCBI (National Centre for Biotechnology Information). The primary structure of the protein was converted into the tertiary structure by the utilization of the trRosetta ( https://yanglab.nankai.edu.cn/trRosetta/ , accessed on 17 October 2022). The tertiary structure of the selected protein was visualized by the Discovery Studio Visualizer [ 12 ]. 2.2. Prediction of Binding Pockets It is significant to identify or predict the binding sites present in the protein for better interaction analysis. For this purpose, COACH was used ( https://zhanggroup.org/COACH/ , accessed on 17 October 2022), which is a meta-server method for the recognition of active sites of the protein. It works on 2 comparative approaches: TM-SITE and S-SITE. These sites identify the ligand-binding templates from the BioLiP protein function database having sequence and substructure profiling. This step revealed the possible binding pockets lying in the protein. The PDB structure of the protein was given as the input and the COACH analysis was accomplished. 2.3. Validation of Tertiary Structure of Virulent Protein The predicted tertiary structure was validated for structural quality by PROCHECK ( https://saves.mbi.ucla.edu , accessed on 18 October 2022) and the Ramachandran Plot was constructed. The ERRAT server was utilized for the estimation of structural quality score of the constructed protein. Therefore, the structural quality was assessed by the presence of Rama-favored regions in the computed RC plot by PROCHECK. The predicted tertiary structure was submitted as input to the PROCHECK server. 2.4. Identification of Compounds Different antiviral drug components and phytochemicals were identified by the literature review. A total of 72 antiviral active components including synthetic, and phytochemicals were selected for the screening purpose. The 3D structures of these compounds were retrieved from PubChem ( https://pubchem.ncbi.nlm.nih.gov/ , accessed on 18 October 2022) and the structures were retrieved in SDF format and saved as PDB format for further utilization (Protein Databank) [ 13 ]. 2.5. Screening of Compounds All the retrieved antiviral active components were screened through multiple ligands docking by PyRx. This is a virtual screening software for computational drug designing and discovery, which screens out libraries of candidates against the drug targets. The compound with the highest binding affinity was selected for further optimizations and the removal of toxic molecules. An Excel file in dsv format was retrieved containing the interaction parameters and binding energy. 2.6. Chain Optimization of the Best-Screened Active Compounds Chain optimization is the most recent and advanced breakthrough in drug design and discovery. The unstable and toxic chains or atoms can be substituted through new chains or atoms which have the ability to stabilize the structure of the drug candidate or to enhance the pharmacophore efficiency of a drug by removing its destabilizing or toxic elements [ 14 ]. The Swiss Bioisostere from the Swiss Drug Design tool kit ( https://www.expasy.org/resources/swissdrugdesign , accessed on 18 October 2022) and it was exploited for the optimization of chains from the 4 C-terminals, 2 Carbons on benzene ring and OH-terminals of the selected component. CH 2 and Cl chains were added to stabilize the structure and improve the efficacy of the antiviral active compounds as reported in the literature. 2.7. Interaction Analysis Autodock Vina 1 revealed the docking analysis of selected best-screened active compounds [ 15 ]. This is a collection of automated docking tools that are integrated for the prediction of the interaction of small molecules with the protein. Preparation of protein and ligand was conducted, followed by the identification of active sites and grid box setting (by default). Docking was performed between the targeted protein and the chain-optimized active compound based on the lowest binding energies. The bond lengths and the types of interactions were predicted by visualizing the docked complex on PyMol. 2.8. Interpretation of Docking Results Discovery Studio Visualizer was accessed for the interpretation of the docking results. It gave the type of atoms, interactions, forces, and length of bonds present in the complex. The docked complex of protein and chain-optimized best active compound was loaded onto the PLIP server and analyzed for the presence of bond types, bond angles, and other energy parameters. 2.9. Exploitation of Host–Pathogen Interaction Network The network of host–pathogen interactions between the human host and the monkeypox virus was created using the PHIST [ 16 ]. It is a Phage–Host interaction search tool that employs accurate genome matches between the viral and host genomes to determine the prokaryotic hosts of viruses. In comparison to current alignment-based tools, alignment-free tools, and CRISPR-based tools, it increases host prediction accuracy at the species level (on average by 3 percentage points) (by 14–20 percentage points) [ 17 , 18 , 19 ]. PHIST is suited for metagenomics studies because it is also two orders of magnitude faster than alignment-based methods. 2.10. Molecular Dynamic Simulation Molecular dynamic simulation of the docked complexes was facilitated by the IMODs server ( http://imods.chaconlab.org/ , accessed on 19 October 2022) [ 20 ]. It gives information about the validation of the interaction analysis and predicts the behavior of proteins when the molecule interacts with it, so that it can be simulated in the body of the host. For this analysis, the docked complex was given as input in PDB format, and the simulations were explored for further results. 2.11. Pre-Clinical Testing SwissADME is an online platform from where the drug candidate can be tested pre-clinically ( http://www.swissadme.ch/ , accessed on 19 October 2022) [ 21 ]. It predicts important features such as absorption, distribution, metabolism, excretion, and toxicity of the drug candidate. [ 19 ] The PDB structure of the chain-optimized best active compound was given in the input and the drug likeliness properties of the drug were calculated. 2.12. Validation of Lipinski's Rule of Five Using the Molinspiration tool, the drug-like characteristics of the chain-optimized best active compound were examined [ 22 ]. The canonical smiles of the compounds were submitted as input. Using the Molinspiration service, the molecular characteristics and bioactivity of the drugs with high affinities were predicted. Molinspiration computes the following parameters such as logP, polar surface area, mass, range of atoms, range of O or N, range of OH, range of rotatable bonds, volume, ion channel modulator, enzymes, and nuclear receptors, as well as a range of Lipinski's rule violations [ 23 ]. 3. Results 3.1. Identification and Preparation of Virulent Protein The MPXVgp169 protein of monkeypox with the accession number of UTG40865.1 containing 182 aa, was retrieved from NCBI (National Centre for the Biotechnology Information) and, further, it was converted into tertiary structure by trRosetta. Model 1 with the highest TM score (template modeling score) 1 and C score (structure confidence score) was 0.9, was selected and visualized by Discovery Studio as shown in Figure 1 . 3.2. Binding Site Identification The PDB structure of protein MPXVgp169 was given to the COACH, meta-server for the identification of actives sites of the protein. Relative methods TM-SITE and S-SITE predicted the sites and detected the ligand binding templates from the BioLiP protein function database with sequence and substructure profiling. The TM-SITE results showed a C-score of 0.21, a cluster size of 3, and the ligands were TYR, MG, and V36 with the residues 29, 88, 89, 90 and 127, respectively. The S-SITE results have a C-score of 0.20, the cluster size is 5, and the ligands are CA, SIA, and UUU with the predicted binding site residues 42, 44, 45, 49, 50, 65, 69, 92, 92, 104, 116, 122 and 161, respectively, as depicted in Figure 2 . 3.3. Validation of Tertiary Structure of Virulent Protein The tertiary structure of the virulent MPXVgp169 protein was validated by the PROCHECK server. The ERRAT sever predicted the overall quality factor 91.9075 and Ramachandran plot statistics showed that 79.3% of residues are in the most favored region, 18.3% residues in the additional allowed region, 1.2% residues in generously allowed regions and 1.2% residues in disallowed regions as shown in Figure 3 . 3.4. Screening of Compounds The antiviral active compounds were selected and then docked against the MPVXgp169 using PyRx to filter out the docking energies of all the 72 antiviral binding energies. The binding affinities between random 72 antiviral active compounds and protein MPXVgp169 range lay between −7.5 to −4.8 Kcal/mol, respectively, as presented in Table 1 . A total of 72 antiviral active compounds were analyzed on the basis of their binding affinities with the MPXVgp169. Screening out the most suitable antiviral compound using PyRx, the lowest binding affinity with MPXVgp169 was −7.5 Kcal/mol for Dolutegravir. The lowest reported binding affinity was −4.8 Kcal/mol with the Amantadine antiviral compound. The prediction of type of bonds and their lengths in docked complex are given in Table 1 . 3.5. Chain Optimization To boost the antiviral active compound's potency and to stabilize the structure, CH 2 , F, and Cl chains were added using Swiss Bioisostere from the Swiss Drug Design tool kit. Unstable or toxic chains were removed and the competence of the target compound which was Dolutegravir was enhanced through chain engineering. The selected chains are shown in red, depicting the point of addition of methyl chains on 5 C-terminals, 1 fluorine atom on the OH chain of the 3rd benzene ring, and the chlorine atom on the 2nd carbon of last benzene ring on benzene ring and the OH-terminals of the selected component as picturized in the Figure 4 . As described in the literature, CH 2 chains, fluorine, and chlorine atoms can be added to the carbonyl chains or carbon atoms of benzene rings of active compounds were added to strengthen the structure, eliminate the toxicity, and hence improve the effectiveness of the antiviral active chemical (25). 3.6. Interaction Analysis Molecular docking was performed between chain-optimized active compound and the targeted virulent protein MPXVgp169 using Autodock Vina 1. The docked model which was selected on the basis of the lowest binding energy retained the binding energy of −9.4 kcal/mol, predicting the more efficient binding with the protein as compared to non-chain engineered active compound whose affinity was −7.4 Kcal/mol. The docked complex of chain-engineered dolutegravir and MPXVgp169 virulent protein is presented in Figure 5 . Interaction analysis was further interpreted through discovery studio to visualize the conventional bonds by predicting the bond length. There were four hydrogen bonds with the length of 2.71, 2.42, 3.12, and 3.15 angstroms, as usually the length of a hydrogen bond is 2.7–3.2 Angstrom. Two van der Waals or hydrophobic bonds were predicted with the length of 3.37 and 3.66 angstroms as hydrophobic interaction (van der Waal bonds) have distances a bit longer, i.e., 3.3–4.0 angstroms, as signified in Table 1 . Figure 6 shows the pictorial representation of the type of conventional bonds and lengths visualized by Discovery Studio. 3.7. Host–Pathogen Interaction Phage–Host Interaction search tool (PHIST) predicted the host of viruses on the basis of exact matches between viral and host genomes. It improved the accuracy of host prediction at the species level. Various interactions between humans and monkey pox were depicted such as cellular interaction of anthrax toxin, cytokine signaling in immune system, IL12 signaling mediated by STAT4, IL12-mediated signaling events, IL23-mediated signaling events, and IL27-mediated signaling events with statistics-based P values as organized in Table 2 . The betweenness centrality graph showed that most of the proteins were targeted between the interaction of human and monkeypox virus with total fraction of 1 indicating the significant interaction as presented in Figure 7 . 3.8. Molecular Dymanics Simulation IMODs computed different parameters for the simulation analysis of the docked complex between chain-engineered active compound and MPXVgp169 virulent protein. The eigenvalue of the complex structure was predicted as 1.784310 × 10 −5 . High co-related regions in the heat map and low RMSD value indicate better interactions of the individual residues as shown in Figure 8 . 3.9. Pre-Clinical Testing Swiss ADMET depicted the drug-like parameters, such as physiochemical properties, water solubility, GI absorption, topological polar surface area, skin permeation, bioavailability score, synthetic accessibility, which are given in Table 3 . According to the International Standard drug-likeness rules there is only one violation predicted, i.e., no. of atoms is greater than 70. The boiled egg model delivered an intuitive, rapid, easily reproducible yet unprecedented and robust method to analyze the brain access of small molecules and the passive gastrointestinal absorption useful for drug discovery and development. If the molecule is present in the white area of the boiled egg model, it depicts gastrointestinal absorption and if the molecule is present in the yellow area in the boiled egg model, it depicts that molecule has access to the blood–brain barrier. The boiled egg model of the chain-engineered dolutegravir showed that this drug will be absorbed in the gastrointestinal tract with efficiency, as presented in Figure 9 . 3.10. Valuation of Lipinski's Rule Molinspiration calculated the parameters of Lipinski's rule such as logP, polar surface area, mass, range of atoms, range of O or N or NH, i.e., hydrogen bond donors and range of noN, i.e., number of hydrogen bond acceptors, and range of rotatable bonds, as shown in Table 4 . The description of ADMET analysis is given in Table 5 . 3.1. Identification and Preparation of Virulent Protein The MPXVgp169 protein of monkeypox with the accession number of UTG40865.1 containing 182 aa, was retrieved from NCBI (National Centre for the Biotechnology Information) and, further, it was converted into tertiary structure by trRosetta. Model 1 with the highest TM score (template modeling score) 1 and C score (structure confidence score) was 0.9, was selected and visualized by Discovery Studio as shown in Figure 1 . 3.2. Binding Site Identification The PDB structure of protein MPXVgp169 was given to the COACH, meta-server for the identification of actives sites of the protein. Relative methods TM-SITE and S-SITE predicted the sites and detected the ligand binding templates from the BioLiP protein function database with sequence and substructure profiling. The TM-SITE results showed a C-score of 0.21, a cluster size of 3, and the ligands were TYR, MG, and V36 with the residues 29, 88, 89, 90 and 127, respectively. The S-SITE results have a C-score of 0.20, the cluster size is 5, and the ligands are CA, SIA, and UUU with the predicted binding site residues 42, 44, 45, 49, 50, 65, 69, 92, 92, 104, 116, 122 and 161, respectively, as depicted in Figure 2 . 3.3. Validation of Tertiary Structure of Virulent Protein The tertiary structure of the virulent MPXVgp169 protein was validated by the PROCHECK server. The ERRAT sever predicted the overall quality factor 91.9075 and Ramachandran plot statistics showed that 79.3% of residues are in the most favored region, 18.3% residues in the additional allowed region, 1.2% residues in generously allowed regions and 1.2% residues in disallowed regions as shown in Figure 3 . 3.4. Screening of Compounds The antiviral active compounds were selected and then docked against the MPVXgp169 using PyRx to filter out the docking energies of all the 72 antiviral binding energies. The binding affinities between random 72 antiviral active compounds and protein MPXVgp169 range lay between −7.5 to −4.8 Kcal/mol, respectively, as presented in Table 1 . A total of 72 antiviral active compounds were analyzed on the basis of their binding affinities with the MPXVgp169. Screening out the most suitable antiviral compound using PyRx, the lowest binding affinity with MPXVgp169 was −7.5 Kcal/mol for Dolutegravir. The lowest reported binding affinity was −4.8 Kcal/mol with the Amantadine antiviral compound. The prediction of type of bonds and their lengths in docked complex are given in Table 1 . 3.5. Chain Optimization To boost the antiviral active compound's potency and to stabilize the structure, CH 2 , F, and Cl chains were added using Swiss Bioisostere from the Swiss Drug Design tool kit. Unstable or toxic chains were removed and the competence of the target compound which was Dolutegravir was enhanced through chain engineering. The selected chains are shown in red, depicting the point of addition of methyl chains on 5 C-terminals, 1 fluorine atom on the OH chain of the 3rd benzene ring, and the chlorine atom on the 2nd carbon of last benzene ring on benzene ring and the OH-terminals of the selected component as picturized in the Figure 4 . As described in the literature, CH 2 chains, fluorine, and chlorine atoms can be added to the carbonyl chains or carbon atoms of benzene rings of active compounds were added to strengthen the structure, eliminate the toxicity, and hence improve the effectiveness of the antiviral active chemical (25). 3.6. Interaction Analysis Molecular docking was performed between chain-optimized active compound and the targeted virulent protein MPXVgp169 using Autodock Vina 1. The docked model which was selected on the basis of the lowest binding energy retained the binding energy of −9.4 kcal/mol, predicting the more efficient binding with the protein as compared to non-chain engineered active compound whose affinity was −7.4 Kcal/mol. The docked complex of chain-engineered dolutegravir and MPXVgp169 virulent protein is presented in Figure 5 . Interaction analysis was further interpreted through discovery studio to visualize the conventional bonds by predicting the bond length. There were four hydrogen bonds with the length of 2.71, 2.42, 3.12, and 3.15 angstroms, as usually the length of a hydrogen bond is 2.7–3.2 Angstrom. Two van der Waals or hydrophobic bonds were predicted with the length of 3.37 and 3.66 angstroms as hydrophobic interaction (van der Waal bonds) have distances a bit longer, i.e., 3.3–4.0 angstroms, as signified in Table 1 . Figure 6 shows the pictorial representation of the type of conventional bonds and lengths visualized by Discovery Studio. 3.7. Host–Pathogen Interaction Phage–Host Interaction search tool (PHIST) predicted the host of viruses on the basis of exact matches between viral and host genomes. It improved the accuracy of host prediction at the species level. Various interactions between humans and monkey pox were depicted such as cellular interaction of anthrax toxin, cytokine signaling in immune system, IL12 signaling mediated by STAT4, IL12-mediated signaling events, IL23-mediated signaling events, and IL27-mediated signaling events with statistics-based P values as organized in Table 2 . The betweenness centrality graph showed that most of the proteins were targeted between the interaction of human and monkeypox virus with total fraction of 1 indicating the significant interaction as presented in Figure 7 . 3.8. Molecular Dymanics Simulation IMODs computed different parameters for the simulation analysis of the docked complex between chain-engineered active compound and MPXVgp169 virulent protein. The eigenvalue of the complex structure was predicted as 1.784310 × 10 −5 . High co-related regions in the heat map and low RMSD value indicate better interactions of the individual residues as shown in Figure 8 . 3.9. Pre-Clinical Testing Swiss ADMET depicted the drug-like parameters, such as physiochemical properties, water solubility, GI absorption, topological polar surface area, skin permeation, bioavailability score, synthetic accessibility, which are given in Table 3 . According to the International Standard drug-likeness rules there is only one violation predicted, i.e., no. of atoms is greater than 70. The boiled egg model delivered an intuitive, rapid, easily reproducible yet unprecedented and robust method to analyze the brain access of small molecules and the passive gastrointestinal absorption useful for drug discovery and development. If the molecule is present in the white area of the boiled egg model, it depicts gastrointestinal absorption and if the molecule is present in the yellow area in the boiled egg model, it depicts that molecule has access to the blood–brain barrier. The boiled egg model of the chain-engineered dolutegravir showed that this drug will be absorbed in the gastrointestinal tract with efficiency, as presented in Figure 9 . 3.10. Valuation of Lipinski's Rule Molinspiration calculated the parameters of Lipinski's rule such as logP, polar surface area, mass, range of atoms, range of O or N or NH, i.e., hydrogen bond donors and range of noN, i.e., number of hydrogen bond acceptors, and range of rotatable bonds, as shown in Table 4 . The description of ADMET analysis is given in Table 5 . 4. Discussion The precise reason for the re-emergence of MPX has not yet been determined. There is currently no known cure or vaccine against MPX; however, as was already indicated, animal research specifies that some smallpox immunizations may be useful against MPX [ 2 ]. Due to their known side effects, particularly in immunocompromised people, replicating smallpox vaccinations should not be used to immunize against MPX. Additionally, in an individual already infected with the virus, the risk of recombination between MPXV and the vaccine strain enhances [ 24 ]. According to the study carried out by Matthew W. et al. for the infections caused by the monkeypox virus, there are no particular therapies. However, because the viruses that cause monkeypox and smallpox are genetically related, medications created to treat smallpox may also be effective against monkeypox, though no specific monkeypox drug exists yet. As antiviral therapies, numerous antiviral drugs have demonstrated some activity against various orthopoxvirus species [ 25 ]. The drug tecovirimat may also be used to treat monkeypox in people, according to limited observational evidence. In a study by Melamed et al. [ 26 ], it was reported that the tecovirimat prevented the release of viruses from the cell and has been licensed for use to treat monkeypox on the basis of results reliant on the drug's effectiveness in pertinent animal models [ 26 ]. However, the risk of effects on human beings remains likely; also the efficacy of the drug remains unpredictable. A lipid compound of the antiviral drug cidofovir, used to treat cytomegalovirus retinitis in AIDS patients, is called brincidofovir. This drug has been used for the treatment of poxviruses; however, its administration is only possible intravenously and has caused side effects such as nephrotoxicity [ 27 ]. When brincidofovir was delivered to prairie dogs at the same dosages as previous animal models, brincidofovir's pharmacokinetics (PK) investigation revealed that plasma exposure (maximum concentration [C max]) was lower, suggesting that inadequate BCV exposure may account for the reduced protective impact on survival [ 28 ]. (Considering that brincidofovir is an oral antiviral, more research in humans is imperative keeping in view the results of the findings. In this study, the screening of the most suitable antiviral active compounds that result in potential and highly competent attachments to the MPXVgp169 were analyzed by docking analysis. Additionally, preclinical testing through ADMET profiling, using computational approaches, was performed. Many significant responses of the drugs already being administered are leading to the issues being raised due to the ineffectiveness of various drugs, as the drugs are not targeted towards monkeypox specifically. A drug designed for smallpox and other orthopoxviruses cannot bind strongly to the target, while the combination and conjugation of the target antiviral compound screened in this study, chain-engineered dolutegravir, has proved to have strong binding energies and can lead to the eradication of virulence since the drug target is a virulence-causing protein of monkeypox. The aspect of the drugs being utilized for the treatment of monkeypox that was absent was the ability to progress the elimination of monkeypox precisely, as this study accomplished. As the spread of the monkeypox virus is growing, the virus is found to evolve side-by-side. Identification and containment of the spreading outbreak depend on pre-symptomatic suspicion, rapid notification of public health authorities, and assessment of high-risk exposures [ 29 ]. A more potent and curative-focused drug has been designed and thus proposed in this paper to overcome the predictive pandemic at an early stage. Conclusively, the studies that were presented in this research contain sufficient computational pharmacological information to allow for the propagation of a specifically targeted monkeypox drug. It would be useful if in vitro research could use this drug for indication of reliability of the proposed design. Due to the noteworthy collaboration of supporting pieces of evidence in this framework, it can close the gaps that were noted in earlier research for drugs for the treatment of monkeypox. 5. Conclusions The methods for preventing, containing, and treating monkeypox must change as our knowledge of the disease does. To guarantee that patients receive the finest care, treatments for monkeypox must keep up with scientific advances and management must be founded on solid, randomized evidence. As a result, it is crucial to design and discover targeted drugs in advance, and this seems to be the most logical course of action. In this study, the experimentation of chain-engineered antiviral active compounds was performed in order to enhance the ability for improvising the efficacy of the drug against MPXVgp169-virulent protein. Dolutegravir, an antiviral active compound, was selected for chain-engineering and the toxic chains, such as chains of benzene rings, were replaced with non-toxic or inert chains, such as methyl, fluorine and chlorine chains, providing it with stronger binding to the targeted receptor and increased effectiveness as a drug. The results abide by the Lipinski rule of five. Moreover, the substantiated ADMET results are additional arguments in favor of chain-engineered dolutegravir's legitimacy as a target medication. Additionally, molecular dynamics apparently offer further credence for the findings. To confirm the outcomes indicated by this study, more in vivo and in vitro experimental assessments are required. In conclusion, the research's findings provide enough computational pharmacological data about antiviral drugs to enable the regulation of a monkeypox treatment that is particularly targeted.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746593/

Adipose tissue: a new target for electroporation-enhanced DNA vaccines

DNA vaccines delivered using electroporation (EP) have had clinical success, but these EP methods generally utilize invasive needle electrodes. Here, we demonstrate the delivery and immunogenicity of a DNA vaccine into subcutaneous adipose tissue cells using noninvasive EP. Using finite element analysis, we predicted that plate electrodes, when oriented properly, could effectively concentrate the electric field within adipose tissue. In practice, these electrodes generated widespread gene expression persisting for at least 60 days in vivo within interscapular subcutaneous fat pads of guinea pigs. We then applied this adipose-EP protocol to deliver a DNA vaccine coding for an influenza antigen into guinea pigs. The resulting host immune responses elicited were of a similar magnitude to those achieved by skin delivery with EP. The onset of the humoral immune response was more rapid when the DNA dose was spread over multiple injection sites, and increasing the voltage of the EP device increased the magnitude of the immune response. This study supports further development of EP protocols delivering gene-based therapies to subcutaneous fat. Introduction The delivery of DNA-encoded vaccine antigens is a promising vaccination strategy that offers several advantages over traditional vaccine methods. Plasmid DNA production can be both rapid and cost effective, and the product may not require cold chain. Because of the plasticity of DNA-based constructs, a plasmid can be specifically designed to code for any known antigen as long as the sequence of the pathogen is known. Unlike viral vector platforms, there is no vector-acquired immunity. This permits repeated administrations of plasmid DNA vaccines to boost immunity, without the requirement to re-engineer the construct for each vaccine. Before the employment of electroporation (EP) as an enabling technology, the success of DNA vaccines has historically been limited because transfection efficiency is poor following simple injection of naked DNA. Overcoming this barrier by increasing the dose is impractical when scaling up to larger animals and humans, and several clinical trials in the past utilizing protocols based on injection of naked plasmid DNA failed to replicate the promising preclinical data. 1 , 2 , 3 To overcome these delivery limitations, the physical application of EP has been established as a key enabling technology for the in vivo delivery of plasmid DNA vectors for vaccination. To perform in vivo EP, brief electrical pulses are applied at the site of drug injection, causing transient permeability of cell membranes because of the electric field generated in the targeted tissue. This permeability allows up to 1000-fold increases in transfection efficiency. 4 , 5 DNA vaccines delivered using EP can lead to antibody responses comparable to leading viral vectors. 6 , 7 , 8 Furthermore, EP-enhanced DNA vaccines have been shown to drive strong cellular immune responses in large animals and humans that can be essential for vaccines against chronic infections and for cancer immunotherapies. 9 , 10 , 11 , 12 Recently, a therapeutic DNA EP vaccine for human papilloma virus was shown to be effective at reducing or eliminating the precancerous lesions and viral loads in patients with human papilloma virus-related cervical intraepithelial neoplasia. 13 This was the first randomized controlled trial demonstrating the clinical efficacy of an EP-enhanced DNA vaccine. Traditionally, EP-enhanced DNA vaccinations are performed intramuscularly (IM-EP) using needle electrodes that are inserted and pulsed at the injection site. Alternatively, intradermal EP (ID-EP) is a less invasive procedure than IM-EP because of shallower penetration and smaller-gauge needles required, and can also promote a potent humoral and cellular immune response. 14 , 15 , 16 , 17 IM-EP is an invasive procedure, but is capable of delivering large volumes of DNA (∼1 ml) and generating long-term gene expression, whereas ID-EP delivers smaller volumes (∼50–100 μl) but targets a more accessible tissue enriched with resident immune cells. Clinically, the subcutaneous injection route is popular because it is generally more tolerable than intramuscular injection, technically simple to administer and suitable for efficacious delivery of many drugs, including biologics and vaccines. 18 Therefore, as an alternative to IM-EP and ID-EP, we sought to investigate subcutaneous adipose tissue as a potential target for EP-enhanced plasmid DNA vaccine delivery by developing a noninvasive adipose-EP method and evaluating its gene expression and immunogenicity. Adipose, or fat, tissue is present in a layer dividing the skin and skeletal muscle. Adipose tissue is a loose connective tissue composed predominantly of adipocytes held together by extracellular matrix proteins. The primary role of fat is to store energy in the form of lipids and act as a cushion to insulate the body. However, far from being an inert storage tissue, adipose has also been shown to be a major endocrine organ, responsible for hormone production and secretion. 19 , 20 In addition, fat also has immune-modulatory functions, and recent research has shown that macrophages within adipose tissue, or even adipocytes themselves, can function as antigen-presenting cells. 21 , 22 In the case of EP-enhanced drug delivery, subcutaneous adipose tissue can act as an electrical barrier that must be physically bypassed using needle electrodes for IM-EP, and has historically been ignored as a potential target tissue for these treatments. ID-EP DNA vaccinations have been shown to transfect some adipocytes within the hypodermis, but the predominant transfected cell types for these treatments are in the dermis and epidermis. 23 To date, the role that adipose tissue itself plays in DNA vaccines remains unexplored. The motivation of the present study is to evaluate a new method of DNA EP vaccination that preferentially targets adipocytes within subcutaneous fat tissue. First, finite element analysis was used to demonstrate that noninvasive plate electrodes can be used to generate electric fields within subcutaneous adipose tissue in a highly specific manner compared with invasive needle EP. Next, a guinea pig model was used to evaluate spatial distribution of transfected adipocytes and kinetics of gene expression within adipose tissue following noninvasive adipose-EP. Finally, the humoral and cellular immunogenicity of several adipose-EP protocols was compared with ID-EP in guinea pigs. Results Finite element analysis In a bid to understand the electrical properties of adipose tissue from an EP perspective, finite element analysis was carried out to quantify the predicted electric field distribution within each tissue type of interest (in this case, skin, muscle and adipose) using the two electrode designs—needles within flat tissue and plates around folded tissue—illustrated in Figure 1 , using a 200 V excitation in both cases. Standard needle electrodes resulted in an electric field gradient distributed equally through skin, adipose and muscle ( Figure 2 , top), with the strongest electric fields at the electrode surface. In comparison, plate electrodes generated a more uniform electric field almost exclusively within adipose tissue ( Figure 2 , bottom), with comparatively small electric fields in skin and muscle. Needle electrodes were predicted to provide field strengths higher than 350 V cm −1 to 12–14% of each tissue, and fields lower than 150 V cm −1 to approximately half of treated tissue. Plate electrodes were predicted to produce electric fields between 150 and 350 V cm −1 in 95% of the treated adipose tissue. Meanwhile, muscle received no electric field above 100 V cm −1 and 87% of skin received less than 150 V cm −1 using the plate electrode design. Based on the results of these simulations, subsequent adipose-EP experiments utilized plate electrodes with voltages up to 200 V. Dye injection studies Although the fluid dynamic properties of a bolus IM or ID injection are well characterized, the distribution of a fluid within in vivo subcutaneous fat was less clear. In addition, the impact of the physical effect of compressing the injection site between plate electrodes was unknown. To investigate these dynamics, dye injection studies were carried out in guinea pigs to allow visualization of the distribution of injected fluid within fat. After subcutaneous injection and squeezing between calipers, dye was visible within intact fat pads as an elongated bolus shape ( Figure 3a ). Upon dissection, dye appeared primarily within the collagenous septa dividing adipose lobes ( Figure 3b ). The blue stain was retained within the fat pad, with little or no stain present in the overlying skin or the underlying muscle (not shown). We performed the same dye analysis using multiple injection sites with the objective of increasing the distribution of DNA throughout the adipose tissue. When five separate 50 μl dye injections were performed and then clamped between electrode plates, five individual dye sites remained visible, although some were more prominent than others ( Figure 3c ). When dissected, each individual injection sites possessed similar dye distribution within the adipose tissue, with dye concentrated along the collagenous septa (not shown). In vivo transfection of adipocytes To assess the in vivo expression of a reporter construct in adipose tissue, 50 μg of plasmid coding for green fluorescent protein (GFP) was injected into guinea pig fat pads and electroporated with voltages ranging from 50 to 200 V using noninvasive plate electrodes as described in 'General adipose-EP treatment procedure' section. The treatment site and EP clamping procedure is shown in Figure 4 . At 3 days after performing these treatments, intact fat pads were removed and imaged at the gross tissue level. No GFP expression was detected in animals receiving plasmid injection without adipose-EP ( Figure 5a ). In adipose-EP-treated fat pads, GFP was expressed exclusively at the injection site within the subcutaneous fat pads in a region ∼5–10 mm in length and 1–2 mm across, and there was no visible difference in signal area or intensity between the EP voltages tested ( Figures 5b–e ). At the microscopic cellular level, adipocytes were distinguished by their large diameter (50–100 μm) and characteristic globular shape because of the lipid droplet occupying the center of the cell volume ( Figures 5f and g ). The fat pads of guinea pigs receiving adipose-EP possessed numerous GFP-expressing adipocytes that were easily distinguishable by their sharp fluorescent outline. In addition, there were regions of strong, diffuse autofluorescence located in the extracellular space between adipocytes, and the collagen septa were also prominently fluorescent. In guinea pigs receiving plasmid DNA injection without adipose-EP, there were no detectable GFP-expressing adipocytes or regions of high autofluorescence, and the collagen septa were visible, but less prominent. Further histological analysis was performed to visualize the distribution of reporter construct through the depth of the fat pad. Again, the strongest and most abundant GFP signal was localized to adipocytes adjacent to the collagenous septa dividing the adipose lobes ( Figure 6 , right). No GFP was detectable in the overlying skin layer (not shown). Gene expression was detectable several millimeters deep into the fat, and was generally consistent with fluid distribution observed in dye injection studies. High-resolution confocal images revealed that GFP was expressed in a distinct punctate manner surrounding each transfected adipocyte ( Figure 7 ). GFP expression was not associated with the numerous nuclei surrounding and in between the adipocytes that are indicative of a smaller, secondary cell population within adipose. This population is known to include preadipocytes, fibroblasts and endothelial cells. Gene expression kinetics and histological analysis To investigate the kinetics of reporter construct expression in an adipocyte population, guinea pig fat pads were removed, sectioned and analyzed at defined time points following 200 V adipose-EP using noninvasive plate electrodes. Gene expression was measurable as early as 24 h following adipose-EP treatment, and expression was sustained throughout the 60 days monitored ( Figure 8 , top). Qualitatively, there was no clear difference in the intensity or distribution of the GFP fluorescence over the first 7 days. The signal appeared more diffuse beginning at day 14, and even weaker and more diffuse at day 60. Each distinct site of GFP expression was on the order of 10 mm in diameter. Histological changes following adipose-EP, as observed through hematoxylin and eosin staining of adipose sections, were noticeable beginning at day 3, continued through day 14 and appeared to mostly resolve by day 60 ( Figure 8 , bottom). No detectable difference in tissue physiology at 3 h (not shown) or 24 h post treatment was observed. At these early time points, adipocytes were well defined, lipid storage droplets were identifiable as empty voids and collagenous septa were visible because of darker eosin staining and numerous nuclei. Beginning at day 3 and persisting through the length of the 60 days of observation, collagenous septa at the treatment site were noticeably more prominent, likely because of the visualization of large numbers of nuclei from infiltrating cells. In regions where the collagenous septa were more prominent, the extracellular space around adipocytes became populated with higher numbers of cells as well. These histological changes were most prominent between 3 and 7 days post treatment. By 60 days post treatment, cellular infiltration into the extracellular space was mild and the cell density within the collagenous septa was still elevated, but less pronounced. Humoral immunogenicity Although expression of reporter gene constructs in adipose tissue was an extremely promising observation, the applicability of this technology for DNA vaccination would only be relevant if that expression was able to drive an immune response. To assess this, guinea pigs were immunized with a construct expressing the influenza A nucleoprotein (PR8) antigen using adipose-EP, with ID-EP for comparison, and binding titers were measured using enzyme-linked immunosorbent assay (ELISA). The adipose-EP experimental groups included high-voltage EP with 1 injection site (HV-1), high-voltage EP with 5 injection sites (HV-5), low-voltage EP with 1 injection site (LV-1) and low-voltage EP with 5 injection sites (LV-5). All guinea pigs received the same total DNA dose. HV adipose-EP and ID-EP resulted in similar antibody response kinetics and magnitude, but LV adipose-EP treatments resulted in highly variable and generally lower antibody responses compared with HV adipose-EP or ID-EP ( Figures 9a–c ). Within HV adipose-EP, 5 injections appeared to generate faster onset of immune response than a single injection site. For LV-treated adipose-EP guinea pigs, there were guinea pigs in both groups that had low or nondetectable titers throughout the study. There were main effects of voltage (F(1, 99)=65.16, P =1.68 × 10 −12 ), time (F(1, 99)=5.32, P =0.023) and number of injection sites (F(1, 99)=4.47, P =0.037) on titers of animals receiving adipose-EP. There was no significant interaction between any of these factors. Because voltage appeared to have a stronger effect upon adipose-EP titers than number of treatment sites, grouped analysis was performed to compare the immunogenicity of ID-EP, HV adipose-EP and LV adipose-EP ( Figure 9b ). There were main effects on titers due to both treatment group (F(2, 122)=43.31, P =6.13 × 10 −15 ) and time (F(2, 122)=7.20, P =0.0083), with no interaction (F(2, 122)=0.035, P =0.97). Although not necessary because of the lack of interaction, simple main effects analysis was also performed. This analysis indicated that the titer difference between HV and LV adipose-EP treatments was significant from week 6 onward (0.0004810 mm thick). The stratum corneum was not included in these models because voltages as low as 50 V will permeabilize the stratum corneum within microseconds, and its contribution to total tissue resistance then becomes negligible. 46 , 47 Furthermore, the thickness of the stratum corneum is on the order of 20 μm, and very thin layers can cause artifacts in finite element analysis. To model tissue clamped between plate electrodes, two square plate electrode geometries with rounded edges and contact area measuring 4 cm 2 were placed on opposite sides of a tissue fold comprising two skin layers, two adipose layers and a small 1 mm muscle layer separating a portion of the two adipose layers ( Figure 1a ). To model penetrating needle electrodes, two 22-gauge needle geometries were placed into the flat tissue geometry with an interelectrode spacing of 10 mm and a penetration depth of 18 mm ( Figure 1b ). The two tissue-electrode assemblies were exported to ANSYS Maxwell 2015.2 (ANSYS Software, Canonsburg, PA, USA) for finite element analysis. Electrical conductivity values for each tissue type were based on literature values, 48 and were assumed to be constant. Conductivity values and general tissue dimensions used in the models are listed in Table 1 . A built-in adaptive meshing algorithm was used to generate the mesh used for analysis. An excitation voltage was assigned to one electrode, whereas the opposing electrode was assigned a voltage of zero, and cross-sections bisecting the electrodes in the x – y analysis plane were created to visualize the electric field distribution. Plasmids Gene expression studies utilized plasmid DNA encoding GFP. Immune studies were carried out using plasmid DNA encoding full-length nucleoprotein from Influenza A (H1N1, A/Puerto Rico/ 8). All plasmid formulations were prepared in saline sodium citrate buffer for a final buffer concentration of 1 ×. Dye injection studies Methylene blue (Sigma-Aldrich, St Louis, MO, USA) was dissolved in deionized water at a concentration of 0.5 mg ml −1 . For single-site injections, guinea pigs were injected subcutaneously with 100 μl of methylene blue solution. For multi-site injections, five separate 50 μl subcutaneous injections were performed, spaced ∼5 mm apart. Following injection, the entire fat pad was gripped tightly between two plate electrodes to simulate the full treatment protocol. Animals were immediately killed and the fat pads were imaged intact, and then dissected along the sagittal plane and imaged again to visualize dye distribution within the tissue. General adipose-EP treatment procedure Treatment sites were shaved and cleaned. Adipose-EP treatments were performed on the subcutaneous fat pad in the interscapular region, whereas skin treatments were performed on the flank. Immediately following DNA injection, two plate electrodes attached to opposing caliper jaws were coated with conductive gel and then used to pinch the tissue surrounding the injection site, and pulses were administered using the Elgen 1000 control unit (Inovio Pharmaceuticals, San Diego, CA, USA). For ID-EP treatments, DNA was injected intradermally followed immediately by electroporation using the surface electroporation device consisting of a 4 × 4 array of needle electrodes. 15 Gross imaging and histological analysis Following adipose-EP using GFP plasmid, intact fat pads were harvested at predetermined time points and imaged using a FluorChem R imaging system (ProteinSimple, San Jose, CA, USA). Then, fat pads were frozen, and samples measuring ∼10 mm × 10 mm were cut from the transfected region of the fat pad and cryosectioned at a thickness of 30 μm either along the transverse plane to view the depth of transfection or along the coronal plane to view the horizontal distribution of transfected cells. Some sections were fixed in 4% formalin, cleared in xylene, stained with either 4',6-diamidino-2-phenylindole or Hoechst 3342 (Life Technologies, Carlsbad, CA, USA), and coverslipped using Fluoromount (eBioscience, San Diego, CA, USA). Other sections were fixed in formalin, cleared in xylene, stained with hematoxylin and eosin and coverslipped using Permount (VWR, Radnor, PA, USA). Sections that were hematoxylin and eosin stained were imaged in brightfield using an Olympus BX51 microscope (Olympus, Center Valley, PA, USA) equipped with a MicroPublisher 3.3 camera (QImaging, Surrey, BC, Canada). Fluorescence images were captured with a Retiga 3000 camera (QImaging). Confocal images were acquired as high-resolution, multi-paneled and auto-stitched z -stacks of the whole tissue using a Zeiss LSM 780 laser scanning confocal microscope (Carl Zeiss, Jena, Germany) and the images were further processed using Zen 2012 (Carl Zeiss) and IMARIS software (Bitplane, Belfast, UK). GFP expression and cellular kinetics Adipose-EP was performed on 14 guinea pigs, using 100 μg of a plasmid coding for GFP and EP parameters of 200 V, 3 pulses, 100 ms duration and 100 ms interpulse delay. As controls, two guinea pigs were treated with EP but did not receive plasmid injection, whereas two additional guinea pigs received the plasmid injection but were not treated with EP. Controls were killed 3 days following treatments, and treated guinea pigs were killed at intervals ( n =2) following the treatment, ranging from 3 h to 14 days post treatment, as well as a long-term follow-up at day 60. Fat pads were imaged intact for GFP expression and then sectioned and stained with hematoxylin and eosin to visualize signs of cellular infiltration at the treatment site. Immunogenicity study Guinea pigs were treated with 25 μg of nucleoprotein plasmid. Four groups of guinea pigs ( n =4) received adipose EP treatments as described above, and each group received either a single 100 μl DNA injection or five separate 50 μl injections. Plasmid injection was immediately followed by a single EP treatment consisting of three 100 ms square wave pulses, 200 ms interpulse delay and voltage of either 50 or 200 V. Guinea pigs vaccinated via ID-EP with the surface electroporation device ( n =3) served as a comparator group for this study as this method has been previously shown to transfect epidermal cells but not subcutaneous adipocytes. 15 The four adipose-EP groups were as follows: HV-1, HV-5, LV-1 and LV-5. For guinea pigs receiving 5 injections, a single EP procedure was performed immediately following the final injection. The total dose of plasmid DNA was identical for all groups. The study design is illustrated in Table 2 . Every 3 weeks for the duration of the study, 300 μl of blood was collected and serum was stored at −20 °C until analysis. Immunizations were administered at weeks 0, 3, 6 and 21. At 18 days following the final immunization, 3 ml of blood was collected and peripheral blood mononuclear cells were separated for ELISpot analysis. ELISA Serum from vaccinated guinea pigs was analyzed using ELISA. ELISAs were performed using 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) coated overnight with 100 μl per well of 0.3 μg ml −1 nucleoprotein antigen (Sino Biological, Beijing, China) in Dulbecco's phosphate-buffered saline (VWR). Plates were washed, blocked with phosphate-buffered saline containing 3% bovine serum albumin (Sigma-Aldrich) and 0.05% Tween-20 (Sigma-Aldrich) at 150 μl per well for 1 h at 37 °C, and then washed again. Serum was serially diluted from 1:50 to 1:2 952 450 in phosphate-buffered saline containing 1% bovine serum albumin and 0.05% Tween-20 (sample dilution buffer) at 100 μl per well and incubated for 2 h at 37 °C. Plates were then washed, and horseradish peroxidase-conjugated goat anti-guinea pig IgG (Sigma-Aldrich, catalog no. A7289) was diluted 1:10 000 with sample dilution buffer and added to each well at 100 μl per well for 1 h at 37 °C. Plates were washed and tetramethylbenzidine substrate solution (VWR) was added to each well at 100 μl per well and the color development was stopped with tetramethylbenzidine stop reagent solution (VWR) after 6 min. Absorbance values at 450 nm in each well were measured using a SpectraMax PLUS 384 plate reader (Molecular Devices, Sunnyvale, CA, USA), and the cutoff for a positive titer was calculated using the method described by Frey et al. , 49 in which the mean absorbance and s.d. of the negative controls—in this case, the prebleed samples—were used to calculate cutoff absorbance values. End point titers were used for all ELISA results presented. ELISpot At 18 days following the final immunization, 3 ml peripheral blood was drawn and collected in EDTA tubes to perform interferon-γ ELISpot, using methods previously developed in-house. The blood was diluted 1:1 with Hanks' balanced salt solution and centrifuged over Ficoll-Paque Plus (GE Healthcare Biosciences, Pittsburgh, PA, USA). The buffy coat was harvested and resuspended at a concentration of 1 × 10 6 live cells per ml in R10 medium, and plated at a density of 1 × 10 5 cells per well on 96-well Millipore IP plates (MilliporeSigma, Burlington, MA, USA) that had been coated overnight with 5 μg ml −1 mouse monoclonal anti-guinea pig interferon-γ antibody (V-E4, a gift from Hubert Schäfer, Berlin, Germany) and blocked with 1 × phosphate-buffered saline containing 10% (w/v) sucrose and 2% (w/v) bovine serum albumin. In triplicate, peripheral blood mononuclear cells were incubated for 18 h with either Concanavalin A or one of three different nucleoprotein antigen peptide pools previously found to be immunostimulatory. 50 Following a wash to remove cells, 0.2 μg biotinylated mouse monoclonal anti-guinea pig interferon-γ antibody (N-G3, a gift from Hubert Schäfer) was added to each well and allowed to incubate for 2 h. Wells were then washed and 100 μl BCIP/NBT detection reagent substrate was added to each well for 15 min. Plates were imaged using a CTL-Immunospot S6 ELISpot plate reader (Cellular Technology Limited, Cleveland, OH, USA), and CTL-Immunospot software (Cellular Technology Limited) was used to process and count the spots. For each animal, spot counts were normalized by subtracting the counts of unstimulated cells. Statistical methods All animal studies were performed once and the number of biological replicates per group were based on availability, and studies were not designed to detect a prespecified effect size as this work is exploratory in nature. For immunogenicity studies, all guinea pigs were born and received on the same date, and cage mates received the same treatments. The investigators were not blinded during these studies. Because of the small sample sizes in the immune study ( n =3 or n =4), tests of normality are uninformative and parametric statistical tests were performed on log-transformed ELISA and ELISpot data. To compare ELISA titer data of adipose-EP-treated groups, factorial ANOVA was performed, using EP voltage, number of injection sites and study week as factors. For grouped comparison between adipose-EP at different voltages and ID-EP, two-way factorial ANOVA was performed with treatment group and study week as factors. For specific comparisons of ELISA titer data between all treatments, data were stratified by study week and then one-way ANOVA was performed, and multiple comparisons were made using Tukey's post hoc testing when the F-test was significant. Because this simple main effect analysis at each time point was meant to generate hypotheses for future testing, we sought to minimize type II error and did not correct for multiple comparisons because of the repeated Tukey's tests across multiple time points. ELISpot data were analyzed first within adipose-EP-treated groups using factorial ANOVA, with EP voltage and number of treatment sites as factors. One-way ANOVA was performed to compare ELISA data for all treatment groups, including ID-EP. The cutoff for significance was defined as P 10 mm thick). The stratum corneum was not included in these models because voltages as low as 50 V will permeabilize the stratum corneum within microseconds, and its contribution to total tissue resistance then becomes negligible. 46 , 47 Furthermore, the thickness of the stratum corneum is on the order of 20 μm, and very thin layers can cause artifacts in finite element analysis. To model tissue clamped between plate electrodes, two square plate electrode geometries with rounded edges and contact area measuring 4 cm 2 were placed on opposite sides of a tissue fold comprising two skin layers, two adipose layers and a small 1 mm muscle layer separating a portion of the two adipose layers ( Figure 1a ). To model penetrating needle electrodes, two 22-gauge needle geometries were placed into the flat tissue geometry with an interelectrode spacing of 10 mm and a penetration depth of 18 mm ( Figure 1b ). The two tissue-electrode assemblies were exported to ANSYS Maxwell 2015.2 (ANSYS Software, Canonsburg, PA, USA) for finite element analysis. Electrical conductivity values for each tissue type were based on literature values, 48 and were assumed to be constant. Conductivity values and general tissue dimensions used in the models are listed in Table 1 . A built-in adaptive meshing algorithm was used to generate the mesh used for analysis. An excitation voltage was assigned to one electrode, whereas the opposing electrode was assigned a voltage of zero, and cross-sections bisecting the electrodes in the x – y analysis plane were created to visualize the electric field distribution. Plasmids Gene expression studies utilized plasmid DNA encoding GFP. Immune studies were carried out using plasmid DNA encoding full-length nucleoprotein from Influenza A (H1N1, A/Puerto Rico/ 8). All plasmid formulations were prepared in saline sodium citrate buffer for a final buffer concentration of 1 ×. Dye injection studies Methylene blue (Sigma-Aldrich, St Louis, MO, USA) was dissolved in deionized water at a concentration of 0.5 mg ml −1 . For single-site injections, guinea pigs were injected subcutaneously with 100 μl of methylene blue solution. For multi-site injections, five separate 50 μl subcutaneous injections were performed, spaced ∼5 mm apart. Following injection, the entire fat pad was gripped tightly between two plate electrodes to simulate the full treatment protocol. Animals were immediately killed and the fat pads were imaged intact, and then dissected along the sagittal plane and imaged again to visualize dye distribution within the tissue. General adipose-EP treatment procedure Treatment sites were shaved and cleaned. Adipose-EP treatments were performed on the subcutaneous fat pad in the interscapular region, whereas skin treatments were performed on the flank. Immediately following DNA injection, two plate electrodes attached to opposing caliper jaws were coated with conductive gel and then used to pinch the tissue surrounding the injection site, and pulses were administered using the Elgen 1000 control unit (Inovio Pharmaceuticals, San Diego, CA, USA). For ID-EP treatments, DNA was injected intradermally followed immediately by electroporation using the surface electroporation device consisting of a 4 × 4 array of needle electrodes. 15 Gross imaging and histological analysis Following adipose-EP using GFP plasmid, intact fat pads were harvested at predetermined time points and imaged using a FluorChem R imaging system (ProteinSimple, San Jose, CA, USA). Then, fat pads were frozen, and samples measuring ∼10 mm × 10 mm were cut from the transfected region of the fat pad and cryosectioned at a thickness of 30 μm either along the transverse plane to view the depth of transfection or along the coronal plane to view the horizontal distribution of transfected cells. Some sections were fixed in 4% formalin, cleared in xylene, stained with either 4',6-diamidino-2-phenylindole or Hoechst 3342 (Life Technologies, Carlsbad, CA, USA), and coverslipped using Fluoromount (eBioscience, San Diego, CA, USA). Other sections were fixed in formalin, cleared in xylene, stained with hematoxylin and eosin and coverslipped using Permount (VWR, Radnor, PA, USA). Sections that were hematoxylin and eosin stained were imaged in brightfield using an Olympus BX51 microscope (Olympus, Center Valley, PA, USA) equipped with a MicroPublisher 3.3 camera (QImaging, Surrey, BC, Canada). Fluorescence images were captured with a Retiga 3000 camera (QImaging). Confocal images were acquired as high-resolution, multi-paneled and auto-stitched z -stacks of the whole tissue using a Zeiss LSM 780 laser scanning confocal microscope (Carl Zeiss, Jena, Germany) and the images were further processed using Zen 2012 (Carl Zeiss) and IMARIS software (Bitplane, Belfast, UK). GFP expression and cellular kinetics Adipose-EP was performed on 14 guinea pigs, using 100 μg of a plasmid coding for GFP and EP parameters of 200 V, 3 pulses, 100 ms duration and 100 ms interpulse delay. As controls, two guinea pigs were treated with EP but did not receive plasmid injection, whereas two additional guinea pigs received the plasmid injection but were not treated with EP. Controls were killed 3 days following treatments, and treated guinea pigs were killed at intervals ( n =2) following the treatment, ranging from 3 h to 14 days post treatment, as well as a long-term follow-up at day 60. Fat pads were imaged intact for GFP expression and then sectioned and stained with hematoxylin and eosin to visualize signs of cellular infiltration at the treatment site. Immunogenicity study Guinea pigs were treated with 25 μg of nucleoprotein plasmid. Four groups of guinea pigs ( n =4) received adipose EP treatments as described above, and each group received either a single 100 μl DNA injection or five separate 50 μl injections. Plasmid injection was immediately followed by a single EP treatment consisting of three 100 ms square wave pulses, 200 ms interpulse delay and voltage of either 50 or 200 V. Guinea pigs vaccinated via ID-EP with the surface electroporation device ( n =3) served as a comparator group for this study as this method has been previously shown to transfect epidermal cells but not subcutaneous adipocytes. 15 The four adipose-EP groups were as follows: HV-1, HV-5, LV-1 and LV-5. For guinea pigs receiving 5 injections, a single EP procedure was performed immediately following the final injection. The total dose of plasmid DNA was identical for all groups. The study design is illustrated in Table 2 . Every 3 weeks for the duration of the study, 300 μl of blood was collected and serum was stored at −20 °C until analysis. Immunizations were administered at weeks 0, 3, 6 and 21. At 18 days following the final immunization, 3 ml of blood was collected and peripheral blood mononuclear cells were separated for ELISpot analysis. ELISA Serum from vaccinated guinea pigs was analyzed using ELISA. ELISAs were performed using 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) coated overnight with 100 μl per well of 0.3 μg ml −1 nucleoprotein antigen (Sino Biological, Beijing, China) in Dulbecco's phosphate-buffered saline (VWR). Plates were washed, blocked with phosphate-buffered saline containing 3% bovine serum albumin (Sigma-Aldrich) and 0.05% Tween-20 (Sigma-Aldrich) at 150 μl per well for 1 h at 37 °C, and then washed again. Serum was serially diluted from 1:50 to 1:2 952 450 in phosphate-buffered saline containing 1% bovine serum albumin and 0.05% Tween-20 (sample dilution buffer) at 100 μl per well and incubated for 2 h at 37 °C. Plates were then washed, and horseradish peroxidase-conjugated goat anti-guinea pig IgG (Sigma-Aldrich, catalog no. A7289) was diluted 1:10 000 with sample dilution buffer and added to each well at 100 μl per well for 1 h at 37 °C. Plates were washed and tetramethylbenzidine substrate solution (VWR) was added to each well at 100 μl per well and the color development was stopped with tetramethylbenzidine stop reagent solution (VWR) after 6 min. Absorbance values at 450 nm in each well were measured using a SpectraMax PLUS 384 plate reader (Molecular Devices, Sunnyvale, CA, USA), and the cutoff for a positive titer was calculated using the method described by Frey et al. , 49 in which the mean absorbance and s.d. of the negative controls—in this case, the prebleed samples—were used to calculate cutoff absorbance values. End point titers were used for all ELISA results presented. ELISpot At 18 days following the final immunization, 3 ml peripheral blood was drawn and collected in EDTA tubes to perform interferon-γ ELISpot, using methods previously developed in-house. The blood was diluted 1:1 with Hanks' balanced salt solution and centrifuged over Ficoll-Paque Plus (GE Healthcare Biosciences, Pittsburgh, PA, USA). The buffy coat was harvested and resuspended at a concentration of 1 × 10 6 live cells per ml in R10 medium, and plated at a density of 1 × 10 5 cells per well on 96-well Millipore IP plates (MilliporeSigma, Burlington, MA, USA) that had been coated overnight with 5 μg ml −1 mouse monoclonal anti-guinea pig interferon-γ antibody (V-E4, a gift from Hubert Schäfer, Berlin, Germany) and blocked with 1 × phosphate-buffered saline containing 10% (w/v) sucrose and 2% (w/v) bovine serum albumin. In triplicate, peripheral blood mononuclear cells were incubated for 18 h with either Concanavalin A or one of three different nucleoprotein antigen peptide pools previously found to be immunostimulatory. 50 Following a wash to remove cells, 0.2 μg biotinylated mouse monoclonal anti-guinea pig interferon-γ antibody (N-G3, a gift from Hubert Schäfer) was added to each well and allowed to incubate for 2 h. Wells were then washed and 100 μl BCIP/NBT detection reagent substrate was added to each well for 15 min. Plates were imaged using a CTL-Immunospot S6 ELISpot plate reader (Cellular Technology Limited, Cleveland, OH, USA), and CTL-Immunospot software (Cellular Technology Limited) was used to process and count the spots. For each animal, spot counts were normalized by subtracting the counts of unstimulated cells. Statistical methods All animal studies were performed once and the number of biological replicates per group were based on availability, and studies were not designed to detect a prespecified effect size as this work is exploratory in nature. For immunogenicity studies, all guinea pigs were born and received on the same date, and cage mates received the same treatments. The investigators were not blinded during these studies. Because of the small sample sizes in the immune study ( n =3 or n =4), tests of normality are uninformative and parametric statistical tests were performed on log-transformed ELISA and ELISpot data. To compare ELISA titer data of adipose-EP-treated groups, factorial ANOVA was performed, using EP voltage, number of injection sites and study week as factors. For grouped comparison between adipose-EP at different voltages and ID-EP, two-way factorial ANOVA was performed with treatment group and study week as factors. For specific comparisons of ELISA titer data between all treatments, data were stratified by study week and then one-way ANOVA was performed, and multiple comparisons were made using Tukey's post hoc testing when the F-test was significant. Because this simple main effect analysis at each time point was meant to generate hypotheses for future testing, we sought to minimize type II error and did not correct for multiple comparisons because of the repeated Tukey's tests across multiple time points. ELISpot data were analyzed first within adipose-EP-treated groups using factorial ANOVA, with EP voltage and number of treatment sites as factors. One-way ANOVA was performed to compare ELISA data for all treatment groups, including ID-EP. The cutoff for significance was defined as P <0.05, and all observations of nonsignificant trends and differences were accompanied by P -values. All plots of ELISA and ELISpot data are expressed as geometric mean±s.e. Conclusions The work here demonstrates that an adipose-targeted DNA vaccine is immunogenic following optimization of DNA delivery and electroporation parameters. This approach provides rapid and sustained immune responses, and does not require invasive needle electrodes. At a fixed dose of DNA, the magnitude and onset of the immune response both improve with electroporation voltage and increasing number of injection sites. Adipose-targeted EP DNA vaccination offers potential safety, tolerability and ease-of-use advantages over IM administration and does not suffer from the dosage or cell turnover limitations of ID treatments. As such, the authors believe that this platform warrants further investigation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3487655/

Interaction of Occupational and Personal Risk Factors in Workforce Health and Safety

Most diseases, injuries, and other health conditions experienced by working people are multifactorial, especially as the workforce ages. Evidence supporting the role of work and personal risk factors in the health of working people is frequently underused in developing interventions. Achieving a longer, healthy working life requires a comprehensive preventive approach. To help develop such an approach, we evaluated the influence of both occupational and personal risk factors on workforce health. We present 32 examples illustrating 4 combinatorial models of occupational hazards and personal risk factors (genetics, age, gender, chronic disease, obesity, smoking, alcohol use, prescription drug use). Models that address occupational and personal risk factors and their interactions can improve our understanding of health hazards and guide research and interventions.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9565420/

Modeling the environmental suitability for Bacillus anthracis in the Qinghai Lake Basin, China

Bacillus anthracis is a gram-positive, rod-shaped and endospore-forming bacterium that causes anthrax, a deadly disease to livestock and, occasionally, to humans. The spores are extremely hardy and may remain viable for many years in soil. Previous studies have identified East Qinghai and neighbouring Gansu in northwest China as a potential source of anthrax infection. This study was carried out to identify conditions and areas in the Qinghai Lake basin that are environmentally suitable for B . anthracis distribution. Anthrax occurrence data from 2005–2016 and environmental variables were spatially modeled by a maximum entropy algorithm to evaluate the contribution of the variables to the distribution of B . anthracis . Principal Component Analysis and Variance Inflation Analysis were adopted to limit the number of environmental variables and minimize multicollinearity. Model performance was evaluated using AUC (area under the curve) ROC (receiver operating characteristics) curves. The three variables that contributed most to the suitability model for B . anthracis are a relatively high annual mean temperature of -2 to 0°C, (53%), soil type classified as; cambisols and kastanozems (35%), and a high human population density of 40 individuals per km 2 (12%). The resulting distribution map identifies the permanently inhabited rim of the Qinghai Lake as highly suitable for B . anthracis . Our environmental suitability map and the identified variables provide the nature reserve managers and animal health authorities readily available information to devise both surveillance strategy and control strategy (administration of vaccine to livestock) in B . anthracis suitable regions to abate future epidemics. Introduction Anthrax is an infectious, often fatal disease of wild and domestic animals and humans that is caused by the endospore-forming, soil-borne and gram-positive Bacillus anthracis . It is primarily a disease found in herbivores but direct or indirect contact with contaminated animals can lead to outbreaks in humans, with potentially serious consequences' [ 1 ]. Herbivorous mammals are infected when grazing on contaminated grass, bitten by tabanid flies with contaminated mouthparts or ingesting contaminated carcasses [ 1 ]. Herders, livestock farmers, workers in abattoirs, meat and fur processing plants and veterinarians are exposed to the disease as an occupational hazard. Recently, the disease has transposed from industry to agriculture affecting farmers and herdsmen in 87.6% of the human cases in China [ 2 ]. B . anthracis , the etiological agent of anthrax, exhibits a bimodal lifestyle consisting of the vegetative and the spore stage [ 3 ]. Bacteria in the vegetative stage are shed by infected animals and may die rapidly in most environmental conditions. After sporulation from the vegetative cells, the B . anthracis can survive in the soil for decades [ 4 ]. The bacillus replicates rapidly in the bloodstream to high concentrations and releases toxins resulting in septicemia, which soon kills the susceptible host. In soil and vegetation, the spore can remain viable and infectious for years until it comes in contact with and enters a new susceptible host where it germinates and begin a new life cycle [ 5 ]. Human anthrax infections are caused by contact with infected animals or animal products, ingestion of undercooked infected meat; or exposure to processing of contaminated hides, wool, and hair in enclosed spaces [ 6 ]. Clinically, there are three forms of anthrax namely; cutaneous, gastrointestinal tract and pulmonary (inhalation). Globally, cutaneous anthrax accounts for over 95% of the human cases with 97.7% recently reported in China [ 2 ] and are rare in livestock and wildlife. Due to B . anthracis' virulence, tenacious anthrax cases and repetitive outbreaks, concerns have been raised across continents in recent years, e.g., sub-Saharan Africa [ 7 ], Asia [ 8 ], Europe [ 9 ], Australia [ 10 ], and the North America [ 11 ]. Also due to its potential use for bioterrorism, anthrax is considered as a global public health threat [ 12 ]. Effective vaccines in livestock have reduced the economic significance of the disease in developed countries where it now occurs sporadically in unvaccinated domestic stock and wildlife populations [ 13 ]. In the Qinghai Province of China, anthrax occurs sporadically and all year round. The prevalence of anthrax in Qinghai rose from 0.35/100,000 in 2012 to 1.17/100,000 in 2016. The incidence is gradually increasing as well [ 14 ]. Qinghai province has recently been identified as one of the potential sources of B . anthracis in a recent study characterizing the distributional patterns of both human and livestock anthrax in China [ 15 , 16 ]. The study used both clinically and laboratory-confirmed cases during 2005–2013, routine surveillance of livestock anthrax was conducted by the Ministry of Agriculture of the People's Republic of China [ 15 ]. Anthrax in livestock has been controlled, but not in wildlife. The behavior of avian and mammalian scavengers and alternative routes (waterborne transmission and flies) has proved unimportant relative to the long-term persistence of anthrax spores in soil and their infection of herbivore hosts [ 17 ]. However, human beings, livestock, and wildlife will invariably encounter each other or share habitats and other resources at the interface areas which would cause a spill back or spillover of the infection. Livestock vaccination and intensive surveillance of disease are essential for anthrax prevention [ 18 ]. However, widespread surveillance is costly, therefore, there is a need to concentrate intensive control measures in high-risk areas by increasing the understanding of B . anthracis ecology. Similar studies in China have identified climatic variables and human population density as good predictors of B . anthracis suitability [ 15 ]. Presence-only modeling algorithms to predict the environmental suitability of B . anthracis have been widely used, including maximum entropy [ 19 ] and GARP (genetic algorithm for the rule-set prediction) [ 10 ]. During comparative model studies, MaxEnt outperformed other algorithms [ 20 ]. We tested the hypothesis that soil type, climatic variables and human population density are significant predictors of B . anthracis suitability in the Qinghai Lake basin. We predicted the distribution of B . anthracis in Qinghai Lake basin using MaxEnt algorithm [ 21 , 22 ]. This study revealed the environmental suitability areas and variables responsible for B . anthracis distribution, which could help local authorities to devise both surveillance and control strategy to forestall future outbreak of anthrax in Qinghai Lake basin. Materials and methods Study area Our area of interest (AOI) is the basin of the Qinghai Lake (98°37' - 101° 45' E and 36° 33' - 39° 14' N) in Qinghai province, Northwest China ( Fig 1 ). The basin is approximately 29,600 km 2 , and the lake about 4,300 km 2 . The water surface is roughly situated at 3,193 meter above the sea level (m a.s.l.), with an average depth of 21m [ 23 ]. Qinghai Lake is situated in a closed-basin (29,661 km 2 ) with no surface water outflow. The entire watershed is in a high-altitude, cold and semiarid climate zone [ 24 ]. More than 40 rivers flow into the Qinghai Lake, but most are intermittent. Qinghai Lake is the largest salt lake in China, an international wetland [ 25 ] and a breeding ground for migratory water fowl. Further, the mountains around the Qinghai Lake are perhaps the last refuge of the endangered Przewalski's gazelle ( Procapra przewalskii ) [ 26 ]. The Qinghai Lake basin has been identified as a modern, highly efficient animal husbandry production area where human beings and nature live in harmony [ 27 ]. The human population in the entire watershed is about 110,000, mainly living around Qinghai Lake [ 28 ]. The mainstay of the rural economy in Qinghai province in China, including the lake basin is livestock husbandry [ 29 ]. Livestock mainly includes sheep, goat and yak, but also some horse, cattle and donkey. Livestock numbers per household varied from dozens to more than 1,000 [ 30 ]. In spring or early summer most livestock are transferred to high-altitude pastures (4850 to 4950 m.a.s.l) where milk is processed and herds gain weight. After returning to the homestead (3190 to 3300 m a.s.l.) in late summer, fodder (oats) and crop residues are provided as principal feed in addition to stubble grazing and grassy patches near the winter residence [ 31 ]. Grassland is the major land cover, accounting for about 63% of the AOI [ 27 ]. The main vegetation types are: needleaved forest in cold temperate, shrubs in plateau valley, alpine shrubs, sandy shrubs, steppe in temperate, alpine steppe, alpine meadow, swamp meadow, subnival vegetation and so on [ 32 ]. Two predominantly grown crops are the oilseed rape and highland barley [ 33 ]. 10.1371/journal.pone.0275261.g001 Fig 1 Area of Interest (AOI): Qinghai Lake basin in Qinghai province, People's Republic of China. ArcGIS was used to process the raster obtained from WorldClim ( http://worldclim.org/version2 ) to draw AOI map, with kind permission of Dr. Stephen Fick, geo-spatial data scientist. The boundary lines should not be reused or misinterpreted for any political reason. Anthrax occurrence data and preprocessing We collected 37 cases of anthrax in human beings (n = 08), livestock (cattle and sheep) (n = 20) and wildlife carcasses (n = 05) from spatial records provided by the World Organization for Animal Health [ 34 ] and publications [ 14 – 16 ] The spatial autocorrelation was minimized by filtering all recorded anthrax locations using SDM Toolbox v1.1c in ArcGIS 10.3 [ 35 ]. Filtering was performed by limiting the minimum distance between each pair of points. In addition, the filtering program plays the role of systematic sampling. It can delete adjacent records to reduce spatial aggregation, which is regarded as the most effective method in correcting sampling bias [ 36 , 37 ]. Environmental variables and preprocessing A total of 68 climatic, 12 incoming solar radiation (ISR) and 8 soil variables were used in our analysis ( S1 Table ). We extracted the climatic variables from WorldClim version 2.1 for data from 1970–2000 at 30 arc–second resolution [ 38 , 39 ]. The categorical variable soil type and continuous soil variables in 1km grids were extracted from soil grids database [ 40 ] including soil organic carbon, clay, silt and sand content as well as cation exchange capacity, soil pH, and land cover/use ( S2 Table ). Livestock (cattle, sheep and goat) population animals/km 2 was obtained from https://livestockdata.org/contributor/gridded-livestock-world-glw3 [ 41 ]. In addition, the human population density from the Asia Continental Population Datasets (2000–2020), which are publicly and freely available both through the WorldPop Dataverse Repository and the WorldPop project website ( http://www.worldpop.org.uk/data/ ), were used as predictor variables in this research. Principal component analysis (PCA) was used to reduce the number of continuous environmental variables [ 37 , 42 , 43 ]. During PCA, we used eigenvalues larger than 0.97 and the scree plot criterion for PCA in item level factoring [ 44 ]. Suppression of unnecessary loading and rotation of factor pattern of climatic variables [ 45 ] were used to retain climatic variables. After variable reduction in PCA, we used VIF (linear regression statistics) in SPSS 22.0 to assess multicollinearity among both the remaining continuous variables and the categorical variables [ 46 ]. A VIF >10 was considered to indicate highly correlated variables, which were thus removed from the input data set. Subsequently, only seven uncorrelated variables were used ( S3 Table ). The Jackknife test, backward stepwise variable elimination, and the variable response curves were selected to identify the relative contribution of predictor variables to the model [ 22 ]. Model development and evaluation A MaxEnt model v3.4.1 was fitted using 100 bootstrap runs, with a 70/30 partition percentage for the training/testing datasets. The advanced options in MaxEnt that were selected include the maximum iteration set to 5000 to allow the models to have enough time to reach convergence at 0.00001 [ 47 ] 90% sensitivity was set within the MaxEnt model for determining suitability. The area under the Receiver Operating Characteristics [ 48 ] was used to assess the accuracy of the model. In the MaxEnt model, the Area Under the Curve (AUC) of the receiver operating characteristic plot was used as an evaluation criterion to assess the accuracy of the model [ 49 ]. The stepwise elimination approach was used to remove variables that contributed less than ten percent (10%) to the model [ 37 ]. Further, a smooth response curve was used as a quality standard [ 22 ]. We reclassified the MaxEnt spatial model output into two environmental suitability classes, namely high and low in ArcGIS v10.3. Study area Our area of interest (AOI) is the basin of the Qinghai Lake (98°37' - 101° 45' E and 36° 33' - 39° 14' N) in Qinghai province, Northwest China ( Fig 1 ). The basin is approximately 29,600 km 2 , and the lake about 4,300 km 2 . The water surface is roughly situated at 3,193 meter above the sea level (m a.s.l.), with an average depth of 21m [ 23 ]. Qinghai Lake is situated in a closed-basin (29,661 km 2 ) with no surface water outflow. The entire watershed is in a high-altitude, cold and semiarid climate zone [ 24 ]. More than 40 rivers flow into the Qinghai Lake, but most are intermittent. Qinghai Lake is the largest salt lake in China, an international wetland [ 25 ] and a breeding ground for migratory water fowl. Further, the mountains around the Qinghai Lake are perhaps the last refuge of the endangered Przewalski's gazelle ( Procapra przewalskii ) [ 26 ]. The Qinghai Lake basin has been identified as a modern, highly efficient animal husbandry production area where human beings and nature live in harmony [ 27 ]. The human population in the entire watershed is about 110,000, mainly living around Qinghai Lake [ 28 ]. The mainstay of the rural economy in Qinghai province in China, including the lake basin is livestock husbandry [ 29 ]. Livestock mainly includes sheep, goat and yak, but also some horse, cattle and donkey. Livestock numbers per household varied from dozens to more than 1,000 [ 30 ]. In spring or early summer most livestock are transferred to high-altitude pastures (4850 to 4950 m.a.s.l) where milk is processed and herds gain weight. After returning to the homestead (3190 to 3300 m a.s.l.) in late summer, fodder (oats) and crop residues are provided as principal feed in addition to stubble grazing and grassy patches near the winter residence [ 31 ]. Grassland is the major land cover, accounting for about 63% of the AOI [ 27 ]. The main vegetation types are: needleaved forest in cold temperate, shrubs in plateau valley, alpine shrubs, sandy shrubs, steppe in temperate, alpine steppe, alpine meadow, swamp meadow, subnival vegetation and so on [ 32 ]. Two predominantly grown crops are the oilseed rape and highland barley [ 33 ]. 10.1371/journal.pone.0275261.g001 Fig 1 Area of Interest (AOI): Qinghai Lake basin in Qinghai province, People's Republic of China. ArcGIS was used to process the raster obtained from WorldClim ( http://worldclim.org/version2 ) to draw AOI map, with kind permission of Dr. Stephen Fick, geo-spatial data scientist. The boundary lines should not be reused or misinterpreted for any political reason. Anthrax occurrence data and preprocessing We collected 37 cases of anthrax in human beings (n = 08), livestock (cattle and sheep) (n = 20) and wildlife carcasses (n = 05) from spatial records provided by the World Organization for Animal Health [ 34 ] and publications [ 14 – 16 ] The spatial autocorrelation was minimized by filtering all recorded anthrax locations using SDM Toolbox v1.1c in ArcGIS 10.3 [ 35 ]. Filtering was performed by limiting the minimum distance between each pair of points. In addition, the filtering program plays the role of systematic sampling. It can delete adjacent records to reduce spatial aggregation, which is regarded as the most effective method in correcting sampling bias [ 36 , 37 ]. Environmental variables and preprocessing A total of 68 climatic, 12 incoming solar radiation (ISR) and 8 soil variables were used in our analysis ( S1 Table ). We extracted the climatic variables from WorldClim version 2.1 for data from 1970–2000 at 30 arc–second resolution [ 38 , 39 ]. The categorical variable soil type and continuous soil variables in 1km grids were extracted from soil grids database [ 40 ] including soil organic carbon, clay, silt and sand content as well as cation exchange capacity, soil pH, and land cover/use ( S2 Table ). Livestock (cattle, sheep and goat) population animals/km 2 was obtained from https://livestockdata.org/contributor/gridded-livestock-world-glw3 [ 41 ]. In addition, the human population density from the Asia Continental Population Datasets (2000–2020), which are publicly and freely available both through the WorldPop Dataverse Repository and the WorldPop project website ( http://www.worldpop.org.uk/data/ ), were used as predictor variables in this research. Principal component analysis (PCA) was used to reduce the number of continuous environmental variables [ 37 , 42 , 43 ]. During PCA, we used eigenvalues larger than 0.97 and the scree plot criterion for PCA in item level factoring [ 44 ]. Suppression of unnecessary loading and rotation of factor pattern of climatic variables [ 45 ] were used to retain climatic variables. After variable reduction in PCA, we used VIF (linear regression statistics) in SPSS 22.0 to assess multicollinearity among both the remaining continuous variables and the categorical variables [ 46 ]. A VIF >10 was considered to indicate highly correlated variables, which were thus removed from the input data set. Subsequently, only seven uncorrelated variables were used ( S3 Table ). The Jackknife test, backward stepwise variable elimination, and the variable response curves were selected to identify the relative contribution of predictor variables to the model [ 22 ]. Model development and evaluation A MaxEnt model v3.4.1 was fitted using 100 bootstrap runs, with a 70/30 partition percentage for the training/testing datasets. The advanced options in MaxEnt that were selected include the maximum iteration set to 5000 to allow the models to have enough time to reach convergence at 0.00001 [ 47 ] 90% sensitivity was set within the MaxEnt model for determining suitability. The area under the Receiver Operating Characteristics [ 48 ] was used to assess the accuracy of the model. In the MaxEnt model, the Area Under the Curve (AUC) of the receiver operating characteristic plot was used as an evaluation criterion to assess the accuracy of the model [ 49 ]. The stepwise elimination approach was used to remove variables that contributed less than ten percent (10%) to the model [ 37 ]. Further, a smooth response curve was used as a quality standard [ 22 ]. We reclassified the MaxEnt spatial model output into two environmental suitability classes, namely high and low in ArcGIS v10.3. Results The filtering selected 25 out of 37 presence records at 10 km rarefying. The PCA delivered five PCs, together accounting for 98.7% of the total variance ( Table 1 , S1 Fig ). After exclusion of unnecessary factor loading, thirteen predictor variables were retained. No multicollinearity was detected with VIF values of 0 to 2 (10% ( Table 2 ) namely, annual mean temperature (53%), soil type (35%), and human population density (12%). The Jackknife test of variables shows that omitting any of these three variables affects the regularization gain, test gain and AUC in the model. The annual mean temperature has the highest training gain when each variable was tested as the only environmental variable (1.2), and the lowest values were observed when analyzing only human population density (0.4). The lowest training gain appeared when the annual mean temperature was excluded from the model, while the model has the highest gain when human population density (1.2) and soil type (1.3) were excluded ( Table 3 ). 10.1371/journal.pone.0275261.t002 Table 2 Contribution of the three environmental predictors to the final suitability model. Variable Contribution (%) Permutation (%) Annual mean temperature 53 83 Soil type 35 4 Human population density 12 13 10.1371/journal.pone.0275261.t003 Table 3 Summary of the Jackknife analysis performed to determine importance per environmental variable. Variable Regularized Training gain Test gain Test AUC Alone Excluded Alone Excluded Alone Excluded Annual mean temperature 1.2 0.8 1.2 0.8 0.86 0.87 Soil type 0.6 1.3 0.4 1.5 0.76 0.91 Human population density 0.4 1.2 0.8 1.0 0.89 0.83 Annual mean temperature has the highest test gain values when used as the only environmental variable and soil type has the least test gain among the variables. Our model has a high training gain value when human population density and soil type were simultaneously excluded from our modeling process. The exclusion of annual mean temperature variable from the model results in a decline of the test gain ( Table 3 ). Observing our Jackknife test for AUC, the three important variables (annual mean temperature, soil type and human population density), when used in isolation were not significant different from each other. The AUC value of our model was excellent when soil type was excluded. There were no significantly differences in AUC values of the other two variables in the model ( Table 3 ). The suitability for the anthrax peaked when the annual mean temperature increased from -2 to 0°C, but declined briefly thereafter and maintained a constant probability across higher temperatures ( Fig 2 ). The soil types with the highest suitability are cambisol and kastanozem, both in their Haplic subtype ( Fig 2 ); Leptosols were unsuitable either in the presence of other variables or in isolation. The human population density response curve shows a gradual upward trend reaching a plateau at 40 individuals per km 2 ( Fig 2 ). Spatially, the highly suitable conditions are primarily found around Qinghai Lake. The northern and western part of the basin was predicted to be unsuitable for B . anthracis ( Fig 3 ). 10.1371/journal.pone.0275261.g002 Fig 2 Response curves of continuous predictor variables (climate and human population density) and bar graph of categorical predictor variable (cover) for B . anthracis distribution in the Qinghai Lake basin. The red lines indicate the mean values while the blue lines denote the standard deviation. 10.1371/journal.pone.0275261.g003 Fig 3 The environmental suitability map of B . anthracis distribution in the Qinghai Lake basin. Predictor variables used for modeling obtained from WorldClim ( http://worldclim.org/version2 ), with kind permission of Dr. Stephen Fick, geo-spatial data scientist. Discussion Our study presents the first assessment and spatial analysis of ecological suitability for B . anthracis in the Qinghai Lake basin. Although, epidemiological analysis had been done in Qinghai province [ 14 ] and anthrax distribution mapping in mainland China [ 15 ]. Here we analyzed over ten years mixed anthrax outbreaks using MaxEnt algorithms to investigate the environment and the geographic distribution of B . anthracis in the Qinghai Lake basin. The identified sets of environmental predictors of B . anthracis may represent factors directly or proxies thereof. This study supports the results of other studies, which have shown that anthrax outbreaks are associated with specific soils types [ 7 ], relatively high temperatures [ 19 ] and high human population density [ 15 ]. We found that relatively high mean annual temperatures within our high alpine environment had greatest predicted probability for anthrax occurrence. Consequently, continued climate warming may increase suitability for anthrax [ 8 ] also in our AOI. Soil types and certain soil characteristics, such as high levels of organic matter, alkaline pH or calcium, were previously thought to facilitate the B . anthracis distribution [ 50 , 51 ]. In our model, anthrax suitability was largely driven by two soil types, namely cambisols and kastanozems while leptosols showed the lowest B . anthracis suitability. The cambisols, occurs on young alluvial deposits in our AOI as well as worldwide. Cambisols are medium-textured and have a good structural stability, a high porosity, good water holding capacity and good internal drainage. Most cambisols have a neutral to weak acidity, a satisfactory chemical fertility and an active soil fauna [ 52 ]. The emergence of humus-rich kastanozems as the second most suitable soil type with the lowest standard deviations may be due to the presence of calcite (carbonate mineral) in its subsurface [ 50 ]. However, soil organic carbon, pH, cation exchange capacity, silt content and calcium were not predictive for B . anthracis distribution in our AOI. We found that the human population density was associated with anthrax which may be caused by the increased in human population. However, sheep, goat, and cattle population density both contributed to the model during the initial modeling but failed to meet the backward stepwise variable elimination criterion in our variable selection mode. The increase in human population density, settlement expansion, and seasonal migration would enhance human, livestock, and wildlife contact which would provide opportunity for B . anthracis transmission. The pastoralist nature of the population in Qinghai Lake basin could establish a human–animal interface either in pasture or in corrals. They also practice mixed farming; rearing animals and crop cultivation (mostly oilseed rape and highland barley) which are often used as fodder, while tillage would transpose dormant spores to the soil surface which increases anthrax emergence rate. In research carried out in Gangcha county (northwest of the lake), the migration of farmers and livestock were assessed that follow a regulation moving between low land and high mountains. The pastures are divided into spring pastures, spring and winter pastures, autumn pastures and summer pastures arranged from south to north. The spring, autumn, spring and winter, and winter pastures have an average altitude of 3190 m, 3890 m, 3890 m, and 4015 m respectively. Sheep, cattle and yak are the most dominant livestock in our AOI, Sheep herding practices include high-altitude summer pasturing which may reduce exposure to B . anthracis at the high anthrax risk zone around the lake during summer months. Although, human population density may act in proxy but there are other factors for B . anthracis risk analysis and assessment such as: type of animal husbandry, the number and density of livestock herds per household, transhumance, and carcasses disposal methods. The later, if not well practice would create 'locally infectious zones' (LIZs) at carcass sites [ 53 ], and establish a demography of their own as these zones appear and fade over time. Rather than passively acting as a fomite, evidence suggests that anthrax carcass sites have a complex set of biotic interactions that determine their persistence and infectiousness within the area [ 11 ]. Other important factors such as the difference in the quality of veterinary surveillance, and anti-epidemic measures during an outbreak of anthrax, ulcers through slaughtering of sick and suspicious livestock, lack of preventive therapy among the rest of the livestock and people working with it could increase frequency of outbreak. The areas with the highest suitability ranking are the low-lying area around the lake. The suitability could be dependent on the alluvial deposits, the various drainage channels from the higher elevation, characterized by the relocation of B . anthracis with soil through water, flooding or rain [ 50 ]. The result of our model with low altitude (around 3200 m.a.s.l) as an essential condition for survival of anthrax is in agreement with most similar studies on anthrax in South Africa [ 54 ], Zimbabwe [ 19 ] and Canada [ 55 ]. Our study reveals that bioclimatic and edaphic factors are fundamental conditions for B . anthracis distribution. Also, human population density and other related activities are specific factors reshaping the spatial distribution of B . anthracis . Our results should be interpreted with the following limitations in mind. First, human and livestock cases could have been under-reported as the surveillance was passive. The size of the occurrence data could be associated with sampling bias such as reporting cases only at where there is higher population density rather than truly being absent [ 46 ]. Broadly, MaxEnt can perform well with small sample sizes [ 46 ]. High success rates and statistical significance has been observed in jackknife tests with sample size as low as five [ 56 ] and ten using MaxEnt algorithm [ 57 ]. The changes in the diagnostic criteria for human anthrax cases since 2008 might have affected the quantity of the reported data. Second, some risk factors were not available to enrich our model, including but not limited to some soil characteristics (organic matter, alkaline pH, calcium etc.), seroprevalence in human and livestock exposure level of people at risk, and the industrialization level of livestock production. These factors may have been influenced by the sample size and spatial resolution of available predictor variables. Conclusion We categorized the Qinghai Lake basin into two suitability classes for B . anthracis distribution i.e., high and low, and revealed that increase annual mean temperature, two specific soil types (cambisols and kastanozems), and a high human population density, were the contributing variables for predicting B . anthracis environmental suitability. Soil type was the only significant categorical variables and second most influential variable overall; this would strengthen the edaphic paradigm for B . anthracis in its role for global B . anthracis suitability and anthrax epidemiology studies. Additionally, disease surveillance, health education, safe disposal of infected animal carcasses, vaccination of livestock, and other anthrax control measure strategies would be essential for disease prevention and can be prioritized for high-risk regions identified in our work. Supporting information S1 Table Bioclimatic, elevation and classical meteorological variables used for initial modeling in MaxEnt software (T-Temperature and P–Precipitation) R. (DOC) Click here for additional data file. S2 Table Edaphic and other factors used in modeling. (DOC) Click here for additional data file. S3 Table Environmental variables used for the final MaxEnt model. (DOC) Click here for additional data file. S4 Table The record of anthrax outbreak with latitude and longitude information of the location. (DOC) Click here for additional data file. S1 Fig Scree plot of climate predictor variables showing a steep decline of eigenvalues across the component numbers. (TIF) Click here for additional data file. S2 Fig Average ROC and related area under the curve (AUC) of the selected model. (TIF) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3026542/

Forward genetic dissection of innate response to infection in inbred mouse strains: selected success stories

Mouse genetics is a powerful tool for the dissection of genes, proteins, and pathways important in biological processes. Application of this approach to study the host response to infection has been a rich source of discoveries that have increased our understanding of the early innate pathways involved in responding to microbial infections. Here we review some of the key discoveries that have arisen from pinpointing the genetic defect in mouse strains with unusual or extreme response to infection and have led to insights into pathogen sensing pathways and downstream effector functions of the early innate immune response.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC156469/

Conjugative Plasmid Transfer in Gram-Positive Bacteria

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer ( tra ) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer ( oriT ), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7558263/

An African Canine Trypanosomosis Case Import: Is There a Possibility of Creating a Secondary Focus of Trypanosoma congolense Infection in France?

African animal trypanosomosis are parasitic diseases caused by several protozoa of the genus Trypanosoma , transmitted by hematophagous insects, essentially tsetse flies, but also, less frequently by Tabanidae and Stomoxidae. They are geolocated in a part of the continent and affect livestock animals and carnivores; dogs are especially sensitive to them. They do not seem to present a zoonotic risk. Despite the chemical prevention with trypanocides for French military working dogs on mission in Côte d'Ivoire, a fatal case induced by Trypanosoma congolense in France after returning from Abidjan raises the question of an imported secondary focus. The clinical case was developed and the causative agent was confirmed by microscopy and PCR methods. The three necessary pillars to create a secondary potential focus are present: the parasite introduction in a new territory, the presence and the propagation vectors, and their proximity with sensitive species. 1. Introduction Animal African trypanosomosis is a group of diseases caused by several flagellated protozoa from the genus Trypanosoma , transmitted by hematophagous insects, essentially tsetse flies, Glossina genus, but also by Tabanidae and Stomoxidae, even less frequently [ 1 , 2 ]. Tsetse flies are the true vectors of trypanosomes. They behave like intermediate hosts for the parasite, which are essential for their biological cycle. Tabanidae and Stomoxidae are simple mechanical vectors (simple regurgitation of the parasite) [ 3 , 4 ]. They affect various mammal species with an acute incidence in livestock and equids. The causative agents are mainly Trypanosoma congolense , Trypanosoma vivax and Trypanosoma evansi [ 2 , 5 ]. Dogs are also susceptible to infection [ 6 , 7 , 8 , 9 , 10 ]. The human African trypanosomiasis (or sleeping sickness) is caused by strictly human parasites ( T. brucei ) but presents vectorial similarities with animal trypanosomosis [ 11 ]. It occurs in areas where tsetse flies are present (especially Glossina morsitans , Glossina tachinoïdes and Glossina palpalis ): in vegetation, by the rivers and lakes, in forest galleries and in shrubby savannah [ 12 ]. This distribution is located between two imaginary lines: the first, from the 14th to the 10th north parallel (Senegal/ Somalia) and the second in the 20th south parallel, north of the Kalahari Desert. This area includes about 10 million square kilometers and covers 37 countries [ 11 ]. In Côte d'Ivoire, an exceptional human case of T. brucei and T. congolense coinfection has been described [ 13 ]. Animal trypanosomosis are classic acute or chronic diseases that cause fever and are accompanied by anemia, oedemas, lacrimation, lymphatic nodes hypertrophy, abortions, reduced fertility, loss of appetite and weight loss, leading to premature death in acute forms or to digestive and/or nervous signs with emaciation and subsequent death in chronic forms. Eye damage is not rare, such as keratitis, conjunctivitis and corneal clouding. These non-specific symptoms make it difficult to diagnose these diseases, for which no vaccine currently exists [ 8 , 9 , 10 , 14 , 15 ]. Canine African trypanosomosis (CAT) due to Trypanosoma congolense is described in Côte d'Ivoire, especially in slaughterhouses [ 6 ]. Cattle are the common hosts and the tsetse fly is the main vector. The French military kennel at Port-Bouët, which has potential breeding sites for Glossina spp. in its forested area, is likely to be exposed to this disease. As a precaution, since 2004, a chemoprevention with isometamidium chloride has been established, for all the military working dogs (MWD) spending time in this kennel, with Trypamidium ® (Boehringer Ingelheim, Lyon, France), one injection of 1 mg/kg every two months [ 16 , 17 ]. A recent imported case of canine trypanosomosis due to T. congolense , on return from the Abidjan region, raised the question of the potential for the creation of a secondary outbreak in France. In this report, we describe the clinical case and evaluate this risk of importation. 2. Case-Report 2.1. Commemoratives and Clinical Description A seven-year-old male Belgian shepherd was based in Côte d'Ivoire from 17 December 2018 to 7 January 2019. From December 18, an injection of Trypamidium ® was administered, in addition to classical MWD prophylactic treatments used in tropical regions, such as collars of deltamethrin, Scalibor ® (MSD Santé Animale, Beaucouze, France), during the mission. On 12 February, it was rushed to the 1st Veterinary Group of the French Army Health Service (Toulon) for convulsions that had appeared suddenly two hours earlier. It was in lateral decubitus, coma, polypnea-tachypnea and its mucous membranes were congested. It also presented a bilateral mydriasis, a flexible abdomen, a temperature of 39.5 °C and a cardiac frequency of 140 bpm. The dog received a treatment against shock and a constant dose-perfusion of analgesic. The coma intensified (eyes rolled back, increased hyperventilation), and the dog was referred to the veterinary clinic Olliolis in Ollioules (Var, France). Additional examinations were performed: medical imaging was normal (scanner, chest radiography, abdominal echography). The blood count showed an anemia, leucopenia, thrombocytopenia, and biochemical analysis showed severe hypoglycemia, moderate hypoalbuminemia and uremia; alkaline phosphatase and alanine transaminase concentrations were increased. Blood smear microscopic examination, after May–Grünwald Giemsa coloration and observation with oil-immersion-objective (×100), highlighted many nucleated elongated shapes. The blood smear also showed an anisocytosis and non-normochromic erythrocytes. The dog died 12 h later without regaining consciousness and without further convulsions. The necropsy was carried out 15 h later on all organs including an opening of the skull and observation of the encephalon. No lesions were detected, with the exception of splenomegaly. The cytology on splenic punctures revealed a reactional spleen with a major lympho-plasma cell hyperplasia. Blood, spleen, liver, kidneys and encephalon samples were sent to the Institut Hospitalo-Universitaire Méditerranée Infection of Marseille (France) for further analyses. 2.2. Microscopic Observation Blood smears were performed and after fixation in methanol; they were stained in eosin for 3 s and in methylene blue for 6 s. The slides were washed twice in a buffer and observed microscopically with an objective (×100). Microscopic observation revealed numerous trypanosomes ( Figure 1 ). 2.3. Molecular Assays DNA was extracted from 200 µL of blood and approximately 20 mg from the spleen, liver, kidney and brain samples after digestion with glass powder and proteinase K (10 µL) at 56 °C overnight. Extraction was performed on BIOROBOT EZ1 (Qiagen, Qiagen, Courtaboeuf, France), using a commercial DNA extraction kit (QIAamp DNA Mini Kit ® , Qiagen, Courtaboeuf, France) following the manufacturer's instructions. DNA was eluted in 200 µL. All DNA were tested by a real-time PCR (qPCR) assay targeting the 5.8S rRNA gene for Trypanosoma spp., with primers F5.8S_Tryp_CAACGTGTCGCGATGGATGA and F5.8S_Tryp_ ATTCTGCAATTGATACCACTTATC and probe S5.8S_Tryp_FAM-GTTGAAGAACGCAGCAAAGGCGAT. All samples were subjected to a conventional PCR targeting ~550 bps of the 28S RNA gene of Kinetoplastida parasites and sequencing by using primers: F2_ ACCAAGGAGTCAAACAGACG and R1_ GACGCCACATATCCCTAAG [ 18 , 19 ]. The qPCR assay was prepared in a final volume of 20 μL as previously described [ 19 ]. Amplification was performed in a CFX96 Real-Time system (BioRad Laboratories, Foster City, CA, USA) according to the following Roche protocol: an incubation step at 50 °C for two minutes and an initial denaturation step at 95 °C for five minutes, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing-extension at 60 °C for 30 s. DNA of Trypanosoma brucei and master mixture were added as positive control and negative control, respectively. Samples were considered positive when the cycle threshold (C t ) was lower than 35 C t . PCR amplifications were performed in a Peltier PTC-200 model thermal cycler (MJ Research Inc., Watertown, MA, USA). Reaction mixtures were prepared in 50 µL volume as previously described [ 18 ]. The thermal cycling protocols were as follows: incubation step at 95 °C for 15 min, 40 cycles of one minute at 95 °C, 30 s at 57 °C and one minute at 72 °C and a final extension step for five minutes at 72 °C. All amplicons were visualized in electrophoresis on 2% agarose gels. Amplicons were then purified using NucleoFast 96 PCR plates (Macherey Nagel EURL, Hoerdt, France) according to the manufacturer's instructions and were then sequenced using the Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA) with an ABI automated sequencer (Applied Biosystems). The obtained electropherograms were assembled and edited using ChromasPro software (ChromasPro 1.7, Technelysium Pty Ltd., Tewantin, Australia) and compared with those available in the GenBank database by National Center for Biotechnology Information (NCBI) BLAST ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). All organs and blood were positive in qPCR with C t (16–26.5) ( Table 1 ) and in PCR ( Figure 2 ). In the blast analysis, the sequences obtained showed 99.33% identity and 100% cover with Trypanosoma congolense riverine/forest-type (acc No. U22319) [ 18 , 19 ]. 2.4. Further Investigations Another dog that completed the same mission and returned to France at the same time as the sick dog, as well as six other dogs that spent four months in Côte d'Ivoire around the time of the sick dog, were tested by PCR for the presence of Trypanosoma spp. All these samples were negative. 2.1. Commemoratives and Clinical Description A seven-year-old male Belgian shepherd was based in Côte d'Ivoire from 17 December 2018 to 7 January 2019. From December 18, an injection of Trypamidium ® was administered, in addition to classical MWD prophylactic treatments used in tropical regions, such as collars of deltamethrin, Scalibor ® (MSD Santé Animale, Beaucouze, France), during the mission. On 12 February, it was rushed to the 1st Veterinary Group of the French Army Health Service (Toulon) for convulsions that had appeared suddenly two hours earlier. It was in lateral decubitus, coma, polypnea-tachypnea and its mucous membranes were congested. It also presented a bilateral mydriasis, a flexible abdomen, a temperature of 39.5 °C and a cardiac frequency of 140 bpm. The dog received a treatment against shock and a constant dose-perfusion of analgesic. The coma intensified (eyes rolled back, increased hyperventilation), and the dog was referred to the veterinary clinic Olliolis in Ollioules (Var, France). Additional examinations were performed: medical imaging was normal (scanner, chest radiography, abdominal echography). The blood count showed an anemia, leucopenia, thrombocytopenia, and biochemical analysis showed severe hypoglycemia, moderate hypoalbuminemia and uremia; alkaline phosphatase and alanine transaminase concentrations were increased. Blood smear microscopic examination, after May–Grünwald Giemsa coloration and observation with oil-immersion-objective (×100), highlighted many nucleated elongated shapes. The blood smear also showed an anisocytosis and non-normochromic erythrocytes. The dog died 12 h later without regaining consciousness and without further convulsions. The necropsy was carried out 15 h later on all organs including an opening of the skull and observation of the encephalon. No lesions were detected, with the exception of splenomegaly. The cytology on splenic punctures revealed a reactional spleen with a major lympho-plasma cell hyperplasia. Blood, spleen, liver, kidneys and encephalon samples were sent to the Institut Hospitalo-Universitaire Méditerranée Infection of Marseille (France) for further analyses. 2.2. Microscopic Observation Blood smears were performed and after fixation in methanol; they were stained in eosin for 3 s and in methylene blue for 6 s. The slides were washed twice in a buffer and observed microscopically with an objective (×100). Microscopic observation revealed numerous trypanosomes ( Figure 1 ). 2.3. Molecular Assays DNA was extracted from 200 µL of blood and approximately 20 mg from the spleen, liver, kidney and brain samples after digestion with glass powder and proteinase K (10 µL) at 56 °C overnight. Extraction was performed on BIOROBOT EZ1 (Qiagen, Qiagen, Courtaboeuf, France), using a commercial DNA extraction kit (QIAamp DNA Mini Kit ® , Qiagen, Courtaboeuf, France) following the manufacturer's instructions. DNA was eluted in 200 µL. All DNA were tested by a real-time PCR (qPCR) assay targeting the 5.8S rRNA gene for Trypanosoma spp., with primers F5.8S_Tryp_CAACGTGTCGCGATGGATGA and F5.8S_Tryp_ ATTCTGCAATTGATACCACTTATC and probe S5.8S_Tryp_FAM-GTTGAAGAACGCAGCAAAGGCGAT. All samples were subjected to a conventional PCR targeting ~550 bps of the 28S RNA gene of Kinetoplastida parasites and sequencing by using primers: F2_ ACCAAGGAGTCAAACAGACG and R1_ GACGCCACATATCCCTAAG [ 18 , 19 ]. The qPCR assay was prepared in a final volume of 20 μL as previously described [ 19 ]. Amplification was performed in a CFX96 Real-Time system (BioRad Laboratories, Foster City, CA, USA) according to the following Roche protocol: an incubation step at 50 °C for two minutes and an initial denaturation step at 95 °C for five minutes, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing-extension at 60 °C for 30 s. DNA of Trypanosoma brucei and master mixture were added as positive control and negative control, respectively. Samples were considered positive when the cycle threshold (C t ) was lower than 35 C t . PCR amplifications were performed in a Peltier PTC-200 model thermal cycler (MJ Research Inc., Watertown, MA, USA). Reaction mixtures were prepared in 50 µL volume as previously described [ 18 ]. The thermal cycling protocols were as follows: incubation step at 95 °C for 15 min, 40 cycles of one minute at 95 °C, 30 s at 57 °C and one minute at 72 °C and a final extension step for five minutes at 72 °C. All amplicons were visualized in electrophoresis on 2% agarose gels. Amplicons were then purified using NucleoFast 96 PCR plates (Macherey Nagel EURL, Hoerdt, France) according to the manufacturer's instructions and were then sequenced using the Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA) with an ABI automated sequencer (Applied Biosystems). The obtained electropherograms were assembled and edited using ChromasPro software (ChromasPro 1.7, Technelysium Pty Ltd., Tewantin, Australia) and compared with those available in the GenBank database by National Center for Biotechnology Information (NCBI) BLAST ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). All organs and blood were positive in qPCR with C t (16–26.5) ( Table 1 ) and in PCR ( Figure 2 ). In the blast analysis, the sequences obtained showed 99.33% identity and 100% cover with Trypanosoma congolense riverine/forest-type (acc No. U22319) [ 18 , 19 ]. 2.4. Further Investigations Another dog that completed the same mission and returned to France at the same time as the sick dog, as well as six other dogs that spent four months in Côte d'Ivoire around the time of the sick dog, were tested by PCR for the presence of Trypanosoma spp. All these samples were negative. 3. Discussion Could such an imported clinical case contaminate other MWD in the army kennel upon its return, and subsequently the livestock or pets surrounding it? This risk assessment justifies our discussion, especially because African trypanosomes represent pathogenic exotic agents in Metropolitan France [ 20 ]. African trypanosomosis does not exist in the territory except imported cases and livestock, pets and wildlife animals in France could be sensitive. Several dozen French military dogs are projected annually in areas where CAT is endemic. They return to France after a period of few days to several months (four most often), in their original kennels, located all over France, with a majority of them in Suippes (Marne, East of France) at the 132 e Cynotechnical Infantry Regiment. The CAT incubation period is variable. In the case of trypanosomosis due to T. congolense , for which dogs are highly sensitive, the incubation period varies from one to three weeks [ 8 ], but may last longer (in our case, it developed the disease at least five to eight weeks after his infection in Abidjan). The creation of a secondary focus requires the establishment of three pillars: the imported parasite, the vectors and the susceptible hosts. Once the pathogen is imported, its spread will be the second step, leading to the creation of an endemic outbreak. This requires early detection and limitation of spread [ 20 ]. We hypothesize that this MWD is a reservoir of infective trypanosomes. Despite the consequent number of MWD movements towards Côte d'Ivoire (about 24 per year), our fatal case constitutes an exception. The last confirmed cases among MWD dates back to the years 2001–2002 [ 16 , 17 ]. During that episode, 19 MWD had been affected with T. congolense , five died: three of them despite the specific treatment administered on the spot and two on their return to France. All of them were post-mortem diagnosed (hematologic and histologic analyses). The remaining fourteen were successfully treated, seven on the spot and seven after their return to France. The molecule chosen was isometamidium chloride of 1 mg/kg (2% solution or 20 mg/L) [ 21 ]. Subsequently to this episode, CAT prevention was systematically administered for the canine military units. The dogs were injected with isometamidium chloride from the first days of their arrival in Côte d'Ivoire and every two months throughout the mission. Since these measures, and up until our dog, no other MWD had developed the disease (over 400 dogs) [ 17 ]. Otherwise, any military dog returning from a mission outside mainland France has been subjected to a quarantine period of at least 21 days. During these three weeks, the dog is hosted in the isolation kennel yard, and never leaves its unit. It is observed every day. At the end of the quarantine, the animal is presented to a military veterinarian and blood tests are performed for exotic diseases (ehrlichiosis, Lyme disease, anaplasmosis, heartworm disease and leishmaniosis). A blood smear is also performed to look for trypanosomes. Further examinations for CAT diagnosis are very important as their clinical picture is not pathognomonic. For T. congolense , the dog is particularly more sensitive than other species, and develops an acute clinical form that can be fatal with a devastating neurological picture, as in our case [ 8 ]. It occurs especially when the dog is naive for the parasite. Any individual, human or animal, regularly exposed to trypanosomes, seems to develop a trypanotolerance [ 22 , 23 ]. The first signs could be just abatement with anorexia and hyperthermia. The evolution can be very rapid, involving different organs with various symptoms: gastro-intestinal and skin disorders, effusions and oedemas, hemorragic and nervous manifestations. Other sensitive animals express less severe clinical forms. Consequently, CAT clinical diagnosis remains difficult [ 8 , 9 ]. The early detection of CAT requires the examination of blood samples. Blood smear coloration is the common test performed by the Military Veterinary Groups. Its low sensibility has led to a preference for the centrifugation technique on capillary tubes. This procedure makes it possible to detect even small infections (the detection limit is around 500 trypanosomes/mL of blood), mostly six to ten days before the parasite detection by direct blood examinations. In case of suspicion or if the CAT must be included in a differential diagnosis when clinical signs are not very evocative, and in the context of recent stay in Côte d'Ivoire, the use of a laboratory reference test such as PCR is therefore systematic [ 19 ]. The introduction of the parasite by the MWD is surveyed, thanks to well-known and well-established procedures, but it remains impossible to completely prevent all events. However, this surveillance can fail, as showed in this case. One scenario previously reported is that of dromedaries imported for breeding purposes in Aveyron (South of France), from the Canary Islands (Spain, 1995 and 2006). This led to a fatal case of T. evansi trypanosomosis in 2007, with the detection of antibodies in two of the imported camels and in three others belonging to the farm for years and in sheep from the same area. This focus did not spread to the entire farm, thanks to the elimination of all positive animals [ 24 ]. Disease spread requires the presence of competent vectors, mainly haematophagous arthropods. Animal African trypanosomosis are mainly transmitted by tsetse flies absent in France, or, in the case of their accidental importation, they cannot survive enough to eat and reproduce in the French climate. Tsetse flies live at temperatures between 20 °C and 30 °C, in high hygrometry level and shady atmospheres, with vegetation as high as possible. Whenever possible, these Diptera flee areas of human disturbance and settle on the leaves at a maximum height of 50 cm. Any excessive climate change is quickly unfavorable regardless of their development stage and can even be fatal. These factors explain the strict limits of the geographical distribution of Glossina [ 2 , 25 , 26 ]. In France, however, the main risk of trypanosomosis transmission is due to bites from the Tabanidae and Stomoxidae [ 3 , 4 ]. Animal trypanosomosis exists in Europe: T. theileri in cattle (transmission by Tabanidae, not very pathogenic). Trypanosomes of birds or amphibians also exist [ 2 ]. Among Tabanidae, only females are hematophagous, males feed on nectar. They are diurnal, but only during the warm season. They are found in intensive farming regions and wooded areas and they can fly for long distances (several kilometers). Tabanids bite by tearing off a piece of tissue. The blood flows and the females easily take the blood meal. Many pathogen agents for humans or animals can be transmitted by tabanids, including virus, bacteria, protozoa and helminthes, causing a panel of diseases such as, for instance, pasteurellosis, tularemia, anthrax, leucosis or equine infectious anemia. They also have a direct pathogenic role through their decomposing action on the epidermis of livestock, especially if they are present in large numbers. Their bite is very painful and has a spoliative effect leading to stunted growth and reduced milk production. These biting flies can therefore play a role in the vector transmission of CAT in Metropolitan France [ 27 ]. Stomoxidae are small flies that look like house flies. They parasitize cows, horses, and other livestock animals. They are common in stables and sheepfolds, where they find feces to lay their eggs. The difference from other blood-sucking insects is that males also need blood for their biological cycle. They are also vectors for several diseases: equine infectious anemia, African swine fever, West Nile and Rift Valley fever viruses, bacteria and parasites. In addition, Stomoxidae are very often resistant to available insecticides. Here again, their action as vectors for trypanosomosis in France is possible [ 3 , 28 ]. However, such a vector role should be limited by an external parameter: MWD receive, throughout the year, a special prevention against flies, and external anti-parasitics of topical use such as permethrin, fipronil or imidacloprid. Tabanidae and Stomoxidae, therefore, certainly do not bite these dogs. Transmission through infected syringes is unlikely, thanks to good practice in veterinary consultations [ 17 ]. Although the probability of importing a case of T. congolense into France is low and its spread by local vectors is unlikely, many animal species in metropolitan France are susceptible to trypanosomosis (cattle, suidae, goats, equine and carnivores). Livestock and wildlife animals would be concerned. Clinical disparities exist and have been developed above. In cattle, the parasitaemia remains too low, allowing mechanical transmission of the vector. These animals are often epidemiological dead ends. 4. Conclusions Preventing the creation of a secondary trypanosomosis outbreak in France, in case of reimportation of MWD, is the collective work of military veterinarians, kennel managers and dog handlers. They alert the health services immediately when a dog presents deteriorated general conditions and systematically after quarantine, upon return from the tropical zones. The case reported here allows all the French military veterinarians to be informed about CAT, their clinical picture entering into a consequent differential diagnosis, and to check blood smear screening procedures. The probability of trypanosomosis spread is very low given the deficiencies of the parasite chain at each link: there are few cases of importation, seasonal and regionalized vectors in France, and military working dogs are treated against them. For these reasons, trypanosome transmission in France remains a negligible risk. On the other hand, it seems reasonable to consider the risk of new threats to animal health. They are becoming possible because of globalization, the movement of products, animals and humans, climate change and changes in the ecosystem that may become favorable to some pathogen vectors. Combined measures for the prevention of infectious diseases (vaccination, chemoprophylaxis, vector control, deworming, quarantine, serology or detection of pathogens or antigens, and clinical monitoring upon return) are essential to control the risks of importation of many infectious diseases by animals travelling between endemic areas and their country of origin.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3615786/

Applying the XForms Standard to Public Health Case Reporting and Alerting

Notifiable condition reporting and alerting are two important public health functions. Today, a variety of methods are used to transfer these types of information. The increasing use of electronic health record systems by healthcare providers makes new types of electronic communication possible. We used the XForms standard and nationally recognized technical profiles to demonstrate the communication of both notifiable condition reports and patient-tailored public health alerts. This demonstration of bi-directional communication took placein a prototypical health information exchange environment. We successfully transferred information between provider electronic health record systems and public health systems for notifiable condition reporting. Patient-specific alerts were successfully sent from public health to provider systems. In this paper we discuss the benefits of XForms, including the use of XML, advanced form controls, form initialization and reduction in scripting. We also review implementation challenges, the maturity of the technology and its suitability for use in public health. INTRODUCTION Surveillance of the public's health depends on the collection, investigation and distribution of data and information about illness and health. Timely reporting of notifiable conditions (e.g., tuberculosis, vibriosis, or chlamydia, among numerous other conditions) to public health agenciesby health care providers (HCPs), health care facilities, laboratories, veterinarians, food service establishments, child day care facilities, and schools supports early detection of risks to the community such as outbreaks of infectious or foodborne diseases. Public health and other government organizations use the information collected in these case report s to prevent and control diseases. Also important to the protection of the population's health is the communication of health information from public health agencies to the community. One specific type of communication is public health alerting , e.g., public health warnings about outbreaks, preventive measures, and recommendations sent to HCPs. Case reporting, also referred to as notifiable condition reporting, and public health alerting are part of a bi-directional transfer of information in which information in the form of case reports are transferred from HCPsto public health agencies and information in the form of public health alerts is transferred from public health agencies to HCPs. In the US, this bi-directional communication is being carried out with varying levels of sophistication and success: approaches topublic health alerting cover a broad range of communication types, including print, fax, email, and text message ( 1 ). Notifiable condition reporting methods are similarly varied, ranging from faxed case-report forms to sophisticated electronic laboratory reporting systems. Public health informaticians recognize the importance of data and information exchange standards which define the structure and syntax for sending and receiving information ( 2 – 5 ). Strengthening the connection between public health and provider systems requires interoperability and the use of standards in both public health and clinical care settings. Although public health organizations in the US have been slow to adopt these standards, public-private partnerships ( 6 – 8 ) have been working to ensure that bi-directional communication between public health and HCPs is incorporated into national health information infrastructure standards. Public health use cases describe the interactions between the various components of an information exchange based on a real-life scenario, thus providing a common focus for the different activities to inform development of specific requirements, architecture, and standards. This paper describes our experience using technical profiles and implementing XForms in a notifiable condition reporting and patient-tailored alerting public health use case. XForms were implemented in a prototypical health information exchange (HIE) demonstration and testing environment. We also explore the feasibility and possible implications of the use of these profiles and standards in public health. BACKGROUND Notifiable Condition Reporting (Case Reporting) The timeliness and completeness of notifiable conditions data which public health agencies rely on to track diseases, target interventions, mitigate harmful exposures, initiate investigations, and develop program activities and policies vary widely. In an analytic literature review, Doyle et al. (2002) found that reporting completeness varied from 9% to 99% and was strongly associated with the specific disease reported. For example, active surveillance systems for certain diseases, like sexually transmitted infections, had higher completeness rates ( 9 ). And while timeliness requirements are often specified by health jurisdictions or state law, measures of timeliness do not always meet the specified standards. ( 10 ) Timely and complete case reports are essential to public health surveillance work. Technological developments such as the adoption of electronic laboratory reporting systems have improved the timeliness and completeness of reported notifiable conditions data ( 11 – 13 ). For example, National Electronic Disease Surveillance System (NEDSS) was introduced in 2000 as a new method for US notifiable condition reporting and surveillance. NEDSS was designed to facilitate electronic surveillance of infectious diseases outbreaks, emerging or reemerging pathogens and to identify possible bioterrorist attacks. This system further evolved to become a reporting system which would allow rapid communication among public health authorities of varying size and technical capacity ( 14 ). NEDSS also prompted some state and municipal health departments to begin researching and building electronic surveillance systems for their regions, resulting in significant improvements in reporting rates and data quality ( 15 ). Many health departments in the US share similar work practices ( 16 ); however, nationally recognized standards for content, collection and delivery of notifiable condition data have not been widely adopted. To accelerate adoption, in 2007 the Centers for Disease Control and Prevention (CDC), in cooperation with the Council for State and Territorial Epidemiologists (CSTE), began work on an Implementation Guide for Health Level 7 (HL7) Version 3 Clinical Document Architecture (CDA). The Implementation Guide provides a framework and related standards for the exchange, integration, sharing, and retrieval of notifiable condition reporting from an electronic health record (EHR) to public health ( 17 ). In 2010 a more general, non-disease-specific model for automated public health case reporting using HL7 version 2.5 was proposed ( 18 ). Rapid development in the areas of messaging and data standards in health care, as well as the ever increasing technical capability of both public health and provider organizations, suggest that electroni c notifiable condition reporting may soon be feasible on a large scale. These same advancements in technology also provide the infrastructure to support changes in the way public health communicates information to providers, such as context-specific public health alerts. Public Health Alerting Alerting systems that facilitate the delivery of public health information to HCPs rely on the interactive contribution of HCPs both prior to and during a public health event. Bi-directional communication between HCPs and public health has been well-documented, particularly since 2001. A systematic review of US disease outbreak detection reported that the coordination necessary for aggregating, analyzing, and sharing data between the clinical health care system and local and state public health agencies was a key component in prompt detection of infectious disease outbreaks ( 19 ). Additionally, many infectious disease agents are initially difficult to identify: signs may be nonspecific and illnesses may be scattered geographically ( 20 ). Increasing numbers of individuals presenting to HCPs, pharmacists, hospital emergency rooms, and others can serve as sentinel events for disease outbreaks in the community ( 21 – 23 ). In the event of a bioterrorist attack, in which there may be a delay between exposure and symptom onset, public health relies on HCPs and laboratories to report cases of unexplained or unusual illness to public health officials who, in turn, may be able to identify specific epidemiologic patterns or characteristics indicative of a bioterrorist act ( 24 ). Several programs are designed to facilitate alert communications between public health agencies and HCPs, however, few specify the appropriate timing of communications or contain details regarding which specific organizations or providers should be contacted in a particular type of emergency. While the 2001 anthrax attacks identified electronic communications systems as high priority in facilitating effective infectious disease surveillance and investigation, it is not known the extent to which systems such as the Epidemic Information Exchange and the CDC's Health Alert Network have improved surveillance or communications. These public health alerting systems use electronic communication methods such as e-mail and broadcast fax to link public health agencies with HCPs and other community groups ( 25 ). However, coverage of messages relayed via these methods is unknown or lower than it could be as the system relies on HCP registries that may contain incomplete, missing or out-of-date e-mail and/or fax contact information. The receipt and assimilation of messages by providers is a pre requisite to any related subsequent action, including enhanced event reporting and responsible communication of information to patients ( 26 ). Though alerts can be communicated using various methods, using EHRs as the communication resource offers the potential to provide both timely and context specific information to HCPs ( 27 ). In Indiana, public health alerts have been added to the current clinical results delivery service in order to integrate the communication into the physician's workflow ( 28 ). Unfortunately, overwhelmed providers often suffer from 'alert fatigue,' dismissing even context-specific alerts from clinical decision support or computerized provider order entry systems ( 29 – 31 ). More research is needed to measure the impact of different types of public health alerts as technological developments offer the chance to augment public health-provider communication. One such development is the XForms standard. XForms Web forms are a common tool used on websites to accept user input for activities such as searches, surveys, file uploads, and purchases. They are also common in other areas where structured communication is important, including public health. Some simple web forms can be built using HTML. The data collected using an HTML form, as a set of name-value pairs, can be submitted to a server or sent using email. However, HTML forms have several limitations: reliance on scripting for managing form behavior and dynamic content; difficulty initializing a form with existing data; and constraints of the formats for encoding HTML form data ( 32 ). HTML forms are appropriate for some simple tasks, but the complex needs of users and the limitations of HTML forms have led to the development of web form alternatives. One alternative is XForms. In 2000, recognizing the limitations of HTML forms, a World Wide Web Consortium Forms Working Group (FWG) was created to help guide the development of new web forms technology ( 33 ). The FWG leveraged existing XML recommendations ( 34 ) in meeting the following development goals: support for structured data; improvement in accessibility; support for interrupted form completion; and decoupling of the data, logic, and presentation of a form ( 33 ). While the FWG goals are compelling, it is important to note that XForms does not represent a document type that can stand alone, but is meant to be integrated into other markup languages such as XHTML. However, XForms was touted as the "next generation of web forms" because of its flexibility, portability, and unique separation of data and presentation ( 35 ). Additional advantages include: the ability to incorporate metadata to describe the history and attributes of a particular form instance; the capability to include validation information for data elements on the form which reduces the amount of script needed for data validation; and the availability of components that allow the user to interact with XForms using either stand-alone programs or a web browser. XForms has been demonstrated, used, and explored in a variety of settings ; XForms specification has proved useful in dynamic query development and enabling exploration of data for those with no knowledge of the structure of the data to be queried ( 36 ). Researchers have described successful implementations of XForms processors across diverse environments using different layout models ( 37 ) and in support of dynamic and adaptable document types ( 38 ). Other work has explored the use of XForms with web services ( 39 ), for enhancing accessibility of web interfaces ( 40 ), and for linking data models to commonly used forms in the insurance industry ( 41 ). XForms is still an under utilized specification with an uncertain future and has been slow to gain acceptance within the healthcare industry. However, some early adopters in public health and provider settings have realized the advantages of using XForms to standardize the presentation of, and data collected by, web forms. In Germany, developers successfully implemented an information system to manage details of a prescription drug formulary using XML and XForms ( 42 ). Researchers in Australia used XForms for decision support system development ( 43 ) and scientists in South Korea proposed a radiology information system using XForms for a report management module ( 44 ). XForms has also been used in clinical and public health systems. One example is its successful implementation within Open MRS, an open source Medical Record System, as an alternative to Microsoft's Info Path web forms solution ( 45 ).XForms has also been considered for use in public health surveillance. A group from the CDC proposed use of XForms as a part of the framework for public health form creation and management ( 46 ). Although the technologies in use today range from primitive to sophisticated, it is clear that improved efficiency, timeliness, and completeness could be gained by improving connections between public health and provider systems. We saw a potential match between XForms' capabilities and the need to facilitate bi-directional electronic communication between public health and provider systems. In this paper we present our experience using the XForms standard in the public health context for provider-initiated notifiable condition reporting and patient-specific public health alerting. Notifiable Condition Reporting (Case Reporting) The timeliness and completeness of notifiable conditions data which public health agencies rely on to track diseases, target interventions, mitigate harmful exposures, initiate investigations, and develop program activities and policies vary widely. In an analytic literature review, Doyle et al. (2002) found that reporting completeness varied from 9% to 99% and was strongly associated with the specific disease reported. For example, active surveillance systems for certain diseases, like sexually transmitted infections, had higher completeness rates ( 9 ). And while timeliness requirements are often specified by health jurisdictions or state law, measures of timeliness do not always meet the specified standards. ( 10 ) Timely and complete case reports are essential to public health surveillance work. Technological developments such as the adoption of electronic laboratory reporting systems have improved the timeliness and completeness of reported notifiable conditions data ( 11 – 13 ). For example, National Electronic Disease Surveillance System (NEDSS) was introduced in 2000 as a new method for US notifiable condition reporting and surveillance. NEDSS was designed to facilitate electronic surveillance of infectious diseases outbreaks, emerging or reemerging pathogens and to identify possible bioterrorist attacks. This system further evolved to become a reporting system which would allow rapid communication among public health authorities of varying size and technical capacity ( 14 ). NEDSS also prompted some state and municipal health departments to begin researching and building electronic surveillance systems for their regions, resulting in significant improvements in reporting rates and data quality ( 15 ). Many health departments in the US share similar work practices ( 16 ); however, nationally recognized standards for content, collection and delivery of notifiable condition data have not been widely adopted. To accelerate adoption, in 2007 the Centers for Disease Control and Prevention (CDC), in cooperation with the Council for State and Territorial Epidemiologists (CSTE), began work on an Implementation Guide for Health Level 7 (HL7) Version 3 Clinical Document Architecture (CDA). The Implementation Guide provides a framework and related standards for the exchange, integration, sharing, and retrieval of notifiable condition reporting from an electronic health record (EHR) to public health ( 17 ). In 2010 a more general, non-disease-specific model for automated public health case reporting using HL7 version 2.5 was proposed ( 18 ). Rapid development in the areas of messaging and data standards in health care, as well as the ever increasing technical capability of both public health and provider organizations, suggest that electroni c notifiable condition reporting may soon be feasible on a large scale. These same advancements in technology also provide the infrastructure to support changes in the way public health communicates information to providers, such as context-specific public health alerts. Public Health Alerting Alerting systems that facilitate the delivery of public health information to HCPs rely on the interactive contribution of HCPs both prior to and during a public health event. Bi-directional communication between HCPs and public health has been well-documented, particularly since 2001. A systematic review of US disease outbreak detection reported that the coordination necessary for aggregating, analyzing, and sharing data between the clinical health care system and local and state public health agencies was a key component in prompt detection of infectious disease outbreaks ( 19 ). Additionally, many infectious disease agents are initially difficult to identify: signs may be nonspecific and illnesses may be scattered geographically ( 20 ). Increasing numbers of individuals presenting to HCPs, pharmacists, hospital emergency rooms, and others can serve as sentinel events for disease outbreaks in the community ( 21 – 23 ). In the event of a bioterrorist attack, in which there may be a delay between exposure and symptom onset, public health relies on HCPs and laboratories to report cases of unexplained or unusual illness to public health officials who, in turn, may be able to identify specific epidemiologic patterns or characteristics indicative of a bioterrorist act ( 24 ). Several programs are designed to facilitate alert communications between public health agencies and HCPs, however, few specify the appropriate timing of communications or contain details regarding which specific organizations or providers should be contacted in a particular type of emergency. While the 2001 anthrax attacks identified electronic communications systems as high priority in facilitating effective infectious disease surveillance and investigation, it is not known the extent to which systems such as the Epidemic Information Exchange and the CDC's Health Alert Network have improved surveillance or communications. These public health alerting systems use electronic communication methods such as e-mail and broadcast fax to link public health agencies with HCPs and other community groups ( 25 ). However, coverage of messages relayed via these methods is unknown or lower than it could be as the system relies on HCP registries that may contain incomplete, missing or out-of-date e-mail and/or fax contact information. The receipt and assimilation of messages by providers is a pre requisite to any related subsequent action, including enhanced event reporting and responsible communication of information to patients ( 26 ). Though alerts can be communicated using various methods, using EHRs as the communication resource offers the potential to provide both timely and context specific information to HCPs ( 27 ). In Indiana, public health alerts have been added to the current clinical results delivery service in order to integrate the communication into the physician's workflow ( 28 ). Unfortunately, overwhelmed providers often suffer from 'alert fatigue,' dismissing even context-specific alerts from clinical decision support or computerized provider order entry systems ( 29 – 31 ). More research is needed to measure the impact of different types of public health alerts as technological developments offer the chance to augment public health-provider communication. One such development is the XForms standard. XForms Web forms are a common tool used on websites to accept user input for activities such as searches, surveys, file uploads, and purchases. They are also common in other areas where structured communication is important, including public health. Some simple web forms can be built using HTML. The data collected using an HTML form, as a set of name-value pairs, can be submitted to a server or sent using email. However, HTML forms have several limitations: reliance on scripting for managing form behavior and dynamic content; difficulty initializing a form with existing data; and constraints of the formats for encoding HTML form data ( 32 ). HTML forms are appropriate for some simple tasks, but the complex needs of users and the limitations of HTML forms have led to the development of web form alternatives. One alternative is XForms. In 2000, recognizing the limitations of HTML forms, a World Wide Web Consortium Forms Working Group (FWG) was created to help guide the development of new web forms technology ( 33 ). The FWG leveraged existing XML recommendations ( 34 ) in meeting the following development goals: support for structured data; improvement in accessibility; support for interrupted form completion; and decoupling of the data, logic, and presentation of a form ( 33 ). While the FWG goals are compelling, it is important to note that XForms does not represent a document type that can stand alone, but is meant to be integrated into other markup languages such as XHTML. However, XForms was touted as the "next generation of web forms" because of its flexibility, portability, and unique separation of data and presentation ( 35 ). Additional advantages include: the ability to incorporate metadata to describe the history and attributes of a particular form instance; the capability to include validation information for data elements on the form which reduces the amount of script needed for data validation; and the availability of components that allow the user to interact with XForms using either stand-alone programs or a web browser. XForms has been demonstrated, used, and explored in a variety of settings ; XForms specification has proved useful in dynamic query development and enabling exploration of data for those with no knowledge of the structure of the data to be queried ( 36 ). Researchers have described successful implementations of XForms processors across diverse environments using different layout models ( 37 ) and in support of dynamic and adaptable document types ( 38 ). Other work has explored the use of XForms with web services ( 39 ), for enhancing accessibility of web interfaces ( 40 ), and for linking data models to commonly used forms in the insurance industry ( 41 ). XForms is still an under utilized specification with an uncertain future and has been slow to gain acceptance within the healthcare industry. However, some early adopters in public health and provider settings have realized the advantages of using XForms to standardize the presentation of, and data collected by, web forms. In Germany, developers successfully implemented an information system to manage details of a prescription drug formulary using XML and XForms ( 42 ). Researchers in Australia used XForms for decision support system development ( 43 ) and scientists in South Korea proposed a radiology information system using XForms for a report management module ( 44 ). XForms has also been used in clinical and public health systems. One example is its successful implementation within Open MRS, an open source Medical Record System, as an alternative to Microsoft's Info Path web forms solution ( 45 ).XForms has also been considered for use in public health surveillance. A group from the CDC proposed use of XForms as a part of the framework for public health form creation and management ( 46 ). Although the technologies in use today range from primitive to sophisticated, it is clear that improved efficiency, timeliness, and completeness could be gained by improving connections between public health and provider systems. We saw a potential match between XForms' capabilities and the need to facilitate bi-directional electronic communication between public health and provider systems. In this paper we present our experience using the XForms standard in the public health context for provider-initiated notifiable condition reporting and patient-specific public health alerting. METHODS/EXPERIENCE Beginning in 2005, we participated in a series of large national development and demonstration projects as a part of the Integrating the Healthcare Enterprise's Connectathon and Interoperability Showcase. We played several parts in the demonstrations, including roles that required the use Interoperability Profiles such as Retrieve Forms for Data Capture (RFD) and use of the XForms standard for notifiable condition reporting and patient-specific public health alerting. Below we describe our experiences in bi-directional communication demonstrations at two large health informatics conferences. Integrating the Healthcare Enterprise Integrating the Healthcare Enterprise (IHE)was formed in 1998 by a group of healthcare and industry professionals with the goal of improving interoperability in healthcare information systems ( 7 ).The organization encourages the adoption of standards by developing, promoting and demonstrating Interoperability Profiles which are implementation guides for incorporating standards and that describe the business rules, specific transactions and standards which can be used in a structured way to address specific clinical and population health use cases. In the public health domain, the standards are typically those identified by the Health Information Technology Standards Panel (HITSP) ( 6 ). Annually, vendors and other participants gather to test interoperability and implementation of profiles during the IHE Connectathon; this testing is followed by the Interoperability Showcase which takes place at the Healthcare Information Management Systems Society annual conference and demonstrates the implementation of standards and IHE Profiles. In addition, the CDC's Public Health Information Network conference has also hosted a smaller-scale, public health focused showcase. The demonstrations use scenarios to tell a story, usually about a patient's experience in the healthcare system. We participated in several population health scenarios and implemented interoperability profiles, including those to support notifiable condition reporting and patient-specific public health alerting ( 47 ). Retrieve Forms for Data Capture and XForms for Public Health The primary profile we used for the demonstration of the reporting and alerting public health use cases was RFD, which uses XForms and enables viewing, pre-population, completion, and submission of forms and form data. The RFD profile specifies how different roles will function during the transaction: forms manager; forms filler; forms receiver; and forms archiver. These roles can be filled by any organization but are fundamentally descriptions of computer systems that are equipped to satisfy the needs of each role. The general exchange of data that takes place in an RFD transaction can be broken down into three steps as illustrated in Figure 1 : retrieve form request; form delivery; and submission of a completed form to the forms receiver and the forms archiver. To further illustrate the details of how XForms functions within the RFD transaction, we describe our role in two public health use cases: notifiable condition reporting and patient-specific public health alerting. 1. Notifiable Condition Reporting In the notifiable condition reporting scenario we used RFD and XForms to enable the capture of provider initiated notifiable condition case-report data from within an EHR. In this scenario, a patient has tested positive for Salmonella, a notifiable condition. The scenario includes several steps: The patient's medical record on the local EHR system is open while the HCP is explaining the test results to the patient. 2) Knowing Salmonella is a notifiable condition, the HCP clicks a "retrieve case-report form" button within the EHR. 3) The EHR system (form filler) sends a message to a local public health system (form manager) requesting a Salmonella case-report form. The form request includes a structured document called a CCD (Continuity of Care Document). 4) The form manager finds the requested form and pre-populates it with data from the CCD. Most of the patient demographic information is pre-populated on the Salmonella case-report form ( Figure 2 ), but some fields about exposure are left blank because they were not included in the CCD. 5) The pre-populated form is returned to the provider, who is able to view the form, complete the empty fields, and click "submit." 6) The form is submitted as an XML document to public health, the form receiver, and to a backup location, the form archiver. This part of the case reporting scenario demonstrates public health as a form manager, i.e., serving as a repository of available case-report forms, as well as a form receiver, i.e., accepting completed case-report forms from providers. In this demonstration, public health used a case-report management system to import, access, and edit the submitted XML instance data. 2. Patient-Specific Public Health Alerting We also used the RFD profile for a demonstration of patient-specific public health alerting. In this scenario, a patient visits her HCP complaining of diarrhea, vomiting, fever, and headache. The scenario includes several steps: After examining the patient, the HCP enters the patient's data into the electronic patient record. Because the symptoms sound like a possible food borne pathogen, the provider clicks a button within the patient record to "check for public health alerts." 2) This mouse-click initiates a retrieve form request to a public health system serving as the form manager. As in the first scenario, a CCD is attached to the request, providing some of the patient's basic demographic and symptom information. The public health system accepts the request for a form and examines several fields within the CCD: patient age, patient zip code, and conditions and dates from the problems section. Using this information, public health's form manager determines the appropriate form to return to the provider. In this case, based on the zip code and symptoms, the "Salmonella Outbreak Alert" form is delivered because public health officials have been made aware of an ongoing outbreak of Salmonella in the area. 3) This form appears within the EHR and provides all relevant details about the current outbreak with recommendations for laboratory testing and treatment as well as contact information for the local public health department. (Note that if no matching alerts were found, an unobtrusive "no current alerts" message would have been sent). 3. Combining notifiable condition reporting and patient-specific public health alerts Because the alert "form" is not asking for any input, the alerting scenario could end after the HCP views and acknowledges the context-specific alert. In the case of an infectious disease outbreak when the disease is also a notifiable condition, public health may want to not only provide alert information, but also collect case information from the provider. To demonstrate this, we included a button on the alert form to "retrieve a case-report form". Clicking this button initiated another "retrieve form request" to public health, this time for a specific form, the Salmonella case-report form. Again, the CCD data were used to pre-populate the case-report form and the provider needed only to complete the empty fields and click "submit," sending the instance data as an XML file back to public health. Integrating the Healthcare Enterprise Integrating the Healthcare Enterprise (IHE)was formed in 1998 by a group of healthcare and industry professionals with the goal of improving interoperability in healthcare information systems ( 7 ).The organization encourages the adoption of standards by developing, promoting and demonstrating Interoperability Profiles which are implementation guides for incorporating standards and that describe the business rules, specific transactions and standards which can be used in a structured way to address specific clinical and population health use cases. In the public health domain, the standards are typically those identified by the Health Information Technology Standards Panel (HITSP) ( 6 ). Annually, vendors and other participants gather to test interoperability and implementation of profiles during the IHE Connectathon; this testing is followed by the Interoperability Showcase which takes place at the Healthcare Information Management Systems Society annual conference and demonstrates the implementation of standards and IHE Profiles. In addition, the CDC's Public Health Information Network conference has also hosted a smaller-scale, public health focused showcase. The demonstrations use scenarios to tell a story, usually about a patient's experience in the healthcare system. We participated in several population health scenarios and implemented interoperability profiles, including those to support notifiable condition reporting and patient-specific public health alerting ( 47 ). Retrieve Forms for Data Capture and XForms for Public Health The primary profile we used for the demonstration of the reporting and alerting public health use cases was RFD, which uses XForms and enables viewing, pre-population, completion, and submission of forms and form data. The RFD profile specifies how different roles will function during the transaction: forms manager; forms filler; forms receiver; and forms archiver. These roles can be filled by any organization but are fundamentally descriptions of computer systems that are equipped to satisfy the needs of each role. The general exchange of data that takes place in an RFD transaction can be broken down into three steps as illustrated in Figure 1 : retrieve form request; form delivery; and submission of a completed form to the forms receiver and the forms archiver. To further illustrate the details of how XForms functions within the RFD transaction, we describe our role in two public health use cases: notifiable condition reporting and patient-specific public health alerting. 1. Notifiable Condition Reporting In the notifiable condition reporting scenario we used RFD and XForms to enable the capture of provider initiated notifiable condition case-report data from within an EHR. In this scenario, a patient has tested positive for Salmonella, a notifiable condition. The scenario includes several steps: The patient's medical record on the local EHR system is open while the HCP is explaining the test results to the patient. 2) Knowing Salmonella is a notifiable condition, the HCP clicks a "retrieve case-report form" button within the EHR. 3) The EHR system (form filler) sends a message to a local public health system (form manager) requesting a Salmonella case-report form. The form request includes a structured document called a CCD (Continuity of Care Document). 4) The form manager finds the requested form and pre-populates it with data from the CCD. Most of the patient demographic information is pre-populated on the Salmonella case-report form ( Figure 2 ), but some fields about exposure are left blank because they were not included in the CCD. 5) The pre-populated form is returned to the provider, who is able to view the form, complete the empty fields, and click "submit." 6) The form is submitted as an XML document to public health, the form receiver, and to a backup location, the form archiver. This part of the case reporting scenario demonstrates public health as a form manager, i.e., serving as a repository of available case-report forms, as well as a form receiver, i.e., accepting completed case-report forms from providers. In this demonstration, public health used a case-report management system to import, access, and edit the submitted XML instance data. 2. Patient-Specific Public Health Alerting We also used the RFD profile for a demonstration of patient-specific public health alerting. In this scenario, a patient visits her HCP complaining of diarrhea, vomiting, fever, and headache. The scenario includes several steps: After examining the patient, the HCP enters the patient's data into the electronic patient record. Because the symptoms sound like a possible food borne pathogen, the provider clicks a button within the patient record to "check for public health alerts." 2) This mouse-click initiates a retrieve form request to a public health system serving as the form manager. As in the first scenario, a CCD is attached to the request, providing some of the patient's basic demographic and symptom information. The public health system accepts the request for a form and examines several fields within the CCD: patient age, patient zip code, and conditions and dates from the problems section. Using this information, public health's form manager determines the appropriate form to return to the provider. In this case, based on the zip code and symptoms, the "Salmonella Outbreak Alert" form is delivered because public health officials have been made aware of an ongoing outbreak of Salmonella in the area. 3) This form appears within the EHR and provides all relevant details about the current outbreak with recommendations for laboratory testing and treatment as well as contact information for the local public health department. (Note that if no matching alerts were found, an unobtrusive "no current alerts" message would have been sent). 3. Combining notifiable condition reporting and patient-specific public health alerts Because the alert "form" is not asking for any input, the alerting scenario could end after the HCP views and acknowledges the context-specific alert. In the case of an infectious disease outbreak when the disease is also a notifiable condition, public health may want to not only provide alert information, but also collect case information from the provider. To demonstrate this, we included a button on the alert form to "retrieve a case-report form". Clicking this button initiated another "retrieve form request" to public health, this time for a specific form, the Salmonella case-report form. Again, the CCD data were used to pre-populate the case-report form and the provider needed only to complete the empty fields and click "submit," sending the instance data as an XML file back to public health. 1. Notifiable Condition Reporting In the notifiable condition reporting scenario we used RFD and XForms to enable the capture of provider initiated notifiable condition case-report data from within an EHR. In this scenario, a patient has tested positive for Salmonella, a notifiable condition. The scenario includes several steps: The patient's medical record on the local EHR system is open while the HCP is explaining the test results to the patient. 2) Knowing Salmonella is a notifiable condition, the HCP clicks a "retrieve case-report form" button within the EHR. 3) The EHR system (form filler) sends a message to a local public health system (form manager) requesting a Salmonella case-report form. The form request includes a structured document called a CCD (Continuity of Care Document). 4) The form manager finds the requested form and pre-populates it with data from the CCD. Most of the patient demographic information is pre-populated on the Salmonella case-report form ( Figure 2 ), but some fields about exposure are left blank because they were not included in the CCD. 5) The pre-populated form is returned to the provider, who is able to view the form, complete the empty fields, and click "submit." 6) The form is submitted as an XML document to public health, the form receiver, and to a backup location, the form archiver. This part of the case reporting scenario demonstrates public health as a form manager, i.e., serving as a repository of available case-report forms, as well as a form receiver, i.e., accepting completed case-report forms from providers. In this demonstration, public health used a case-report management system to import, access, and edit the submitted XML instance data. 2. Patient-Specific Public Health Alerting We also used the RFD profile for a demonstration of patient-specific public health alerting. In this scenario, a patient visits her HCP complaining of diarrhea, vomiting, fever, and headache. The scenario includes several steps: After examining the patient, the HCP enters the patient's data into the electronic patient record. Because the symptoms sound like a possible food borne pathogen, the provider clicks a button within the patient record to "check for public health alerts." 2) This mouse-click initiates a retrieve form request to a public health system serving as the form manager. As in the first scenario, a CCD is attached to the request, providing some of the patient's basic demographic and symptom information. The public health system accepts the request for a form and examines several fields within the CCD: patient age, patient zip code, and conditions and dates from the problems section. Using this information, public health's form manager determines the appropriate form to return to the provider. In this case, based on the zip code and symptoms, the "Salmonella Outbreak Alert" form is delivered because public health officials have been made aware of an ongoing outbreak of Salmonella in the area. 3) This form appears within the EHR and provides all relevant details about the current outbreak with recommendations for laboratory testing and treatment as well as contact information for the local public health department. (Note that if no matching alerts were found, an unobtrusive "no current alerts" message would have been sent). 3. Combining notifiable condition reporting and patient-specific public health alerts Because the alert "form" is not asking for any input, the alerting scenario could end after the HCP views and acknowledges the context-specific alert. In the case of an infectious disease outbreak when the disease is also a notifiable condition, public health may want to not only provide alert information, but also collect case information from the provider. To demonstrate this, we included a button on the alert form to "retrieve a case-report form". Clicking this button initiated another "retrieve form request" to public health, this time for a specific form, the Salmonella case-report form. Again, the CCD data were used to pre-populate the case-report form and the provider needed only to complete the empty fields and click "submit," sending the instance data as an XML file back to public health. RESULTS Through this work we have demonstrated that notifiable condition reporting and patient-specific public health alerting can be accomplished with a set of technical profiles that use nationally identified standards. The flexibility of the RFD profile was essential in implementing these two use cases. We found that the versatility of both RFD and XForms were beneficial, but significant challenges arose with use of XForms technology in RFD. Retrieve Forms for Data Capture The RFD interoperability profile provides a method for collecting data from within one system while meeting the requirements of an external system and enables interoperability with other systems that have implemented RFD. We provided the URL of our form manager to participating vendors and this URL served as the endpoint for the vendor form requests. Although CCD is an optional component of RFD, the ability of the form manager to use the CCD was an important part of the success of these demonstrations. Including CCD data in the form request allows for both the pre-population of case-report forms and tailoring public health alerts to a particular patient. Most EHR vendors participating in these demonstrations have the ability to create CDA documents, including CCDs, but without this capability, much of the benefit of using RFD is lost. XForms At the time of this publication, XForms are specified within the RFD profile. XForms were included in this profile because of their ability to negotiate issues such as partial completion of forms, series of forms, and forms filled out across different user sessions. We benefited from the ability of XForms to support series of forms when we combined the alerting and case reporting use cases. We also found some of XForms' fundamental traits to be useful in our implementation. For our project the most important feature of XForms was the use of XML to define form data. The use of XML documents to not only build a form, but also to store and transport form instance data, combined with the near universality of XML, made this one of the key benefits to using XForms. Another major benefit is the ease of use of advanced controls available in XForms. One control available in XForms is the range-selection control, adding a volume-control like slider to a form for ease of user data input. Range selection only recently became available for HTML forms. The reduction in the need for scripting to add logic to form controls also reduced development time for some components of the work, but the barriers we encountered were significant as was the time spent on XForms-related problems. Challenges of XForms Implementation We experienced significant challenges related to the development and implementation of XForms. First, two of the most common browsers, Internet Explorer and Mozilla Firefox, do not include native support for XForms. They require plug-ins in order to display the forms, and, when the same form is displayed in different browsers, different issues arise. In some cases, an XForms document displays properly in one browser but is not recognized as a form in the other enabled browser. This issue necessitates scripting within the forms to identify the browser and specify conditional code. Although it was not an issue for this demonstration, form developers need to be mindful of the complexities of plug-in installation when browsers are used by forms fillers. Second, although several XForms editors were tested, none provided adequate support and hand-coding was necessary for all of our form development. Without the help of an editor, and with little online support, the coding of forms was significantly more time-consuming than developing HTML forms. Third, though XML is ubiquitous in computing today, XForms is not and vendors are often unwilling to integrate support for XForms. This limited the number of partners for our demonstration, and could have more significant implications for use in practice. Overall, our demonstrations were successful. The RFD profile, including the XForms standard, was implemented by our team and by participating vendors. Use of XForms had benefits, including its use of XML, availability of advanced form controls and reduction in necessary scripting for form behavior. However, our experience developing and using XForms, and the challenges we encountered, such as compatibility issues and time-consuming development, indicate that this technology may not yet be mature enough for widespread use for public health form development. Retrieve Forms for Data Capture The RFD interoperability profile provides a method for collecting data from within one system while meeting the requirements of an external system and enables interoperability with other systems that have implemented RFD. We provided the URL of our form manager to participating vendors and this URL served as the endpoint for the vendor form requests. Although CCD is an optional component of RFD, the ability of the form manager to use the CCD was an important part of the success of these demonstrations. Including CCD data in the form request allows for both the pre-population of case-report forms and tailoring public health alerts to a particular patient. Most EHR vendors participating in these demonstrations have the ability to create CDA documents, including CCDs, but without this capability, much of the benefit of using RFD is lost. XForms At the time of this publication, XForms are specified within the RFD profile. XForms were included in this profile because of their ability to negotiate issues such as partial completion of forms, series of forms, and forms filled out across different user sessions. We benefited from the ability of XForms to support series of forms when we combined the alerting and case reporting use cases. We also found some of XForms' fundamental traits to be useful in our implementation. For our project the most important feature of XForms was the use of XML to define form data. The use of XML documents to not only build a form, but also to store and transport form instance data, combined with the near universality of XML, made this one of the key benefits to using XForms. Another major benefit is the ease of use of advanced controls available in XForms. One control available in XForms is the range-selection control, adding a volume-control like slider to a form for ease of user data input. Range selection only recently became available for HTML forms. The reduction in the need for scripting to add logic to form controls also reduced development time for some components of the work, but the barriers we encountered were significant as was the time spent on XForms-related problems. Challenges of XForms Implementation We experienced significant challenges related to the development and implementation of XForms. First, two of the most common browsers, Internet Explorer and Mozilla Firefox, do not include native support for XForms. They require plug-ins in order to display the forms, and, when the same form is displayed in different browsers, different issues arise. In some cases, an XForms document displays properly in one browser but is not recognized as a form in the other enabled browser. This issue necessitates scripting within the forms to identify the browser and specify conditional code. Although it was not an issue for this demonstration, form developers need to be mindful of the complexities of plug-in installation when browsers are used by forms fillers. Second, although several XForms editors were tested, none provided adequate support and hand-coding was necessary for all of our form development. Without the help of an editor, and with little online support, the coding of forms was significantly more time-consuming than developing HTML forms. Third, though XML is ubiquitous in computing today, XForms is not and vendors are often unwilling to integrate support for XForms. This limited the number of partners for our demonstration, and could have more significant implications for use in practice. Overall, our demonstrations were successful. The RFD profile, including the XForms standard, was implemented by our team and by participating vendors. Use of XForms had benefits, including its use of XML, availability of advanced form controls and reduction in necessary scripting for form behavior. However, our experience developing and using XForms, and the challenges we encountered, such as compatibility issues and time-consuming development, indicate that this technology may not yet be mature enough for widespread use for public health form development. DISCUSSION We have identified and demonstrated technology that enables two public health functions, case-reporting and patient-specific public health alerting, where communication between public health and provider is essential. Using electronic information exchange from provider to public health for case-reporting, and from public health to provider for alerting, the RFD profile and the XForms standard were sufficient to meet the simplified set of user needs represented in our demonstration scenarios. The use of RFD and XForms not only helped integrate case-reporting into the provider's workflow, but it also leveraged a standard XML representation of patient data to initialize the case-report form, thus demonstrating the potential to reduce the burden of reporting for the provider. The use of this technology also has the potential to positively influence timeliness and completeness of reported data. Implementations in practice settings are called for in order to quantify these effects. Implementing a system such as this for case-reporting would be a significant change to the way many providers currently go about notifiable condition reporting activities. If EHR-integrated, provider-initiated case-reporting is to be successful, provider and public health practitioner workflow should be further studied, and used to inform system design. Our demonstration of patient-specific public health alerting is, in practice, more similar to the way clinical decision support is currently implemented than the way most public health alerts are distributed. Today, public health alerting is rarely context or patient-specific; the alerts we demonstrated represent a significant change to current practice. Before patient-specific public health alerts are implemented in the field, it will be important to assess the information needs, preferences, resources and capacity of both public health and provider organizations. Both of our scenarios used provider initiated events, i.e., public health's action was triggered by an event within the provider's system, in both cases a mouse-click. This trigger could be any type of event; instead of the provider asking for the case-repot form, the form's appearance might be triggered by code running in the background to check patient characteristics. Similarly, instead of the provider asking if there are any relevant public health alerts, these alerts could appear before a patient's record is closed or after a problem list is updated. Though the ideal format for this type of information exchange has not yet been established, enabling bi-directional information flow between providers and public health is becoming increasingly important. Interoperability between provider systems and public health systems is emphasized in parts of the US Health Information Technology for Economic and Clinical Health Act of 2009. Under this act, Medicare and Medicaid will provide financial incentives for the "meaningful use" of EHRs. Published rules indicate that communication of public health information from providers to public health, as well as patient-specific decision support services will be among the criteria used to certify and assess EHR systems ( 48 ). Standards are one important part of achieving interoperable public health and clinical systems. Determining which of the existing standards will be adopted is a challenging task. By participating in IHE's Interoperability Showcase we've engaged with one of the largest groups of vendors actively testing and demonstrating specific use cases and standards. We believe that our participation has helped encourage a more prominent role for public health in the scenario development process, and has increased our awareness of some of the benefits and drawbacks of use of RFD and XForms. XForms technology is still immature and its trajectory is uncertain. Recently, the development of HTML5 has led to questions about the future of web forms. Though HTML5, the most recent update to HTML, offers simpler support for more complex multimedia elements, it's most important overlap with XForms is in the area of more advanced validation and controls. HTML5 does not replace XForms, in fact some XForms implementations use HTML and will be able to take advantage of some of the new HTML5 features. We believe that as XForms or a similar standard is used and tested, as support becomes more widely available, and as we gain a better understanding of provider and public health preferences related to these functions, it is likely that tools using forms standards to facilitate bi-directional communication between EHR systems and public health information systems will become more practical. LIMITATIONS Our findings are limited in several ways. First, the environment in which we implemented XForms was unique in that the collaborating EHR vendors are, as evidenced by their participation in the Interoperability Showcase, early-adopters, and therefore this sample may not be representative of all vendors. Future work should engage with vendors who do not participate in the Interoperability Showcase or similar venues. Despite engaging with the public health practice community regarding the impact of implementation of XForms on work practice, we may have encountered different challenges if this technology was implemented in a state or local health department. Future work needs to explore the barriers and facilitators to XForms implementation and the impact of this technology on current HCP and public health practices. It is well-known that health departments across the US have heterogeneous work practices, thus potentially limiting the generalizability of our conclusions. Because this work took place as part of a demonstration project, we were not able to fully explore user information needs related to bi-directional communication, workflow in the HCP office and public health departments, or the suitability of the technology for other use cases, for instance other notifiable conditions. We suggest that future work regarding XForms and other data capture technology for public health reporting and alerting continue to explore the use of standards and nationally recognized profiles, but also explores the information needs and workflow of the users in public health practice and the healthcare providers targeted. Lastly, our report is limited by the fast pace of technology development and adoption. New tools, e.g., HTML5, became available after our initial demonstration. A side-by-side comparison of XForms and some of the newer tools would be a useful artifact. and a comparison between these tools and the XForms technology described here would have been useful. CONCLUSION Although XForms present significant development and implementation challenges, we believe the benefits of using a standardized method for representing form content, presentation, and logic is an important step for public health. We suggest that health departments with some system development capacity consider exploring the use of XForms or similar technologies to use and re-use XML documents for notifiable condition reporting and patient-specific public health alerting. Early projects making use of XForms should, if possible, measure the impact of the technology on timeliness and completeness of reporting and on effectiveness of context-specific alerting. Most resource-constrained organizations will benefit from continued migration of data toward an XML model. However, we suggest these organizations wait to implement XForms until the technology is more mature, tools for development have been proven, and the capacity within local EHRs has reached a level that will make the investment worthwhile.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC139364/

Our own petards

Several international treaties on chemical and biological weapons expressly prohibit the development, production, stockpiling or acquisition of such weapons. All too often, when troops go into battle, they are attacked by weapons we ourselves conceived and manufactured.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6806190/

SERS-Based Immunoassays for the Detection of Botulinum Toxins A and B Using Magnetic Beads

Rapid and sensitive detection of botulinum neurotoxins (BoNTs) is important for immediate treatment with proper antitoxins. However, it is difficult to detect BoNTs at the acute phase of infection, owing to its rarity and ambiguous symptoms. To resolve this problem, we developed a surface-enhanced Raman scattering (SERS)-based immunoassay technique for the rapid and sensitive detection of BoNTs. Magnetic beads and SERS nanotags as capture substrates and detection probes, respectively, and Nile Blue A (NBA) and malachite green isothiocyanate (MGITC) as Raman reporter molecules were used for the detection of two different types of BoNTs (types A and B), respectively. The corresponding limits of detection (LODs) were determined as 5.7 ng/mL (type A) and 1.3 ng/mL (type B). Total assay time, including that for immunoreaction, washing, and detection, was less than 2 h. 1. Introduction Botulinum neurotoxins (BoNTs) are regarded as one of the most serious high-risk biological agents used in bioterrorism. Considering their high toxicity (LD 50 ∼1 ng/kg), ease of handling, and low price, BoNTs are listed as "Category A" bio-threat agents together with anthrax, plague, smallpox, tularemia, and viral hemorrhagic fevers by the US Centers for Disease Control and Prevention (CDCP) [ 1 , 2 , 3 ]. BoNTs have also been designated as lethal infectious agents for bio-terror by the Korea Centers for Disease Control and Prevention (KCDC). Seven different BoNT serotypes (serotype A–G) are known, of which the A, B, E, and F types are considered harmful to humans [ 4 ]. Intoxication with these BoNTs causes flaccid paralysis owing to the inhibition of neuromuscular signal transmission, which is identical for all serotypes. Muscle paralysis starts from the face, spreads to the whole body, and in severe cases, leads to death due to respiratory paralysis [ 5 , 6 ]. Immediate treatment with a specific antitoxin for a given toxin type is the only way to relieve the symptoms before the toxin enters nerve terminals [ 7 , 8 ]. Therefore, it is urgent to develop a rapid and accurate detection technique for toxic serotypes of BoNTs. The gold standard method for the detection and identification of BoNTs is the mouse toxicity and neutralization bioassay (e.g., mouse bioassay), which is the only FDA-approved method to confirm the presence of active BoNTs [ 9 , 10 ]. However, this method has several drawbacks, including labor intensiveness, cost ineffectiveness, animal use, and time consumption (longer than four days). To resolve these problems, herein, we developed a surface-enhanced Raman scattering (SERS)-based immunoassay technique using magnetic beads [ 11 , 12 , 13 , 14 , 15 ]. The SERS-based bioassay technique has recently received great attention, owing to its high sensitivity and multiplex detection capability. Raman active sites called "hot junctions" show promise in their ability to overcome the low sensitivity problem associated with conventional Raman or fluorescence spectroscopy [ 16 , 17 , 18 , 19 , 20 ]. Herein, magnetic beads and gold nanoparticles (AuNPs) were used to capture antibody-supporting materials and detect antibody-conjugated sensing probes, respectively. This magnetic bead-based assay offers several advantages over conventional SERS assays using two-dimensional substrates [ 21 , 22 , 23 ]. First, the loading density of capture antibodies could be improved, as the three-dimensional magnetic beads have larger surface-to-volume ratio than any two-dimensional surface. Second, the assay does not require an extended incubation time because the fast molecular diffusion near three-dimensional beads facilitates faster kinetics of antibody–antigen assays. Finally, more reproducible detection is possible, as SERS signals are measured for the average ensembles of AuNPs in solution phase. In the present study, the feasibility of our SERS-based magnetic immunoassay was investigated for the rapid and sensitive detection of BoNT types A and B. The limit of detection (LOD), sensitivity, and assay time were compared with those of an enzyme-linked immunosorbent assay (ELISA) for validation. 2. Materials and Methods 2.1. Materials Gold (III) chloride trihydrate (HAuCl 4 ·3H 2 O), tri-sodium citrate (Na 3 -citrate), 1-ethyl-3-(3-[dimethylamino]propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), thiol-PEG-COOH (HS-PEG-COOH, MW∼3500), bovine serum albumin (BSA), Nile blue A (NBA), 3,3',5,5'-tetramethylbenzidine (TMB), liquid substrate system for ELISA, horseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonal antibody, and HRP-conjugated goat anti-mouse polyclonal antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Malachite green isothiocyanate (MGITC), phosphate-buffered saline (PBS) (10×, pH 7.4), and carboxylic acid-activated magnetic beads (Dynabeads MyOne TM ) were obtained from Invitrogen (Eugene, OR, USA), whereas HRP conjugation kit (ab102890) was supplied by Abcam (Cambridge, UK). We procured 3,3',5,5'-tetramethylbenzidine (TMB) buffer from GenDEPOT (Katy, TX, USA) and inactivated botulinum toxins A and B from the Korea Center for Disease Control and Prevention (KCDCP). Anti-botulinum toxin antibody sets were also provided by KCDCP. Commercial carboxylic acid-conjugated magnetic beads (Dynabeads ® MyOne) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ultrapure water (18 MΩ·cm −1 ) used in this work was obtained from Milli-Q water purification system (Billerica, MA, USA). 2.2. ELISA Test Sandwich ELISA was performed to test the antibody-binding capability of BoNTs. For BoNT/A assay, monoclonal capture antibodies (100 μL, 0.5 μg/mL) in Na 2 CO 3 -NaHCO 3 buffer (pH 9.6) were immobilized on the surface of a 96-well plate and incubated overnight at 4 °C. The wells were blocked with 200 μL of PBS buffer (containing 1% BSA) to reduce non-specific binding. After 2 h, BoNT/A (100 μL) in the range of 0–1 μg/mL was added to different wells and allowed to react for 2 h. Polyclonal detection antibodies and HRP-linked secondary antibodies were sequentially added at an interval of 2 h. After incubation for another 2 h, TMB solution was added to induce TMB-HRP enzymatic reaction. PBST containing 0.05% (v/v) Tween-20 (200 μL, thrice) was used for washing. TMB stop buffer was added to terminate the reaction and absorbance was measured at 450 nm wavelength. For BoNT/B assay, HRP-conjugated polyclonal detection antibodies were used instead of secondary antibodies to avoid any cross-reaction between capture antibodies and secondary antibodies. An HRP conjugation kit was used to attach HRP to detection polyclonal antibodies, as per the manufacturer's instructions. Other steps were the same as those for BoNT/A. 2.3. Preparation of Antibody-Conjugated SERS Nanotags AuNPs were prepared as previously reported with the seeded-growth method [ 24 ]. Briefly, 75 mL of 2.2 mM sodium citrate solution was heated to its boiling point under vigorous stirring and mixed with 0.5 mL of 25 mM chloroauric acid (HAuCl 4 ) upon boiling. The change in the color of the solution from light yellow to bluish gray and then to soft pink was noted in 15 min. The resulting gold seed solution was cooled to 90 °C and sequentially treated with 0.5 mL of 60 mM sodium citrate and 0.5 mL of 25 mM HAuCl 4 12 times at an interval of 2 min. The color of the solution finally changed from pink to deep red. The solution was stirred for 30 min at 90 °C and cooled to room temperature. The shape and size distribution of AuNPs were characterized with dynamic light scattering (DLS) and transmission electron microscopy (TEM). Antibody-conjugated SERS nanotags were also prepared, as previously reported. Two Raman reporters, 2.0 μL of 10 −5 M MGITC and 1.5 μL of 10 −4 M NBA, were added to 1.0 mL of AuNP solution and allowed to react for 30 min under constant stirring (500 rpm). In total, 40 μL of 12.5 μM HS-PEG-COOH linkers were added to each solution to facilitate their immobilization on the surface of AuNPs via Au-S bonds. After stirring for 3 h at room temperature, the PEGylated AuNPs were washed twice with DI water. To activate -COOH terminal groups on the surface of AuNPs, 4 μL of 0.5 mM EDC and 4 μL of 0.5 mM NHS were sequentially added. After 30 min, excess EDC/NHS was washed twice with DI water and the NHS-activated AuNPs were reacted with 30 μL of 0.1 mg/mL detection antibodies overnight at 4 °C. Approximately 100 μL of 1% (w/v) BSA aqueous solution was added to block any unbound sites on the surface of AuNPs. The mixture was shaken for additional 30 min and centrifuged at 5000 rcf for 10 min to remove unbound proteins. After discarding the supernatant, the pellets were re-dispersed in PBS buffer. 2.4. Preparation of Antibody-Conjugated Magnetic Beads The magnetic property of commercial beads is superparamagnetic, and the average diameter size is estimated to be 1 μm. Core material is composed of Fe 3 O 4 and the surface was homogeneously coated with highly cross-linked polystyrene and hydrophilic layer of glycidyl ether. Their surface was conjugated with carboxylic acids for antibody immobilization. In brief, 200 μL of 0.5 mg/mL carboxylated magnetic beads were prepared using 15 mM MES buffer (pH 6). After washing thrice with MES buffer, the solution was incubated with 2.5 μL of 0.1 M EDC and 2.5 μL of 0.1 M NHS for 30 min, followed by washing of the beads thrice with MES buffer. The beads were treated with 5 μL of 1 mg/mL mouse anti-BoNT monoclonal antibody overnight at 4 °C with continuous shaking. After washing three times with PBS buffer, the reaction mixture was treated with 20 μL of 1% (w/v) BSA aqueous solution for 30 min at room temperature. Unreacted reagents were removed by washing the beads thrice with PBS. The final product was stored in PBS at 4 °C for further use. 2.5. SERS-based Immunoassay of BoNT/A and BoNT/B Parallel sandwich immunoassays for BoNT/A and BoNT/B were performed with spiked samples at eight different concentrations. First, 40 μL of SERS nanotags and 20 μL of BoNT-spiked samples were mixed and allowed to react under constant stirring. After 30 min, 20 μL of antibody-conjugated magnetic beads were added and allowed to react for 1 h. The mixture was washed thrice with PBST and the immunocomplexes were resuspended in PBS and transferred to a capillary tube for Raman measurements. 2.6. Instrumentation DLS measurement was performed with a Nano-ZS90 instrument (Malvern, UK) and TEM images were acquired using a JEOL JEM 2100F instrument at an accelerating voltage of 200 kV. Size distribution of AuNPs was calculated using ImageJ software. ELISA was performed using a microplate reader (Synergy H1 Hybrid Multi-Mode Reader, BioTek, Winooski, VT, USA) equipped with a 96-well plate. Raman spectra were measured with a Renishaw inVia Raman microscope system (Renishaw, New Mills, UK). A He-Ne laser at 633 nm was used as the excitation source with a power of 20 mW. The Rayleigh line was removed using an edge filter located in the collection path. Raman scattering signal was collected using a charge-coupled device (CCD) camera at a spectral resolution of 1 cm −1 . BoNT immunocomplexes from microtube were transferred to a capillary tube, and their Raman scattering signals were measured by focusing a laser spot on the tube using a 20× objective lens. Baseline correction for each Raman spectrum was performed using Renishaw WIRE 4.0 software. 2.1. Materials Gold (III) chloride trihydrate (HAuCl 4 ·3H 2 O), tri-sodium citrate (Na 3 -citrate), 1-ethyl-3-(3-[dimethylamino]propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), thiol-PEG-COOH (HS-PEG-COOH, MW∼3500), bovine serum albumin (BSA), Nile blue A (NBA), 3,3',5,5'-tetramethylbenzidine (TMB), liquid substrate system for ELISA, horseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonal antibody, and HRP-conjugated goat anti-mouse polyclonal antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Malachite green isothiocyanate (MGITC), phosphate-buffered saline (PBS) (10×, pH 7.4), and carboxylic acid-activated magnetic beads (Dynabeads MyOne TM ) were obtained from Invitrogen (Eugene, OR, USA), whereas HRP conjugation kit (ab102890) was supplied by Abcam (Cambridge, UK). We procured 3,3',5,5'-tetramethylbenzidine (TMB) buffer from GenDEPOT (Katy, TX, USA) and inactivated botulinum toxins A and B from the Korea Center for Disease Control and Prevention (KCDCP). Anti-botulinum toxin antibody sets were also provided by KCDCP. Commercial carboxylic acid-conjugated magnetic beads (Dynabeads ® MyOne) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ultrapure water (18 MΩ·cm −1 ) used in this work was obtained from Milli-Q water purification system (Billerica, MA, USA). 2.2. ELISA Test Sandwich ELISA was performed to test the antibody-binding capability of BoNTs. For BoNT/A assay, monoclonal capture antibodies (100 μL, 0.5 μg/mL) in Na 2 CO 3 -NaHCO 3 buffer (pH 9.6) were immobilized on the surface of a 96-well plate and incubated overnight at 4 °C. The wells were blocked with 200 μL of PBS buffer (containing 1% BSA) to reduce non-specific binding. After 2 h, BoNT/A (100 μL) in the range of 0–1 μg/mL was added to different wells and allowed to react for 2 h. Polyclonal detection antibodies and HRP-linked secondary antibodies were sequentially added at an interval of 2 h. After incubation for another 2 h, TMB solution was added to induce TMB-HRP enzymatic reaction. PBST containing 0.05% (v/v) Tween-20 (200 μL, thrice) was used for washing. TMB stop buffer was added to terminate the reaction and absorbance was measured at 450 nm wavelength. For BoNT/B assay, HRP-conjugated polyclonal detection antibodies were used instead of secondary antibodies to avoid any cross-reaction between capture antibodies and secondary antibodies. An HRP conjugation kit was used to attach HRP to detection polyclonal antibodies, as per the manufacturer's instructions. Other steps were the same as those for BoNT/A. 2.3. Preparation of Antibody-Conjugated SERS Nanotags AuNPs were prepared as previously reported with the seeded-growth method [ 24 ]. Briefly, 75 mL of 2.2 mM sodium citrate solution was heated to its boiling point under vigorous stirring and mixed with 0.5 mL of 25 mM chloroauric acid (HAuCl 4 ) upon boiling. The change in the color of the solution from light yellow to bluish gray and then to soft pink was noted in 15 min. The resulting gold seed solution was cooled to 90 °C and sequentially treated with 0.5 mL of 60 mM sodium citrate and 0.5 mL of 25 mM HAuCl 4 12 times at an interval of 2 min. The color of the solution finally changed from pink to deep red. The solution was stirred for 30 min at 90 °C and cooled to room temperature. The shape and size distribution of AuNPs were characterized with dynamic light scattering (DLS) and transmission electron microscopy (TEM). Antibody-conjugated SERS nanotags were also prepared, as previously reported. Two Raman reporters, 2.0 μL of 10 −5 M MGITC and 1.5 μL of 10 −4 M NBA, were added to 1.0 mL of AuNP solution and allowed to react for 30 min under constant stirring (500 rpm). In total, 40 μL of 12.5 μM HS-PEG-COOH linkers were added to each solution to facilitate their immobilization on the surface of AuNPs via Au-S bonds. After stirring for 3 h at room temperature, the PEGylated AuNPs were washed twice with DI water. To activate -COOH terminal groups on the surface of AuNPs, 4 μL of 0.5 mM EDC and 4 μL of 0.5 mM NHS were sequentially added. After 30 min, excess EDC/NHS was washed twice with DI water and the NHS-activated AuNPs were reacted with 30 μL of 0.1 mg/mL detection antibodies overnight at 4 °C. Approximately 100 μL of 1% (w/v) BSA aqueous solution was added to block any unbound sites on the surface of AuNPs. The mixture was shaken for additional 30 min and centrifuged at 5000 rcf for 10 min to remove unbound proteins. After discarding the supernatant, the pellets were re-dispersed in PBS buffer. 2.4. Preparation of Antibody-Conjugated Magnetic Beads The magnetic property of commercial beads is superparamagnetic, and the average diameter size is estimated to be 1 μm. Core material is composed of Fe 3 O 4 and the surface was homogeneously coated with highly cross-linked polystyrene and hydrophilic layer of glycidyl ether. Their surface was conjugated with carboxylic acids for antibody immobilization. In brief, 200 μL of 0.5 mg/mL carboxylated magnetic beads were prepared using 15 mM MES buffer (pH 6). After washing thrice with MES buffer, the solution was incubated with 2.5 μL of 0.1 M EDC and 2.5 μL of 0.1 M NHS for 30 min, followed by washing of the beads thrice with MES buffer. The beads were treated with 5 μL of 1 mg/mL mouse anti-BoNT monoclonal antibody overnight at 4 °C with continuous shaking. After washing three times with PBS buffer, the reaction mixture was treated with 20 μL of 1% (w/v) BSA aqueous solution for 30 min at room temperature. Unreacted reagents were removed by washing the beads thrice with PBS. The final product was stored in PBS at 4 °C for further use. 2.5. SERS-based Immunoassay of BoNT/A and BoNT/B Parallel sandwich immunoassays for BoNT/A and BoNT/B were performed with spiked samples at eight different concentrations. First, 40 μL of SERS nanotags and 20 μL of BoNT-spiked samples were mixed and allowed to react under constant stirring. After 30 min, 20 μL of antibody-conjugated magnetic beads were added and allowed to react for 1 h. The mixture was washed thrice with PBST and the immunocomplexes were resuspended in PBS and transferred to a capillary tube for Raman measurements. 2.6. Instrumentation DLS measurement was performed with a Nano-ZS90 instrument (Malvern, UK) and TEM images were acquired using a JEOL JEM 2100F instrument at an accelerating voltage of 200 kV. Size distribution of AuNPs was calculated using ImageJ software. ELISA was performed using a microplate reader (Synergy H1 Hybrid Multi-Mode Reader, BioTek, Winooski, VT, USA) equipped with a 96-well plate. Raman spectra were measured with a Renishaw inVia Raman microscope system (Renishaw, New Mills, UK). A He-Ne laser at 633 nm was used as the excitation source with a power of 20 mW. The Rayleigh line was removed using an edge filter located in the collection path. Raman scattering signal was collected using a charge-coupled device (CCD) camera at a spectral resolution of 1 cm −1 . BoNT immunocomplexes from microtube were transferred to a capillary tube, and their Raman scattering signals were measured by focusing a laser spot on the tube using a 20× objective lens. Baseline correction for each Raman spectrum was performed using Renishaw WIRE 4.0 software. 3. Results and Discussion Figure S1a shows the TEM image of AuNPs synthesized with the seeded-growth method. The average diameter was estimated to be 45 ± 5 nm. Figure S1b shows the size distribution of AuNPs, as determined with DLS measurements. The error bars indicate standard deviations from three measurements. Figure 1 demonstrates the process for the preparation of two different types of detection antibody-conjugated SERS nanotags. In Figure 1 a, two Raman reporter molecules (MGITC and NBA) were adsorbed on the surfaces of AuNPs. BoNT antibodies (types A and B) were subsequently immobilized on the surfaces of AuNPs using HS-PEG-COOH. Optimization of the amount of HS-PEG-COOH was important to retain the stability of SERS nanotags. To determine the optimum concentration, six different concentrations of HS-PEG-COOH were tested with the colloidal solution containing AuNPs. After centrifugation, the pellet was re-suspended in PBS and the aggregation properties were investigated. Figure 2 a shows the UV/Vis spectra of PEGylated AuNPs for various concentrations of HS-PEG-COOH in the range of 63 nM to 2.0 μM. Figure 2 b shows the corresponding images of PEGylated AuNPs. As shown in these figures, aggregation was observed from a concentration of 250 nM HS-PEG-COOH along with a change in the color of the solution to deep purple at concentrations lower than 125 nM. In the presence of low concentration of HS-PEG-COOH, the negative charge of citrate ions on the surface of AuNPs is neutralized by the salts in PBS. Therefore, AuNPs aggregate; however, higher concentrations of HS-PEG-COOH stabilize the surface of AuNPs. Hence, the optimum concentration of HS-PEG-COOH for the stabilization of SERS nanotags was determined to be 0.50 μM. Detection BoNT/A and BoNT/B antibodies were conjugated on AuNPs using EDC/NHS coupling reactions. No significant change of Raman signal intensities was observed after antibody conjugation. Capture antibody-conjugated magnetic beads were prepared with a similar method. Carboxylic acid-functionalized magnetic beads were activated with EDN/NHS and immobilized with capture BoNT antibodies. UV/Vis spectral data in Figure 1 b show that the surface plasmon bands for both BoNT/A and BoNT/B were slightly shifted from 527 nm to 531 nm upon immobilization of the corresponding antibodies on the surfaces. These spectral changes confirm that both BoNT antibodies were successfully immobilized on the surfaces of AuNPs. Raman spectra of BoNT/A (i) and BoNT/B (ii) SERS nanotags in Figure 1 c also demonstrate the successful adsorption of the Raman reporter molecules on the surfaces of AuNPs. Figure S2a shows the sequential process for the preparation of capture BoNT antibody-conjugated magnetic beads. Carboxylic acid-functionalized magnetic beads were used for the fabrication of capture substrates. The surfaces of magnetic beads were activated with the NHS/EDC coupling reaction, and then immobilized with BoNT antibodies. The remaining sites were treated with BSA to prevent any nonspecific binding. The conjugation was determined with the enzyme-catalyzed reactions. Anti-mouse IgG HRP conjugates were incubated with bare and antibody-conjugated magnetic beads. The addition of TMB and TMB stop buffer to each well resulted in a change in the color of antibody-conjugated magnetic beads from colorless to yellow. Enzyme immunoassays frequently incorporate the use of HRP as the enzyme label. This enzyme usually catalyzes the oxidation of a chromogen which can be quantified after termination of the enzyme reaction. A chromogen widely used for this purpose is TMB. The absorbance at 450 nm was measured to quantify the target BoNT toxins. However, no color change was detected for bare magnetic beads. Figure S2b demonstrates the difference in the relative absorption intensities at 450 nm for BoNT/A (i) and BoNT/B (ii) antibody-conjugated magnetic beads. Herein, the histogram for control indicates the absorption intensity of bare magnetic beads. Figure 3 shows the schematic illustration of the sequential process of SERS-based immunoassays using magnetic beads and SERS nanotags. Target BoNT toxins were mixed with detection antibody-conjugated SERS nanotags in a microtube, and then captured by the capture antibody-conjugated magnetic beads through antigen-antibody immunoreactions. We used a magnetic bar to collect magnetic sandwich immunocomplexes on the wall of the microtube, and then washed the supernatant solution with PBST three times using a micropipette. The magnetic immunocomplexes were re-dispersed in PBS and the solution was transferred into a capillary tube for the analysis of Raman signals. In the presence of target BoNT toxins, strong Raman signals were observed; however, Raman signals were relatively weak in the absence of BoNT toxins. In the present work, the strongest Raman peak intensities at 1614 cm −1 (MGITC) and 1639 cm −1 (NBA) were used for the quantitative evaluation of BoNT/A and BoNT/B toxins, respectively. Figure 4 a,b shows the Raman spectra for various concentrations of BoNT/A and BoNT/B, respectively. Their concentrations varied from 0 ng/mL to 1.0 μg/mLml. In the absence of BoNTs, weak Raman signals were observed owing to the formation of few immunocomplexes in the microtube; hence, most SERS nanotags in the supernatant solution were removed during washing. An increase in BoNT concentration, however, led to the formation of more immunocomplexes, as evident from the corresponding increase in Raman signal intensities. Figure 4 c,d demonstrates the corresponding calibration curves for BoNT/A and BoNT/B, as constructed from the intensity variations at 1614 cm −1 (MGITC) and 1639 cm −1 (NBA), respectively. The error bars indicate standard deviations from the measurements for three immunocomplex replicas. The LODs, as determined with the SERS-based immunoassay method, were 5.7 ng/mL (R 2 = 0.999) and 1.3 ng/mL (R 2 = 0.999) for BoNT/A and BoNT/B, respectively. ELISAs were also performed for both BoNT toxins to evaluate the SERS-based immunoassays using magnetic beads and SERS nanotags. Figure 5 shows the ELISA standard curves and images of 96-well plates for different concentrations of BoNT/A (a) and BoNT/B (b). The dynamic ranges for both BoNT toxins varied from 0 ng/mL to 1.0 μg/mL. The increase in BoNT concentration led to a change in the color from colorless to deep yellow. The related LODs determined by ELISA were 0.5 ng/mL (R 2 = 0.999) and 5.0 ng/mL (R 2 = 0.999) for BoNT/A and BoNT/B, respectively. 4. Conclusions In the present study, we developed a SERS immunoassay technique for the rapid and sensitive detection of BoNT/A and BoNT/B using SERS nanotags and magnetic beads. Two different types of detection antibody-conjugated SERS nanotags, labeled with MGITC and NBA, and capture antibody-conjugated magnetic beads were fabricated for the dual detection of BoNT/A and BoNT/B. Total assay time was less than 2 h, including immunoreaction, washing, and detection, and the sample volume needed was lower than that for ELISA.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962947/

A therapeutic nanoparticle vaccine against Trypanosoma cruzi in a BALB/c mouse model of Chagas disease

ABSTRACT Chagas disease, caused by Trypanosoma cruzi , results in an acute febrile illness that progresses to chronic chagasic cardiomyopathy in 30% of patients. Current treatments have significant side effects and poor efficacy during the chronic phase; therefore, there is an urgent need for new treatment modalities. A robust T H 1-mediated immune response correlates with favorable clinical outcomes. A therapeutic vaccine administered to infected individuals could bolster the immune response, thereby slowing or stopping the progression of chagasic cardiomyopathy. Prior work in mice has identified an efficacious T. cruzi DNA vaccine encoding Tc24. To elicit a similar protective cell-mediated immune response to a Tc24 recombinant protein, we utilized a poly(lactic-co-glycolic acid) nanoparticle delivery system in conjunction with CpG motif-containing oligodeoxynucleotides as an immunomodulatory adjuvant. In a BALB/c mouse model, the vaccine produced a T H 1-biased immune response, as demonstrated by a significant increase in antigen-specific IFNγ-producing splenocytes, IgG2a titers, and proliferative capacity of CD8 + T cells. When tested for therapeutic efficacy, significantly reduced systemic parasitemia was seen during peak parasitemia. Additionally, there was a significant reduction in cardiac parasite burden and inflammatory cell infiltrate. This is the first study demonstrating immunogenicity and efficacy of a therapeutic Chagas vaccine using a nanoparticle delivery system.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1876623/

Identification and Analysis of Novel Amino-Acid Sequence Repeats in Bacillus anthracis str. Ames Proteome Using Computational Tools

We have identified four repeats and ten domains that are novel in proteins encoded by the Bacillus anthracis str. Ames proteome using automated in silico methods. A "repeat" corresponds to a region comprising less than 55-amino-acid residues that occur more than once in the protein sequence and sometimes present in tandem. A "domain" corresponds to a conserved region with greater than 55-amino-acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 57-amino-acid-residue PxV domain, (2) 122-amino-acid-residue FxF domain, (3) 111-amino-acid-residue YEFF domain, (4) 109-amino-acid-residue IMxxH domain, (5) 103-amino-acid-residue VxxT domain, (6) 84-amino-acid-residue ExW domain, (7) 104-amino-acid-residue NTGFIG domain, (8) 36-amino-acid-residue NxGK repeat, (9) 95-amino-acid-residue VYV domain, (10) 75-amino-acid-residue KEWE domain, (11) 59-amino-acid-residue AFL domain, (12) 53-amino-acid-residue RIDVK repeat, (13) (a) 41-amino-acid-residue AGQF repeat and (b) 42-amino-acid-residue GSAL repeat. A repeat or domain type is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure. 1. INTRODUCTION The anthrax is a disease of herbivores and other mammals including humans, caused by the Bacillus anthracis str. Ames , a Gram-positive, rod-shaped, nonmotile, spore-forming bacterium [ 1 ]. It is an endospore-forming bacterium that causes inhalational anthrax. During the course of disease, endospores are taken up by alveolar macrophages where they germinate in the phagolysosomal compartment. Vegetative cells then escape from the macrophage, eventually infecting blood. Expression of the major plasmid-encoded virulence determinants, tripartite toxin, and a poly-D-glutamic acid capsule is essential for full pathogenicity [ 2 ]. Key virulence genes found on plasmids are pXO1 and pXO2 [ 1 ]. The 60 MDa plasmid pXO2 carries genes required for the synthesis of an antiphagocytic poly-D-glutamic acid capsule [ 3 ]. The 110 MDa plasmid pXO1 [ 4 ] is required for the synthesis of the anthrax proteins, edema factor, lethal factor, and protective antigen. These proteins act in binary combinations to produce two anthrax toxins: edema toxin (a protective antigen and edema factor) and lethal toxin (a protective antigen and lethal factor) [ 5 ]. The chromosome encodes potential virulence factors that include haemolysins, enterotoxins, phospholipases, proteases, metalloproteases, and iron-acquisition proteins. The chromosome of B. anthracis str. Ames contains three homologues of sortase transpeptidase that is responsible for attachment of secreted proteins to peptidoglycan on the cell surface of Gram-positive bacteria [ 6 ]. A range of important surface proteins, including enzymes and virulence-related MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) are anchored to the cell wall in Gram-positive bacteria by sortase, a transpeptidase in Staphylococcus aureus , that cleaves polypeptides at a conserved LPxTG motif near the carboxyl terminus and covalently links them to penta-glycine crossbridges in peptidoglycan [ 7 , 8 ]. Nearly 34 candidate surface proteins which have sortase attachment sites and SLH domains were identified. Two putative B. anthracis str. Ames sortase attached genes have internalin like repeats [ 9 ]. The chromosome of B. anthracis str. Ames also contains the csaAB genes for binding of proteins with S-layer homology (SLH) domains to polysaccharide. The SLH domain is a repetitive modular element that is present in several bacterial cell surface proteins and is involved in noncovalent association with peptidoglycan associated polymers [ 10 ]. The SLH domain comprises 55-amino-acid residues [ 11 ] and the potential role of most proteins with SLH domains on the surface of B. anthracis str. Ames is unknown at present [ 12 ]. However, these surface proteins may mediate unknown interactions between B. anthracis str. Ames and its external environment and could be targets for vaccine and drug design. Read et al. [ 12 ] reported the complete genome sequence of B. anthracis str. Ames . It comprises 5 227 293 base pairs and 5508 genes with an overall G+C content of 35.4%. Of these, 2762 are functional genes, 1212 are conserved hypothetical genes, 657 genes are of unknown function, and 877 genes are annotated as hypothetical proteins. As the complete genome sequence of B. anthracis str. Ames is available [ 12 ], we intended to systematically identify and analyze all the amino-acid sequence repeats in this proteome. In a general context, a "repeat" corresponds to a region comprising less than 55-amino-acid residues that occur more than once, sometimes in tandem along the primary sequence, examples are the YVTN repeats in various cell surface proteins and the WD repeats present in proteins that perform a variety of functions. On the other hand, a "domain" refers to a region of the protein comprising greater than 55-amino-acid residues and does not contain internal sequence repeats. According to the crystallographer definition, a domain represents a region of the protein capable of folding independently as a stable unit. A domain can also exist in multiple copies and there can be several different domains per protein, examples are the SH2, SH3, and PH domains present in signal transduction proteins. The repeats and domains are characterized by conserved sequence motifs that may be identified according to the conservation of individual amino-acid residues at equivalent positions derived from multiple sequence alignments. In the absence of experimental data, the structural information can be obtained from secondary structure or fold prediction studies in silico. Information about the identified domains and repeats is represented in databases such as SMART, INTERPRO and PFAM. SMART (simple modular architecture research tool) allows the identification and annotation of genetically mobile domains and the analysis of domain architectures [ 13 ]. INTERPRO is a searchable database that provides information on sequence, function, and annotation. It is an integrated documentation resource for protein families, domains, and sites [ 14 ]. PFAM is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. This can be used to view the domain organization of proteins [ 15 ]. We believe that a systematic sequence analysis will provide information on the novel repeats and domains present in B. anthracis str. Ames proteome that are not identified so far. The B. anthracis str. Ames proteome consists of several known repeats and domains. Some of these domains are as follows. (1) BRCT (breast cancer carboxy terminal) domain was first identified as 100-amino-acid tandem repeat at the C-terminus of the tumor suppressor gene product BRCA1, in which the germline mutations lead to nearly 50% familial breast cancer. Most BRCT domains containing proteins participate in DNA damage checkpoint or DNA repair pathways and transcription regulation [ 16 ]. The BRCT is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. (2) Excalibur (extracellular calcium binding) domain consists of a conserved DxDxDGxxCE motif, which is strikingly similar to the Ca 2+ binding loop of the calmodulin like EF hand domains, suggesting an evolutionary relationship. (3) Cna_B domain forms a stalk in Streptococcus aureus collagen-binding protein that presents the ligand binding domain away from the bacterial cell surface. (4) CBS (cystathionine beta synthase) domain is a small intracellular module with 60-amino-acid residues, mostly found in two or four copies within a protein and occurs in several proteins in all kingdoms of life. Tandem pairs of CBS domains can act as binding domains for adenosine derivatives. In some cases, CBS domains may act as sensors of cellular energy status by being activated by AMP and inhibited by ATP. (5) Par B (par B like nuclease) domain cleaves single stranded DNA, nicks supercoiled plasmid DNA, and exhibits 5′-3′ exonuclease activity. (6) KH (K homology) domain comprises 70-amino-acids residues and is involved in RNA binding. (7) PAS and PAC domains comprising 300 and 45-amino-acid residues, respectively, mediate signal transduction. (8) PASTA domain is an extracellular module comprising 70-amino-acids residues that fold into a globular architecture consisting of 3 β -strands and an α -helix which aids in penicillin binding. (9) NEAT (near transporter) domain is a 125-amino-acid residue conserved region consisting mainly β -strands. The NEAT domain appears to be associated with iron transport in several Gram-positive species, some of them are pathogenic. (10) SLH domain is present in several bacterial cell surface proteins and is involved in noncovalent association with peptidoglycan associated polymers. It comprises 55-amino-acid residues and the predicted secondary structure comprises two α -helices flanking a short β -strand [ 11 ]. The repeats present in B. anthracis str. Ames proteome are as follows. (1) RHS repeats are 21-amino-acids residues long and are involved in carbohydrate binding. (2) TPR (tetratricopeptide) repeats are 34-amino-acids residues long and are involved in protein-protein interactions. (3) EZ − HEAT repeats are 37–47-amino-acid residues long and occur in tandem in a number of cytoplasmic proteins that are involved in intracellular transport processes. Arrays of HEAT repeats consist of 3 to 36 units forming a rod-like helical structure and appear to function as protein-protein interaction surfaces. (4) Ankyrin repeats are about 33-amino-acid residues long and occur in at least four consecutive copies; the core of the repeat appears as a helix-loop-helix structure and is involved in protein-protein interactions. (5) LRR (lecuine rich repeats) are 20-amino-acids residues long, each repeat consists of a β -strand and α -helix, that are oriented in an antiparallel manner. The function of LRRs includes signal transduction, transmembrane receptors, DNA repair, cell adhesion, and extracellualr matrix proteins [ 17 ]. Andrade et al. [ 18 ] reviewed methods to identify repeats in proteins and the relationship between repeat sequences and their associated functions. Repeats may be identified by manual examination, if the sequence similarity is very high and present in tandem. Repeats are thought to arise due to gene duplication and recombination events. Protein domains may exist either as single or multiple copies and repeats always exist as multiple copies [ 18 , 19 ]. Programs such as BLASTP [ 20 ] are also useful in detecting internal and homologous repeats in a protein database. By using the BLAST program, the presence of repeats in a query protein sequence can be identified if (a) the same region of the query is aligned against two or more distinct regions of a second protein; and (b) different regions of the query are being aligned against the same region of a second protein [ 18 ]. Several web-based methods are available for ab initio identification of sequence repeats in proteins. For example, RADAR (rapid automatic detection and alignment of repeats) [ 21 ] uses an automatic algorithm, for segmenting a query sequence into repeats; it identifies short composition biased as well as gapped approximate repeats and complex repeat architectures involving many different types of repeats in a query sequence. Rep program [ 22 ] uses an iterative algorithm based on score distributions from profile analysis. This procedure allows the identification of homologues at alignment scores lower than the highest optimal alignment score for nonhomologous sequences. The PROSPERO program [ 23 ] is ideal for large scale self-comparison of protein sequences. It uses a formula that accurately assesses the significance of protein repeat similarities, allowing for existence of gaps, and also takes into account sequence length and composition. TRUST (tracking repeats using significance and transitivity) program [ 24 ] exploits the concept of transitivity of alignments as well as a statistical scheme optimized for the evaluation of repeat significance. Starting from significant local suboptimal alignments, the application of transitivity allows to (1) identify distant repeat homologues for which no alignments were found; (2) gain confidence about consistently well-aligned regions; and (3) recognize and reduce the contribution of nonhomologous repeats. This assessment step will enable to derive a virtually noise-free profile representing a generalized repeat with high fidelity. It has been demonstrated by the authors that TRUST is a useful and reliable tool for mining tandem and nontandem repeats in protein sequence databases, to predict multiple repeat types with varying intervening segments within a single sequence. Once statistically significant repeats are detected, construction of a multiple sequence alignment provides insight into the extent of sequence homology among members of the new protein family and identification of the conserved sequence motifs. We have implemented TRUST on a personal computer in our laboratory and used it to identify amino-acid sequence repeats in the proteins of B. anthracis str. Ames proteome. We have identified four repeats and ten domains that are novel in the proteome of B. anthracis str. Ames . Further analysis corresponding to searches of the completed and unfinished genome databases identified some of these to be present in other bacterial genomes. 2. METHODS We have downloaded the entire proteome of B. anthracis str. Ames from the website http://www.ncbi.nlm.nih.gov in the FASTA format. The TRUST program was downloaded from the website and installed on the local Pentium IV computers on the Linux platform. The TRUST server together with the source code is available at http://ibivu.cs.vu.nl/programs/trustwww . The TRUST program was run for all the sequences in this proteome. Based on the size of the TRUST output file, the protein sequences with no internal repeats were discarded automatically; that is, only those protein sequences which comprise repeats were retained. The lengths of repeats and domains currently annotated in the INTERPRO database often comprise greater than 25-amino-acid residues; therefore, in this work, we have considered the repeats with greater than 25-amino-acid residues alone for further analysis. Thus selected proteins were submitted to SMART online ( http://smart.embl-heidelberg.de/smart/batch.pl ) [ 13 ] program in batch mode. Manual inspections of the SMART results identified proteins comprising known repeats or domains and were therefore discarded. Only those repeats that are not identified by SMART database are retained for further analysis. We have downloaded NCBI NR (release date: April 22, 2005) and UNIPROT (release date: April 23, 2005) databases and installed BLAST-2.2.10 on the local Linux computers (OS: Fedora Core-2, Pentium-IV 3.00 GHz, 1 GB RAM, 80 GB hard disk). Using automatic shell scripts, these protein sequences were then blasted using PSI-BLAST program [ 25 ] for three iterations against the NCBI NR database and using BLASTALL program against UNIPROT database. The proteins confirmed to comprise repeats by the BLAST program were retained and were tested for presence in the offline versions of INTERPRO (Database: iprscan_DATA_10.0, Applications: iprscan_V4.1, iprscan_binn4.x_Linux) and PFAM (release date: April 26, 2005) databases. A final check was made using online versions of INTERPRO and PFAM. These series of steps are given in the flowchart as shown in Figure 1 . The repeats which are not present in any of these databases were considered to be novel repeats or domains, depending upon (1) the number of times they occur in the protein sequences, and (2) length of the amino-acid sequence region. The novel repeats and domains thus identified in B. anthracis str. Ames proteome were subjected to PSI-BLAST analysis in order to identify other proteins from databases that comprise these repeats and domains. Multiple sequence alignment program, ClustalW [ 26 ], was used to detect the extent of sequence conservation and the secondary structure prediction was carried out using PHD [ 27 ] method. 3. RESULTS AND DISCUSSION From the analysis of B. anthracis str. Ames proteome using TRUST program, we identified 905 proteins comprising of amino-acid sequence repeats. SMART database analysis identified that 302 entries do not have a SMART description. Based on their absence in the INTERPRO and PFAM databases and the length of repeat sequence (greater than 25-amino-acid residues), we have identified about 120 proteins (data not shown) in the B. anthracis str. Ames proteome to comprise novel amino-acid sequence repeats. We have added an additional constraint that the repeats identified by TRUST program should also be identified as a repeat by the BLAST program. Subsequent online INTERPRO and PFAM searches confirmed that these domains and repeats have not been reported before. In this work, we have identified four repeats and ten domains, that are not within or part of previously reported repeats and our findings are therefore novel. Further analysis identified some of these in the proteins of other bacterial genomes. The conserved amino-acid residues observed from multiple sequence alignments using the CLUSTALW program were used to describe sequence motifs characteristic of these novel repeats and domains. Often, more than one sequence motif is associated with repeats or domains and the amino-acid sequence patterns characteristic of these repeats are represented according to the PROSITE description [ 28 ]. Ponting et al. [ 29 ], have earlier used a similar approach to identify novel domains and repeats in Drosophila melanogaster . In this work, we identified four repeats and ten domains that have not been reported before in the B. anthracis str. Ames proteome. The repeats and domains described in 1 to 6 and 9 are also present in some bacterial organisms, 7, 8, 10 and 11 are Bacillus -specific, 12 and 13 are Bacillus anthracis str. Ames specific. Lists of the proteins containing these novel repeats and domains are shown in Tables 1a to 1k . These tables indicate the protein identifiers (Gene or Swall_ID), the number of amino-acid residues in the protein, a description of the protein, and other well-characterized repeats and domains present in the protein. Some sequences representing these repeats or domains share lower than 15% pairwise sequence identity. However, these sequences retain the conserved motifs and the positions of secondary structure elements in the multiple sequence alignment. For all the proteins, the amino-acid sequence corresponding to each representative repeat are shown in the multiple sequence alignments (see Figures from 2 to 14 ). 1 Conservation of the position of secondary structural elements is indicated from the multiple sequence alignment. The schematic figures used to represent these repeats and domains are shown in Figures 15 to 27 . These figures (drawn to an approximate scale) reflect the relative proximity and location of individual repeats and domains along the primary sequence. We discuss each of these novel repeats and domains below. 3.1. 57-amino-acid-residue PxV domain The 251-amino-acid-residue protein corresponding to the GENE_ID BA2292 and described as hypothetical protein comprises of a 57-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (65–121) as a query identified 24 proteins that are described as hypothetical (see Table 1(a) ). This region occurs as four copies in proteins from Shewanella amazonensis, and Haloarcula marismortui , as two copies in proteins from B. anthracis, B. cereus, B. halodurans, B. thuringiensis, B. thuringiensis serovar, Thermus thermopilus, Chloroflexus aurantiacus, Chloroflexus aggregans Exiguobacterium sp., Bacillus weihenstephanensis, Roseiflexus castenholzii, Clostridium novyi, Herpetosiphon aurantiacus , and as single copy in Anabaena variabilis ; we therefore describe this region as a domain. The length of proteins varied between 196 to 488-amino-acid residues. The multiple sequence alignment corresponding to this domain is associated with PxV sequence motif where x is any amino-acid residue and is shown in Figure 2 . The pairwise identities between sequences corresponding to PxV domain varied between 15–96%. The secondary structure corresponding to PxV domain is predicted to comprise four β -strands as shown in Figure 2 . The representative domain architecture corresponding to proteins comprising the PxV domain is shown in Figure 15 . 3.2. 122-amino-acid-residue FxF domain The 293-amino-acid-residue protein corresponding to the GENE_ID BA0881 and described as conserved domain protein comprises a 122-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (55–176) as a query identified 10 proteins (see Table 1(b) ). The proteins comprising this region are described as either conserved or hypothetical proteins. This region occurs as two copies in the proteins of B. anthracis, B. cereus, B. thuringiensis , Geobacillus kaustophilus, Clostridium tetani , Clostridium novyi , and Desulfotomaculum reducens genomes. The length of proteins varied between 262 to 305-amino-acid residues. The multiple sequence alignment corresponding to this domain is associated with characteristic sequence motif FxF ( Figure 3 ) and we refer to this as the FxF domain. The pairwise sequence identities corresponding to this domain varies between 18–97%. The secondary structure corresponding to FxF domain is predicted to comprise one α -helix and five β -strands, and the representative domain architecture of proteins comprising this domain is shown in Figure 16 . 3.3. 111-amino-acid-residue YEFF domain The 510-amino-acid-residue protein corresponding to the GENE_ID BA3695 and described as a S-layer protein comprises a 111-amino-acid-residue region that is present as two copies. Further BLAST searches, using sequence corresponding to the region (247–357) as a query, identified 13 proteins (see Table 1(c) ), that are described as S-layer proteins, hypothetical, or lipoproteins and correspond to the B. anthracis str. Ames and A2012, B. cereus , B. thuringiensis, B. thuringiensis serovar israelensis , and Enterococcus faecalis genomes. The length of proteins varied between 321 to 510-amino-acid residues. Five proteins corresponding to the GENE_ID BA3695 and Bant_01004347 of B. anthracis , BCE_G9241_3590, and BCZK3337 of B. cereus and BT9727_3386 of B. thuringiensis comprise three copies of SLH domain, indicating a cell surface role for these proteins. This domain is characterized by conserved sequence motifs; YEFF, RGD, FTY, GKD, and FVEH. We refer to this 111-amino-acid region as the YEFF domain. The pairwise sequence identities corresponding to the YEFF domain varied between 36–96%. The consensus secondary structure predicted for this domain suggests mainly β -strands and the conserved sequence motifs, that is, YEFF and FTY are associated with β -strands; see Figure 4 . The representative domain architecture of proteins comprising this domain is shown in Figure 17 . It is intriguing that each domain comprises RGD sequence motif which is found in the proteins of extracellular matrix. Many viruses enter their host cells via the RGD motif—integrin interaction and synthetic peptides containing this RGD motif are active modulators of cell adhesion [ 30 ] . The RGD motif was originally identified as the sequence within fibronectin that mediates cell attachment. This motif has now been found in numerous other proteins and supports cell adhesion. The integrins, a family of cell surface proteins, act as receptors for cell adhesion molecules. A subset of the integrins recognizes the RGD motif within their ligands, the binding of which mediates both cell substratum and cell-cell interactions [ 31 ]. The presence of RGD motif and SLH domain implies that the YEFF domain comprising proteins is also present on the cell surface and mediates protein-protein interactions. 3.4. 109-amino-acid-residue IMxxH domain The 266-amino-acid-residue protein corresponding to the GENE_ID BA1021 and described as hypothetical protein comprises a 109-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (4–112) as a query identified 22 proteins (see Table 1(d) ) that are described as either conserved or hypothetical proteins. This domain region occurs as two copies in all the proteins of B. anthracis, B. cereus, B. thuringiensis, Bacillus weihenstephanensis C. acetobutylicum, C. perfringens, C. tetani, C. thermocellum, Desulfitobacterium hafniense, Clostridium phytofermentans, and Alkaliphilus metalliredigenes, and as single domain in the 171-amino-acid-residue protein BcerKBAB4DRAFT_0307. The length of proteins varied between 171 to 321 amino acid residues. The multiple sequence alignment corresponding to this domain identified the characteristic sequence motifs; IMxxH, REA, and we refer to this as the IMxxH domain. The IMxxH sequence motif occurs at the N-terminal region of the domain. The pairwise sequence identities corresponding to the IMxxH domain varies between 5–98%. The secondary structure corresponding to IMxxH domain is predicted to comprise four α -helices as shown in Figure 5 . The representative domain architecture corresponding to proteins comprising this domain is shown in Figure 18 . 3.5. 103-amino-acid-residue VxxT domain The 349-amino-acid-residue protein corresponding to the GENE_ID BA4716 and described as germination protein comprises a 103-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (67–169) as query identified 23 proteins (see Table 1(e) ). The proteins comprising this domain are described as germination proteins as the Bacillus anthracis is an endospore-forming bacterium. This domain region occurs twice in proteins of B. anthracis str. Ames, B. cereus, B. clausii, B. thuringiensis , B. thuringiensis serovar israelensis , Alkaliphilus metalliredigene , and Bacillus weihenstephanensis genomes and only once in the proteins of Syntrophomonas wolfei str. Goettingen, Moorella thermoacetica, Clostridium thermocellum, B. subtilis , and Pelotomaculum thermopropionicum genomes. The length of proteins varied between 195 to 377-amino-acid residues. The multiple sequence alignment corresponding to this domain identified VxxT as sequence motif. This sequence motif occurs in the N-terminal region of each protein and the pairwise sequence identity varied between 11–98%. The secondary structure is predicted to comprise two α -helices and three β -strands as shown in Figure 6 . The representative domain architecture corresponding to proteins comprising this domain is shown in Figure 19 . 3.6. 84-amino-acid-residue ExW domain The 246-amino-acid-residue protein corresponding to the GENE_ID BA4310 and described as hypothetical protein comprises an 84-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the domain (45–128) as a query identified 25 proteins ( Table 1(f) ) that are described as either conserved or hypothetical proteins. This domain region occurs as two copies in proteins of B. anthracis str. Ames, B. cereus, B. halodurans (GENE_ID BH0678), B. thuringiensis , B. thuringiensis serovar israelensis , Geobacillus kaustophil us, Bacillus weihenstephanensis , and Exiguobacterium sibiricum genomes and as single copy in proteins of B. clausii , B. halodurans (GENE_ID BH0983), B. licheniformis, B. subtilis, Exiguobacterium sp. , and Oceanobacillus ihenyensis genomes. The length of proteins varied between 142 to 273-amino-acid residues. The multiple sequence alignment corresponding to this domain identified ExW sequence motif. The pairwise sequence identities corresponding to the ExW domain varied between 14–98%. The secondary structure of this domain is predicted to comprise five β -strands and the conserved sequence motif is associated with one of the β -strands as shown in Figure 7 . The representative domain architecture corresponding to proteins comprising this domain is shown in Figure 20 . 3.7. 104-amino-acid-residue NTGFIG domain The 232-amino-acid-residue protein corresponding to the GENE_ID BA2665 and described as a hypothetical protein comprises a 104-amino-acid-residue region as two copies in tandem. Further BLAST searches using sequence corresponding to the region (16–119) as query identified 9 hypothetical proteins comprising this domain from organisms such as B. anthracis , B. thuringiensis, Bacillus weihenstephanensis , and B. cereus . The protein corresponding to the GENE_ID BCZK2413 of B. cereus is described as group-specific protein. The list of 9 proteins comprising this domain is shown in Table 1(g) . The length of proteins varied between 232 to 236-amino-acid residues. This domain occurs twice in every protein of the bacillus species as shown in Table 1(g) . We refer to this as the NTGFIG domain based on the conserved sequence motif that is present at the N-terminal part. The pairwise sequence identities between sequences corresponding to this domain varied between 31–99%. The secondary structure corresponding to this domain is predicted to comprise three α -helices and two β -strands as shown in Figure 8 . The representative domain architecture corresponding to proteins comprising this domain is shown in Figure 21 . 3.8. 36-amino-acid-residue NxGK repeat The 193-amino-acid-residue protein corresponding to GENE_ID BA3686 and described as hypothetical cytosolic protein comprises a 36-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (94–129) as query identified 9 hypothetical proteins comprising this repeat region from the organisms B. anthracis , B. thuringiensis , B. thuringiensis serovar israelensis, Bacillus weihenstephanensis , and B. cereus (see Table 1(h) ). The length of proteins varied between 189 to 193-amino-acid residues, and also consists a SAP domain at the N-terminus, in addition to the novel repeat described here. A SAP domain consists of two α -helices and is a DNA-binding motif that is involved in chromosomal organization [ 32 ]. Therefore, we believe that these repeats might also participate in a similar function. The multiple sequence alignment corresponding to this repeat identified NxGK sequence motif ( Figure 9 ). The pairwise sequence identities between sequences corresponding to NxGK repeats varied between 36–97%. The secondary structure is predicted to comprise a α -helix and the conserved sequence motif described above is also associated with α -helix. The representative domain architecture corresponding to proteins comprising the NxGK repeats is shown in Figure 22 . 3.9. 95-amino-acid-residue VYV domain The 225-amino-acid-residue protein corresponding to the GENE_ID BA1701 and described as a hypothetical protein comprises a 95-amino-acid-residue region, as two copies in tandem. Further BLAST searches using sequence corresponding to the region (31–125) as query identified BAS1577 protein of B. anthracis , RBTH_03882 protein of Bacillus thuringiensis serovar israelensis , and DSY3134 of Desulfitobacterium hafniense Y51 that are described as hypothetical proteins. The length of proteins varied between 227 to 1674-amino-acid residues (see Table 1(i) ). In RBTH_03882, this region occurs ten times and in tandem. The multiple sequence alignment corresponding to this domain identified characteristic sequence motifs; GDxV, VYV (see Figure 10 ). For the sake of simplicity, we refer to this 95-amino-acid region as VYV domain. The pairwise sequence identities between sequences corresponding to VYV domains varied between 29–95%. The secondary structure corresponding to VYV domain is predicted to comprise five β -strands. The representative domain architecture corresponding to proteins comprising the VYV domains is shown in Figure 23 . 3.10. 75-amino-acid-residue KEWE domain The 262-amino-acid-residue protein corresponding to the GENE_ID BA3147 and described as a hypothetical protein comprises a 75-amino-acid-residue region as three copies in tandem. Further BLAST searches using the sequence corresponding to the region (34–108) as query identified this domain in 6 proteins that are described as hypothetical proteins (see Table 1(j) ). This domain may exist as 2, 3, or 4 copies in these proteins. The length of proteins identified varied between 178 to 344-amino-acid residues. The pairwise sequence identities between sequences corresponding to these regions varied between 22–69%. These domains are present in tandem and associated with SPY, MIN, LYP, KEWE, and FWT conserved sequence motifs as indicated in the multiple sequence alignment (see Figure 11 ). We refer to these as the KEWE domain, and this sequence motif occurs at the C-terminus of the domain. The secondary structure corresponding to KEWE domain is predicted to comprise three α -helices as shown in Figure 11 . The representative domain architecture corresponding to proteins comprising the KEWE domain is shown in Figure 24 . 3.11. 59-amino-acid-residue AFL domain The 290-amino-acid-residue protein corresponding to the GENE_ID BA3065 and described as hypothetical protein comprises a 59-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (13–71) as query identified that this region occurs twice in the proteins with GENE_ID's: BAS2851 and Bant_01003715 of B. anthracis strains, the protein with GENE_ID: BcerKBAB4DRAFT_1832 of Bacillus weihenstephanensis, and once in the protein with GENE_ID: RBTH_02124 of Bacillus thuringiensis serovar israelensis (see Table 1(K) ). The lengths of the proteins varied between 145 to 297-amino-acid residues and are described as hypothetical proteins. The multiple sequence alignment corresponding to this domain identified two characteristic sequence motifs: RFxI and AFL (see Figure 12 ). We refer to this as the AFL domain. The sequence identities shared between AFL domains varied between 38–91%. The secondary structure corresponding to the AFL domain is predicted to comprise of one α -helix and two β -strands and the conserved sequence motif AFL is a part of the α -helix. The representative domain architecture corresponding to protein comprising the AFL domain is shown in Figure 25 . 3.12. 53-amino-acid-residue RIDVK repeat The 159-amino-acid-residue protein corresponding to the GENE_ID BA0482 and described as a conserved domain protein comprises a 53-amino-acid region as two copies. BLAST did not identify this repeat in any other proteins; therefore this repeat is unique to B. anthracis str. Ames . The multiple sequence alignment corresponding to this repeat identified three characteristic sequence motifs: ITV, IGD, and RIDVK ( Figure 13 ). We refer to this as the RIDVK repeat. The sequence identity shared between this RIDVK repeats in BA0482 is 45%. The secondary structure corresponding to the RIDVK repeat is predicted to comprise three β -strands. The representative domain architecture corresponding to protein comprising the RIDVK repeat is shown in Figure 26 . 3.13. (a) 41-amino-acid-residue AGQF repeat and (b) 42-amino-acid-residue GSAL repeat The protein corresponding to the GENE_ID BA4081 comprises 462 amino acid residues and described as conserved domain protein contains two novel repeat types. The sequence length corresponding to repeat types are 41 and 42 amino acid residues and are present as two copies in BA4081. BLAST searches identified these repeats to be specific to this protein alone. (a) The sequence alignment corresponding to 41-amino-acid-residue repeat identified two characteristic sequence motifs: DLG and AGQF ( Figure 14(a) ). We refer to this as the AGQF repeat. The motif occurs at the C-terminal part of the repeat region. The sequence homology shared between this AGQF repeats is about 34%. The secondary structure corresponding to the AGQF repeat is predicted to comprise one α -helix. The representative domain architecture corresponding to protein comprising the AGQF repeat is shown in Figure 27 . (b) The sequence alignment corresponding to the 42-amino-acid-residue tandem repeat identified three characteristic sequence motifs: GYI, GSAL, and TING ( Figure 14(b) ) and is a glycine-rich repeat. We refer to this as the GSAL repeat. The sequence homology shared between this GSAL repeats is 52%. The secondary structure corresponding to the GSAL repeat is predicted to comprise one α -helix and one β -strand. The representative domain architecture corresponding to protein comprising the GSAL repeat is shown in Figure 27 . This protein is associated with a 27-amino-acid residue Ribosomal_S7 region that is sandwiched between the 41-amino-acid-residue AGQF repeat and the 42-amino-acid-residue GSAL repeat. These two repeats are specific to this protein alone and are therefore B. anthracis str. Ames specific. From the analysis of the B. anthracis proteome, we observed that the novel repeats and domains are present in all the strains, such as Ames , Ames ancestor, Sterne, and A2012, that have been sequenced so far. This indicates that these strains of B. anthracis have diverged recently. We also observed that the domains PxV, FxF, YEFF, VxxT, ExW, and VYV are present in proteins from several bacterial organisms. The domains NTGFIG, KEWE, AFL, and the repeats NxGK are specific to bacillus. It is interesting to note that the domains VYV and AFL are present in all the B. anthracis species while absent in B. cereus genomes. The repeats RIDVK, AGQF, and GSAL are also specifically present only in all the strains of B. anthracis . This analysis explains some differences in the closely related B. anthracis and B. cereus genomes. The identification of these novel domains and repeats in subsequently sequenced genomes will add value to their annotation. 3.1. 57-amino-acid-residue PxV domain The 251-amino-acid-residue protein corresponding to the GENE_ID BA2292 and described as hypothetical protein comprises of a 57-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (65–121) as a query identified 24 proteins that are described as hypothetical (see Table 1(a) ). This region occurs as four copies in proteins from Shewanella amazonensis, and Haloarcula marismortui , as two copies in proteins from B. anthracis, B. cereus, B. halodurans, B. thuringiensis, B. thuringiensis serovar, Thermus thermopilus, Chloroflexus aurantiacus, Chloroflexus aggregans Exiguobacterium sp., Bacillus weihenstephanensis, Roseiflexus castenholzii, Clostridium novyi, Herpetosiphon aurantiacus , and as single copy in Anabaena variabilis ; we therefore describe this region as a domain. The length of proteins varied between 196 to 488-amino-acid residues. The multiple sequence alignment corresponding to this domain is associated with PxV sequence motif where x is any amino-acid residue and is shown in Figure 2 . The pairwise identities between sequences corresponding to PxV domain varied between 15–96%. The secondary structure corresponding to PxV domain is predicted to comprise four β -strands as shown in Figure 2 . The representative domain architecture corresponding to proteins comprising the PxV domain is shown in Figure 15 . 3.2. 122-amino-acid-residue FxF domain The 293-amino-acid-residue protein corresponding to the GENE_ID BA0881 and described as conserved domain protein comprises a 122-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (55–176) as a query identified 10 proteins (see Table 1(b) ). The proteins comprising this region are described as either conserved or hypothetical proteins. This region occurs as two copies in the proteins of B. anthracis, B. cereus, B. thuringiensis , Geobacillus kaustophilus, Clostridium tetani , Clostridium novyi , and Desulfotomaculum reducens genomes. The length of proteins varied between 262 to 305-amino-acid residues. The multiple sequence alignment corresponding to this domain is associated with characteristic sequence motif FxF ( Figure 3 ) and we refer to this as the FxF domain. The pairwise sequence identities corresponding to this domain varies between 18–97%. The secondary structure corresponding to FxF domain is predicted to comprise one α -helix and five β -strands, and the representative domain architecture of proteins comprising this domain is shown in Figure 16 . 3.3. 111-amino-acid-residue YEFF domain The 510-amino-acid-residue protein corresponding to the GENE_ID BA3695 and described as a S-layer protein comprises a 111-amino-acid-residue region that is present as two copies. Further BLAST searches, using sequence corresponding to the region (247–357) as a query, identified 13 proteins (see Table 1(c) ), that are described as S-layer proteins, hypothetical, or lipoproteins and correspond to the B. anthracis str. Ames and A2012, B. cereus , B. thuringiensis, B. thuringiensis serovar israelensis , and Enterococcus faecalis genomes. The length of proteins varied between 321 to 510-amino-acid residues. Five proteins corresponding to the GENE_ID BA3695 and Bant_01004347 of B. anthracis , BCE_G9241_3590, and BCZK3337 of B. cereus and BT9727_3386 of B. thuringiensis comprise three copies of SLH domain, indicating a cell surface role for these proteins. This domain is characterized by conserved sequence motifs; YEFF, RGD, FTY, GKD, and FVEH. We refer to this 111-amino-acid region as the YEFF domain. The pairwise sequence identities corresponding to the YEFF domain varied between 36–96%. The consensus secondary structure predicted for this domain suggests mainly β -strands and the conserved sequence motifs, that is, YEFF and FTY are associated with β -strands; see Figure 4 . The representative domain architecture of proteins comprising this domain is shown in Figure 17 . It is intriguing that each domain comprises RGD sequence motif which is found in the proteins of extracellular matrix. Many viruses enter their host cells via the RGD motif—integrin interaction and synthetic peptides containing this RGD motif are active modulators of cell adhesion [ 30 ] . The RGD motif was originally identified as the sequence within fibronectin that mediates cell attachment. This motif has now been found in numerous other proteins and supports cell adhesion. The integrins, a family of cell surface proteins, act as receptors for cell adhesion molecules. A subset of the integrins recognizes the RGD motif within their ligands, the binding of which mediates both cell substratum and cell-cell interactions [ 31 ]. The presence of RGD motif and SLH domain implies that the YEFF domain comprising proteins is also present on the cell surface and mediates protein-protein interactions. 3.4. 109-amino-acid-residue IMxxH domain The 266-amino-acid-residue protein corresponding to the GENE_ID BA1021 and described as hypothetical protein comprises a 109-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (4–112) as a query identified 22 proteins (see Table 1(d) ) that are described as either conserved or hypothetical proteins. This domain region occurs as two copies in all the proteins of B. anthracis, B. cereus, B. thuringiensis, Bacillus weihenstephanensis C. acetobutylicum, C. perfringens, C. tetani, C. thermocellum, Desulfitobacterium hafniense, Clostridium phytofermentans, and Alkaliphilus metalliredigenes, and as single domain in the 171-amino-acid-residue protein BcerKBAB4DRAFT_0307. The length of proteins varied between 171 to 321 amino acid residues. The multiple sequence alignment corresponding to this domain identified the characteristic sequence motifs; IMxxH, REA, and we refer to this as the IMxxH domain. The IMxxH sequence motif occurs at the N-terminal region of the domain. The pairwise sequence identities corresponding to the IMxxH domain varies between 5–98%. The secondary structure corresponding to IMxxH domain is predicted to comprise four α -helices as shown in Figure 5 . The representative domain architecture corresponding to proteins comprising this domain is shown in Figure 18 . 3.5. 103-amino-acid-residue VxxT domain The 349-amino-acid-residue protein corresponding to the GENE_ID BA4716 and described as germination protein comprises a 103-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (67–169) as query identified 23 proteins (see Table 1(e) ). The proteins comprising this domain are described as germination proteins as the Bacillus anthracis is an endospore-forming bacterium. This domain region occurs twice in proteins of B. anthracis str. Ames, B. cereus, B. clausii, B. thuringiensis , B. thuringiensis serovar israelensis , Alkaliphilus metalliredigene , and Bacillus weihenstephanensis genomes and only once in the proteins of Syntrophomonas wolfei str. Goettingen, Moorella thermoacetica, Clostridium thermocellum, B. subtilis , and Pelotomaculum thermopropionicum genomes. The length of proteins varied between 195 to 377-amino-acid residues. The multiple sequence alignment corresponding to this domain identified VxxT as sequence motif. This sequence motif occurs in the N-terminal region of each protein and the pairwise sequence identity varied between 11–98%. The secondary structure is predicted to comprise two α -helices and three β -strands as shown in Figure 6 . The representative domain architecture corresponding to proteins comprising this domain is shown in Figure 19 . 3.6. 84-amino-acid-residue ExW domain The 246-amino-acid-residue protein corresponding to the GENE_ID BA4310 and described as hypothetical protein comprises an 84-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the domain (45–128) as a query identified 25 proteins ( Table 1(f) ) that are described as either conserved or hypothetical proteins. This domain region occurs as two copies in proteins of B. anthracis str. Ames, B. cereus, B. halodurans (GENE_ID BH0678), B. thuringiensis , B. thuringiensis serovar israelensis , Geobacillus kaustophil us, Bacillus weihenstephanensis , and Exiguobacterium sibiricum genomes and as single copy in proteins of B. clausii , B. halodurans (GENE_ID BH0983), B. licheniformis, B. subtilis, Exiguobacterium sp. , and Oceanobacillus ihenyensis genomes. The length of proteins varied between 142 to 273-amino-acid residues. The multiple sequence alignment corresponding to this domain identified ExW sequence motif. The pairwise sequence identities corresponding to the ExW domain varied between 14–98%. The secondary structure of this domain is predicted to comprise five β -strands and the conserved sequence motif is associated with one of the β -strands as shown in Figure 7 . The representative domain architecture corresponding to proteins comprising this domain is shown in Figure 20 . 3.7. 104-amino-acid-residue NTGFIG domain The 232-amino-acid-residue protein corresponding to the GENE_ID BA2665 and described as a hypothetical protein comprises a 104-amino-acid-residue region as two copies in tandem. Further BLAST searches using sequence corresponding to the region (16–119) as query identified 9 hypothetical proteins comprising this domain from organisms such as B. anthracis , B. thuringiensis, Bacillus weihenstephanensis , and B. cereus . The protein corresponding to the GENE_ID BCZK2413 of B. cereus is described as group-specific protein. The list of 9 proteins comprising this domain is shown in Table 1(g) . The length of proteins varied between 232 to 236-amino-acid residues. This domain occurs twice in every protein of the bacillus species as shown in Table 1(g) . We refer to this as the NTGFIG domain based on the conserved sequence motif that is present at the N-terminal part. The pairwise sequence identities between sequences corresponding to this domain varied between 31–99%. The secondary structure corresponding to this domain is predicted to comprise three α -helices and two β -strands as shown in Figure 8 . The representative domain architecture corresponding to proteins comprising this domain is shown in Figure 21 . 3.8. 36-amino-acid-residue NxGK repeat The 193-amino-acid-residue protein corresponding to GENE_ID BA3686 and described as hypothetical cytosolic protein comprises a 36-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (94–129) as query identified 9 hypothetical proteins comprising this repeat region from the organisms B. anthracis , B. thuringiensis , B. thuringiensis serovar israelensis, Bacillus weihenstephanensis , and B. cereus (see Table 1(h) ). The length of proteins varied between 189 to 193-amino-acid residues, and also consists a SAP domain at the N-terminus, in addition to the novel repeat described here. A SAP domain consists of two α -helices and is a DNA-binding motif that is involved in chromosomal organization [ 32 ]. Therefore, we believe that these repeats might also participate in a similar function. The multiple sequence alignment corresponding to this repeat identified NxGK sequence motif ( Figure 9 ). The pairwise sequence identities between sequences corresponding to NxGK repeats varied between 36–97%. The secondary structure is predicted to comprise a α -helix and the conserved sequence motif described above is also associated with α -helix. The representative domain architecture corresponding to proteins comprising the NxGK repeats is shown in Figure 22 . 3.9. 95-amino-acid-residue VYV domain The 225-amino-acid-residue protein corresponding to the GENE_ID BA1701 and described as a hypothetical protein comprises a 95-amino-acid-residue region, as two copies in tandem. Further BLAST searches using sequence corresponding to the region (31–125) as query identified BAS1577 protein of B. anthracis , RBTH_03882 protein of Bacillus thuringiensis serovar israelensis , and DSY3134 of Desulfitobacterium hafniense Y51 that are described as hypothetical proteins. The length of proteins varied between 227 to 1674-amino-acid residues (see Table 1(i) ). In RBTH_03882, this region occurs ten times and in tandem. The multiple sequence alignment corresponding to this domain identified characteristic sequence motifs; GDxV, VYV (see Figure 10 ). For the sake of simplicity, we refer to this 95-amino-acid region as VYV domain. The pairwise sequence identities between sequences corresponding to VYV domains varied between 29–95%. The secondary structure corresponding to VYV domain is predicted to comprise five β -strands. The representative domain architecture corresponding to proteins comprising the VYV domains is shown in Figure 23 . 3.10. 75-amino-acid-residue KEWE domain The 262-amino-acid-residue protein corresponding to the GENE_ID BA3147 and described as a hypothetical protein comprises a 75-amino-acid-residue region as three copies in tandem. Further BLAST searches using the sequence corresponding to the region (34–108) as query identified this domain in 6 proteins that are described as hypothetical proteins (see Table 1(j) ). This domain may exist as 2, 3, or 4 copies in these proteins. The length of proteins identified varied between 178 to 344-amino-acid residues. The pairwise sequence identities between sequences corresponding to these regions varied between 22–69%. These domains are present in tandem and associated with SPY, MIN, LYP, KEWE, and FWT conserved sequence motifs as indicated in the multiple sequence alignment (see Figure 11 ). We refer to these as the KEWE domain, and this sequence motif occurs at the C-terminus of the domain. The secondary structure corresponding to KEWE domain is predicted to comprise three α -helices as shown in Figure 11 . The representative domain architecture corresponding to proteins comprising the KEWE domain is shown in Figure 24 . 3.11. 59-amino-acid-residue AFL domain The 290-amino-acid-residue protein corresponding to the GENE_ID BA3065 and described as hypothetical protein comprises a 59-amino-acid-residue region as two copies. Further BLAST searches using sequence corresponding to the region (13–71) as query identified that this region occurs twice in the proteins with GENE_ID's: BAS2851 and Bant_01003715 of B. anthracis strains, the protein with GENE_ID: BcerKBAB4DRAFT_1832 of Bacillus weihenstephanensis, and once in the protein with GENE_ID: RBTH_02124 of Bacillus thuringiensis serovar israelensis (see Table 1(K) ). The lengths of the proteins varied between 145 to 297-amino-acid residues and are described as hypothetical proteins. The multiple sequence alignment corresponding to this domain identified two characteristic sequence motifs: RFxI and AFL (see Figure 12 ). We refer to this as the AFL domain. The sequence identities shared between AFL domains varied between 38–91%. The secondary structure corresponding to the AFL domain is predicted to comprise of one α -helix and two β -strands and the conserved sequence motif AFL is a part of the α -helix. The representative domain architecture corresponding to protein comprising the AFL domain is shown in Figure 25 . 3.12. 53-amino-acid-residue RIDVK repeat The 159-amino-acid-residue protein corresponding to the GENE_ID BA0482 and described as a conserved domain protein comprises a 53-amino-acid region as two copies. BLAST did not identify this repeat in any other proteins; therefore this repeat is unique to B. anthracis str. Ames . The multiple sequence alignment corresponding to this repeat identified three characteristic sequence motifs: ITV, IGD, and RIDVK ( Figure 13 ). We refer to this as the RIDVK repeat. The sequence identity shared between this RIDVK repeats in BA0482 is 45%. The secondary structure corresponding to the RIDVK repeat is predicted to comprise three β -strands. The representative domain architecture corresponding to protein comprising the RIDVK repeat is shown in Figure 26 . 3.13. (a) 41-amino-acid-residue AGQF repeat and (b) 42-amino-acid-residue GSAL repeat The protein corresponding to the GENE_ID BA4081 comprises 462 amino acid residues and described as conserved domain protein contains two novel repeat types. The sequence length corresponding to repeat types are 41 and 42 amino acid residues and are present as two copies in BA4081. BLAST searches identified these repeats to be specific to this protein alone. (a) The sequence alignment corresponding to 41-amino-acid-residue repeat identified two characteristic sequence motifs: DLG and AGQF ( Figure 14(a) ). We refer to this as the AGQF repeat. The motif occurs at the C-terminal part of the repeat region. The sequence homology shared between this AGQF repeats is about 34%. The secondary structure corresponding to the AGQF repeat is predicted to comprise one α -helix. The representative domain architecture corresponding to protein comprising the AGQF repeat is shown in Figure 27 . (b) The sequence alignment corresponding to the 42-amino-acid-residue tandem repeat identified three characteristic sequence motifs: GYI, GSAL, and TING ( Figure 14(b) ) and is a glycine-rich repeat. We refer to this as the GSAL repeat. The sequence homology shared between this GSAL repeats is 52%. The secondary structure corresponding to the GSAL repeat is predicted to comprise one α -helix and one β -strand. The representative domain architecture corresponding to protein comprising the GSAL repeat is shown in Figure 27 . This protein is associated with a 27-amino-acid residue Ribosomal_S7 region that is sandwiched between the 41-amino-acid-residue AGQF repeat and the 42-amino-acid-residue GSAL repeat. These two repeats are specific to this protein alone and are therefore B. anthracis str. Ames specific. From the analysis of the B. anthracis proteome, we observed that the novel repeats and domains are present in all the strains, such as Ames , Ames ancestor, Sterne, and A2012, that have been sequenced so far. This indicates that these strains of B. anthracis have diverged recently. We also observed that the domains PxV, FxF, YEFF, VxxT, ExW, and VYV are present in proteins from several bacterial organisms. The domains NTGFIG, KEWE, AFL, and the repeats NxGK are specific to bacillus. It is interesting to note that the domains VYV and AFL are present in all the B. anthracis species while absent in B. cereus genomes. The repeats RIDVK, AGQF, and GSAL are also specifically present only in all the strains of B. anthracis . This analysis explains some differences in the closely related B. anthracis and B. cereus genomes. The identification of these novel domains and repeats in subsequently sequenced genomes will add value to their annotation. 4. CONCLUSIONS A systematic analysis using computational tools identified four novel repeats and ten domains corresponding to the B. anthracis str. Ames proteome. Further database searches identified that some novel repeats and domains are also present in other bacterial genomes. The NxGK repeats are associated with SAP domain. The SAP domain is a DNA-binding motif that is involved in chromosomal organization. Therefore, we believe that these repeats also participate in similar function. The YEFF domain containing proteins are associated with RGD motif and may be involved in cell adhesion. The identification of novel repeats and domains corresponding to B. anthracis proteome may be useful for annotation. From the presence of VYV and AFL domains in all the B. anthracis species and their absence in B. cereus genomes, we identified some differences in these two genomes that are otherwise closely related.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3885664/

Toxin-Independent Virulence of Bacillus anthracis in Rabbits

The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. In the guinea pig (GP) model we have previously shown that deletion of all three toxin components results in a relatively moderate attenuation in virulence, indicating that B. anthracis possesses an additional toxin-independent virulence mechanism. To characterize this toxin-independent mechanism in anthrax disease, we developed a new rabbit model by intravenous injection (IV) of B. anthracis encapsulated vegetative cells, artificially creating bacteremia. Using this model we were able to demonstrate that also in rabbits, B. anthracis mutants lacking the toxins are capable of killing the host within 24 hours. This virulent trait depends on the activity of AtxA in the presence of pXO2, as, in the absence of the toxin genes, deletion of either component abolishes virulence. Furthermore, this IV virulence depends mainly on AtxA rather than the whole pXO1. A similar pattern was shown in the GP model using subcutaneous (SC) administration of spores of the mutant strains, demonstrating the generality of the phenomenon. The virulent strains showed higher bacteremia levels and more efficient tissue dissemination; however our interpretation is that tissue dissemination per se is not the main determinant of virulence whose exact nature requires further elucidation. Introduction The tripartite toxin and the poly-D-glutamic acid capsule are considered the major virulence factors of Bacillus anthracis , the etiological agent of anthrax. The capsule, composed of poly-D-glutamic acid and encoded by the pXO2 plasmid, allows unrestrained bacilli growth in the infected host, since it inhibits phagocytosis of the vegetative cells by the innate immunity system (macrophages and neutrophils). The tripartite toxin consists of lethal factor (LF), edema factor (EF) and protective antigen (PA), (encoded by lef, cya and pag genes respectively, located in a pathogenicity island on the pXO1 plasmid). Toxic activity is expressed only when PA is combined with LF (forming the lethal toxin, LT) or EF (forming the edema toxin, ET) [1] . In vitro , toxin and capsule production are optimal when cells are grown in inductive conditions (CO 2 and 37°C) reflecting the normal mammalian host environment [1] . AtxA, a B. anthracis global regulator, encoded by atxA and located in the pathogenicity island on pXO1, was shown to mediate the interaction between environmental conditions and the metabolic state of the bacterium and the expression of the virulence factors [3] , [4] , [5] . Curing pXO1 from B. anthracis results in complete loss of virulence, indicating the possible essential role of the toxins in pathogenicity. In previous studies we evaluated the assumption that LT and ET play major roles in the pathogenicity of B. anthracis by a systematic genetic study deleting the pag , lef and cya genes and their combinations [6] , [7] , [8] . The effects of the mutations on virulence were tested in rabbits and guinea pigs (GP) using two routes of infection, subcutaneous injection (SC) and intranasal instillation (IN). The results demonstrated that while the toxins are necessary for optimal virulence (full mortality and wild type mean time to death) in all models tested, one toxin is sufficient for efficient pathogenicity. These results were corroborated in a rabbit model of inhalational anthrax [9] . In the rabbit model, the toxins play a major role in virulence, as the deletion of either pag alone or lef and cya completely attenuates the strain when introduced IN or SC, similar to the effect of pXO1 curing. On the other hand, in the GP model no major role could be attributed to the toxins. In GP, deletion of pag or the three toxin components resulted only in moderate attenuation and prolonged mean time to death (MTTD), whereas the pXO1-cured mutant showed complete attenuation. These results may indicate that B . anthracis possesses an additional virulence mechanism, exhibited in GP, which is toxin independent but pXO1 dependent [6] . In previous studies we and others have suggested that toxins play a major role in the early stages of the infection, enabling the organism to overcome innate host defenses, whereas the death of the animals relates to bacteremia and organ bacterial burden rather than systemic toxemia [6] , [9] , [10] . This assumption was further supported by the finding that passive immunization with anti-PA antibodies could prevent the establishment of disease in animals exposed to B. anthracis spores, but could not cure (and save) bacteremic animals [11] , [12] , [13] , [14] . To characterize the toxin-independent virulence trait as a possible cause of death, we established a new rabbit model; artificially creating bacteremia by intravenously injecting B. anthracis encapsulated vegetative cells. In this manuscript we show using this model that in rabbits, similarly to previous data from GP, B. anthracis mutants lacking the toxins, even though partially attenuated still maintain significant virulence, killing the host within 24 hr. We also demonstrate that this toxin independent virulence trait depends on the activity of AtxA in the presence of pXO2, as deletion of either component abolishes virulence. The same pattern was shown in the GP model using SC administration of spores of the mutant strains, demonstrating the potential generality of the phenomenon. Materials and Methods Bacterial strains, media and growth conditions Bacterial strains used in this study are listed in Table 1 . B. anthracis and Escherichia coli strains were cultivated in Terrific broth [15] at 37°C with vigorous shaking (250 rpm). For the induction of toxins and capsule production, a modified DMEM (supplemented with 10% normal rabbit serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid) was used. Sporulation was carried out using G broth, as previously described [16] . E. coli strains were used for the facilitation of plasmid construction. Antibiotic concentrations used for selection in Mueller Hinton (MH) agar (Difco)/Terrific broth were: for E. coli strains, ampicillin (Amp, 100 µg ml −1 ); for B. anthracis strains, kanamycin (Kn, 10 µg ml −1 ), and erythromycin (Ery, 5 µg ml −1 ). 10.1371/journal.pone.0084947.t001 Table 1 Bacterial strains, plasmids and oligonucleotide primers used in this study. Description/characteristics Source Strain B. anthracis Vollum ATCC 14578 IIBR collection VollumΔ pag Δ cya Δ lef Complete deletion of the pag , lef and cya genes [6] VollumΔ pag Δ cya Δ lef Δ atxA Complete deletion of the atxA gene in the VollumΔ pag Δ cya Δ lef mutant This study VollumΔ pag Δ cya Δ lef Δ bslA Complete deletion of the bslA gene in the VollumΔ pag Δ cya Δ lef mutant This study VollumΔpXO1 BA2805 :: atxA Genome insertion of the atxA gene replacing major parts of the PlyPH ( BA2805 ) [31] in the VollumΔpXO1 This study VollumΔ pag Δ cya Δ lef ΔpXO2 Curing of the pXO2 plasmid in the VollumΔ pag Δ cya Δ lef mutant This study VollumΔpXO1 Vollum pXO1−, pXO2+ IIBR Collection VollumΔpXO2 Vollum pXO1+, pXO2− IIBR Collection VollumΔpXO1ΔpXO2 Vollum pXO1−, pXO2− IIBR Collection E. coli DH5α endA1 recA1 IIBR collection GM2929 dam::Tn9 (CmR) dcm-6 NEB Plasmids pEGS Allelic replacement vector platform [8] pEGS-atxA This study pEGS-bslA This study pEGS-BA2805::atxA This study pEGS-CAP This study Primer Sequence a Complementary Position ( A UG = 1) ATX1 TCTTCAATGTCTTGTAAATTAATT −550 ATX2 tttgcggccgcTTTCTCCTGGCTTTCTTTTAGGTA −410 ATX3c tgtactagtGTCTATAATTGATTCTCCTTTCCT −23 ATX4 ataactagtATGCCCTTTAAATATTTGTTTAAT 1428 ATX5c ggcgcgccATAAAAACGACATATAAATATGTC 1900 ATX6c CTCAATAAACTCAAAACTAATTGT 2119 ATXs1 ATTAATTTACTACACTTTATCAAT 42 ATXs2 CAGTTTCATGTAATGTAACGCCGA 742 PX901 GTGGGTTAAATGGTGG −482 PX902 gacgcgcggccgcAGGATATGCCCACG −430 PX903c ggagtagtGCGTTTTCTCTGTGTGC −39 PX904 ggactagtGTAACCCTAAACC 1280 PX905c ttggcgcgccCATATATAATAGTACCTCC 2230 PX906c AACGTTTCACTTGCC 2332 2805 2 gacgcgcggccgcAAAGCACGGCTACCG −287 2805 3c ggactagtCCCATAACTTAACACCTCC 5 2805 4 ggactagtTTTCGGTATGG 381 2805 5c ttggcgcgccCGCTCCCATAACATCTGGTG 663 ATXcomp1 ataactagtTATACTCACCAAAAATTTCAAGGT −913 ATXcomp2c ataactagtTTATATTATCTTTTTGATTTCATG 27 past ter. a The homology region to the coding sequence is marked in capital letters. Plasmid and strain construction Plasmids and oligonucleotide primers used in this study are summarized in Table 1 . The oligonucleotide primers were designed according to the genomic sequence of B. anthracis Sterne strain. Genomic DNA (containing the chromosomal DNA and the native plasmids, pX01 and pX02) for cloning the target gene fragments was extracted from the Vollum strain as previously described [6] . Polymerase chain reaction (PCR) amplifications were performed using the AccuTaq LA systems (Sigma). Prior to transformation into the Vollum strain, all plasmids were first propagated in the methylation deficient E. coli strain GM2929 ( Table 1 ). B. anthracis cells were electrotransformed as described [18] . Target genes were disrupted by homologous recombination, using a previously described method [8] , [19] . In general, gene deletion was accomplished by a marker-less allelic exchange technique that replaced the complete coding region with the Spe I restriction site. At the end of the procedure the resulting mutants did not code for any foreign sequences and the only modification is the null mutation of the target gene. To construct a VollumΔpXO1 strain that encodes a genomic copy of the atxA gene, we used the plasmid pEGS 2805, which we previously used to inactivate the BA2805 in the wild type Vollum strain (no effect in virulence in guinea pigs, data not shown). The atxA gene with the upstream ORF were amplified using primers ATXcomp1 and ATXcomp2c and cloned into the Spe I site of the pEGS2805 located between the sequences with homology to the 5′ (primers 2805 2-3c) and the sequences homologous to the 3′ (primers 2805 4-5c) of the BA2805 gene. The different mutations were verified by PCR for the deletion of the target gene, and that no major rearrangements occurred in the area of the deletion. All the mutants were tested for their ability to produce capsule by incubation in modified DMEM in 10% CO 2 atmosphere. The capsule was visualized by negative staining with India ink. To cure the pXO2 from the VollumΔ pag Δ lef Δ cya mutant, the pEGS CAP was inserted, by single cross over into the capA – capD region of the pXO2 plasmid (primers 5′ gacgcgcggccgcCAAGGGGGTGAGAGG and 3′ ttggcgcgccGGGGCAGATATTATTGTGG). The resulting GFP positive clone was cultured in 2 ml terrific broth in 15 ml falcon tube at 40°C 250 rpm. Every 24 hr 1 µl of the culture was plated on LB plate and the culture was diluted 1:20 into fresh media. Dark non florescent colonies were scored following over night incubation at 37°C. These colonies were tested for erythromycin sensitivity and the presence of pXO1 and absence of pXO2 by PCR. The absence of pXO2 was confirmed by the absence of a typical capsule following induction, as previously described. The genotype and phenotype of the VollumΔpXO1 or ΔpXO2 were verified by PCR, capsule production and ELISA for the secretion of PA. In in-vitro studies we demonstrated that the growth and sporulation profiles (data not shown) as well as capsule production of all mutants were indistinguishable from those observed for the parental wild type Vollum strain DNA preparation and PCR DNA was purified from Bacillus cultures or colony collections as described previously [17] . For fast colony screening, each colony was resuspended in 50 µl of sterile double distilled water (DDW) in a 0.2 ml PCR tube. The tube was then placed in a thermocycler for two cycles of 95°C for 10 min. The tubes were then centrifuged in a minifuge, at maximal speed for 1 min at room temperature. The clear supernatant was transferred to a clean tube and 5 µl were used for the PCR reactions. All PCR reactions (25 µl) were performed in 1xPCR buffer (3.5 mM MgCl2); dNTPs (0.2 mM each); 0.04 U/μl of TaKaRaTaq DNA polymerase (all from TaKaRa Bio Inc. R001A) and ∼2 ng of DNA. The general thermocycling program for the PCR reaction was 95°C for 30 sec followed by 40 cycles of 94°C for 1 min; 55°C for 30 sec; 72°C for 1 min, and then one cycle of 72°C for 5 min. The PCR products were separated on 1.1% agarose gel using 1× TBE as running buffer. Infection of rabbits and guinea pigs Female New Zealand white rabbits (Charles River Laboratories or Harlan Laboratories), 2.2–2.5 kg were used to test the virulence of the wild-type and mutant Vollum strains. Spores were germinated by incubation in Terrific broth for 1 hr at 37°C, and then incubated in modified DMEM in 10% CO 2 atmosphere for 2 hr at 37°C, to induce capsule formation. The capsule was visualized by negative staining with India ink. The capsulated vegetative cells were injected IV via the ear vein and a remaining sample was plated for total viable counts (CFU/ml). The animals were observed daily for 14 days or for the indicated period. Upon death, blood samples were plated and DNA was extracted, followed by PCR analysis in order to determine the identity of the strain responsible for the animals' death. Female Hartley guinea pigs (Charles River Laboratories), weighing 220–250 g were used. The animals were infected with spore preparations of either the mutant strains or the parental Vollum strain. Prior to infecting the animals, the spore preparations were heat-shocked (70°C, 20 min) and serially diluted in saline to produce spore suspensions within the range 10 2 –10 9 per milliliter. A spore dose of 0.1 ml was administered subcutaneously (SC) to each animal. The remaining spore dose suspensions were plated for total viable counts (CFU/ml). The animals were observed daily for 14 days or for the indicated period. Upon death, blood samples were plated and DNA was extracted, followed by PCR analysis in order to confirm the identity of the strain responsible for the animals' death. This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Research Council. The protocols were approved by the Committee on the Ethics of Animal Experiments of the IIBR (permit numbers GP-08-2012, RB-06-2012, RB-24-2012, RB-25-2013). Animals were euthanized when one of the following symptoms was detected: severe respiratory distress or the loss of righting reflex. Guinea pigs were sacrificed by CO 2 inhalation and rabbits by the sodium pentabarbitone injection. Determination of bacterial burden in tissues Infected rabbits were euthanized either 5 or 24 hr post-infection with Pental (sodium pentobarbitone) and various organs were harvested. The organs: spleen, lungs, brain, kidneys and liver, were immediately homogenized and serial dilutions of the homogenate were plated on agar plates to determine the bacterial level. Production of convalescent sera and passive immunization Convalescent sera were prepared as previously described [21] . Rabbits were intranasally inoculated with 10 7 Vollum spores. Thirty hours later the rabbits were bled for bacteremia determination and immediately afterwards antibiotic treatment (ciprofloxacin) was initiated. Rabbits that were bacteremic at the beginning of treatment and survived the antibiotic treatment were bled 30 days post inoculation and the convalescent serum was frozen till use. Rabbits prior to inoculation and rabbits that did not show any bacteremia at the beginning of treatment were bled to prepare control sera. For passive immunization, 20 ml of sera were injected IV to naïve rabbits 2 hours prior to IV inoculation with capsulated vegetative Vollum wild type or mutant cells. Since the challenge strain is missing the toxins – LF, EF and PA, the presence of antibodies against these proteins does not have any neutralizing effect. Protein extraction and Immunoblotting Spores (2.5×10 7 CFU) were germinated by incubation in Terrific broth for 1 hr at 37°C, and then incubated in 10 ml modified DMEM in 10% CO 2 atmosphere for 16 hr at 37°C. The culture pellet was resuspended in 0.5 ml of 6× protein loading buffer and incubated of 15 min in 99°C. 20 µl of the clear supernatant was loaded on 10% SDS PAGE, transferred to PVDF membrane and blotted with specific antisera. Statistical analysis The vegetative bacteria lethal dose required to kill 50% of the animals (LD 50 ) was calculated by the method of Reed and Muench [20] . The bacteremia and bacterial organ load were grouped according to the sample time (5 or 24 hr post infection) and whether the infection was lethal or not. The groups were plotted as the distribution of each of the animals in the group (scattered). The significance of the differences in the blood bacterial burden between the virulent and non virulent mutants was determined by t test comparing the log10 values of the CFU/ml, using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com ). The mean time to death (MTTD) was calculated for each mutant as the sum of the days till death of all the animals that succumbed divided by their number. No score was given to animals that survived the infection. Bacterial strains, media and growth conditions Bacterial strains used in this study are listed in Table 1 . B. anthracis and Escherichia coli strains were cultivated in Terrific broth [15] at 37°C with vigorous shaking (250 rpm). For the induction of toxins and capsule production, a modified DMEM (supplemented with 10% normal rabbit serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid) was used. Sporulation was carried out using G broth, as previously described [16] . E. coli strains were used for the facilitation of plasmid construction. Antibiotic concentrations used for selection in Mueller Hinton (MH) agar (Difco)/Terrific broth were: for E. coli strains, ampicillin (Amp, 100 µg ml −1 ); for B. anthracis strains, kanamycin (Kn, 10 µg ml −1 ), and erythromycin (Ery, 5 µg ml −1 ). 10.1371/journal.pone.0084947.t001 Table 1 Bacterial strains, plasmids and oligonucleotide primers used in this study. Description/characteristics Source Strain B. anthracis Vollum ATCC 14578 IIBR collection VollumΔ pag Δ cya Δ lef Complete deletion of the pag , lef and cya genes [6] VollumΔ pag Δ cya Δ lef Δ atxA Complete deletion of the atxA gene in the VollumΔ pag Δ cya Δ lef mutant This study VollumΔ pag Δ cya Δ lef Δ bslA Complete deletion of the bslA gene in the VollumΔ pag Δ cya Δ lef mutant This study VollumΔpXO1 BA2805 :: atxA Genome insertion of the atxA gene replacing major parts of the PlyPH ( BA2805 ) [31] in the VollumΔpXO1 This study VollumΔ pag Δ cya Δ lef ΔpXO2 Curing of the pXO2 plasmid in the VollumΔ pag Δ cya Δ lef mutant This study VollumΔpXO1 Vollum pXO1−, pXO2+ IIBR Collection VollumΔpXO2 Vollum pXO1+, pXO2− IIBR Collection VollumΔpXO1ΔpXO2 Vollum pXO1−, pXO2− IIBR Collection E. coli DH5α endA1 recA1 IIBR collection GM2929 dam::Tn9 (CmR) dcm-6 NEB Plasmids pEGS Allelic replacement vector platform [8] pEGS-atxA This study pEGS-bslA This study pEGS-BA2805::atxA This study pEGS-CAP This study Primer Sequence a Complementary Position ( A UG = 1) ATX1 TCTTCAATGTCTTGTAAATTAATT −550 ATX2 tttgcggccgcTTTCTCCTGGCTTTCTTTTAGGTA −410 ATX3c tgtactagtGTCTATAATTGATTCTCCTTTCCT −23 ATX4 ataactagtATGCCCTTTAAATATTTGTTTAAT 1428 ATX5c ggcgcgccATAAAAACGACATATAAATATGTC 1900 ATX6c CTCAATAAACTCAAAACTAATTGT 2119 ATXs1 ATTAATTTACTACACTTTATCAAT 42 ATXs2 CAGTTTCATGTAATGTAACGCCGA 742 PX901 GTGGGTTAAATGGTGG −482 PX902 gacgcgcggccgcAGGATATGCCCACG −430 PX903c ggagtagtGCGTTTTCTCTGTGTGC −39 PX904 ggactagtGTAACCCTAAACC 1280 PX905c ttggcgcgccCATATATAATAGTACCTCC 2230 PX906c AACGTTTCACTTGCC 2332 2805 2 gacgcgcggccgcAAAGCACGGCTACCG −287 2805 3c ggactagtCCCATAACTTAACACCTCC 5 2805 4 ggactagtTTTCGGTATGG 381 2805 5c ttggcgcgccCGCTCCCATAACATCTGGTG 663 ATXcomp1 ataactagtTATACTCACCAAAAATTTCAAGGT −913 ATXcomp2c ataactagtTTATATTATCTTTTTGATTTCATG 27 past ter. a The homology region to the coding sequence is marked in capital letters. Plasmid and strain construction Plasmids and oligonucleotide primers used in this study are summarized in Table 1 . The oligonucleotide primers were designed according to the genomic sequence of B. anthracis Sterne strain. Genomic DNA (containing the chromosomal DNA and the native plasmids, pX01 and pX02) for cloning the target gene fragments was extracted from the Vollum strain as previously described [6] . Polymerase chain reaction (PCR) amplifications were performed using the AccuTaq LA systems (Sigma). Prior to transformation into the Vollum strain, all plasmids were first propagated in the methylation deficient E. coli strain GM2929 ( Table 1 ). B. anthracis cells were electrotransformed as described [18] . Target genes were disrupted by homologous recombination, using a previously described method [8] , [19] . In general, gene deletion was accomplished by a marker-less allelic exchange technique that replaced the complete coding region with the Spe I restriction site. At the end of the procedure the resulting mutants did not code for any foreign sequences and the only modification is the null mutation of the target gene. To construct a VollumΔpXO1 strain that encodes a genomic copy of the atxA gene, we used the plasmid pEGS 2805, which we previously used to inactivate the BA2805 in the wild type Vollum strain (no effect in virulence in guinea pigs, data not shown). The atxA gene with the upstream ORF were amplified using primers ATXcomp1 and ATXcomp2c and cloned into the Spe I site of the pEGS2805 located between the sequences with homology to the 5′ (primers 2805 2-3c) and the sequences homologous to the 3′ (primers 2805 4-5c) of the BA2805 gene. The different mutations were verified by PCR for the deletion of the target gene, and that no major rearrangements occurred in the area of the deletion. All the mutants were tested for their ability to produce capsule by incubation in modified DMEM in 10% CO 2 atmosphere. The capsule was visualized by negative staining with India ink. To cure the pXO2 from the VollumΔ pag Δ lef Δ cya mutant, the pEGS CAP was inserted, by single cross over into the capA – capD region of the pXO2 plasmid (primers 5′ gacgcgcggccgcCAAGGGGGTGAGAGG and 3′ ttggcgcgccGGGGCAGATATTATTGTGG). The resulting GFP positive clone was cultured in 2 ml terrific broth in 15 ml falcon tube at 40°C 250 rpm. Every 24 hr 1 µl of the culture was plated on LB plate and the culture was diluted 1:20 into fresh media. Dark non florescent colonies were scored following over night incubation at 37°C. These colonies were tested for erythromycin sensitivity and the presence of pXO1 and absence of pXO2 by PCR. The absence of pXO2 was confirmed by the absence of a typical capsule following induction, as previously described. The genotype and phenotype of the VollumΔpXO1 or ΔpXO2 were verified by PCR, capsule production and ELISA for the secretion of PA. In in-vitro studies we demonstrated that the growth and sporulation profiles (data not shown) as well as capsule production of all mutants were indistinguishable from those observed for the parental wild type Vollum strain DNA preparation and PCR DNA was purified from Bacillus cultures or colony collections as described previously [17] . For fast colony screening, each colony was resuspended in 50 µl of sterile double distilled water (DDW) in a 0.2 ml PCR tube. The tube was then placed in a thermocycler for two cycles of 95°C for 10 min. The tubes were then centrifuged in a minifuge, at maximal speed for 1 min at room temperature. The clear supernatant was transferred to a clean tube and 5 µl were used for the PCR reactions. All PCR reactions (25 µl) were performed in 1xPCR buffer (3.5 mM MgCl2); dNTPs (0.2 mM each); 0.04 U/μl of TaKaRaTaq DNA polymerase (all from TaKaRa Bio Inc. R001A) and ∼2 ng of DNA. The general thermocycling program for the PCR reaction was 95°C for 30 sec followed by 40 cycles of 94°C for 1 min; 55°C for 30 sec; 72°C for 1 min, and then one cycle of 72°C for 5 min. The PCR products were separated on 1.1% agarose gel using 1× TBE as running buffer. Infection of rabbits and guinea pigs Female New Zealand white rabbits (Charles River Laboratories or Harlan Laboratories), 2.2–2.5 kg were used to test the virulence of the wild-type and mutant Vollum strains. Spores were germinated by incubation in Terrific broth for 1 hr at 37°C, and then incubated in modified DMEM in 10% CO 2 atmosphere for 2 hr at 37°C, to induce capsule formation. The capsule was visualized by negative staining with India ink. The capsulated vegetative cells were injected IV via the ear vein and a remaining sample was plated for total viable counts (CFU/ml). The animals were observed daily for 14 days or for the indicated period. Upon death, blood samples were plated and DNA was extracted, followed by PCR analysis in order to determine the identity of the strain responsible for the animals' death. Female Hartley guinea pigs (Charles River Laboratories), weighing 220–250 g were used. The animals were infected with spore preparations of either the mutant strains or the parental Vollum strain. Prior to infecting the animals, the spore preparations were heat-shocked (70°C, 20 min) and serially diluted in saline to produce spore suspensions within the range 10 2 –10 9 per milliliter. A spore dose of 0.1 ml was administered subcutaneously (SC) to each animal. The remaining spore dose suspensions were plated for total viable counts (CFU/ml). The animals were observed daily for 14 days or for the indicated period. Upon death, blood samples were plated and DNA was extracted, followed by PCR analysis in order to confirm the identity of the strain responsible for the animals' death. This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Research Council. The protocols were approved by the Committee on the Ethics of Animal Experiments of the IIBR (permit numbers GP-08-2012, RB-06-2012, RB-24-2012, RB-25-2013). Animals were euthanized when one of the following symptoms was detected: severe respiratory distress or the loss of righting reflex. Guinea pigs were sacrificed by CO 2 inhalation and rabbits by the sodium pentabarbitone injection. Determination of bacterial burden in tissues Infected rabbits were euthanized either 5 or 24 hr post-infection with Pental (sodium pentobarbitone) and various organs were harvested. The organs: spleen, lungs, brain, kidneys and liver, were immediately homogenized and serial dilutions of the homogenate were plated on agar plates to determine the bacterial level. Production of convalescent sera and passive immunization Convalescent sera were prepared as previously described [21] . Rabbits were intranasally inoculated with 10 7 Vollum spores. Thirty hours later the rabbits were bled for bacteremia determination and immediately afterwards antibiotic treatment (ciprofloxacin) was initiated. Rabbits that were bacteremic at the beginning of treatment and survived the antibiotic treatment were bled 30 days post inoculation and the convalescent serum was frozen till use. Rabbits prior to inoculation and rabbits that did not show any bacteremia at the beginning of treatment were bled to prepare control sera. For passive immunization, 20 ml of sera were injected IV to naïve rabbits 2 hours prior to IV inoculation with capsulated vegetative Vollum wild type or mutant cells. Since the challenge strain is missing the toxins – LF, EF and PA, the presence of antibodies against these proteins does not have any neutralizing effect. Protein extraction and Immunoblotting Spores (2.5×10 7 CFU) were germinated by incubation in Terrific broth for 1 hr at 37°C, and then incubated in 10 ml modified DMEM in 10% CO 2 atmosphere for 16 hr at 37°C. The culture pellet was resuspended in 0.5 ml of 6× protein loading buffer and incubated of 15 min in 99°C. 20 µl of the clear supernatant was loaded on 10% SDS PAGE, transferred to PVDF membrane and blotted with specific antisera. Statistical analysis The vegetative bacteria lethal dose required to kill 50% of the animals (LD 50 ) was calculated by the method of Reed and Muench [20] . The bacteremia and bacterial organ load were grouped according to the sample time (5 or 24 hr post infection) and whether the infection was lethal or not. The groups were plotted as the distribution of each of the animals in the group (scattered). The significance of the differences in the blood bacterial burden between the virulent and non virulent mutants was determined by t test comparing the log10 values of the CFU/ml, using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com ). The mean time to death (MTTD) was calculated for each mutant as the sum of the days till death of all the animals that succumbed divided by their number. No score was given to animals that survived the infection. Results Establishment of the IV inoculation model To characterize the toxin-independent virulence trait as a possible cause of death, we established an IV inoculation model in rabbits. Vegetative encapsulated bacilli were cultured in inductive medium (M&M) and injected IV to test their ability to cause lethal disease. Upon IV inoculation, the encapsulated vegetative bacteria disperse and are diluted in the circulation. The bacterial dose was calculated, according to an estimate of blood volume of 10% of the body weight, to a final concentration in the range of 10 4 CFU/ml, that we have previously shown to be treatable by antibiotics [21] . In reality, 30 minutes post inoculation the bacteremia detected was about a tenth of the expected concentration (data not shown), suggesting rapid clearance from circulation. Additional doses were used for the different mutants depending on the strains' virulence. The results, shown in Figure 1A , demonstrate that injection of the fully virulent Vollum strain (pXO1+pXO2+) caused death within 24–48 hr, whereas the VollumΔpXO1ΔpXO2 strain was completely avirulent in the rabbit model. IV inoculation of encapsulated vegetative Vollum bacilli resulting in bacterial levels of less than 1 CFU/ml (total dose of ∼10 CFU) is sufficient to cause lethality in rabbits ( Table S1 ). 10.1371/journal.pone.0084947.g001 Figure 1 Virulence of mutants or wild type Vollum strains following IV inoculation of rabbits. Rabbits were inoculated IV with vegetative cells of the wild type and mutants strains. A . Survival of rabbits inoculated with the fully virulent Vollum (n = 4), the capsular VollumΔpXO1 (n = 4), toxinogenic VollumΔpXO2 (n = 6) or the non-capsular non-toxinogenic VollumΔpXO1ΔpXO2 strains (n = 4). Strain name and inculcation doses are as marked. B . Survival of rabbits inoculated with different doses of the wild type Vollum (n = 2 to 4) or the VollumΔ pag Δ lef Δ cya (n = 3 to 4) strains (also see Table S1 ). Are B. anthracis toxins essential for virulence? Using the IV inoculation model, we evaluated the involvement of pXO1 and the toxins in the bacterial capacity to cause death in inoculated rabbits. As can be seen in Figure 1A , curing pXO1 from B. anthracis results in the complete loss of virulence. On the other hand, the mutant strain lacking toxins but containing pXO1 (VollumΔ pag Δ cya Δ lef ) shows significant virulence ( Figure 1B ), efficiently killing the IV inoculated rabbits. This mutant exhibited strong attenuation compared to the wild type (a difference in LD50 of >4 orders of magnitude; LD 50 mut = 2×10 5 vs. LD 50 wt = 14 Vollum ΔpXO1 pXO1 − pXO2 + 10 8 0/5 >14 Vollum Δ pag Δ cya Δ lef Complete deletion of the pag, lef and cya genes 10 6 2/3 2.5 10 5 4/6 5.75 Vollum Δ pag Δ cya Δ lef Δ atxA Complete deletion of the pag, lef , cya and atxA genes 10 8 0/4 >14 10 7 0/4 Vollum ΔpXO2Δ pag Δ cya Δ lef Complete deletion of pXO2 and the pag, lef and cya genes 10 8 0/4 >14 Vollum ΔpXO2 pXO1 + pXO2 − 10 8 4/5 7.75 10 7 0/5 >14 For the sake of completeness, the toxinogenic non-capsulated strain, VollumΔpXO2, or VollumΔ atxA which are completely attenuated when administrated SC, were also tested using this model. As can be seen in Figure 1A and Table S2 , both mutants caused the death of 50% of the injected rabbits, with a longer MTTD. In an additional attempt to elucidate the underlying virulence mechanism, exploring the possibility of protection using antibodies, we attempted passive immunization using sera from convalescent rabbits (material and methods). It is our experience that this serum contain antibodies against cellular and secreted components, conferring full protection to the convalescent rabbit from a challenge with the wild type Vollum strain spore [21] . As the deletion of all toxin components does not affect virulence, we assumed that the accepted PA-based vaccination (or passive immunization with sera from a PA-vaccinated animal) is not applicable in this model. While infecting naïve rabbits IV with 10 7 VollumΔ pag Δ cya Δ lef encapsulated vegetative cells results in death within 24 hr, the described passive immunization with sera from convalescent rabbits ( Figure 3 A ) was able to fully protect rabbits from the same infection dose. This passive protection was specific to convalescent sera since pretreatment with pre- immune sera (or sera from rabbits that were exposed to spores and prophylacticly treated with antibiotics) did not confer protection under the same IV vegetative cell challenge conditions (n = 4). 10.1371/journal.pone.0084947.g003 Figure 3 Passive protection of rabbits using convalescent sera against IV challenge with the VollumΔ pag Δ cya Δ lef mutant. A . 20 ml of sterile convalescent or control sera were administered IV to rabbits (n = 4) three hours before an IV challenge with 5×10 6 CFU of the vegetative VollumΔ pag Δ cya Δ lef mutant cells. B . Cellular antigens recognized by the protective sera. Proteins were extracted by boiling of different encapsulated B. anthracis mutants. The blots were immune-blotted by a representative protective serum. 1. VollumΔpXO1. 2. VollumΔ pag Δ cya Δ lef . 3. VollumΔ pag Δ cya Δ lef Δ atxA . 4. VollumΔ pag Δ cya Δ lef Δ bslA . The size marker in kDa is marked on the left. The arrowhead marks the location of the BslA protein. In a previous study [6] we have shown that GP are susceptible to the spores of the VollumΔ pag Δ cya Δ lef strain, similar to the finding with the rabbit IV model. Therefore, using the GP SC spore injection model, we decided to test whether a similar dependence of virulence on AtxA activity can be demonstrated. As can be seen in Table 2 , there is a clear parallel pattern in the virulence exhibited by the various Vollum strains in the IV rabbit model and the GP SC model. In both models, the virulence of the Vollum strains is dependent on the presence of atxA on the pXO1 plasmid, but does not require the toxins. Here again, it seems that both AtxA and the pXO2 plasmid are required for the efficient exertion of virulence, as deletion of pXO2 abolishes virulence ( Table 2 , Table S1 ). These results raised the question whether the difference between rabbit and GP reflects a fundamental difference between administration routes or rather a sensitivity difference between animal models. In order to clarify this point we tested the susceptibility of rabbits to SC injection of high doses of VollumΔ pag Δ cya Δ lef spores. Whereas a dose of 10 7 CFU spores injected SC did not kill the infected rabbits (0/8), an inoculum of 10 8 VollumΔ pag Δ cya Δ lef spores resulted in a lethality rate of 75% (6/8). Therefore, we can conclude that while GP are more susceptible than rabbits, the basic mechanisms can be demonstrated in both models. Does capsule production and function reflect pXO2 activation? The observed differences in virulence between the Vollum strains may be related to AtxA exerting its activity on pXO2. The main function of pXO2 in virulence is assumed to be the generation of the capsule, which inhibits phagocytosis of vegetative cells by innate immune system cells, like macrophages and neutrophils. Therefore we compared the various B. anthracis strains for their ability to produce/generate the capsule, and the survival of their encapsulated vegetative cells in the circulation. As can be seen in Figure 4 , no qualitative difference could be detected in capsule morphology (following two hours incubation in the inductive growth media) between the virulent strains Vollum-wt and VollumΔ pag Δ cya Δ lef , and the non-virulent strains, VollumΔ pag Δ cya Δ lef Δ atxA and VollumΔpXO1. 10.1371/journal.pone.0084947.g004 Figure 4 Capsule production by the different mutants. Vegetative bacteria were incubated for 2 hs in inductive medium, and the capsules were negatively stained with India ink. No difference could be detected between virulent strains and non-virulent strains. (A) Vollum-wt, (B) VollumΔ pag Δ cya Δ lef , (C) VollumΔ pag Δ cya Δ lef Δ atxA, (D) VollumΔpXO1. On the other hand, determination of bacteremia in the rabbit circulation 5 hr and 22–24 hr post IV injection with inoculums of 10 7 CFU ( Figure 5 ) shows a clear tendency of the virulent strains (red shapes) to maintain higher bacterial levels (P = 0.0092) than the non-virulent strains (blue shapes). In all rabbits tested ( Figure 5 ), both virulent strains -Vollum-wt and VollumΔ pag Δ cya Δ lef , showed higher bacteremia (>10 3 CFU/ml) than the non-virulent strains - VollumΔ pag Δ cya Δ lef Δ atxA and VollumΔpXO1 (4 orders of magnitude; LD 50 mut = 2×10 5 vs. LD 50 wt = 14 Vollum ΔpXO1 pXO1 − pXO2 + 10 8 0/5 >14 Vollum Δ pag Δ cya Δ lef Complete deletion of the pag, lef and cya genes 10 6 2/3 2.5 10 5 4/6 5.75 Vollum Δ pag Δ cya Δ lef Δ atxA Complete deletion of the pag, lef , cya and atxA genes 10 8 0/4 >14 10 7 0/4 Vollum ΔpXO2Δ pag Δ cya Δ lef Complete deletion of pXO2 and the pag, lef and cya genes 10 8 0/4 >14 Vollum ΔpXO2 pXO1 + pXO2 − 10 8 4/5 7.75 10 7 0/5 >14 For the sake of completeness, the toxinogenic non-capsulated strain, VollumΔpXO2, or VollumΔ atxA which are completely attenuated when administrated SC, were also tested using this model. As can be seen in Figure 1A and Table S2 , both mutants caused the death of 50% of the injected rabbits, with a longer MTTD. In an additional attempt to elucidate the underlying virulence mechanism, exploring the possibility of protection using antibodies, we attempted passive immunization using sera from convalescent rabbits (material and methods). It is our experience that this serum contain antibodies against cellular and secreted components, conferring full protection to the convalescent rabbit from a challenge with the wild type Vollum strain spore [21] . As the deletion of all toxin components does not affect virulence, we assumed that the accepted PA-based vaccination (or passive immunization with sera from a PA-vaccinated animal) is not applicable in this model. While infecting naïve rabbits IV with 10 7 VollumΔ pag Δ cya Δ lef encapsulated vegetative cells results in death within 24 hr, the described passive immunization with sera from convalescent rabbits ( Figure 3 A ) was able to fully protect rabbits from the same infection dose. This passive protection was specific to convalescent sera since pretreatment with pre- immune sera (or sera from rabbits that were exposed to spores and prophylacticly treated with antibiotics) did not confer protection under the same IV vegetative cell challenge conditions (n = 4). 10.1371/journal.pone.0084947.g003 Figure 3 Passive protection of rabbits using convalescent sera against IV challenge with the VollumΔ pag Δ cya Δ lef mutant. A . 20 ml of sterile convalescent or control sera were administered IV to rabbits (n = 4) three hours before an IV challenge with 5×10 6 CFU of the vegetative VollumΔ pag Δ cya Δ lef mutant cells. B . Cellular antigens recognized by the protective sera. Proteins were extracted by boiling of different encapsulated B. anthracis mutants. The blots were immune-blotted by a representative protective serum. 1. VollumΔpXO1. 2. VollumΔ pag Δ cya Δ lef . 3. VollumΔ pag Δ cya Δ lef Δ atxA . 4. VollumΔ pag Δ cya Δ lef Δ bslA . The size marker in kDa is marked on the left. The arrowhead marks the location of the BslA protein. In a previous study [6] we have shown that GP are susceptible to the spores of the VollumΔ pag Δ cya Δ lef strain, similar to the finding with the rabbit IV model. Therefore, using the GP SC spore injection model, we decided to test whether a similar dependence of virulence on AtxA activity can be demonstrated. As can be seen in Table 2 , there is a clear parallel pattern in the virulence exhibited by the various Vollum strains in the IV rabbit model and the GP SC model. In both models, the virulence of the Vollum strains is dependent on the presence of atxA on the pXO1 plasmid, but does not require the toxins. Here again, it seems that both AtxA and the pXO2 plasmid are required for the efficient exertion of virulence, as deletion of pXO2 abolishes virulence ( Table 2 , Table S1 ). These results raised the question whether the difference between rabbit and GP reflects a fundamental difference between administration routes or rather a sensitivity difference between animal models. In order to clarify this point we tested the susceptibility of rabbits to SC injection of high doses of VollumΔ pag Δ cya Δ lef spores. Whereas a dose of 10 7 CFU spores injected SC did not kill the infected rabbits (0/8), an inoculum of 10 8 VollumΔ pag Δ cya Δ lef spores resulted in a lethality rate of 75% (6/8). Therefore, we can conclude that while GP are more susceptible than rabbits, the basic mechanisms can be demonstrated in both models. Does capsule production and function reflect pXO2 activation? The observed differences in virulence between the Vollum strains may be related to AtxA exerting its activity on pXO2. The main function of pXO2 in virulence is assumed to be the generation of the capsule, which inhibits phagocytosis of vegetative cells by innate immune system cells, like macrophages and neutrophils. Therefore we compared the various B. anthracis strains for their ability to produce/generate the capsule, and the survival of their encapsulated vegetative cells in the circulation. As can be seen in Figure 4 , no qualitative difference could be detected in capsule morphology (following two hours incubation in the inductive growth media) between the virulent strains Vollum-wt and VollumΔ pag Δ cya Δ lef , and the non-virulent strains, VollumΔ pag Δ cya Δ lef Δ atxA and VollumΔpXO1. 10.1371/journal.pone.0084947.g004 Figure 4 Capsule production by the different mutants. Vegetative bacteria were incubated for 2 hs in inductive medium, and the capsules were negatively stained with India ink. No difference could be detected between virulent strains and non-virulent strains. (A) Vollum-wt, (B) VollumΔ pag Δ cya Δ lef , (C) VollumΔ pag Δ cya Δ lef Δ atxA, (D) VollumΔpXO1. On the other hand, determination of bacteremia in the rabbit circulation 5 hr and 22–24 hr post IV injection with inoculums of 10 7 CFU ( Figure 5 ) shows a clear tendency of the virulent strains (red shapes) to maintain higher bacterial levels (P = 0.0092) than the non-virulent strains (blue shapes). In all rabbits tested ( Figure 5 ), both virulent strains -Vollum-wt and VollumΔ pag Δ cya Δ lef , showed higher bacteremia (>10 3 CFU/ml) than the non-virulent strains - VollumΔ pag Δ cya Δ lef Δ atxA and VollumΔpXO1 (<10 3 CFU/ml) at the two time points tested. Corroborating this finding, IV inoculation with VollumΔ pag Δ cya Δ lef encapsulated vegetative cells of rabbits passively immunized with sera from convalescent rabbits (using fully protective conditions, as described above), showed a decreased bacteremia, similar to the non-virulent strains. These differences in the levels of circulating bacteria could affect the dissemination of the bacteria to different host tissues. 10.1371/journal.pone.0084947.g005 Figure 5 Bacteremia following intravenous injection of vegetative cells of the different mutants. Vegetative capsulated bacteria (10 7 ) were inoculated IV and the bacteremia levels in individual animals, were determined 5 and 24 hr post injection. Virulent strains (Vollum-wt - red square and Vollum-Δ pag Δ cya Δ lef - red circle) showed higher bacteremia than non-virulent strains (VollumΔ pag Δ cya Δ lef Δ atxA - blue triangle and VollumΔpXO1 – inverted blue triangle). Does bacterial dissemination reflect variations in virulence? To determine whether the observed differences in circulating bacteremia between the B. anthracis strains reflect a difference in their ability to spread systemically, a small-scale study was conducted to compare the bacterial burden in the different tissues at two different time points. Rabbits, IV inoculated with 10 7 CFU of the Vollum strains, were sacrificed 5 hr and 24 hr p.i. and the bacterial content of the spleen, lungs, brain, liver and kidneys was determined. As can be seen in Figure 6 , virulent strains seem to exhibit higher tissue bacterial burdens than the non-virulent strains, creating seemingly similar pattern to that seen in circulating bacteria levels. 10.1371/journal.pone.0084947.g006 Figure 6 Organ bacterial load following intravenous injection of vegetative cells of the different mutants. Vegetative capsulated bacteria (10 7 ) were inoculated IV and the bacterial burden, were determined 5 and 24 hr post injection at various tissues of individual animals. Virulent strains (Vollum-wt - red square and Vollum-Δ pag Δ cya Δ lef - red circle) showed a tendency to exhibit higher bacterial levels than non-virulent strains (VollumΔ pag Δ cya Δ lef Δ atxA - blue triangle and VollumΔpXO1 – inverted blue triangle). These findings indicate that the virulence of B. anthracis strains may be related to their ability to spread to different tissues. Questioning the relevance of this assumption, we found that passive immunization with sera from convalescent rabbits, while saving naïve rabbits inoculated IV with VollumΔ pag Δ cya Δ lef encapsulated vegetative cells, did not seem to significantly affect bacterial tissue dissemination (data not shown). Based on the findings of the passive immunization, an attempt was made to identify the antigen responsible for the protective activity of the sera of convalescent rabbits. Bacterial extracts were prepared from virulent and non-virulent strains and were compared by Western-blotting with protective sera. As can be seen in Figure 3B , the protective sera recognized among others, a distinct major band of about 70 Kd present in the VollumΔ pag Δ cya Δ lef extract but not in the Vollum ΔpXO1 and VollumΔ pag Δ cya Δ lef Δ atxA extracts. Deletion of the pXO1-90 (BslA – B. anthracis S-layer protein A), which is regulated by AtxA, from the VollumΔ pag Δ cya Δ lef resulted in the disappearance of this band from the Western-blot ( Figure 3B ). This mutation (Δ bslA ) only slightly attenuated the pathogenicity of the mutant ( Table S1 ) and did not affect tissue dissemination (data not shown). The finding that the toxin-independent virulent trait is not mediated by BslA is corroborated by the finding that AtxA is the only pXO1 gene required for the exhibition of this virulence (see below). Is pXO1 essential for the display of the toxin-independent virulence? The toxin-independent virulent trait, exhibited following IV inoculation of encapsulated VollumΔ pag Δ cya Δ lef vegetative cells, depends on the activity of AtxA in the presence of pXO2, as deletion of either component abolishes virulence ( Figure 7 ). This finding can be interpreted as if the AtxA-dependent virulence results from its regulation of pXO2-borne elements, rather than regulating pXO1- and genome-borne elements. The non-essential role of pXO1 in this virulent trait was demonstrated by inserting the atxA gene into the genome of the non-virulent Vollum ΔpXO1, creating the VollumΔ pXO 1 ba2805::atxA . This insertion resulted in the recovery of the virulent trait ( Figure 7 , Table S2 ). These results demonstrate that atxA , in the background of pXO2, is sufficient for the exhibition of the toxin-independent virulent trait. 10.1371/journal.pone.0084947.g007 Figure 7 Genomic copy of atxA restores IV virulence to the VollumΔpXO1 mutant. The atxA gene with the necessary upstream regulatory elements were inserted into the VollumΔpXO1 strain replacing the BA2805 ORF. Rabbits (n = 4) were inoculated IV with 1×10 7 CFU of vegetative cells of the different mutants (also see Table S2 ). Discussion The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. This hypothesis was based mainly on studies carried out in mice using the attenuated Sterne strain (pXO1 + pXO2 − ). In previous studies, we tested this assumption by a systematic genetic study deleting the toxin genes in a fully virulent strain, testing the virulence of the mutant strains in GP and rabbits. In both models, full virulence requires both toxins, LT and ET, but either one is sufficient for virulence [7] , [8] . In the GP SC infection model, deletion of the all three toxin components, pag, lef and cya genes, results only in a relative moderate attenuation (approximately a hundred fold increase in LD 50 ). These findings suggest that B. anthracis possesses an additional toxin independent virulence mechanism, since significant residual virulence is exhibited after fully deleting toxin components. Both mechanisms are pXO1 dependent, as the encapsulated non-toxinogenic (pXO1 − pXO2 + ) mutant shows complete attenuation. On the other hand, rabbits were shown to be less sensitive to this toxin-independent virulence mechanism with toxins still required for the establishment of an effective (lethal) infection [6] . The ability of toxin-deficient mutants to effectively kill experimental hosts, coupled with abundant data regarding the function of these toxin on various systems, and especially the immune system, leads to the hypothesis that the toxins play a major role in the early stages of infection. These include the initial confrontation of the spores/bacteria with innate host defenses and their neutralization/evasion by the pathogen [10] . In order to test this hypothesis, we developed an assay designed to bypass these stages. Intravenous inoculation with encapsulated vegetative bacteria artificially creates bacteremia that resembles the septic stage of the disease, allowing the hematogenous spread of the bacilli and causing lethality in rabbits. IV injection of Vollum-wt encapsulated vegetative bacilli leads to death of infected rabbits in a time course similar to that seen during the bacteremic phase of intranasal- or SC-initiated infections [22] . This finding indicates that the development of bacteremia and hematogenous spread are an intermediate step in the progression of the anthrax disease, validating the significance of this assay. In addition, we observed that the results obtained with the rabbit IV inoculation assay parallel those obtained for GP infected SC. Therefore, the IV inoculation assay was used for initial characterization of the factors involved in the regulation of the toxin-independent virulence mechanism. In both models, our results demonstrate that curing pXO1 from B. anthracis results in complete loss of virulence. On the other hand, deletion of the toxin genes, pag, lef and cya , from pXO1 of Vollum-wt (creating VollumΔ pag Δ cya Δ lef ) results only in moderate attenuation while maintaining significant virulence. In addition, this virulence was shown to depend on AtxA activity in the presence of pXO2, as deletion of either atxA (VollumΔ pag Δ cya Δ lef Δ atxA ) or pXO2 (VollumΔpXO2) results in complete loss of virulence. AtxA, a global B. anthracis virulence regulator is a 476 amino-acid-long protein encoded by the activator gene atxA , located in the pXO1 pathogenicity island. Although the mechanisms by which AtxA exerts its regulation are not yet fully understood, AtxA was shown to control the expression of more than a hundred genes residing on all genetic elements (the chromosome and the two virulence plasmids) [3] . For AtxA to activate additional genes of unknown genomic location required for exerting this novel virulent trait, the prolonged survival of the bacteria in the circulation of a naïve host may be needed. This type of protection can be exerted by pXO2, through the capsule, or pXO1, through the toxins. This assumption is supported by the results with VollumΔpXO2 ( Figure 1A ), which also induces partial lethality in IV inoculated rabbits, though with longer MTTD. This demonstrates that a toxinogenic strain lacking pXO2 is sufficiently protected in the circulation by the toxins, thus remaining able to exhibit the virulent trait. On the other hand, systemic toxemia has been shown to cause death of mice, rats (Fisher) and GP [23] , and therefore we cannot conclude which virulence mechanism was crucial in this strain's ability to kill the host. As the virulence exerted by the VollumΔ pag Δ cya Δ lef encapsulated vegetative bacilli depends both on AtxA and pXO2, we studied the effect of AtxA activity on the generation and function of the main product of pXO2, the capsule. Using a simple India-ink negative stain, no qualitative difference could be detected between the capsules of the assumed AtxA-activated bacilli and the bacteria lacking AtxA ( Figure 4 ). Several studies have shown that atxA mutant bacilli are less or non-encapsulated in vitro [2] , [24] , however this mutation did not affect capsule production in vivo [25] . It seems that our ex vivo conditions mimic the in vivo situation. Variations in capsule function may result in differences in bacterial survival in the circulation. Indeed, bacteremia levels determined for encapsulated AtxA-activated strains (virulent strains) were higher than those determined for the atxA mutant strains (non-virulent strains, Figure 5 ). The variation in bacteremia levels in the circulation can result in differences in bacterial dissemination to the tissues. Comparison of the tissue bacterial burden shows that virulent strains seem to exhibit higher levels than the non-virulent strains ( Figure 6 ). Although not statistically significant, these differences correlate with the bacteremia pattern and may indicate that the virulence of B. anthracis strains may be related to their ability to spread to different tissues. However, it should be emphasized that while this difference may reflect a leading cause for the development of the disease towards host mortality, it may also prove to be irrelevant. Passive immunization with sera from convalescent rabbits, while saving naïve rabbits inoculated IV with VollumΔ pag Δ cya Δ lef encapsulated vegetative cells, did not affect bacterial systemic spread. This indicates that tissue dissemination per se is not the main determinant of virulence. The convalescent serum was further used to try and identify the antigen responsible for the protective activity. Using Western-blot, the protective sera recognized a distinct band of about 70 Kd present in the bacterial extract of the virulent strains, but not in the extract of the non-virulent strains, which was identified as pXO1-90 (BslA – B. anthracis S-layer protein A). BslA, a putative surface layer immunoreactive protein [26] was studied and shown to mediate adherence of the Sterne vegetative bacteria to host cells [27] , [28] , [29] , including to the blood-brain barrier endothelial cells, promoting penetration during the pathogenesis of anthrax meningitis [30] . However, deletion of the bslA gene in the VollumΔ pag Δ cya Δ lef background had a minor effect on the pathogenicity of the mutant ( Table S1 ), and no effect on tissue dissemination. In order to evaluate the contribution of pXO1 to the toxin-independent virulence mechanism, we inserted the atxA gene into the genome of the VollumΔ pXO 1 mutant, creating VollumΔ pXO 1 BA2805::atxA . The virulence exhibited by the new mutant indicates that AtxA, in the background of pXO2, is sufficient to induce the virulent trait. Furthermore, as deletion of atxA from the virulent strain VollumΔ pag Δ cya Δ lef results in loss of virulence, and insertion of atxA into the non-virulent VollumΔ pXO 1 results in recovery of the virulent trait ( Fig 7 ), we can conclude that atxA is the only gene from pXO1 required for the exhibition of the IV virulence. Further studies to genetically define the toxin independent virulence should include, among other, identification of components on pXO2 that might be regulated, directly or indirectly by AtxA, such as the capBCADE structural genes and acpA/acpB regulatory genes, as well as testing additional B. anthracis strains and relevant animal models. To conclude, in this study we demonstrate that the toxin-independent virulence mechanism, demonstrated previously in GP, is a general trait of B. anthracis . Strains lacking the three toxin-genes, previously shown to kill GP when injected SC as spores [6] , were now shown to be able to kill rabbit hosts when injected IV as encapsulated vegetative cells. This mechanism of virulence was shown to be AtxA dependent. This novel virulence mechanism should be further explored, as it may prove to be a fundamental virulent trait of B. anthracis . The fact that artificially-induced VollumΔ pag Δ cya Δ lef bacteremia in animals could be cured by passive immunization indicates that bacterial antigens other than PA induce this immune response. Therefore we assume that the main findings described in this work may have major implications on future research both on B. anthracis pathogenicity and on vaccine development. Supporting Information Table S1 Susceptibility of rabbits to Vollum strains in the IV injection model. Rabbits were inoculated IV with different doses of vegetative cells of the wild type and mutants strains. (DOCX) Click here for additional data file. Table S2 Effect of atxA gene on the toxin independent virulent trait in rabbits IV injection model. Rabbits were inoculated IV with different doses of vegetative cells of the wild type and mutants strains. (DOCX) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5069959/

Multiplexed Metagenomic Deep Sequencing To Analyze the Composition of High-Priority Pathogen Reagents

Both the integrity and reproducibility of experiments using select agents depend in large part on unbiased validation to ensure the correct identity and purity of the species in question. Metagenomic deep sequencing (MDS) provides the required level of validation by allowing for an unbiased and comprehensive assessment of all the microbes in a laboratory stock. INTRODUCTION Virology laboratories must maintain constant vigilance regarding the provenance of strains in their collections. In addition to confirming that experiments are being performed on the intended virus, unbiased methods that can detect viral coinfections and/or other microbial contaminants are critical for ensuring that experiments are faithfully reporting on the biology of a particular virus and not on a polymicrobial exposure. Stock assurance is exceptionally important when working with viruses which require a high level of biocontainment, given the increased security requirements and higher costs and higher stakes of the research. The validity and reproducibility of experiments, in addition to security and tracking concerns, demand an approach that can provide not only simple viral identification but also the entire spectrum of single nucleotide variants (SNVs) and their respective frequencies within a given stock. Molecular subtyping was critical in determining the source of the 2001 bioterrorism-associated anthrax outbreak ( 1 ). Furthermore, the recent missteps in the handling of Bacillus anthracis , influenza virus, and smallpox virus highlight the potential for inadvertent laboratory contamination and the need to improve isolate-tracking methods ( 2 , 3 ). In this study, we sequenced virus stocks of six distinct negative-sense RNA viruses, each of them with a unique passage history: (i) mycoplasma-contaminated Ebola virus (EBOV), (ii) mycoplasma-contaminated La Crosse virus (LACV) grown on cells with a known lymphocytic choriomeningitis virus (LCMV) contamination, (iii) vesicular stomatitis virus (VSV) grown on LCMV-contaminated cells, (iv) recombinant human respiratory syncytial virus (rHRSV) expressing enhanced green fluorescent protein (EGFP) from an additional transcription unit (ATU) ( 4 ), (v) recombinant canine distemper virus (rCDV) expressing Venus fluorescent protein from an ATU ( 5 ), and recombinant measles virus (rMV) expressing EGFP from an ATU ( 6 ). The investigators performing the sequence analysis were blinded to the virus isolates and the origins and known contaminations of the virus stocks. Here, we demonstrate the utility of metagenomic deep sequencing (MDS) for comprehensively assessing multiple viral isolates. We show that MDS and custom bioinformatics pipelines utilizing publicly available and free software packages can detect a wide range of bacterial and viral contaminants in high-priority virus stocks and that SNV analysis can successfully discriminate between two closely related EBOV isolates. We also demonstrate how dual-indexed barcodes dramatically decrease the false-positive assignment of sequencing reads to an input sample in multiplexed high-titer virus MDS libraries. RESULTS Importance of dual-index barcoding. In many sequencing applications, including MDS, it is critical to be able to distinguish low-level true-positive samples from false positives that result from assignment of sequencing reads to the wrong index ("index bleed-through"). Chimeric molecules produced during amplification steps in library preparation or on the sequencer during cluster generation are particularly susceptible to such misassignment. Creating library molecules with barcode sequences on both ends of the molecule (dual indexing) has been shown to reduce the rate at which this misassignment occurs by requiring concordance between the two barcode sequences ( 7 ). To corroborate the benefit of minimizing misassignment of sequences by using dual indexing in the context of MDS, we analyzed the rate of index bleed-through in an independently generated sequencing data set that was demultiplexed using one or both index reads. We measured the rate of bleed-through in a set of 50 samples that included two samples that were positive for a recently discovered reptarenavirus. The other 48 samples were negative by quantitative reverse transcription PCR (qRT-PCR). We aligned reads from all 50 pooled samples to a virus genome segment present in the two positive samples ( 8 ). The data sets contained a median of 2.75 × 10 6 read pairs, and the two positive samples contained 65,284 and 203,565 virus-mapping reads ( Fig. 1 ). Index bleed-through was determined to have occurred when reads identified as one of the 48 virus-negative samples by barcode actually aligned to this particular reptarenavirus genome segment. The median rate of bleed-through in the data sets that were demultiplexed using a single index was 17.9 per million filtered reads ( Fig. 1 ). The median rate in dual-index demultiplexed data sets was 0.48 per million reads. Thus, in our data set, dual indexing reduced the median rate of read misassignment by 37-fold. The absolute number of mismapping reads decreased from a median of 50 reads to one read per data set. FIG 1 Dual indexing decreases the median rate of read misassignment by nearly 40-fold. Libraries from 50 samples were pooled and sequenced together. Two of the samples (indicated by arrows) were positive for snake arenavirus, and the other 48 were negative. Data sets were demultiplexed using a single index sequence (black squares) or dual index sequences (red circles), and reads from each data set were mapped with high stringency to the virus sequence. The number of virus mapping reads per million quality-filtered reads is indicated. Some dual-index-demultiplexed data sets had no misassigned reads. In these cases, red circles are not shown. Identification of microbes. The MDS of total RNA from each sample yielded 150-nucleotide (nt) paired-end sequences ( Table 1 ). The data were analyzed using a rapid computational pipeline developed at the University of California, San Francisco (UCSF), to classify MDS reads and identify potential pathogens by comparison to the entire NCBI nucleotide reference database as described in Materials and Methods ( 9 ). TABLE 1 Metagenomic deep sequencing results a Sample Total no. of sequencing reads No. of unique nonhuman reads Target virus Other viral sequence(s) Bacterial sequence EBOV 47,328,387 17,777,406 EBOV (3,342,624) None Mycoplasma hyorhinis (12,215,655) rMV 7,129,497 802,549 MV (159,184) HHV4 (905) Negative rHRSV 6,274,384 1,165,688 HRSV (673,096) HPV18 (121) Negative LACV 6,217,368 1,389,113 LACV (1,122,903) LCMV (5,388), Syrian hamster IAP H10 (45), hamster gammaretrovirus (11) Mycoplasma arginini (23,919) rCDV 6,165,559 900,609 CDV (582,074) Fowlpox (1,138), LACV (7) Negative VSV 10,164,946 2,130,521 VSV (1,481,456) LCMV (3,315) Negative a Number of sequences aligning to each microbe are in parentheses. Abbreviations: EBOV, Ebola virus; rMV, recombinant measles virus; HHV4, human herpesvirus 4; rHRSV, recombinant human respiratory syncytial virus; LACV, La Crosse virus; HPV18, human papillomavirus 18; IAP, intracisternal A particle; LCMV, lymphocytic choriomeningitis virus; rCDV, recombinant canine distemper virus; VSV, vesicular stomatitis virus. The results are summarized in Table 1 . The EBOV and LACV samples were found to have significant contamination with Mycoplasma spp., with 3.65 and 0.02 Mycoplasma reads per viral read, respectively. The rMV sample contained more than 900 nonredundant, paired-end Epstein-Barr virus (EBV) sequences (0.013% of total reads) that were expected to come from the EBV-transformed human B-lymphoblastoid cell line (B-LCL) on which the virus was grown ( 10 ). Similarly, the rHRSV sample contained 121 paired-end sequences (0.0019% of total reads) that aligned to human papillomavirus 18 (HPV18). This was also unsurprising, since HEp-2 cells are the standard cell line used to culture HRSV and these are known to be a HeLa‑contaminated cell line; HeLa cells were recently shown to have HPV18 DNA integrated into their genome ( 11 ). The LACV sample had evidence of Syrian hamster retroviruses, and it was confirmed that the virus had been grown on baby hamster kidney (BHK) cells. The rCDV sample contained sequence reads mapping to fowlpox virus, which had been used as part of the process to generate rCDV from plasmid. Because these RNA extractions were not DNase treated, we cannot rule out that some of the EBV, HPV18, and fowlpox virus sequences were remnants of genomic DNA. Last, the LACV and VSV samples were found to be contaminated with LCMV. None of the isolates had evidence of fungal contamination. As evidence of the minimal index bleed-through in these dual-indexed samples, only seven viral read pairs unique to LACV were found in the rCDV sample, and no other sample cross-contamination was present in any of the other samples in this set, despite being processed in parallel. Regardless, the fact that low-level index bleed-through may still occur, despite dual indexing, highlights the need for additional layers of error correction. Future iterations of this technology will likely include added features that will further reduce bleed-through of multiplexed samples, such as unique molecular identifier (UMI) barcodes, in which each cDNA molecule is uniquely indexed at the time of first-strand synthesis ( 12 ). SNV experiment. All of the viruses contained several SNVs compared to their reference sequences that ranged in frequency, including some consensus-level SNVs, as listed in Table 2 . At a frequency of greater than 0.005, rCDV had 427 SNVs (170 of those being nonsynonymous), EBOV had 143 SNVs (70 nonsynonymous), the LACV L segment had 61 SNVs (45 nonsynonymous), the LACV M segment had 31 SNVs (21 nonsynonymous), the LACV S segment had 6 SNVs (4 nonsynonymous), rMV had 122 SNVs (80 nonsynonymous), rHRSV had 115 SNVs (71 nonsynonymous), and VSV strain Indiana (VSV IN ) had 109 SNVs (72 nonsynonymous). A majority of SNVs are rare variants, having a frequency of less than 0.10. All six viruses had nonsynonymous and synonymous SNVs at frequencies of greater than 0.10. Four viruses had nonsynonymous SNVs at a frequency of at least 0.90, while five viruses had synonymous SNVs at a frequency of at least 0.90. Figure 2A displays the distribution of the SNV frequency on each genomic segment. TABLE 2 SNVs in each virus genome segment that are at least 0.5% of population a Viral genome Mean coverage No. of SNVs: Total ≥1% ≥10% ≥50% ≥90% EBOV 8,334 143 (70) 49 (19) 3 (1) 2 (1) 1 (1) MV 1,622 122 (80) 72 (42) 13 (2) 11 (1) 11 (1) HRSV 5,429 115 (71) 54 (27) 2 (1) 2 (1) 2 (1) LACV L segment 5,539 61 (45) 27 (18) 4 (1) 3 (0) 2 (0) LACV M segment 10,077 31 (21) 11 (8) 6 (4) 0 (0) 0 (0) LACV S segment 14,906 6 (4) 4 (2) 1 (0) 1 (0) 1 (0) CDV 4,741 427 (170) 297 (91) 30 (6) 3 (0) 1 (0) VSV 13,401 109 (72) 53 (31) 24 (10) 24 (10) 24 (10) a Mean coverage is the mean number of non-PCR-duplicate reads that mapped to each base of the virus genome segment. Each SNV column is listed as the number of total SNVs, followed by the number of nonsynonymous SNVs in parentheses, which are at least the percentage of the population listed in the header. Abbreviations: EBOV, Ebola virus; MV, measles virus; HRSV, human respiratory syncytial virus; LACV, La Crosse virus; CDV, canine distemper virus; VSV, vesicular stomatitis virus. FIG 2 Distribution of single nucleotide variants (SNVs) for each virus and for two Ebola virus strains. For each plot, the y axis is the log 10 (SNV frequency) in order to display the range of low-frequency SNVs more accurately. (A) SNV analysis for each virus sample using its reference genome. Abbreviations: CDV, canine distemper virus; EBOV, Ebola virus; LACV, La Crosse virus; MV, measles virus; HRSV, human respiratory syncytial virus; VSV, vesicular stomatitis virus. (B) SNV analysis of 2,501,8050 filtered read-pairs revealed 223 SNVs in the reads that mapped to Ebola virus Mayinga with a frequency of ≥0.90, while there was only one SNV with a frequency of ≥0.90 in the reads that mapped to Ebola virus Kikwit. The investigators were blinded to the exact isolate of EBOV since the major goal of this study was to identify a state-of-the-art approach which could precisely characterize a virus reagent with isolate specificity in an unbiased fashion. As described in Materials and Methods, SNV analysis was performed to determine the EBOV isolate. Reads were mapped to two different reference genomes: (i) Zaire ebolavirus isolate Ebola virus/ Homo sapiens -tc/COD/1976/Yambuku-Mayinga, complete genome strain (GenBank accession no. NC_002549 ), and (ii) Zaire ebolavirus strain Kikwit, complete genome (GenBank accession no. JQ352763 ). Comparing rates of correctly mapped reads did not reveal isolate identity. The same 2,501,8050 filtered read-pairs mapped at virtually identical percentages, 21.03% to EBOV Mayinga and 21.05% to EBOV Kikwit. However, SNV analysis revealed 223 SNVs in the reads that mapped to EBOV Mayinga with a frequency of ≥0.90, while there was only one SNV with a frequency of ≥0.90 in the reads that mapped to EBOV Kikwit ( Fig. 2B ). Thus, we correctly concluded that the precise isolate sequenced in this experiment was EBOV Kikwit. Importance of dual-index barcoding. In many sequencing applications, including MDS, it is critical to be able to distinguish low-level true-positive samples from false positives that result from assignment of sequencing reads to the wrong index ("index bleed-through"). Chimeric molecules produced during amplification steps in library preparation or on the sequencer during cluster generation are particularly susceptible to such misassignment. Creating library molecules with barcode sequences on both ends of the molecule (dual indexing) has been shown to reduce the rate at which this misassignment occurs by requiring concordance between the two barcode sequences ( 7 ). To corroborate the benefit of minimizing misassignment of sequences by using dual indexing in the context of MDS, we analyzed the rate of index bleed-through in an independently generated sequencing data set that was demultiplexed using one or both index reads. We measured the rate of bleed-through in a set of 50 samples that included two samples that were positive for a recently discovered reptarenavirus. The other 48 samples were negative by quantitative reverse transcription PCR (qRT-PCR). We aligned reads from all 50 pooled samples to a virus genome segment present in the two positive samples ( 8 ). The data sets contained a median of 2.75 × 10 6 read pairs, and the two positive samples contained 65,284 and 203,565 virus-mapping reads ( Fig. 1 ). Index bleed-through was determined to have occurred when reads identified as one of the 48 virus-negative samples by barcode actually aligned to this particular reptarenavirus genome segment. The median rate of bleed-through in the data sets that were demultiplexed using a single index was 17.9 per million filtered reads ( Fig. 1 ). The median rate in dual-index demultiplexed data sets was 0.48 per million reads. Thus, in our data set, dual indexing reduced the median rate of read misassignment by 37-fold. The absolute number of mismapping reads decreased from a median of 50 reads to one read per data set. FIG 1 Dual indexing decreases the median rate of read misassignment by nearly 40-fold. Libraries from 50 samples were pooled and sequenced together. Two of the samples (indicated by arrows) were positive for snake arenavirus, and the other 48 were negative. Data sets were demultiplexed using a single index sequence (black squares) or dual index sequences (red circles), and reads from each data set were mapped with high stringency to the virus sequence. The number of virus mapping reads per million quality-filtered reads is indicated. Some dual-index-demultiplexed data sets had no misassigned reads. In these cases, red circles are not shown. Identification of microbes. The MDS of total RNA from each sample yielded 150-nucleotide (nt) paired-end sequences ( Table 1 ). The data were analyzed using a rapid computational pipeline developed at the University of California, San Francisco (UCSF), to classify MDS reads and identify potential pathogens by comparison to the entire NCBI nucleotide reference database as described in Materials and Methods ( 9 ). TABLE 1 Metagenomic deep sequencing results a Sample Total no. of sequencing reads No. of unique nonhuman reads Target virus Other viral sequence(s) Bacterial sequence EBOV 47,328,387 17,777,406 EBOV (3,342,624) None Mycoplasma hyorhinis (12,215,655) rMV 7,129,497 802,549 MV (159,184) HHV4 (905) Negative rHRSV 6,274,384 1,165,688 HRSV (673,096) HPV18 (121) Negative LACV 6,217,368 1,389,113 LACV (1,122,903) LCMV (5,388), Syrian hamster IAP H10 (45), hamster gammaretrovirus (11) Mycoplasma arginini (23,919) rCDV 6,165,559 900,609 CDV (582,074) Fowlpox (1,138), LACV (7) Negative VSV 10,164,946 2,130,521 VSV (1,481,456) LCMV (3,315) Negative a Number of sequences aligning to each microbe are in parentheses. Abbreviations: EBOV, Ebola virus; rMV, recombinant measles virus; HHV4, human herpesvirus 4; rHRSV, recombinant human respiratory syncytial virus; LACV, La Crosse virus; HPV18, human papillomavirus 18; IAP, intracisternal A particle; LCMV, lymphocytic choriomeningitis virus; rCDV, recombinant canine distemper virus; VSV, vesicular stomatitis virus. The results are summarized in Table 1 . The EBOV and LACV samples were found to have significant contamination with Mycoplasma spp., with 3.65 and 0.02 Mycoplasma reads per viral read, respectively. The rMV sample contained more than 900 nonredundant, paired-end Epstein-Barr virus (EBV) sequences (0.013% of total reads) that were expected to come from the EBV-transformed human B-lymphoblastoid cell line (B-LCL) on which the virus was grown ( 10 ). Similarly, the rHRSV sample contained 121 paired-end sequences (0.0019% of total reads) that aligned to human papillomavirus 18 (HPV18). This was also unsurprising, since HEp-2 cells are the standard cell line used to culture HRSV and these are known to be a HeLa‑contaminated cell line; HeLa cells were recently shown to have HPV18 DNA integrated into their genome ( 11 ). The LACV sample had evidence of Syrian hamster retroviruses, and it was confirmed that the virus had been grown on baby hamster kidney (BHK) cells. The rCDV sample contained sequence reads mapping to fowlpox virus, which had been used as part of the process to generate rCDV from plasmid. Because these RNA extractions were not DNase treated, we cannot rule out that some of the EBV, HPV18, and fowlpox virus sequences were remnants of genomic DNA. Last, the LACV and VSV samples were found to be contaminated with LCMV. None of the isolates had evidence of fungal contamination. As evidence of the minimal index bleed-through in these dual-indexed samples, only seven viral read pairs unique to LACV were found in the rCDV sample, and no other sample cross-contamination was present in any of the other samples in this set, despite being processed in parallel. Regardless, the fact that low-level index bleed-through may still occur, despite dual indexing, highlights the need for additional layers of error correction. Future iterations of this technology will likely include added features that will further reduce bleed-through of multiplexed samples, such as unique molecular identifier (UMI) barcodes, in which each cDNA molecule is uniquely indexed at the time of first-strand synthesis ( 12 ). SNV experiment. All of the viruses contained several SNVs compared to their reference sequences that ranged in frequency, including some consensus-level SNVs, as listed in Table 2 . At a frequency of greater than 0.005, rCDV had 427 SNVs (170 of those being nonsynonymous), EBOV had 143 SNVs (70 nonsynonymous), the LACV L segment had 61 SNVs (45 nonsynonymous), the LACV M segment had 31 SNVs (21 nonsynonymous), the LACV S segment had 6 SNVs (4 nonsynonymous), rMV had 122 SNVs (80 nonsynonymous), rHRSV had 115 SNVs (71 nonsynonymous), and VSV strain Indiana (VSV IN ) had 109 SNVs (72 nonsynonymous). A majority of SNVs are rare variants, having a frequency of less than 0.10. All six viruses had nonsynonymous and synonymous SNVs at frequencies of greater than 0.10. Four viruses had nonsynonymous SNVs at a frequency of at least 0.90, while five viruses had synonymous SNVs at a frequency of at least 0.90. Figure 2A displays the distribution of the SNV frequency on each genomic segment. TABLE 2 SNVs in each virus genome segment that are at least 0.5% of population a Viral genome Mean coverage No. of SNVs: Total ≥1% ≥10% ≥50% ≥90% EBOV 8,334 143 (70) 49 (19) 3 (1) 2 (1) 1 (1) MV 1,622 122 (80) 72 (42) 13 (2) 11 (1) 11 (1) HRSV 5,429 115 (71) 54 (27) 2 (1) 2 (1) 2 (1) LACV L segment 5,539 61 (45) 27 (18) 4 (1) 3 (0) 2 (0) LACV M segment 10,077 31 (21) 11 (8) 6 (4) 0 (0) 0 (0) LACV S segment 14,906 6 (4) 4 (2) 1 (0) 1 (0) 1 (0) CDV 4,741 427 (170) 297 (91) 30 (6) 3 (0) 1 (0) VSV 13,401 109 (72) 53 (31) 24 (10) 24 (10) 24 (10) a Mean coverage is the mean number of non-PCR-duplicate reads that mapped to each base of the virus genome segment. Each SNV column is listed as the number of total SNVs, followed by the number of nonsynonymous SNVs in parentheses, which are at least the percentage of the population listed in the header. Abbreviations: EBOV, Ebola virus; MV, measles virus; HRSV, human respiratory syncytial virus; LACV, La Crosse virus; CDV, canine distemper virus; VSV, vesicular stomatitis virus. FIG 2 Distribution of single nucleotide variants (SNVs) for each virus and for two Ebola virus strains. For each plot, the y axis is the log 10 (SNV frequency) in order to display the range of low-frequency SNVs more accurately. (A) SNV analysis for each virus sample using its reference genome. Abbreviations: CDV, canine distemper virus; EBOV, Ebola virus; LACV, La Crosse virus; MV, measles virus; HRSV, human respiratory syncytial virus; VSV, vesicular stomatitis virus. (B) SNV analysis of 2,501,8050 filtered read-pairs revealed 223 SNVs in the reads that mapped to Ebola virus Mayinga with a frequency of ≥0.90, while there was only one SNV with a frequency of ≥0.90 in the reads that mapped to Ebola virus Kikwit. The investigators were blinded to the exact isolate of EBOV since the major goal of this study was to identify a state-of-the-art approach which could precisely characterize a virus reagent with isolate specificity in an unbiased fashion. As described in Materials and Methods, SNV analysis was performed to determine the EBOV isolate. Reads were mapped to two different reference genomes: (i) Zaire ebolavirus isolate Ebola virus/ Homo sapiens -tc/COD/1976/Yambuku-Mayinga, complete genome strain (GenBank accession no. NC_002549 ), and (ii) Zaire ebolavirus strain Kikwit, complete genome (GenBank accession no. JQ352763 ). Comparing rates of correctly mapped reads did not reveal isolate identity. The same 2,501,8050 filtered read-pairs mapped at virtually identical percentages, 21.03% to EBOV Mayinga and 21.05% to EBOV Kikwit. However, SNV analysis revealed 223 SNVs in the reads that mapped to EBOV Mayinga with a frequency of ≥0.90, while there was only one SNV with a frequency of ≥0.90 in the reads that mapped to EBOV Kikwit ( Fig. 2B ). Thus, we correctly concluded that the precise isolate sequenced in this experiment was EBOV Kikwit. DISCUSSION We describe the successful application of a multiplexed MDS assay and bioinformatics pipelines for comprehensively interrogating the provenance of six virus isolates in a blinded fashion. With regard to multiplexing, the frequency of reads that are assigned an incorrect barcode is a function of the abundance of those reads in the correct barcode library and the degree of multiplexing. So, for high-titer viral stock-derived data sets, where viral reads are abundant, bleed-through occurs at a higher frequency. We demonstrated that dual indexing reduces the misassignment of sequencing reads by 37-fold in a pilot experiment and saw essentially no intersample cross-contamination in our multiplexed assay despite very-high-titer virus isolates. It is important to note that most commercially available dual-indexing kits do not provide this benefit because the reduction in misassignment depends on samples being identified by unique index pairs. Most commercially available kits do not actually provide unique index pairs. Instead, they mix and match indexes to create pairs with repeated index sequences, for instance, by permuting eight i5 indexes with 12 i7 indexes to achieve 96 pairs. We also found that using dual indexing decreased the total number of reads per data set by ~6% due to the removal of unassigned read pairs. However, this is a relatively small tradeoff for the large decrease in misassigned reads. Contaminating sequences were accurately identified, including bacteria ( Mycoplasma spp.) and six viruses across the six virus stocks. Four of these viral contaminating sequences (i.e., EBV, HPV18, hamster gammaretrovirus, and LCMV [ n = 2]) reflected the cells on which the viruses were cultured ( 10 , 11 , 13 , 14 ). The fowlpox virus present in the rCDV sample was a remnant of the procedure used to generate rCDV, which uses a recombinant fowlpox virus to express T7 RNA polymerase ( 5 ). This is unsurprising given the low passage number of the rCDV. The results from MDS of different virus stocks highlight the utility of this approach for in-depth analysis of virus sequences. There were different contaminating pathogens in each of the stocks, and these biological "fingerprints" provided precise information about their origin and passage history. Such information is invaluable for virologists embarking on time-consuming and expensive studies in biocontainment but is also useful for a broad range of microbiologists who would benefit from sample assurance. For rMV and rHRSV, there was evidence of the cell lines used to propagate the viruses, and for rCDV, there was evidence of the helper virus used for reverse genetics. For viruses with an extensive propagation history, the cell type in which the virus had been passaged could be inferred based on contaminating sequences (i.e., hamster retrovirus sequences) as well as the additional, nonmicrobial sequences that can be assembled to characterize aspects of the host cell genome. This suggests that MDS is useful for virus forensic analysis, helping to identify the manner and cell type in which the virus was cultured or recovered. The SNV analysis identified numerous synonymous and nonsynonymous mutations present in each of the isolates. The presence and frequency of such mutations in longitudinal samples may be used to monitor inevitable virus adaptation rigorously in the laboratory setting and the quality of seed stocks ( 15 ) as well as to define particular isolates ( 16 ). Here, SNV analysis allowed the precise determination of the particular EBOV isolate ( Fig. 2B ). Last, detailed cataloging of SNVs for each isolate allows rapid identification and tracking of high-priority pathogens in the event of an accidental or nonaccidental release of virus into the environment and could be an integral part of any microbial source-tracking program. Viral genome sequencing including SNV analysis has become an important tool to monitor viral spread, viral evolution, and routes of transmission in an outbreak situation as exemplified by the recent EBOV disease outbreak in West Africa ( 17 – 26 ). If a validated reference sequence does not exist, we recommend that the first step be a de novo assembly. However, this should be followed with a remapping of all the reads back to the assembled reference and a LoFreq* or similar SNV analysis, as we have demonstrated here. The latter analysis will reveal the presence of variations or even a mixture of strains that would be obscured or lost through an assemble-and-BLAST strategy alone. In summary, we show that a single multiplex MDS assay can comprehensively assess (i) virus and isolate identity, (ii) SNVs in virus populations, (iii) the presence of viral coinfection, and (iv) the presence of bacterial contamination and (v) can provide information about the cell line used to propagate the virus. MATERIALS AND METHODS Viruses and RNA purification. All work with infectious EBOV was performed under biosafety level 4 (BSL-4) conditions at the Integrated Research Facility, Rocky Mountain Laboratories (RML), Division of Intramural Research, NIAID, NIH, Hamilton, MT. Vero E6 cells (ATCC CRL 1586) were infected with EBOV at a multiplicity of infection (MOI) of 1, and virus-containing supernatants were clarified by low-speed centrifugation at 4 days postinfection (p.i.). Total RNA from supernatant (140 µl) was purified using the Qiagen viral RNA minikit, according to the RML standard operating procedures for virus inactivation. Purified RNA was eluted in H 2 O. The EBOV stock used for this analysis contained a known mycoplasma contamination. To generate rMV Khartoum, Sudan (rMV KS ) stocks, B‑LCL cells were infected at an MOI of 0.01. After 4 days, when viral cytopathic effect was maximal, the stock was subjected to one freeze-thaw cycle to release the highly cell-associated measles virus into the medium. Cell debris was removed by low-speed centrifugation, and the supernatant was stored at −80°C as virus stock. To generate rCDV Rhode Island (rCDV RI ) stocks, Vero cells expressing the CDV receptor canine CD150 were infected at an MOI of 0.01. After 2 days, when viral cytopathic effect was maximal, the stock was subjected to one freeze-thaw cycle to release cell‑associated virus into the medium. Cell debris was removed by low-speed centrifugation, and the supernatant was stored at −80°C as virus stock. To generate rHRSV B05 stocks, HEp‑2 cells were infected at an MOI of 0.01. After 3 days, when viral cytopathic effect was maximal, the medium was transferred to 50-ml tubes, and cell debris was removed by low-speed centrifugation. The supernatant was stored at −80°C as virus stock. The LACV H78 strain was grown on BHK cells ( 27 ). BHK cells were infected with passage 3 LACV H78 at an MOI of 0.01. Virus was propagated for 48 h before medium was removed. Cell debris was removed by low-speed centrifugation, and individual aliquots of virus were frozen for future analysis. The LACV stock contained a known mycoplasma contamination. For growth of the VSV Indiana (VSV IN ) strain, BHK cells were infected with VSV IN at an MOI of 0.01. Virus was propagated for 24 h before medium was removed. Cell debris was removed by low-speed centrifugation, and individual aliquots of virus were frozen for future analysis. For rMV, rCDV, rHRSV, LACV, and VSV, 250 µl of virus-containing supernatant was mixed with 750 µl of Trizol-LS (Ambion), and RNA was isolated according to the manufacturer's protocol. Purified RNA was resuspended in H 2 O. Comparison of single and dual indexing. A library containing a pool of 50 dual-indexed libraries was created as previously described ( 8 ). The library was sequenced on an Illumina HiSeq 2500 instrument at the UCSF Center for Advanced Technology. The 150-nt read-length, paired-end, dual-indexed data set was manually demultiplexed using either one or both index reads. To measure the rate of read misassignment, reads were first processed to remove low-quality and adapter sequences as described below. Trimmed reads were then aligned using the Bowtie2 tool to the sequence of a reptarenavirus genome segment (GenBank accession no. KP071661.1 ) actually present in two of the 50 samples ( 8 , 28 ). Bowtie2 was run with parameters –local –qc-filter –score-min C,120,1. The number of aligning reads for each data set was counted. Confirming virus identity and detecting contamination. RNA of six individual viral isolates extracted from supernatants of infected cells was provided for MDS. The investigators preparing the MDS libraries and performing the bioinformatics analysis were blinded to the presence or absence of any known microbial contaminants and also to the strain and isolate identity of each virus. The six viral isolates were LACV strain H78, EBOV, rCDV RI , rMV KS , rHRSV B05 , and the VSV IN strain. Samples were processed for MDS analysis as previously described ( 9 ). Samples were randomly amplified to double-stranded cDNA using the NuGEN Ovation v.2 kit (NuGEN, San Carlos, CA), and MDS libraries were constructed using the Nextera protocol (Illumina, San Diego, CA). Each library was dual indexed in the same manner as described above. Samples were pooled into a single library before library size and concentration were determined using the Blue Pippin (Sage Science, Beverly, MA) and Kapa universal quantitative PCR (qPCR) kit (Kapa Biosystems, Woburn, MA), respectively. Samples were sequenced on an Illumina HiSeq 2500 instrument using 150-nt paired-end sequencing. The paired-end sequences were analyzed for microbes using a rapid computational pipeline for microbial detection. Bioinformatic pipeline. Paired-end reads were quality filtered using PriceSeqFilter (version 1.2, parameters –rqf 95 0.98), a component of the paired-read iterative contig extension (PRICE) assembler ( 29 ), followed by removal of human sequences by alignment to a combined reference genome including human genome build 38 (hg38) and chimpanzee ( Pan troglodytes ) using the Spliced Transcripts Alignment to a Reference (STAR) aligner ( 30 ). Unaligned reads that were at least 95% identical were compressed by cd-hit-dup (v4.6.1) ( 31 , 32 ). After a second Bowtie2 alignment to remove residual human sequences (using an hg38 reference database), these reads were then used as queries to search the NCBI nt database (July 2015) using gsnapl (Genentech, v2015-09-29) ( 33 ). SNV analysis. Illumina data from all the samples were processed in the same way for variant analysis. First reads were quality filtered using PriceSeqFilter (version 1.2) set to remove any read with less than 95% of nucleotides having a 0.98 probability of being correct (-rqf 95 0.98), any read with less than 90% called nucleotides, and any read that matched to the Illumina adapter sequences ( 29 ). Filtered high-quality reads were then aligned to reference genomes using GSNAP (version 2015-09-29) using default settings ( 33 ). Reference genome accession numbers were as follows: EBOV, JQ352763 ; LACV long (L) segment, NC_004108 ; LACV medium (M) segment, NC_004109 ; LACV short (S) segment, NC_004110 ; MV, HM439386 ; rHRSV, KF640637 ; VSV IN , J02428 . The rCDV RI sequence (unpublished) was supplied by W. Paul Duprex (Boston University). According to the recommended guidelines for the variant caller, PCR-duplicate reads were removed using Picard tools (version 2.2.4). Variants were called using LoFreq* (version 2.1.2) ( 34 ) using default settings with the exception of a conservative lower cutoff of ≥0.005 in frequency. LoFreq* is a sequencing read quality aware variant caller that models each location in the genome as a Poisson-binomial distribution of the number of nonreference bases. It also tests for common sequencing errors that may result in false positives, such as testing for the strand bias of a variant. It is also designed to take advantage of high-coverage (>500× coverage) data sets, such as those that we have ( Table 2 ). It uses high-coverage data sets to call true-positive low-frequency variants while maintaining a low false-positive rate but still shows reasonable sensitivity at low coverage levels (50× coverage) ( 34 ). Variants were determined to be synonymous or nonsynonymous using a custom Python script. Accession number(s). The 150-nt paired-end sequences are located in the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA accession number SRP076690 ). Viruses and RNA purification. All work with infectious EBOV was performed under biosafety level 4 (BSL-4) conditions at the Integrated Research Facility, Rocky Mountain Laboratories (RML), Division of Intramural Research, NIAID, NIH, Hamilton, MT. Vero E6 cells (ATCC CRL 1586) were infected with EBOV at a multiplicity of infection (MOI) of 1, and virus-containing supernatants were clarified by low-speed centrifugation at 4 days postinfection (p.i.). Total RNA from supernatant (140 µl) was purified using the Qiagen viral RNA minikit, according to the RML standard operating procedures for virus inactivation. Purified RNA was eluted in H 2 O. The EBOV stock used for this analysis contained a known mycoplasma contamination. To generate rMV Khartoum, Sudan (rMV KS ) stocks, B‑LCL cells were infected at an MOI of 0.01. After 4 days, when viral cytopathic effect was maximal, the stock was subjected to one freeze-thaw cycle to release the highly cell-associated measles virus into the medium. Cell debris was removed by low-speed centrifugation, and the supernatant was stored at −80°C as virus stock. To generate rCDV Rhode Island (rCDV RI ) stocks, Vero cells expressing the CDV receptor canine CD150 were infected at an MOI of 0.01. After 2 days, when viral cytopathic effect was maximal, the stock was subjected to one freeze-thaw cycle to release cell‑associated virus into the medium. Cell debris was removed by low-speed centrifugation, and the supernatant was stored at −80°C as virus stock. To generate rHRSV B05 stocks, HEp‑2 cells were infected at an MOI of 0.01. After 3 days, when viral cytopathic effect was maximal, the medium was transferred to 50-ml tubes, and cell debris was removed by low-speed centrifugation. The supernatant was stored at −80°C as virus stock. The LACV H78 strain was grown on BHK cells ( 27 ). BHK cells were infected with passage 3 LACV H78 at an MOI of 0.01. Virus was propagated for 48 h before medium was removed. Cell debris was removed by low-speed centrifugation, and individual aliquots of virus were frozen for future analysis. The LACV stock contained a known mycoplasma contamination. For growth of the VSV Indiana (VSV IN ) strain, BHK cells were infected with VSV IN at an MOI of 0.01. Virus was propagated for 24 h before medium was removed. Cell debris was removed by low-speed centrifugation, and individual aliquots of virus were frozen for future analysis. For rMV, rCDV, rHRSV, LACV, and VSV, 250 µl of virus-containing supernatant was mixed with 750 µl of Trizol-LS (Ambion), and RNA was isolated according to the manufacturer's protocol. Purified RNA was resuspended in H 2 O. Comparison of single and dual indexing. A library containing a pool of 50 dual-indexed libraries was created as previously described ( 8 ). The library was sequenced on an Illumina HiSeq 2500 instrument at the UCSF Center for Advanced Technology. The 150-nt read-length, paired-end, dual-indexed data set was manually demultiplexed using either one or both index reads. To measure the rate of read misassignment, reads were first processed to remove low-quality and adapter sequences as described below. Trimmed reads were then aligned using the Bowtie2 tool to the sequence of a reptarenavirus genome segment (GenBank accession no. KP071661.1 ) actually present in two of the 50 samples ( 8 , 28 ). Bowtie2 was run with parameters –local –qc-filter –score-min C,120,1. The number of aligning reads for each data set was counted. Confirming virus identity and detecting contamination. RNA of six individual viral isolates extracted from supernatants of infected cells was provided for MDS. The investigators preparing the MDS libraries and performing the bioinformatics analysis were blinded to the presence or absence of any known microbial contaminants and also to the strain and isolate identity of each virus. The six viral isolates were LACV strain H78, EBOV, rCDV RI , rMV KS , rHRSV B05 , and the VSV IN strain. Samples were processed for MDS analysis as previously described ( 9 ). Samples were randomly amplified to double-stranded cDNA using the NuGEN Ovation v.2 kit (NuGEN, San Carlos, CA), and MDS libraries were constructed using the Nextera protocol (Illumina, San Diego, CA). Each library was dual indexed in the same manner as described above. Samples were pooled into a single library before library size and concentration were determined using the Blue Pippin (Sage Science, Beverly, MA) and Kapa universal quantitative PCR (qPCR) kit (Kapa Biosystems, Woburn, MA), respectively. Samples were sequenced on an Illumina HiSeq 2500 instrument using 150-nt paired-end sequencing. The paired-end sequences were analyzed for microbes using a rapid computational pipeline for microbial detection. Bioinformatic pipeline. Paired-end reads were quality filtered using PriceSeqFilter (version 1.2, parameters –rqf 95 0.98), a component of the paired-read iterative contig extension (PRICE) assembler ( 29 ), followed by removal of human sequences by alignment to a combined reference genome including human genome build 38 (hg38) and chimpanzee ( Pan troglodytes ) using the Spliced Transcripts Alignment to a Reference (STAR) aligner ( 30 ). Unaligned reads that were at least 95% identical were compressed by cd-hit-dup (v4.6.1) ( 31 , 32 ). After a second Bowtie2 alignment to remove residual human sequences (using an hg38 reference database), these reads were then used as queries to search the NCBI nt database (July 2015) using gsnapl (Genentech, v2015-09-29) ( 33 ). SNV analysis. Illumina data from all the samples were processed in the same way for variant analysis. First reads were quality filtered using PriceSeqFilter (version 1.2) set to remove any read with less than 95% of nucleotides having a 0.98 probability of being correct (-rqf 95 0.98), any read with less than 90% called nucleotides, and any read that matched to the Illumina adapter sequences ( 29 ). Filtered high-quality reads were then aligned to reference genomes using GSNAP (version 2015-09-29) using default settings ( 33 ). Reference genome accession numbers were as follows: EBOV, JQ352763 ; LACV long (L) segment, NC_004108 ; LACV medium (M) segment, NC_004109 ; LACV short (S) segment, NC_004110 ; MV, HM439386 ; rHRSV, KF640637 ; VSV IN , J02428 . The rCDV RI sequence (unpublished) was supplied by W. Paul Duprex (Boston University). According to the recommended guidelines for the variant caller, PCR-duplicate reads were removed using Picard tools (version 2.2.4). Variants were called using LoFreq* (version 2.1.2) ( 34 ) using default settings with the exception of a conservative lower cutoff of ≥0.005 in frequency. LoFreq* is a sequencing read quality aware variant caller that models each location in the genome as a Poisson-binomial distribution of the number of nonreference bases. It also tests for common sequencing errors that may result in false positives, such as testing for the strand bias of a variant. It is also designed to take advantage of high-coverage (>500× coverage) data sets, such as those that we have ( Table 2 ). It uses high-coverage data sets to call true-positive low-frequency variants while maintaining a low false-positive rate but still shows reasonable sensitivity at low coverage levels (50× coverage) ( 34 ). Variants were determined to be synonymous or nonsynonymous using a custom Python script. Accession number(s). The 150-nt paired-end sequences are located in the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA accession number SRP076690 ).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439391/

Pleiotropic cytotoxicity of VacA toxin in host cells and its impact on immunotherapy

Introduction: In the recent decades, a number of studies have highlighted the importance of Helicobacter pylori in the initiation and development of peptic ulcer and gastric cancer. Some potential virulence factors (e.g., urease, CagA, VacA, BabA) are exploited by this microorganism, facilitating its persistence through evading human defense mechanisms. Among these toxins and enzymes, vacuolating toxin A (VacA) is of a great importance in the pathogenesis of H. pylori. VacA toxin shows different pattern of cytotoxicity through binding to different cell surface receptors in various cells. Methods: To highlight attempts in treatment for H. pylori infection, here, we discussed the VacA potential as a candidate for development of vaccine and targeted immunotherapy. Furthermore, we reviewed the related literature to provide key insights on association of the genetic variants of VacA with the toxicity of the toxin in cells. Results: A number of investigations on the receptor(s) binding of VacA toxin confirmed the pleiotropic nature of VacA that uses a unique mechanism for internalization through some membrane components such as lipid rafts and glycophosphatidylinositol (GPI)-anchored proteins (GPI-AP). Considering the high potency of VacA toxin in the clinical presentations in infection and assisting persistence and colonization of H. pylori , it is considered as one of the pivotal components in production vaccines and monoclonal antibodies (mAbs). Conclusion: It is possible to generate mAbs with a considerable potential to convert into secretory immunoglobulins that could penetrate into the niche of H. pylori and inhibit its normal functionalities. Further, conjugation of H. pylori targeting Ab fragments with the toxic agents or drug delivery systems (DDSs) offers new generation of H. pylori treatments. Introduction Long-term colonization of Helicobacter pylori in the human stomach is often associated with the high incidence of development of peptic ulcers, gastric lymphoma, and gastric adenocarcinoma. Gastric cancer is considered as one of the most prevalent malignancies worldwide, especially in the developing countries. It is largely related to the high colonization of H. pylori , which shows a high rate of morality, in large part because of diagnosis at the late stages of the disease. 1 - 3 H. pylori is classified as type 1 carcinogenic bacteria because more than 60% of the non-cardiac gastric cancers are related to H. pylori infection. 4 - 6 Although the presence of H. pylori is not enough for the initiation of cancer, it is necessary for such phenomenon. H. pylori -associated gastric cancer seems to initiate by the loss of acidic juice secretion due to the persistent atrophic gastritis. 7 H. pylori can survive and colonize in the human stomach through the functional expression of VacA that is produced by all strains, even though with different levels of expression. VacA toxin creates a selective ion channel in order to utilize the electrolyte and metabolites of the host cells for the survival of H. pylori in the mucosal layer. 8 The effects of VacA toxin on the epithelial cells are mediated by diverse mechanisms. Endocytosis of VacA associates with the pattern alteration of intracellular endocytic pathways, resulting in formation of vacuoles, and the impairment of the mitochondrial membrane potential and hence induction of apoptosis. 9 , 10 Besides, VacA binds to variable cell surface receptors and triggers several intracellular signaling pathways, in particular, mitogen-activated protein kinase (MAPK)/p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2), which lead to recruitment of cytokines initiating inflammatory response. 11 - 13 The active VacA toxin is made up of two functional (p33) and binding (p55) domains. Based on sequence diversities in these domains, H. pylori is classified into 4 strains, including: s1m1, s1m2, s2m2 and s2m1. These strains were shown to pose different extent of vacuolation in various cells. 13 , 14 Depending on the cell type and H. pylori strain, the VacA interaction with cells occur through various receptors such as receptor protein tyrosine phosphatase beta (RPTPβ), sphingomyelin, epidermal growth factor receptor (EGFR) and heparin/heparan sulfate. Of these, the RPTPβ is the main receptor for VacA binding, which guaranties survival of H. pylori within the host cells. 15 , 16 The influence of cell surface receptors, in particular RPTPβ, on the VacA-induced cell vacuolation in vitro is still controversial. Collectively, the VacA toxin is considered as a multi-receptor toxin that imposes cellular vacuolization . 17 In addition to the effect of VacA on long-term persistence of H. pylori , VacA is deemed to be a marker for gastric cancer diagnosis, which is also a determinant for the initiation of metastasis in infectious patients. 18 , 19 Former reviews on this subject have not covered the exact roles and effects of multiple receptors on binding and in vitro cytotoxicity of VacA and its influence on immunotherapeutical approaches. Hence, the foremost goal of the current review is to discuss the genotype of VacA toxin and articulate the cell specific toxicity on epithelial cells elicited by VacA and its potential as a candidate for development of vaccine against H. pylori . Structure and expression of VacA The VacA gene is a monosistronic gene with the length of 3860-3940 base pairs. All strains of H. pylori possess the VacA gene, but with different levels of expression leading to different degrees of cytotoxicity. Expression level of the VacA toxin can be affected by several factors, including: (a) promoter strength, (b) stem-loop structures at the 3´ end of mRNA resulting in mRNA endurance, (c) presence of activators or inhibitor sequences, and (d) amino acid sequence differences found between H. pylori strains leading to distinct secretion pattern. 20 , 21 The expression of VacA gene produces a 1287 amino acids (aa), a 140 KDa length pre-protoxin, that is processed to a mature toxin through two cleavages at the N- and C-terminal ends of the toxin by membrane-associated proteases. Similar to autotransporter proteins (ATs), the first proteolysis is occurred by LepB peptidase at the N-terminal signal sequence (33 aa) responsible for transporting toxin to the bacterial periplasm. 22 - 24 The second proteolysis at the C-terminal domain, which happens simultaneously with the secretion of toxin to the outer membrane, produces a 88 KDa mature toxin, a 12 KDa secreted peptide (linker peptide), and a 33 KDa C-Ter autotransporter domain remaining associated with the bacterial membrane. 22 , 25 , 26 The complete processing of VacA produces the mature VacA toxin that is considered as an A-B type toxin ( Fig. 1 ). 27 , 28 The mature VacA toxin is consisted of p33 and p55 domains, which are essential for its activity and binding to the host cells and is performed by various residues confirmed by several studies ( Table 1 ). Fig. 1 The molecular structure of vacuolating toxin of Helicobacter pylori . A) The vacuolating toxin of Helicobacter pylori is expressed as pre-protoxin inside bacteria containing p33 and p55 domains, a linker domain and autotransporter (AT) domain responsible for toxin secretion from bacteria. Maturation of the toxin occurs through two enzymatic cleavages. First, spontaneous N-ter cleavage site leads to breakage of signal sequence (ss), which is a determinant of directing of toxin to periplasmic space. Next, cleavage of the AT domain occurs simultaneously with the secretion of mature toxin to external environment of bacteria. The linker domain degrades by exposure to the environment and mature toxin is produced including two major regions known as p33 and p55. B) There are two diverse regions inside the mature toxin called s in p33 domain and m in p55 domain. The s region is located in the signal sequence of toxin in p33 with two types of s1 and s2 and an extra 21 amino acid extension sequence. The other divergent portion in p55 is also classified in two types, m1 and m2, with 21 amino acid insertion and shows different cell binding capacity to various receptors. Based on these two portions, VacA is classified into four groups of s1m1, s1m2, s2m2 and s2m1, all of which determine various pathogenicity based on their genotype. Table 1 Function of various domains of the VacA toxin Domain Amino acid sequence Role of sequence in toxin activity Ref. P33 6-27 Vacuolation-apoptosisoligomerization 27 , 29 1-32 Ion channel formations 30 49-57 Oligomerization 30 P55 346-347 Vacuolating-depolarizationoligomerization 29 599-628 Folding and secretion of protein 31 312-478 Interaction with p33 29 342-361 Cleavage site 32 313-478 P33/p55 interaction 32 The function of VacA is not fully restricted to the domains introduced for, and each domain- p33 and p55- is participating in the role of the other domain. P55 domain The P55 domain shows different binding specificity to various cells, which is related to a divergent region known as "m region" with the least recombination between strains. Based on the m region, VacA toxin is classified into two types of m1 and m2, which are different in receptor binding and interaction with the host cells as well as the expression level. 33 , 34 Unlike the m1 VacA, the m2 VacA has an additional 23 aa insert located at residues of 460-496, a region that has no effect on the toxin cytotoxicity and/or cell specificity. 5 , 22 Differences in the m region contributes to both different degrees of cytotoxicity in various cells and discrete incidence of gastritis and gastric cancer, even though no relation has been observed between the m region and incidence of peptic ulcer in patients. 5 , 35 , 36 P33 domain The p33 domain (i.e., residues 1 to 311) is a functional domain of the VacA toxin that causes cell vacuolation through formation of anion selective channels. 37 , 38 A hydrophobic 32 aa residual region with a-helix structure is located at the vicinity of the N-terminus of the p33 domain which assumes to be critical for the toxicity of VacA and is designated as s region. 39 Further, some researches have confirmed the importance of the transmembrane dimerization sequence consisted of three tandem repeats of GXXXG motifs for the channel and vacuole formation. 40 Based upon the sequence diversity in the "s region", VacA toxin has been classified into two types of s1 and s2. These two types -like m types- impose the toxicity potential of the toxin via (a) increased expression of the BAbA2 and cagPAI and VacA, (b) enhanced efficiency of signal sequence that facilitates toxin transformation, and (c) elevated capacity of anion selective channels. All these result in phenotypic manifestation of the toxin like peptic ulcer and gastric inflammation. 41 The s1 region shows hydrophobic characteristics that mediates dimerization of toxin leading to channel formation. The s2 region possesses a hydrophilic sequence extension of 12 aa providing a distinct cleavage site from s1, which results in inhibition of cell vacuolation. 42 , 43 Other polymorphic regions inside VacA Besides two mentioned divergent regions inside VacA toxin, two additional regions are identified known as intermediate (i) and deletion (d). The i region, which is located between s and m is classified in 3 diverse types known as i1, i2 and i3. This region is considered to be responsible for increased potential of carcinogenesis and expression of CagA, 44 though one study confirmed that the presence of i is not as efficient as s and m regions on the incidence of disease. 44 - 46 The type of i region is somehow dependent on the presence of types of s and m, so that presence of s1m1 increases the incidence of i1 to be expressed and result in an increased outcome in severity of the disease. 47 The deletion of 81 residues between i and m regions is another polymorphic region inside VacA toxin, which is located at the N-terminus of p55 and is considered to be related to the vacuolating activity and binding to host cells, gastric mucusal atrophy and neutrophil infiltration. Despite the influence of s, m and i regions diversity on toxicity of VacA, it seems that different d regions provide the same function(s). 45 In general, it is believed that various types of VacA toxin are classified based on their polymorphic regions and distributed around the world. They are responsible for various types of diseases with different severity due to their phenotypes expressed and also the effect of host genetic background and environmental effects. 45 P55 domain The P55 domain shows different binding specificity to various cells, which is related to a divergent region known as "m region" with the least recombination between strains. Based on the m region, VacA toxin is classified into two types of m1 and m2, which are different in receptor binding and interaction with the host cells as well as the expression level. 33 , 34 Unlike the m1 VacA, the m2 VacA has an additional 23 aa insert located at residues of 460-496, a region that has no effect on the toxin cytotoxicity and/or cell specificity. 5 , 22 Differences in the m region contributes to both different degrees of cytotoxicity in various cells and discrete incidence of gastritis and gastric cancer, even though no relation has been observed between the m region and incidence of peptic ulcer in patients. 5 , 35 , 36 P33 domain The p33 domain (i.e., residues 1 to 311) is a functional domain of the VacA toxin that causes cell vacuolation through formation of anion selective channels. 37 , 38 A hydrophobic 32 aa residual region with a-helix structure is located at the vicinity of the N-terminus of the p33 domain which assumes to be critical for the toxicity of VacA and is designated as s region. 39 Further, some researches have confirmed the importance of the transmembrane dimerization sequence consisted of three tandem repeats of GXXXG motifs for the channel and vacuole formation. 40 Based upon the sequence diversity in the "s region", VacA toxin has been classified into two types of s1 and s2. These two types -like m types- impose the toxicity potential of the toxin via (a) increased expression of the BAbA2 and cagPAI and VacA, (b) enhanced efficiency of signal sequence that facilitates toxin transformation, and (c) elevated capacity of anion selective channels. All these result in phenotypic manifestation of the toxin like peptic ulcer and gastric inflammation. 41 The s1 region shows hydrophobic characteristics that mediates dimerization of toxin leading to channel formation. The s2 region possesses a hydrophilic sequence extension of 12 aa providing a distinct cleavage site from s1, which results in inhibition of cell vacuolation. 42 , 43 Other polymorphic regions inside VacA Besides two mentioned divergent regions inside VacA toxin, two additional regions are identified known as intermediate (i) and deletion (d). The i region, which is located between s and m is classified in 3 diverse types known as i1, i2 and i3. This region is considered to be responsible for increased potential of carcinogenesis and expression of CagA, 44 though one study confirmed that the presence of i is not as efficient as s and m regions on the incidence of disease. 44 - 46 The type of i region is somehow dependent on the presence of types of s and m, so that presence of s1m1 increases the incidence of i1 to be expressed and result in an increased outcome in severity of the disease. 47 The deletion of 81 residues between i and m regions is another polymorphic region inside VacA toxin, which is located at the N-terminus of p55 and is considered to be related to the vacuolating activity and binding to host cells, gastric mucusal atrophy and neutrophil infiltration. Despite the influence of s, m and i regions diversity on toxicity of VacA, it seems that different d regions provide the same function(s). 45 In general, it is believed that various types of VacA toxin are classified based on their polymorphic regions and distributed around the world. They are responsible for various types of diseases with different severity due to their phenotypes expressed and also the effect of host genetic background and environmental effects. 45 Cell surface receptors for VacA toxin Receptor-like protein tyrosine phosphatase β (RPTPβ) RPTPβ is considered as the major cell receptor for binding of VacA toxin. Similar to inhibitory ligand of RPTPβ, pleiotrophin, the VacA prohibits the phosphatase activity of RPTPβ via interacting with QTTQP motif located at 747-751 containing two O-glycosylation sites that is distinct from the binding site of pleiotrophin. 48 Inhibitory effect of VacA on RPTPβ leads to vacuolation-independent cytotoxicity through phosphorylation of Git-1 that triggers several intracellular signal transductions related to pro-inflammation and ulcerogenesis. 48 Phosphorylation of Git-1 initiates signaling cascades, leading to actin cytoskeleton impairment and focal adhesion segregation known as the first steps in ulcerogenesis. 49 , 50 Further, VacA induces the activation of MAPK pathways through the activation of p38 and ERK1/2 messengers, resulting in expression of various chemokines and inflammatory agents specially interleukins. 12 Several studies have been carried out to investigate whether RPTPβ is related to the VacA induced cellular vacuolation. There is somewhat controversy about relation between the presence of RPTPβ on the cell surface and the toxicity of VacA. It has been shown that the VacA-induced vacuolation in the RPTPβ-positive human promyelocytic leukemia HL-60 cells can be prohibited by antisense oligonucleotide against RPTPβ receptor. 51 , 52 Adversely, the vacuolation, induced by VacA on RPTPβ expressing rabbit kidney epithelial RK13 cells, was not inhibited by phorbol 12-myristate 13-acetate (PMA). In addition, in vivo study with RPTPβ-deficient mice (RPTP-/- strains) indicated that the vacuolation is not dependent on the presence of RPTPβ. 53 - 55 Consequently, RPTPβ is not necessary for the vacuolating activity of VacA, but rather for signal induction. Dependency of the vacuolation to RPTP receptors in various cells is shown in Table 2 . These findings highlight the necessity of further justification of a pattern for the receptor-dependent vacuolation in different cells. Table 2 Phenotype specificity of VacA in relation with RPTPs Cell line Type of receptor Toxic strain of VacA RK13 RPTPβ m1 and m2 Az-521 RPTPβ and RPTPα m1 and m2 AGS RPTPβ and RPTPα m1 HeLa RPTPα m1 G401 RPTPα m1 and m2 HL60 - No toxicity Phenotype specificity caused by the m region of VacA toxin is related to the functional expression of RPTPs. 54 HeLa and G401 cell lines expressing one type of receptor (RPTPα) demonstrate different toxicity responses. Difference in the post-transcriptional processing of RPTPα causes formation of smaller RPTPα in HeLa cell line and reacting solely to the m1, while RPTPα in G401 responds to both m1 and m2. 55 In some cell lines, lack of RPTP is the reason of VacA untoxic behavior. HL-60 cell line with no RPTP receptor and no impression by VacA intoxication responded to VacA m1 when treated with PMA, but still unaffected by m2. 51 , 55 Also two AGS and AZ521 cell lines expressing both RPTP receptors show various reaction to VacA treatment. Additionally, in spite of expressing just RPTPβ in RK13 cell line both m1 and m2 types are considered as toxic strains. As a result, although m1 and m2 both respond to RPTPβ, it seems that in all cell lines presence of RPTPβ does not guarantee the vacuolation potential of VacA. Receptor-like protein tyrosine phosphatase α (RPTPα) RPTPα is a 140-KDa protein receptor (p140) recognized on Wilm's tumor G-401 cell line for the first time. 56 RPTPα, unlike RPTPβ, has shorter glycosylated extracellular domain - necessary for the binding to VacA. In fact, attenuating VacA-induced vacuolation on G401 cells through neuraminidase-α glycosidase treatment indicated the necessity of sialic acid and glycosylation for the binding of VacA. 49 Early studies showed that the function of RPTPα in signaling cascades related to pathogenesis of VacA is still unclear. 48 Epidermal Growth Factor Receptor (EGFR) VacA was shown to be internalized by HeLa cells through EGFR receptor inducing vacuolation. This finding was confirmed through inhibiting the vacuolation by anti-EGFR antibody as well as immunoprecipitaion of two protein fragments (p33 and p55) equivalent to the VacA domains with anti-EGFR antibody. 57 Furthermore, interfering VacA with EGFR-mediated signaling pathways has been shown by ERK1/2 signaling path, actin stress fiber formation, and wound healing. These findings suggest that VacA imposes its impact(s) on HeLa cell through EGFR-mediated signaling pathways leading to inhibition of wound re-epithelialization, renewal of the gastric mucosa and cell proliferation. 58 , 59 Lipid raft/glycosylphosphatidylinositol-anchored proteins (GPI-AP) Lipid raft as a cholesterol-enriched area acts as an accumulation center in the plasma membrane. It appears to be the main pathway, which enables VacA toxin to form oligomerized structure necessary for the flow of electrolytes and ions. 39 , 60 Experiments about low-affinity binding of VacA toxin to the membranes containing low-cholesterol has confirmed the cholesterol-dependent nature of the VacA association to lipid rafts. 61 Furthermore, GPI-AP was shown to be possibly involved with the VacA intoxication through recruitment of GPI-AP-associated VacA by lipid rafts. 62 Incubation of Hep-2 cells with phosphatidylinositol-specific phospholipase C (PI-PLC) was shown to diminish the sensitivity to VacA, supporting the GPI-AP involvement. Also removal of GPI-AP from HeLa cells has confirmed that the localization of VacA into lipid rafts is not a GPI dependent process, but the flux of chloride ions through the membrane is affected. 63 , 64 There are evidences that the translocation of VacA to lipid raft is important for the vacuolation-dependent cytotoxicity. Further, addition of methyl-β-cyclodextrin (MCD), a –cholesterol depleting drug, to HeLa cells and human gastric carcinoma AZ-521 cells was shown to interfere with the VacA internalization and vacuolation, but not with its binding to cells. Thus, it can be conquered that lipid rafts are necessary for the intoxication process. Also, treatment of HeLa and AZ-521 cells by PI-PLC was shown to inhibit the internalization and vacuolation supporting the importance of GPI-AP in the VacA-induced cytotoxicity. 65 Conversely, another study using GPI-AP-deficient mutant Chinese hamster ovarian CHO-LA1 cells has revealed that lipid rafts might play an alternative role of GPI-AP for the VacA toxin. 66 In addition to intoxication, lipid raft influences signaling cascades induced through the binding of VacA toxin to the host surface receptors as well as GPI-APs. Treatment of AZ-521 cells with PI-PLC and MCD was shown to inhibit the p38/MAPK signaling pathway, in large part due to the inhibition of RPTPβ/VacA translocation to lipid raft assisting GPI-AP. As a result, VacA and RPTPβ complex can be translocated to lipid rafts, which is the most important step in triggering the RPTPβ-mediated signaling cascade while the presence of GPI-AP is contributory for the accumulation of VacA in lipid rafts. 65 Sphingomyelin (SM) SM serves as another receptor of the VacA-mediating interaction of toxin with the plasma membrane. The importance of SM in vacuolation was shown by the inhibition of the vacuolation in multiple cell lines (e.g., HeLa, human Caucasian gastric adenocarcinoma AGS cells as well as AZ-521 cells) treated with sphingomyelinase C. Enhanced VacA-induced vacuolation has been shown through exogenously overexpressed SM onto the plasma membrane of HeLa cells, which further indicates the role of the SM in cell vacuolation. In addition, the fate of intracellular pathway in which VacA is involved could be strictly affected by the length of SM carbon chain. In cells with long chain SM (C16 and C18), the VacA intoxication occurs via a cdc42-dependent pinocytic pathway/pinocytosis trafficking to Rab7-containing late endosomal compartment essential for the vacuolating activity of the VacA toxin. However, in the short acyl chains (C2 and C4), the VacA is internalized through a cdc42-independent pathway directing to Rab11-containing compartments of the eukaryotic cells. Interestingly, the length of acyl chain affects the distribution of VacA into lipid or non-lipid raft and also it's uptake by GPI-AP in the plasma membrane. 67 , 68 Fibronectin (FN) FN, a 440 KDa extracellular matrix (ECM) glycoprotein, functions as a receptor for the VacA toxin that binds through its RGD (Arg-Gly-Asp) motif in a dose-dependent manner. 69 Adhesion of the VacA to FN or any other ECM proteins can initiate several mechanisms in favor of bacteria survival, in large part through retarding the clearance of H. pylori and preserving the infection via invasion and penetration into the intracellular junction. 70 , 71 In addition, the VacA binding to FN was shown to impose cell disruption and changes in cell morphology through cytoskeletal reorganization. 72 Heparin (H) and heparan sulfate (HS) The H and HS as two types of receptors are cellular surface and extra cellular matrix-associated proteoglycans and present different binding sites for m1 and m2 strain of VacA in favor of H. pylori survival. 73 It should be also stated that H. pylori evades immune response of the host by binding of VacA to H/HS that, in return, recruits and inactivates complement components, C3b and C4b, known as one of the first barriers of the native immune system. 74 Further, attachment of H/HS onto the surface of microorganism enables H. pylori to avoid phagocytosis. 75 Low-density Lipoprotein Receptor-related Protein-1 (LRP1) LRP1, an endocytic receptor from LDL receptor family, is thought to be the receptor mediating VacA-dependent autophagy. The study investigating the influence of LRP1 was carried out on AZ-521 cell. LRP1 gene silencing by siRNA and confocal microscopy results in the AZ-521 cells determined dependency of binding and internalization of toxin to LRP1. Furthermore, VacA-induced apoptosis occurs through autophagy and caspase independent via binding to LRP1. Interestingly, formation of small vacuoles via binding to LRP1 is exhibited by formation of phagosomes, which is different from the RPTPβ-mediated vacuolation. Overall, LRP1 is considered as one of the main receptors for binding and internalization leading to autophagy dependent apoptosis and vacuolation. 76 CD18 In addition of various receptors on epithelial cells for binding of VacA, one receptor has been identified as a ligand for VacA on T cells named CD18 and known as PKC-associated internalization. Similar to the epithelial cells internalization, the mechanism of intoxication after concentrating within the lipid raft is clathrin-independent due to phosphorylation of cytoplasmic domain of CD18, PKC, by the regulation of Rac1 and cdc-42. 77 As a result, T-cell proliferation and IL-2 signaling pathway is prohibited by inhibition of nuclear factor of activated T-cell (NFAT) making it an immunomodulator toxin. 78 Another immunosuppressive effect of VacA has been observed on B cells, causing interference with antigens (Ags) presenting cells (APCs). 79 Receptor-like protein tyrosine phosphatase β (RPTPβ) RPTPβ is considered as the major cell receptor for binding of VacA toxin. Similar to inhibitory ligand of RPTPβ, pleiotrophin, the VacA prohibits the phosphatase activity of RPTPβ via interacting with QTTQP motif located at 747-751 containing two O-glycosylation sites that is distinct from the binding site of pleiotrophin. 48 Inhibitory effect of VacA on RPTPβ leads to vacuolation-independent cytotoxicity through phosphorylation of Git-1 that triggers several intracellular signal transductions related to pro-inflammation and ulcerogenesis. 48 Phosphorylation of Git-1 initiates signaling cascades, leading to actin cytoskeleton impairment and focal adhesion segregation known as the first steps in ulcerogenesis. 49 , 50 Further, VacA induces the activation of MAPK pathways through the activation of p38 and ERK1/2 messengers, resulting in expression of various chemokines and inflammatory agents specially interleukins. 12 Several studies have been carried out to investigate whether RPTPβ is related to the VacA induced cellular vacuolation. There is somewhat controversy about relation between the presence of RPTPβ on the cell surface and the toxicity of VacA. It has been shown that the VacA-induced vacuolation in the RPTPβ-positive human promyelocytic leukemia HL-60 cells can be prohibited by antisense oligonucleotide against RPTPβ receptor. 51 , 52 Adversely, the vacuolation, induced by VacA on RPTPβ expressing rabbit kidney epithelial RK13 cells, was not inhibited by phorbol 12-myristate 13-acetate (PMA). In addition, in vivo study with RPTPβ-deficient mice (RPTP-/- strains) indicated that the vacuolation is not dependent on the presence of RPTPβ. 53 - 55 Consequently, RPTPβ is not necessary for the vacuolating activity of VacA, but rather for signal induction. Dependency of the vacuolation to RPTP receptors in various cells is shown in Table 2 . These findings highlight the necessity of further justification of a pattern for the receptor-dependent vacuolation in different cells. Table 2 Phenotype specificity of VacA in relation with RPTPs Cell line Type of receptor Toxic strain of VacA RK13 RPTPβ m1 and m2 Az-521 RPTPβ and RPTPα m1 and m2 AGS RPTPβ and RPTPα m1 HeLa RPTPα m1 G401 RPTPα m1 and m2 HL60 - No toxicity Phenotype specificity caused by the m region of VacA toxin is related to the functional expression of RPTPs. 54 HeLa and G401 cell lines expressing one type of receptor (RPTPα) demonstrate different toxicity responses. Difference in the post-transcriptional processing of RPTPα causes formation of smaller RPTPα in HeLa cell line and reacting solely to the m1, while RPTPα in G401 responds to both m1 and m2. 55 In some cell lines, lack of RPTP is the reason of VacA untoxic behavior. HL-60 cell line with no RPTP receptor and no impression by VacA intoxication responded to VacA m1 when treated with PMA, but still unaffected by m2. 51 , 55 Also two AGS and AZ521 cell lines expressing both RPTP receptors show various reaction to VacA treatment. Additionally, in spite of expressing just RPTPβ in RK13 cell line both m1 and m2 types are considered as toxic strains. As a result, although m1 and m2 both respond to RPTPβ, it seems that in all cell lines presence of RPTPβ does not guarantee the vacuolation potential of VacA. Receptor-like protein tyrosine phosphatase α (RPTPα) RPTPα is a 140-KDa protein receptor (p140) recognized on Wilm's tumor G-401 cell line for the first time. 56 RPTPα, unlike RPTPβ, has shorter glycosylated extracellular domain - necessary for the binding to VacA. In fact, attenuating VacA-induced vacuolation on G401 cells through neuraminidase-α glycosidase treatment indicated the necessity of sialic acid and glycosylation for the binding of VacA. 49 Early studies showed that the function of RPTPα in signaling cascades related to pathogenesis of VacA is still unclear. 48 Epidermal Growth Factor Receptor (EGFR) VacA was shown to be internalized by HeLa cells through EGFR receptor inducing vacuolation. This finding was confirmed through inhibiting the vacuolation by anti-EGFR antibody as well as immunoprecipitaion of two protein fragments (p33 and p55) equivalent to the VacA domains with anti-EGFR antibody. 57 Furthermore, interfering VacA with EGFR-mediated signaling pathways has been shown by ERK1/2 signaling path, actin stress fiber formation, and wound healing. These findings suggest that VacA imposes its impact(s) on HeLa cell through EGFR-mediated signaling pathways leading to inhibition of wound re-epithelialization, renewal of the gastric mucosa and cell proliferation. 58 , 59 Lipid raft/glycosylphosphatidylinositol-anchored proteins (GPI-AP) Lipid raft as a cholesterol-enriched area acts as an accumulation center in the plasma membrane. It appears to be the main pathway, which enables VacA toxin to form oligomerized structure necessary for the flow of electrolytes and ions. 39 , 60 Experiments about low-affinity binding of VacA toxin to the membranes containing low-cholesterol has confirmed the cholesterol-dependent nature of the VacA association to lipid rafts. 61 Furthermore, GPI-AP was shown to be possibly involved with the VacA intoxication through recruitment of GPI-AP-associated VacA by lipid rafts. 62 Incubation of Hep-2 cells with phosphatidylinositol-specific phospholipase C (PI-PLC) was shown to diminish the sensitivity to VacA, supporting the GPI-AP involvement. Also removal of GPI-AP from HeLa cells has confirmed that the localization of VacA into lipid rafts is not a GPI dependent process, but the flux of chloride ions through the membrane is affected. 63 , 64 There are evidences that the translocation of VacA to lipid raft is important for the vacuolation-dependent cytotoxicity. Further, addition of methyl-β-cyclodextrin (MCD), a –cholesterol depleting drug, to HeLa cells and human gastric carcinoma AZ-521 cells was shown to interfere with the VacA internalization and vacuolation, but not with its binding to cells. Thus, it can be conquered that lipid rafts are necessary for the intoxication process. Also, treatment of HeLa and AZ-521 cells by PI-PLC was shown to inhibit the internalization and vacuolation supporting the importance of GPI-AP in the VacA-induced cytotoxicity. 65 Conversely, another study using GPI-AP-deficient mutant Chinese hamster ovarian CHO-LA1 cells has revealed that lipid rafts might play an alternative role of GPI-AP for the VacA toxin. 66 In addition to intoxication, lipid raft influences signaling cascades induced through the binding of VacA toxin to the host surface receptors as well as GPI-APs. Treatment of AZ-521 cells with PI-PLC and MCD was shown to inhibit the p38/MAPK signaling pathway, in large part due to the inhibition of RPTPβ/VacA translocation to lipid raft assisting GPI-AP. As a result, VacA and RPTPβ complex can be translocated to lipid rafts, which is the most important step in triggering the RPTPβ-mediated signaling cascade while the presence of GPI-AP is contributory for the accumulation of VacA in lipid rafts. 65 Sphingomyelin (SM) SM serves as another receptor of the VacA-mediating interaction of toxin with the plasma membrane. The importance of SM in vacuolation was shown by the inhibition of the vacuolation in multiple cell lines (e.g., HeLa, human Caucasian gastric adenocarcinoma AGS cells as well as AZ-521 cells) treated with sphingomyelinase C. Enhanced VacA-induced vacuolation has been shown through exogenously overexpressed SM onto the plasma membrane of HeLa cells, which further indicates the role of the SM in cell vacuolation. In addition, the fate of intracellular pathway in which VacA is involved could be strictly affected by the length of SM carbon chain. In cells with long chain SM (C16 and C18), the VacA intoxication occurs via a cdc42-dependent pinocytic pathway/pinocytosis trafficking to Rab7-containing late endosomal compartment essential for the vacuolating activity of the VacA toxin. However, in the short acyl chains (C2 and C4), the VacA is internalized through a cdc42-independent pathway directing to Rab11-containing compartments of the eukaryotic cells. Interestingly, the length of acyl chain affects the distribution of VacA into lipid or non-lipid raft and also it's uptake by GPI-AP in the plasma membrane. 67 , 68 Fibronectin (FN) FN, a 440 KDa extracellular matrix (ECM) glycoprotein, functions as a receptor for the VacA toxin that binds through its RGD (Arg-Gly-Asp) motif in a dose-dependent manner. 69 Adhesion of the VacA to FN or any other ECM proteins can initiate several mechanisms in favor of bacteria survival, in large part through retarding the clearance of H. pylori and preserving the infection via invasion and penetration into the intracellular junction. 70 , 71 In addition, the VacA binding to FN was shown to impose cell disruption and changes in cell morphology through cytoskeletal reorganization. 72 Heparin (H) and heparan sulfate (HS) The H and HS as two types of receptors are cellular surface and extra cellular matrix-associated proteoglycans and present different binding sites for m1 and m2 strain of VacA in favor of H. pylori survival. 73 It should be also stated that H. pylori evades immune response of the host by binding of VacA to H/HS that, in return, recruits and inactivates complement components, C3b and C4b, known as one of the first barriers of the native immune system. 74 Further, attachment of H/HS onto the surface of microorganism enables H. pylori to avoid phagocytosis. 75 Low-density Lipoprotein Receptor-related Protein-1 (LRP1) LRP1, an endocytic receptor from LDL receptor family, is thought to be the receptor mediating VacA-dependent autophagy. The study investigating the influence of LRP1 was carried out on AZ-521 cell. LRP1 gene silencing by siRNA and confocal microscopy results in the AZ-521 cells determined dependency of binding and internalization of toxin to LRP1. Furthermore, VacA-induced apoptosis occurs through autophagy and caspase independent via binding to LRP1. Interestingly, formation of small vacuoles via binding to LRP1 is exhibited by formation of phagosomes, which is different from the RPTPβ-mediated vacuolation. Overall, LRP1 is considered as one of the main receptors for binding and internalization leading to autophagy dependent apoptosis and vacuolation. 76 CD18 In addition of various receptors on epithelial cells for binding of VacA, one receptor has been identified as a ligand for VacA on T cells named CD18 and known as PKC-associated internalization. Similar to the epithelial cells internalization, the mechanism of intoxication after concentrating within the lipid raft is clathrin-independent due to phosphorylation of cytoplasmic domain of CD18, PKC, by the regulation of Rac1 and cdc-42. 77 As a result, T-cell proliferation and IL-2 signaling pathway is prohibited by inhibition of nuclear factor of activated T-cell (NFAT) making it an immunomodulator toxin. 78 Another immunosuppressive effect of VacA has been observed on B cells, causing interference with antigens (Ags) presenting cells (APCs). 79 Internalization and trafficking of VacA toxin Generally, VacA is released from bacteria in two types, including: (a) VacA associated with vesicles known as outer membrane vesicles (OMV), and (b) free VacA. The biological activity of free VacA is triggered immediately after the internalization (known as the early internalization), while the VacA-containing OMVs remain intact for about 72 hours after the internalization (known as the late internalization). 80 Both types of VacA are internalized through clathrin-independent pathway that is believed to be regulated by the small GTPase proteins, Rack1 and Cdc42. Upon internalization, the VacA toxin is located beneath the membrane in GPI-AP early endosome compartment (GEECs). The tubulovesicular compartments containing GPI-AP are part of the GPI recycling system. Once in the early and late endosomes, the toxin either is directed to mitochondria and induces apoptosis or is oligomerized on the vacuole membranes and causes vacuolation. 65 , 81 , 82 Similar to the diphtheria and tetanus toxins, VacA intoxication through lipid rafts is a pH- dependent process. An increased interaction of VacA with lipid raft at an acidic pH is attributed to the structural alteration of p55 domain. Although the effect of low pH on the p33 domain has been shown, its impact(s) on the p55 domain appears to be higher on the toxin binding because the interaction of p33 with lipid raft is weaker/lower than that of the p55. The low pH-induced conformational changes were shown to increase the surface exposure of the hydrophobic domain of toxin in favor of its binding to the lipid rafts, which facilitates the membrane internalization. 80 Pathogenesis of VacA and signaling pathways VacA can trigger intracellular signal transductions related to pro-inflammation, vacuolation and apoptosis. In general, the behaviour of the host cells in relation with VacA toxin is relevant to the type of toxin-cell interaction. Binding of the VacA toxin to the host cell membrane through its receptors can activate several signaling pathways leading to pro-inflammatory responses. On the other hand, the uptake of VacA toxin by cells can direct the toxin to the mitochondria and induce apoptosis or vacuolation ( Fig. 2 ). 23 Fig. 2 Pathogenicity of VacA toxin is exhibited on epithelial cells. A) After the pinocytosis of VacA into intracellular environment, it is directed towards early endosomal compartment GEEC, EE and LE respectively by the aid of F-actin filaments. Next, VacA is oligomerized on the LE membrane acting as the channel discharging chloride ions that results in LE swelling since water molecules are imported due to overpopulation of ammonium resulted from the change in chloride ions. Consequently, over-hydration results in vacuolation of the cell. B) Following aforementioned trafficking of the vacA to the endosomal compartments, its transportation to the mitochondria by N-ter signal sequence of the toxin can commence the programmed cell death (apoptosis). Importing of VacA through TOM complex and oligomerizing alters membrane potential of the mitochondria which recruit pro-apoptotic Bax and Bak complexes indirectly leading to release of cytochrome c. C) Binding of the VacA to cell surface receptor RPTPβ activates the MAPK signaling cascades independently or in cooperation with CagA. The activation of MAPK/p38 pathway induces stimulation of transcription factor ATF-2 that increases the expression of PGE2, known as the angiogenic factor and mitotic signal, via binding to COX-2 promoter. Additionally, VacA phosphorylates ERK1/2 that activates the downstream component c-fos, while CagA activates the other components of AP-1 heterodimer, c-jun, by phosphorylation of JNK. Interaction of c-fos and c-jun together would produce the AP-1 heterodimer which is counted as the transcription factor for several cytokines such as IL-1, IL-6 and TNFα leading to inflammation of the gastric mucosa. A- Vacuolation After pinocytic-dependent and clathrin-independent endocytosis of the VacA toxin through GEECs, they are directed to the early endosomal (EE) compartments containing EEA1 as a marker with assistance of the F-actin and CD2AP as VacA-F-actin interaction mediator. 15 Vesicular trafficking of early endosomes to the LAMP1-containing late endosomes (LE) is the next step that initiates the oligomerization of VacA on the late endosomal compartment membrane. The oligomerized VacA acts as an ion selective channel altering chloride concentration and enhancing proton pump activity that results in LE swelling and vacuole formation. 24 , 83 B- Programmed Cell Death (apoptosis) In addition to the vacuolation of the host cells caused by the VacA, the internalization through endocytosis can elicit apoptosis via mitochondrial-dependent cell death mechanism. Internalization to the mitochondria occurs via TOM complex through the recognition of N-terminus of VacA, which is believed to be futile for the channel formation but important for directing toxin to the mitochondrial membrane. VacA, containing LE compartment and mitochondrial membrane interaction, is also able to initiate the programmed cell death through membrane potential fluctuations. 84 , 85 The release of cytochrome C through translocation of the pro-apoptotic compounds- Bax and Bak caused by the alterations of membrane potential is the basic mechanism for leading cells towards apoptosis. 86 , 87 C- MAPK signaling pathways Furthermore, VacA can impose its effects on the host cell through binding to certain membrane receptors and inducing p38 and ERK1/2 signaling cascades. 84 Activation of p38/MAPK pathway through binding of the VacA initiates the activating transcription factor-2 (ATF-2), which functions as a regulator on the Cox-2 by binding to its promoter via TLR2/9. Induction of Cox-2 expression was shown to enhance the expression of PGE2 - known as a mitogenic signal - and contributes to angiogenesis through up-regulation of the vascular endothelial growth factor (VEGF). 16 , 88 VacA also contributes to the activation of ERK1/2 via its phosphorylation and formation of AP-1 that contains heterodimer of c-fos and c-jun. While the c-fos expression is regulated by the phosphorylated ERK1/2, the other part of the AP-1 complex is activated via JNK phosphorylation through binding of cagA that is another toxin of H. pylori -. As a result, AP-1 regulates the expression and recruitment of cytokines such as IL-8, IL-6, TNF-α and NF-κB through binding to their promoters. 12 , 89 D- Toxin oligomerization on plasma membrane In the host cells, the VacA toxin is functionalized in two forms of monomer and oligomer. The latter can reshape as "rosettes" in the solution and form low-conductance, anion-selective and voltage-dependent bio-structures that are water-soluble 30 nm-diameter channels as two single-layered hexameric and heptameric flowers and bilayered shapes consisting of 6-9 and 12-14 monomers, respectively. 40 , 90 , 91 A study predicting structure of oligomerized VacA by deep-etch electron microscopy (EM) viewed 3 possible forms for hexameric structure, including: (i) the flower shape, a dodecamer constructed of two hexameric flat forms with two symmetrical six-subunit arrays, (ii) the flat form composed of an individual six-petal structure known as half of a dodecamer with central ring and counterclockwise chirality, and (iii) the other flat form with no central ring and clockwise chirality with a more abundance than the former and limited accessibility in the environment ( Fig. 3 ). 91 Fig. 3 Freeze-dried transmission electron microscopy micrographs of H. pylori VacA toxin. A) Rotary replicas of oligomerized structures of VacA toxin. The first, second and third rows respectively show the flower shaped dodecamer form of VacA toxin, the flat form of VacA in oligomerized form, and the seven-membered structure of toxin. B) Three structures predicted based on the studies on the oligomerized forms of VacA with deep-etch electron microscopy analysis. The right, middle and left images respectively show the symmetrical pair of hexameric VacA flower placed face-to-face that is a complete dodecamer constructed, the minor flat form included half of a dodecamer with the central ring that resembles the complete VacA structure, and the more common flat form of oligomerized VacA with no central ring that is sandwiched in the center of dodecamer. The data were adopted with permission from a published work conducted by Cover et al. 91 Affluence of the heptameric structure seems to be dependent on the length of the loop region located between p33 and p55. Since the m1 type possesses longer residue than the m2 type, the heptameric structure is more abundant. These multi-shaped structures are generated in the presence of two interacting sites and environmental effect of pH, which aids the assembly of monomeric structures in the neutral pH. 92 , 93 The oligomerized VacA on the host cells are able to permeabilize the membrane through rosette form channels that can efflux/transfer the metabolites and ions of the host cell to the extracellular environment in favor of survival of bacteria. 40 A- Vacuolation After pinocytic-dependent and clathrin-independent endocytosis of the VacA toxin through GEECs, they are directed to the early endosomal (EE) compartments containing EEA1 as a marker with assistance of the F-actin and CD2AP as VacA-F-actin interaction mediator. 15 Vesicular trafficking of early endosomes to the LAMP1-containing late endosomes (LE) is the next step that initiates the oligomerization of VacA on the late endosomal compartment membrane. The oligomerized VacA acts as an ion selective channel altering chloride concentration and enhancing proton pump activity that results in LE swelling and vacuole formation. 24 , 83 B- Programmed Cell Death (apoptosis) In addition to the vacuolation of the host cells caused by the VacA, the internalization through endocytosis can elicit apoptosis via mitochondrial-dependent cell death mechanism. Internalization to the mitochondria occurs via TOM complex through the recognition of N-terminus of VacA, which is believed to be futile for the channel formation but important for directing toxin to the mitochondrial membrane. VacA, containing LE compartment and mitochondrial membrane interaction, is also able to initiate the programmed cell death through membrane potential fluctuations. 84 , 85 The release of cytochrome C through translocation of the pro-apoptotic compounds- Bax and Bak caused by the alterations of membrane potential is the basic mechanism for leading cells towards apoptosis. 86 , 87 C- MAPK signaling pathways Furthermore, VacA can impose its effects on the host cell through binding to certain membrane receptors and inducing p38 and ERK1/2 signaling cascades. 84 Activation of p38/MAPK pathway through binding of the VacA initiates the activating transcription factor-2 (ATF-2), which functions as a regulator on the Cox-2 by binding to its promoter via TLR2/9. Induction of Cox-2 expression was shown to enhance the expression of PGE2 - known as a mitogenic signal - and contributes to angiogenesis through up-regulation of the vascular endothelial growth factor (VEGF). 16 , 88 VacA also contributes to the activation of ERK1/2 via its phosphorylation and formation of AP-1 that contains heterodimer of c-fos and c-jun. While the c-fos expression is regulated by the phosphorylated ERK1/2, the other part of the AP-1 complex is activated via JNK phosphorylation through binding of cagA that is another toxin of H. pylori -. As a result, AP-1 regulates the expression and recruitment of cytokines such as IL-8, IL-6, TNF-α and NF-κB through binding to their promoters. 12 , 89 D- Toxin oligomerization on plasma membrane In the host cells, the VacA toxin is functionalized in two forms of monomer and oligomer. The latter can reshape as "rosettes" in the solution and form low-conductance, anion-selective and voltage-dependent bio-structures that are water-soluble 30 nm-diameter channels as two single-layered hexameric and heptameric flowers and bilayered shapes consisting of 6-9 and 12-14 monomers, respectively. 40 , 90 , 91 A study predicting structure of oligomerized VacA by deep-etch electron microscopy (EM) viewed 3 possible forms for hexameric structure, including: (i) the flower shape, a dodecamer constructed of two hexameric flat forms with two symmetrical six-subunit arrays, (ii) the flat form composed of an individual six-petal structure known as half of a dodecamer with central ring and counterclockwise chirality, and (iii) the other flat form with no central ring and clockwise chirality with a more abundance than the former and limited accessibility in the environment ( Fig. 3 ). 91 Fig. 3 Freeze-dried transmission electron microscopy micrographs of H. pylori VacA toxin. A) Rotary replicas of oligomerized structures of VacA toxin. The first, second and third rows respectively show the flower shaped dodecamer form of VacA toxin, the flat form of VacA in oligomerized form, and the seven-membered structure of toxin. B) Three structures predicted based on the studies on the oligomerized forms of VacA with deep-etch electron microscopy analysis. The right, middle and left images respectively show the symmetrical pair of hexameric VacA flower placed face-to-face that is a complete dodecamer constructed, the minor flat form included half of a dodecamer with the central ring that resembles the complete VacA structure, and the more common flat form of oligomerized VacA with no central ring that is sandwiched in the center of dodecamer. The data were adopted with permission from a published work conducted by Cover et al. 91 Affluence of the heptameric structure seems to be dependent on the length of the loop region located between p33 and p55. Since the m1 type possesses longer residue than the m2 type, the heptameric structure is more abundant. These multi-shaped structures are generated in the presence of two interacting sites and environmental effect of pH, which aids the assembly of monomeric structures in the neutral pH. 92 , 93 The oligomerized VacA on the host cells are able to permeabilize the membrane through rosette form channels that can efflux/transfer the metabolites and ions of the host cell to the extracellular environment in favor of survival of bacteria. 40 VacA and CagA interaction Besides VacA-induced pathogenesis on the host cell in gastric mucusa, it is proved that the VacA association with CagA is a key regulator for the disease severity. Phosphorylated and unphosphorylated CagAs are able to function as the inhibitor of intracellular trafficking of VacA and the simulator of anti-apoptotic BCL2 in order to prevent initiation of apoptosis induced by VacA, respectively. 94 , 95 Besides this manifestation of CagA, it affects the internalization of the VacA to the host cells to which bacteria is attached in order to assist the survival of the bacteria. It is determined that the binding of VacA to distant cells out of access of H. pylori induces vacuolation and apoptosis, thus the intracellular nutrients become accessible. 96 , 97 VacA as a candidate for molecular therapy For decades, the first line treatment of H. pylori infection is a triple drug combination, including: proton pump inhibitor (PPI), clarithromycin and amoxicillin. However, this traditional regimen often associate with some drawbacks, including: an increased resistance to antibiotic, low patients compliance against the long course of treatment with significant side effects, and relapse of the infection. 98 , 99 Thus, new generation molecular therapies need to be alternatively implemented since they impose less detrimental impacts on the healthy tissues with lower side effects, thereby improving the efficacy of treatment. In several experiments, few vaccines and immunotherapies have successfully been developed against various H. pylori Ags related to infection and colonization of the bacteria and incidence of peptic ulcer and gastric cancer. The candidate Ags for molecular therapy include flagella, hsp60, urease, adhesions, cagA and VacA. 100 Of these, VacA plays a pivotal role in pathogenesis of H. pylori. It contributes to the persistent colonization of bacteria, leading to the longevity of infection through escaping from the autophagy and immunosurveillance of host. 101 VacA is considered as one of the main virulence factors involving in ulcerogenesis and gastric cancer. Further, the severity of disease symptoms among patients suffering from the gastritis and peptic ulcer disease appears to be VacA strain-dependent. 102 Vaccine development Generally, based on multiple studies on developing an effective vaccine against H. pylori, there are two types of vaccines, including: (a) the prophylactic vaccine that prevents the infection by reducing or clearing bacteria in the healthy or previously infected individuals, and (b) the therapeutic vaccine that treats the infection in patients via stimulating immune system different from the infection-induced immunity. 103 , 104 To control this formidable diseases, however, multi-component vaccines need to be developed. To achieve an effective vaccine against H. pylori , multiple virulence factors mediating various clinical manifestations should be targeted. Although multivalent/polyvalent vaccines would be helpful, development of multi-antigen vaccines against H. pylori seems to be inevitable. For this purpose, the vaccine could be designed based on the H. pylori whole cell (HWC) or antigen cocktail which is composite of two or more major virulence factors of bacterium. 105 Although approaches exploiting HWC in mouse models or different clinical trials confirmed the efficacy of these vaccines, in some cases, these types of vaccines show low quality of Ags and possible cross-reactivity and immunologic reactions. 106 , 107 Therefore, an efficient vaccine against H. pylori should contain a series of virulent Ags presented in all strains that are involved in bacterial adhesion and intoxication. Owing to its function in pathogenicity of H. pylori, VacA has a potential ability of being a suitable candidate for vaccine development. This notion has further been verified because of the high amount of IgGs detected in patient's serum, and the high expression rate in most H. pylori strains specially those causing the peptic ulcer. Furthermore, establishing protection with no need to native conformation by VacA toxin makes it an appropriate Ag for development of vaccine and immunotherapy. 104 , 108 A couple of studies have reported the efficacy of the VacA-containing vaccine in vitro and animal models. For example, formaldehyde-inactivated VacA vaccine was shown to elicit immune response(s) in mice as compared to that of the native toxin. 109 It should be noted that formaldehyde is a detoxifying agent via interacting with L-lysinin residues of the toxin, and hence can be exploited as an adjuvant in vaccination. Since toxin inactivation by formaldehyde also causes distortion of some epitopes that do not present in the native Ags, the mutant type of heat-labile enterotoxin of E. coli, LTK63, was utilized as a suitable adjuvant for the vaccine development against H. pylori. 110 In addition, VacA has been exploited for the production of multi-component vaccines. Intragastric immunization of mice infected with H. pylori SPM326 by the therapeutic vaccine consisting of recombinant VacA and CagA along with LTK63 adjuvant was shown to eradicate H. pylori for 3 months and also provide tenacity of eradication for 2 months after infection rehearsal. 111 Immunization of Beagle dog suffering from gastritis with multi-component vaccine, VacA-CagA- neutrophil-activating protein (NAP), supplemented with adjuvant Al(OH) 3 , resulted in an increased level of antibody (Ab) and hence protection for 4 months post-vaccination. Despite such therapeutic impacts, the gastritis was recurred after 29 weeks indicating the partial periodic effect of vaccine. 112 In contrary to mice, Beagle dogs, as animal model for the human H. pylori infection, provide researchers with high ability of tandem biopsy with no need to sacrifice the animal, which helps to gain a better understanding of the overall process of infection and immunity. Further, a prophylactic vaccine containing three Ags (VacA, CagA and NAP) with adjuvant Al(OH) 3 has been developed and examined in the non-infected individuals through a phase 1 clinical trial. This intramuscular booster vaccination demonstrated satisfactory safety and immunogenicity with anamnestic antibody and cellular responses 18–24 months post-vaccination. Long memory immunity was originated from VacA- and CagA-driven interferon ɤ production, which lasted 4.5 months post-immunization. After the third immunization, all volunteers responded to one or two Ags (mostly VacA and NAP), while 86% responded to all three Ags. VacA induced Ab response after two immunizations, whereas CagA showed cell-immune response after three immunizations. 113 Yet, there exist some debates to figure out what portion of the VacA toxin is more efficient for the vaccine development. A study using different recombinant fragments of VacA confirmed that the highest titer of Ab was produced against 297-317 residues including both p33 and p55 regions of the toxin. 114 Nonetheless, another effort was carried out to study the VacA neutralizing activity of 10 monoclonal antibodies (mAbs) produced in mice via epitope mapping using a panel of VacA deletion mutants and VacA chimera. Among these, two mAbs were able to neutralize the cytotoxic activity of VacA through recognition of amino acids 685 to 821 located at the receptor binding region. 115 In the following context, mAbs against H. pylori infection will be discussed. Monoclonal antibody Development of mAbs has provided new insights in the diagnosis and therapy of various diseases, in particular infections and malignancies. As a result, commercial therapeutic mAbs have made their way to the therapeutic markets in the early 1980s. 116 Passive administration of mAb directed at protective Ags such as VacA and urease might be particularly relevant as a substitute to the current therapies and diagnosis methods. Thus, VacA toxin has become a target for the production of therapeutic mAbs to neutralize the cytotoxin activity. Accordingly, various studies reported that some variants of VacA are highly associated with an increased risk of symptomatic gastroduodenal disease. Two anti-VacA mAbs, known as V36E and V41, were shown to be able to neutralize the vacuolation of rabbit kidney cell line RK13 through binding to the native structure of VacA. 117 Another experiment was conducted to produce mAbs against small and large subunits of urease. As a result, two Abs designated as S2 and L2 were determined based on their strong interactions respectively with small and large subunits, and showed significant blockage of the enzyme activity. 118 Besides application of mAbs in the field of therapy, it seems that they are promising tools for increasing accuracy of diagnosis. Common diagnostic methods of H. pylori infection have been developed on the basis of biopsy, which is painful. Further, various diagnostic methods such as Urea Breath Test (UBT) and serological techniques may provide false negative and positive results specially in asymptomatic patients, thus leading to failure of accurate diagnosis and efficient treatments. 119 Gamma-glutamyl transpeptidase (GGT) antigen is a virulence factor, necessary for colonization and cell apoptosis of H. pylori , which appears to be a promising Ag for development of mAb. In 2014, a phase 1 clinical trial (ID: NCT02123771) was successfully carried out to detect the H. pylori infection in the stool of patients using ELISA and anti-GGT mAb. Given that several toxins of H. pylori participate in the pathogenesis and survival of bacteria, a multi-component Ab cocktail may provide the next generation immunotherapy of H. pylori infection, similar to Ab cocktail used for treatment of Antrax. 120 Ab cocktail is a combination of several Abs that can recognize different virulence factors, through which the treatment outcome can be maximized by expanding the spectrum of protection and enhancing the protective efficacy. Production of Ab cocktail to recognize the major virulence factors H. pylori (i.e., VacA, urease, CagA, GGT and flagellin) appears to be very desirable for the treatment of H. pylori infection. Use of class immunoglobulin A (IgA) in Ab cocktail against H. pylori seems to provide an improved immunotherapy modality, in large part because IgA secreting cells often react with H. pylori membrane proteins (e.g., flagellin and urease) in H. pylori -infected patients. 121 Given the high stability and avidity of IgA in the gastric environment and habituating H. pylori as a noninvasive organism in gastric mucosa, passive immunotherapeutic approaches using secretory IgA (sIgA) might provide a robust treatment modality. For example, in a study, a scFv polypeptide specific to H. pylori urease subunit A was compared with its reformatted polymeric IgA (IgAp/d) and sIgA for the degree of enzyme activity inhibition. Due to increased avidity, the sIgA and IgAp/d were found to be able to efficiently block the enzymatic activity of free and membrane-associated ureases. 122 , 123 Although several antibody fragments have been designed against the main virulence factor of H. pylori , urease, 124 no antibody fragment has been developed against domain of VacA by phage display ever. New approaches for production of antibodies, such as phage display technique, 125 , 126 have been developed in the recent decades that provide a convenient and economic procedure for production of monoclonal antibodies. To the best of our knowledge, we are among the first teams who work on the development of mAb fragments by phage display against VacA toxin. 127 , 128 Vaccine development Generally, based on multiple studies on developing an effective vaccine against H. pylori, there are two types of vaccines, including: (a) the prophylactic vaccine that prevents the infection by reducing or clearing bacteria in the healthy or previously infected individuals, and (b) the therapeutic vaccine that treats the infection in patients via stimulating immune system different from the infection-induced immunity. 103 , 104 To control this formidable diseases, however, multi-component vaccines need to be developed. To achieve an effective vaccine against H. pylori , multiple virulence factors mediating various clinical manifestations should be targeted. Although multivalent/polyvalent vaccines would be helpful, development of multi-antigen vaccines against H. pylori seems to be inevitable. For this purpose, the vaccine could be designed based on the H. pylori whole cell (HWC) or antigen cocktail which is composite of two or more major virulence factors of bacterium. 105 Although approaches exploiting HWC in mouse models or different clinical trials confirmed the efficacy of these vaccines, in some cases, these types of vaccines show low quality of Ags and possible cross-reactivity and immunologic reactions. 106 , 107 Therefore, an efficient vaccine against H. pylori should contain a series of virulent Ags presented in all strains that are involved in bacterial adhesion and intoxication. Owing to its function in pathogenicity of H. pylori, VacA has a potential ability of being a suitable candidate for vaccine development. This notion has further been verified because of the high amount of IgGs detected in patient's serum, and the high expression rate in most H. pylori strains specially those causing the peptic ulcer. Furthermore, establishing protection with no need to native conformation by VacA toxin makes it an appropriate Ag for development of vaccine and immunotherapy. 104 , 108 A couple of studies have reported the efficacy of the VacA-containing vaccine in vitro and animal models. For example, formaldehyde-inactivated VacA vaccine was shown to elicit immune response(s) in mice as compared to that of the native toxin. 109 It should be noted that formaldehyde is a detoxifying agent via interacting with L-lysinin residues of the toxin, and hence can be exploited as an adjuvant in vaccination. Since toxin inactivation by formaldehyde also causes distortion of some epitopes that do not present in the native Ags, the mutant type of heat-labile enterotoxin of E. coli, LTK63, was utilized as a suitable adjuvant for the vaccine development against H. pylori. 110 In addition, VacA has been exploited for the production of multi-component vaccines. Intragastric immunization of mice infected with H. pylori SPM326 by the therapeutic vaccine consisting of recombinant VacA and CagA along with LTK63 adjuvant was shown to eradicate H. pylori for 3 months and also provide tenacity of eradication for 2 months after infection rehearsal. 111 Immunization of Beagle dog suffering from gastritis with multi-component vaccine, VacA-CagA- neutrophil-activating protein (NAP), supplemented with adjuvant Al(OH) 3 , resulted in an increased level of antibody (Ab) and hence protection for 4 months post-vaccination. Despite such therapeutic impacts, the gastritis was recurred after 29 weeks indicating the partial periodic effect of vaccine. 112 In contrary to mice, Beagle dogs, as animal model for the human H. pylori infection, provide researchers with high ability of tandem biopsy with no need to sacrifice the animal, which helps to gain a better understanding of the overall process of infection and immunity. Further, a prophylactic vaccine containing three Ags (VacA, CagA and NAP) with adjuvant Al(OH) 3 has been developed and examined in the non-infected individuals through a phase 1 clinical trial. This intramuscular booster vaccination demonstrated satisfactory safety and immunogenicity with anamnestic antibody and cellular responses 18–24 months post-vaccination. Long memory immunity was originated from VacA- and CagA-driven interferon ɤ production, which lasted 4.5 months post-immunization. After the third immunization, all volunteers responded to one or two Ags (mostly VacA and NAP), while 86% responded to all three Ags. VacA induced Ab response after two immunizations, whereas CagA showed cell-immune response after three immunizations. 113 Yet, there exist some debates to figure out what portion of the VacA toxin is more efficient for the vaccine development. A study using different recombinant fragments of VacA confirmed that the highest titer of Ab was produced against 297-317 residues including both p33 and p55 regions of the toxin. 114 Nonetheless, another effort was carried out to study the VacA neutralizing activity of 10 monoclonal antibodies (mAbs) produced in mice via epitope mapping using a panel of VacA deletion mutants and VacA chimera. Among these, two mAbs were able to neutralize the cytotoxic activity of VacA through recognition of amino acids 685 to 821 located at the receptor binding region. 115 In the following context, mAbs against H. pylori infection will be discussed. Monoclonal antibody Development of mAbs has provided new insights in the diagnosis and therapy of various diseases, in particular infections and malignancies. As a result, commercial therapeutic mAbs have made their way to the therapeutic markets in the early 1980s. 116 Passive administration of mAb directed at protective Ags such as VacA and urease might be particularly relevant as a substitute to the current therapies and diagnosis methods. Thus, VacA toxin has become a target for the production of therapeutic mAbs to neutralize the cytotoxin activity. Accordingly, various studies reported that some variants of VacA are highly associated with an increased risk of symptomatic gastroduodenal disease. Two anti-VacA mAbs, known as V36E and V41, were shown to be able to neutralize the vacuolation of rabbit kidney cell line RK13 through binding to the native structure of VacA. 117 Another experiment was conducted to produce mAbs against small and large subunits of urease. As a result, two Abs designated as S2 and L2 were determined based on their strong interactions respectively with small and large subunits, and showed significant blockage of the enzyme activity. 118 Besides application of mAbs in the field of therapy, it seems that they are promising tools for increasing accuracy of diagnosis. Common diagnostic methods of H. pylori infection have been developed on the basis of biopsy, which is painful. Further, various diagnostic methods such as Urea Breath Test (UBT) and serological techniques may provide false negative and positive results specially in asymptomatic patients, thus leading to failure of accurate diagnosis and efficient treatments. 119 Gamma-glutamyl transpeptidase (GGT) antigen is a virulence factor, necessary for colonization and cell apoptosis of H. pylori , which appears to be a promising Ag for development of mAb. In 2014, a phase 1 clinical trial (ID: NCT02123771) was successfully carried out to detect the H. pylori infection in the stool of patients using ELISA and anti-GGT mAb. Given that several toxins of H. pylori participate in the pathogenesis and survival of bacteria, a multi-component Ab cocktail may provide the next generation immunotherapy of H. pylori infection, similar to Ab cocktail used for treatment of Antrax. 120 Ab cocktail is a combination of several Abs that can recognize different virulence factors, through which the treatment outcome can be maximized by expanding the spectrum of protection and enhancing the protective efficacy. Production of Ab cocktail to recognize the major virulence factors H. pylori (i.e., VacA, urease, CagA, GGT and flagellin) appears to be very desirable for the treatment of H. pylori infection. Use of class immunoglobulin A (IgA) in Ab cocktail against H. pylori seems to provide an improved immunotherapy modality, in large part because IgA secreting cells often react with H. pylori membrane proteins (e.g., flagellin and urease) in H. pylori -infected patients. 121 Given the high stability and avidity of IgA in the gastric environment and habituating H. pylori as a noninvasive organism in gastric mucosa, passive immunotherapeutic approaches using secretory IgA (sIgA) might provide a robust treatment modality. For example, in a study, a scFv polypeptide specific to H. pylori urease subunit A was compared with its reformatted polymeric IgA (IgAp/d) and sIgA for the degree of enzyme activity inhibition. Due to increased avidity, the sIgA and IgAp/d were found to be able to efficiently block the enzymatic activity of free and membrane-associated ureases. 122 , 123 Although several antibody fragments have been designed against the main virulence factor of H. pylori , urease, 124 no antibody fragment has been developed against domain of VacA by phage display ever. New approaches for production of antibodies, such as phage display technique, 125 , 126 have been developed in the recent decades that provide a convenient and economic procedure for production of monoclonal antibodies. To the best of our knowledge, we are among the first teams who work on the development of mAb fragments by phage display against VacA toxin. 127 , 128 Concluding remarks VacA is considered as one of the main virulence factors in pathogenesis of H. pylori in the stomach with variety of cytotoxicity, in which the toxicity is assumed to be dependent on different variants of the toxin and various types of receptors and cells. Despite some attempts to justify the binding pattern of VacA, the exact receptor(s) of host cells responsible for the binding of VacA toxin is yet to be fully addressed. In general, it is believed that, regardless of the receptor involved (e.g., RPTPβ or EGFR), the process of VacA internalization occurs in the regions enriched with lipid rafts with the aid of cholesterol, GPI-AP and sphingomyelin. Notwithstanding unprecedented volume of investigations on the H. pylori infection worldwide, its effective eradication remains as a dilemma. Failure of the currently applied therapies, consisting of antibiotic regimens, is largely attributed to inaccurate treatment causing point mutations in H. pylori . New molecular therapies such as vaccines and mAbs seem to offer much more effective treatment modalities with much higher patient compliance and minimal side effects. Although successful vaccination in animal models has been reported, attempts for translation of such therapies to human cases are still under investigation. Among various types of mAbs produced against H. pylori , IgA class of Abs may be considered as one of the best candidates for passive immunotherapies since the infection of gastric mucosa by H. pylori recruit cells that are responsible for the production of IgA specific for a variety of virulence factors. Besides, sIgA was shown to prevent the cellular attachment of H. pylori and its infection. In this review, we discussed genetic diversity and receptors of VacA toxin on epithelial cells and tried to justify whether VacA cytotoxicity is dependent on the type of receptor and genetic diversity for the first time. It is obvious that receptor and genetic diversity are regulators of H. pylori pathogenesis regardless of the genetic background and environmental issues affecting the severity of the disease worldwide and also the effect of cagA on the potential of VacA. Overall, the cytotoxicity of the VacA in vivo is exhibited based on the position/condition of the host cells. Non vacuolating toxicity (e.g., peptic ulcer and gastritis) is resultant from the pathogenicity for adjacent cells with attached H. pylori and vacuolation is observed in distant cells in order to feed on cellular nutrients. Collectively, due to growing trend of mAb applications, recombinant Ab fragments (e.g., scFv, Fab, single domain Abs and bispecific Abs), multivalent Abs and Ab-conjugates with higher avidity as a cocktail of IgG and sIgA Abs as well as multivallent vaccines may be our best shot to tackle this formidable disease. Ethical approval Not applicable. Competing interests The authors declare no conflict of interests. Acknowledgment The authors are grateful for the financial support (Grant No: RCPN-93010) provided by Tabriz University of Medical Sciences. Review Highlights What is current knowledge? √ Application of VacA in production of a vaccine for H. pylori treatment has been confirmed, and used as a part of multicomponent vaccine including several virulence factors of the bacterium. √ Efficiency of therapeutic vaccines has been investigated in various in vivo and in vitro experiments, as well as clinical trials. √ Despite success in the treatment of infection in various studies, relapse of infection after several months appears as a challenging issue. What is new here? √ Besides production of systemic immunoglobulins confronting H. pylori infection, local antibody secreting cells (ASCs) appear to be the major class of Abs representing the state of infection in patients. √ New multivalent vaccines may provide novel treatment modalities against H. pylori. √ New immunotherapies could be designed based on the IgG and IgA Ab cocktail. √ Novel methods of producing recombinant Ab fragments (scFv and Fab) such as display technologies are able to produce mAbs with higher abilities of multimerization into secretory forms in comparison to traditional full length Abs. What is current knowledge? √ Application of VacA in production of a vaccine for H. pylori treatment has been confirmed, and used as a part of multicomponent vaccine including several virulence factors of the bacterium. √ Efficiency of therapeutic vaccines has been investigated in various in vivo and in vitro experiments, as well as clinical trials. √ Despite success in the treatment of infection in various studies, relapse of infection after several months appears as a challenging issue. What is new here? √ Besides production of systemic immunoglobulins confronting H. pylori infection, local antibody secreting cells (ASCs) appear to be the major class of Abs representing the state of infection in patients. √ New multivalent vaccines may provide novel treatment modalities against H. pylori. √ New immunotherapies could be designed based on the IgG and IgA Ab cocktail. √ Novel methods of producing recombinant Ab fragments (scFv and Fab) such as display technologies are able to produce mAbs with higher abilities of multimerization into secretory forms in comparison to traditional full length Abs.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711318/

Progress and Opportunities for Strengthening Global Health Security

Prevent Preventing the emergence and spread of infectious disease threats requires prevention and control of antimicrobial resistance, zoonotic diseases, vaccine-preventable diseases (VPDs), and their potential spread across international borders. Global efforts aimed at building national capacities for IHR compliance are complemented by targeted disease and country-specific efforts. For example, the Enhanced Gonococcal Antimicrobial Surveillance Program aims to inform country-specific treatment guidelines and enhance prevention and control efforts ( 8 ), and the President's Malaria Initiative's collaboration with the Antimalarial Resistance Monitoring in Africa Network supports the early detection of Plasmodium falciparum resistance to facilitate appropriate interventions ( 9 ). Zoonoses prevention and control programs use a One Health approach with multisectoral collaboration between human and animal health. Several countries have conducted One Health prioritization exercises to identify their major zoonotic diseases ( 10 ), a critical step in efforts to control endemic zoonotic diseases ( 11 ). Ethiopia, the Democratic Republic of the Congo, and Georgia are all developing successful integrated zoonotic prevention and control programs ( 12 ). Maintaining high population-wide vaccine coverage is a crucial prevention activity, particularly for VPD health security threats. For example, the failure to sustain high vaccine coverage led to a nationwide measles epidemic in Mongolia, a country previously verified as measles-free ( 13 ). VPD strategies include data improvement teams, which visit district health facilities and can result in improved vaccine administration data through evaluations and training efforts ( 14 ). Another approach for VPD is the Latin American Pertussis Project, a collaboration among 6 countries to address pertussis, a poorly controlled VPD in the region ( 15 ). Finally for antimicrobial resistance, zoonotic diseases, VPDs, and other public health threats, border health efforts aimed at preventing spread of communicable diseases across international boundaries are essential and have been used successfully ( 16 , 17 ). Detect Global health security relies on all countries having > 3 capacities: 1) an adequate national public health laboratory capacity to safely transport and accurately evaluate biologic specimens with appropriate diagnostic testing methods, 2) a sustained and timely public health surveillance system, and 3) a trained competent workforce to conduct essential outbreak investigations. Although rapid laboratory confirmation of public health threats is a complex endeavor, requiring long-term technical assistance and major resources, many countries are advancing key components. For example, Ghana conducted a national public health laboratory system assessment in support of the Second Year of Life initiative for sustaining adequate vaccine coverage through 24 months of age and for monitoring GHSA-sponsored public health laboratory enhancement efforts ( 18 ). South Korea is enhancing its public health laboratory to meet the standards of the US Laboratory Response Network, which will facilitate the ability of this country to rapidly determine the etiology of most public emergencies ( 19 ). Surveillance is the cornerstone for rapidly detecting public health threats. The WHO Early Warning Alert and Response Network is a major tool for conducting public health surveillance in humanitarian emergencies, including in refugee and displaced person camps ( 20 ). Other vital global health security assets are CDC regional Global Disease Detection centers in 10 countries that have provided novel public health surveillance and informatics contributions alongside laboratory research since 2001 ( 21 ). Other disease- and country-specific efforts have also informed best practices, including enhancing anthrax surveillance programs in anthrax-endemic countries ( 22 ) and use of alternative surveillance approaches, such as burial permit reviews, to describe cholera mortality rates in Tanzania during a 2016 epidemic ( 23 ). Rapid detection of public health emergencies also requires an adequate public health workforce, particularly trained field epidemiologists, who can conduct timely and appropriate field investigations. The CDC international 2-year Field Epidemiology Training Program (FETP) began 35 years ago and has established 65 FETP programs in 90 countries, with >3,900 graduates of the 2-year field epidemiologist training ( 24 ). To meet the global health need for more trained field epidemiologists, particularly at the district level, training has been expanded in many countries to include a 3-month FETP Frontline program ( 25 ). In 2014–2016, a total of 24 new FETP Frontline programs were initiated with >1,860 participants ( 4 ). Respond Efficiently responding to public health emergencies is essential for preventing further disease spread and controlling outbreaks at their source. Outbreak responses worldwide have demonstrated the need for a structured incident management system, which is a critical component for a highly functional and efficient emergency operation center. Many countries, particularly GHSA partner countries, have enhanced their emergency response capacity by establishing emergency operation centers with a strong incident management system foundation ( 26 , 27 ). Complex humanitarian emergencies frequently involve the most difficult settings, including fragile states and areas of conflict, and recent case studies illustrate the difficulty of supporting a sustained response in such settings ( 28 ). After the 2014–2016 West Africa Ebola epidemic, CDC established the Global Rapid Response Team (GRRT) to ensure a ready force of trained responders. In the first 16 months, GRRT members deployed 291 times to 35 countries ( 29 ). Medical countermeasures, which are medical interventions aimed at controlling public health emergencies, can be essential for rapid response and containment and include using vaccination during outbreaks of cholera, typhoid, yellow fever, and Ebola virus disease ( 30 ). Conclusions Global health security relies on IHR compliance by all countries and, as such, remains an unfinished journey ( 31 ). Although much has been accomplished through the first years of GHSA implementation, JEEs around the world highlight numerous prevent, detect, and respond capabilities that still need strengthening. Also lacking is an evidence base of the most effective, timely, and cost-effective approaches to building national capacities for IHR 2005 compliance. As countries and partners continue their work to build health security capabilities, there will be useful opportunities to evaluate different implementation strategies and to document the impact of newly acquired capacities. Continuing this work and thereby sustaining this momentum toward IHR 2005 compliance is critical for protecting Americans and other persons worldwide.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3547565/

History, ethics, advantages and limitations of experimental models for hepatic ablation

Numerous techniques developed in medicine require careful evaluation to determine their indications, limitations and potential side effects prior to their clinical use. At present this generally involves the use of animal models which is undesirable from an ethical standpoint, requires complex and time-consuming authorization, and is very expensive. This process is exemplified in the development of hepatic ablation techniques, starting experiments on explanted livers and progressing to safety and efficacy studies in living animals prior to clinical studies. The two main approaches used are ex vivo isolated non-perfused liver models and in vivo animal models. Ex vivo non perfused models are less expensive, easier to obtain but not suitable to study the heat sink effect or experiments requiring several hours. In vivo animal models closely resemble clinical subjects but often are expensive and have small sample sizes due to ethical guidelines. Isolated perfused ex vivo liver models have been used to study drug toxicity, liver failure, organ transplantation and hepatic ablation and combine advantages of both previous models.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3688554/

Direct Proteolytic Cleavage of NLRP1B Is Necessary and Sufficient for Inflammasome Activation by Anthrax Lethal Factor

Inflammasomes are multimeric protein complexes that respond to infection by recruitment and activation of the Caspase-1 (CASP1) protease. Activated CASP1 initiates immune defense by processing inflammatory cytokines and by causing a rapid and lytic cell death called pyroptosis. Inflammasome formation is orchestrated by members of the nucleotide-binding domain and leucine-rich repeat (NLR) or AIM2-like receptor (ALR) protein families. Certain NLRs and ALRs have been shown to function as direct receptors for specific microbial ligands, such as flagellin or DNA, but the molecular mechanism responsible for activation of most NLRs is still poorly understood. Here we determine the mechanism of activation of the NLRP1B inflammasome in mice. NLRP1B, and its ortholog in rats, is activated by the lethal factor (LF) protease that is a key virulence factor secreted by Bacillus anthracis , the causative agent of anthrax. LF was recently shown to cleave mouse and rat NLRP1 directly. However, it is unclear if cleavage is sufficient for NLRP1 activation. Indeed, other LF-induced cellular events have been suggested to play a role in NLRP1B activation. Surprisingly, we show that direct cleavage of NLRP1B is sufficient to induce inflammasome activation in the absence of LF. Our results therefore rule out the need for other LF-dependent cellular effects in activation of NLRP1B. We therefore propose that NLRP1 functions primarily as a sensor of protease activity and thus could conceivably detect a broader spectrum of pathogens than just B. anthracis . By adding proteolytic cleavage to the previously established ligand-receptor mechanism of NLR activation, our results illustrate the remarkable flexibility with which the NLR architecture can be deployed for the purpose of pathogen-detection and host defense. Introduction Recognition of pathogens is an essential first step in the initiation of protective host immune responses. Recognition of pathogens has been shown to be mediated by several families of germ-line encoded receptors that include the Toll-like receptors (TLRs), Nucleotide-binding domain and Leucine-rich Repeat containing proteins (NLRs), and RIG-I-like receptors (RLRs) [1] . Most TLRs, NLRs, and RLRs for which activation mechanisms have been defined appear to function as "pattern recognition receptors" [2] that directly bind to molecular structures called pathogen-associated molecular patterns (PAMPs) that are broadly conserved among many microbes. In addition to detection of PAMPs, it has been previously proposed that the innate immune system might also respond to 'Patterns of Pathogenesis', the virulence-associated activities that pathogens utilize to invade or manipulate their hosts [3] . Detection of pathogen-associated activities might be a beneficial innate immune strategy, complementary to PAMP recognition, as it could allow the innate immune system to discriminate pathogenic from non-pathogenic microbes, and scale responses appropriately, despite the fact that pathogenic and non-pathogenic microbes often share the same PAMPs. However, few instances of a molecular mechanism by which a pathogen-encoded activity could be detected have been described in mammals. For example, a previous study showed how pathogen-induced inhibition of protein synthesis by Legionella pneumophila could be detected, leading to a specific cytokine response [4] , [5] . Disruption of the actin cytoskeletal signaling by bacterial toxins was also found to lead to a protective innate immune response [6] , [7] Overall, however, there is still considerable uncertainty as to whether or how 'patterns of pathogenesis' are sensed by the innate immune system. Anthrax lethal toxin (LeTx) is a critical virulence factor secreted by Bacillus anthracis . LeTx is composed of two proteins: protective antigen (PA) and lethal factor (LF). PA binds to anthrax toxin receptors on host cells, and subsequently translocates the zinc-metalloprotease, LF, into the cytosol. The canonical proteolytic substrates of LF are mitogen-activated protein kinase kinases (MAPKKs) 1–4 and 6–7 [8] , [9] . Cleavage by LF inactivates MAPKKs and results in the disruption of signaling pathways involved in host defense [10] , [11] . Macrophages from certain strains of mice and rats respond to LeTx by undergoing a rapid and lytic form of Caspase-1 (CASP1)-dependent cell death called pyroptosis [12] – [16] . The ability to undergo pyroptosis in response to LeTx was genetically mapped to the Nlrp1b gene in mice [13] , and subsequently to the orthologous Nlrp1 gene in rats [16] . Importantly, mice harboring an allele of Nlrp1b that is responsive to LeTx are protected from challenge with B. anthracis spores [17] , [18] . This protection correlates with enhanced production of IL-1β, recruitment of neutrophils to the site infection, and decreased bacterial counts, and these processes depend on expression of the interleukin-1 receptor [17] , [18] . Despite the importance of NLRP1B in host defense against B. anthracis , the mechanism of NLRP1B activation by LF remains unclear. NLRP1B belongs to the NLR family of innate immune sensors [19] – [21] . Several NLRs, including NLRP1, have been found to assemble into oligomeric complexes, called 'inflammasomes' [22] , in response to a variety of infectious or noxious stimuli [21] . The primary function of inflammasomes appears to be to form a platform for activation of inflammatory caspase proteases, most notably CASP1, but the molecular mechanism by which NLRs are activated is poorly understood [21] . Although NLRP1 proteins contain NBD and LRR domains, as do all other NLRs, the domain organization of NLRP1 differs from other NLRs in two respects. First, NLRP1 proteins contain a C-terminal Caspase Activation and Recruitment Domain (CARD), whereas the CARDs in other NLRs are usually N-terminal. The second unique feature of NLRP1 proteins is that they contain an unusual domain called the 'function-to-find' (FIIND) domain [19] . The FIIND is located between the LRRs and the C-terminal CARD, and was recently shown to undergo an auto-proteolytic processing event that results in the C-terminal CARD being cleaved from the rest of the NLRP1 protein [23] . It is believed that the N- and C-terminal auto-processed fragments of mature NLRP1B remain associated with each other despite cleavage of the polypeptide chain [24] . The FIIND auto-processing event occurs constitutively, prior to NLRP1B activation by LF, but for reasons that remain unclear, is required for the ability of NLRP1 to activate CASP1 [24] , [25] . Several inflammasomes have been suggested to be activated upon direct binding to specific bacterial ligands. For example, another NLR-family member, NAIP5, assembles into an inflammasome upon binding to flagellin, whereas the related NAIP2 inflammasome assembles upon binding to the inner rod proteins from a variety of bacterial type III secretion systems [26] , [27] . A direct receptor-ligand model also applies to the ALR-family AIM2 inflammasome, which is activated upon direct binding to microbial DNA [28] – [31] . In contrast, certain inflammasomes, notably the NLRP3 inflammasome, are believed not to bind directly to bacterial ligands, but have instead been proposed to respond to virally encoded ion channels [32] , bacterial toxins, or other cellular stresses, via indirect mechanisms [21] . However, the molecular basis for how these stresses are sensed by NLRP3 remains unclear. By contrast, the molecular basis for indirect pathogen recognition by plant NLRs has been well-established [33] , [34] . For example, the plant NLR RPS2 has been shown to be maintained in an inactive state by its association with RIN4, a host protein that is targeted for degradation by a bacterial protease [35] , [36] . RPS2 thus detects the activity of a bacterial protease indirectly by monitoring or 'guarding' the integrity of the protease substrate. Direct proteolytic cleavage of a plant NLR by a pathogen-encoded protease has not been described. NLRP1B responds to the protease activity of LF, as catalytically inactive forms of LF do not activate NLRP1 [37] , [38] . This suggests that NLRP1 does not recognize LF via simple receptor-ligand binding, such as that occurs with the NAIP or AIM2 inflammasomes. Boyden and Dietrich initially hypothesized that LF could cleave and activate NLRP1B [13] , but evidence for this simple model of NLRP1B activation was not provided. In fact, several groups have demonstrated that the activity of the proteasome is specifically required for this inflammasome and not for other inflammasomes such as the NAIP5/NLRC4 inflammasome [37] , [39] . In addition, inhibitors of the N-end rule degradation pathway block NLRP1B activation but do not affect the ability of LF to cleave MAPKKs [40] . In contrast to a model in which NLRP1B is activated upon cleavage by LF, these observations suggest that LF might activate NLRP1B by cleaving and destabilizing a negative regulator of NLRP1B. This 'indirect' model resembles the activation of the certain NLRs, e.g., RPS2, in plants. Recently, however, it was shown that the NLRP1 proteins from Fischer rats and BALB/c mice can be directly cleaved near their N-termini by LF [41] , [42] . Mutation of the cleavage site in rat NLRP1 rendered NLRP1 resistant to cleavage by LF and also prevented NLRP1 activation in response to LF. These results suggest that cleavage of rat NLRP1 by LF is essential for NLRP1 activation, but it is difficult to rule out the possibility that mutation of the cleavage site disrupted the fold of NLRP1, or rendered NLRP1 non-functional for other reasons. In addition, the site at which LF cleaves rat NLRP1 is not conserved in the mouse [42] , and moreover, the functional effects of mutating the mouse cleavage site have not been assessed. Lastly, and most importantly, existing studies have not ruled out the involvement of other LF-dependent cellular events in NLRP1B activation, as cleavage of NLRP1 was not shown to be sufficient for its activation. Here, we present data that suggest that murine NLRP1B requires LF-dependent cleavage for its activation. We further demonstrate that cleavage is sufficient for NLRP1B inflammasome activation in the absence of LF, which rules out a requirement for cleavage of other LF substrates in activation of NLRP1B. Our results provide evidence for a simple direct mechanism by which an innate immune sensor detects a pathogen-encoded activity. In addition, our results open the possibility that NLRP1 could function as a direct cytosolic sensor of other pathogen-derived proteases. More broadly, by adding direct proteolytic cleavage to the existing ligand-receptor models for NLR activation, our results also illustrate the remarkable adaptability of the NLR architecture to function as pathogen-detectors in host defense. Results Mouse NLRP1B is cleaved by LF The N-termini of mouse NLRP1B and rat NLRP1 were recently reported to be cleaved by LF [41] , [42] . Interestingly, these proteins do not exhibit much similarity in the region surrounding the cleavage site ( Fig. 1A ), whereas the rest of the protein is highly conserved between mice and rats (37% amino acid identity from position 1–54 vs. 70% identity from residue 55 to the C-terminus). The N-terminal fragment produced by LF is under 10 kDa and appears to be unstable, making it difficult to detect by conventional western blotting techniques in cell lysates. Thus, to confirm that mouse NLRP1B is cleaved by LF, we augmented the mass of the putative N-terminal fragment by 29 kDa by fusing full-length NLRP1B to enhanced green fluorescent protein (EGFP) and a hemagglutinin (HA) affinity-tag. We transfected this construct into HEK 293T cells and then treated the cells with LeTx. As reported previously, NLRP1B constitutively but only partially auto-processes its FIIND domain in untreated cells, resulting in a loss of 29 kDa from the C-terminus, and producing a doublet of 140 kDa and 169 kDa that we will refer to as the 'processed' and 'unprocessed' forms of NLRP1B, respectively ( Fig. 1B and 1D ) [24] . After LeTx addition, an N-terminal fragment smaller than 37 kDa, but larger than EGFP-HA alone (29 kDa), begins to accumulate inside cells. To distinguish LF-dependent cleavage from auto-processing of the FIIND domain, we will refer to the LF-dependent fragments as 'cleaved' (as opposed to 'processed') NLRP1B ( Fig. 1D ). With kinetics corresponding to the appearance of the cleaved N-terminal fragment, the amount of the detectable uncleaved NLRP1B decreased over time, consistent with removal of the N-terminal tag. The LF-dependent cleavage of NLRP1B is not complete even after 6 hours, and thus occurs much more slowly than the LF-dependent cleavage of the MAP kinase kinase MEK2, a canonical LF substrate, which appears complete within 2 hours ( Fig. 1B ). 10.1371/journal.ppat.1003452.g001 Figure 1 Murine NLRP1B from 129S1 mice is cleaved directly by LeTx. A) Protein sequence alignment of the N-terminal region of murine NLRP1B (129S1 allele) and rat NLRP1 (Fischer/CDF allele) was determined by ClutalW with a BLOSUM series matrix. The LF cleavage motif and cleavage site are identified in the rat allele by the bar and arrow above the rat sequence. B) GFP-HA-NLRP1B was transfected into HEK 293T cells and then treated with 1 µg/ml LeTx over the indicate time points followed by immunoblotting (IB) for HA on non-boiled lysates, and boiled lysates when probed with MEK2 and beta-actin antibodies. The arrow-head refers to the LeTx-dependent N-terminal cleavage fragment. C) HA-NLRP1B expressed in 293T cells, immunoprecipitated (IP) with anti-HA beads, and treated with recombinant LF (rLF) for 2 h, followed by immunoblotting for HA. D) Graphic representation of the GFP-HA-NLRP1B construct and annotated functional domains. The different forms of NLRP1B observed are shaded in gray along with their predicted molecular weights, when immunoblotted with an anti-HA antibody. To test if LF cleaves NLRP1B directly, without the GFP fusion, we expressed an HA-tagged NLRP1B in 293T cells, immunoprecipitated NLRP1B, and then treated the purified protein with recombinant LF in vitro . In the sample treated with LF, a fragment smaller than 10 kDa is produced ( Fig. 1C ), suggesting that LF can cleave mouse NLRP1B directly near the N-terminus, confirming recent findings [42] . Cleavage is required for LF activation of NLRP1B Even though the cleavage site in rat NLRP1 is not well-conserved in mouse NLRP1B ( Fig. 1A ), two sequences can be found in the N-terminus of mouse NLRP1B that partially fit the previously established consensus specificity of LF [42] ( Fig. S1A ). For clarity, we refer to the putative site nearest to the N-terminus (cleavage after K38) as site-1, and the C-terminal site (cleavage after K44) as site-2 ( Fig. 1D and S1A ). These two sites were also identified as putative LF cleavage sites in a recent study [42] , but their functional importance was not addressed. We attempted to generate cleavage resistant (CR) forms of NLRP1B by mutating each site. We made a variety of amino acid substitutions at site-1 (CR1A-D) and site-2 (CR2A-C) ( Fig. S1A ), utilizing residues that have previously been used to render MKK3 and MKK6 cleavage resistant [43] , or residues not found in LF consensus sites [44] , [45] . These mutants were transfected into 293T cells, and cells were then treated with LeTx and assayed for cleavage. Mutation of cleavage site-2 produced a cleavage-resistant form, despite the fact that site-1 is intact in this mutant ( Fig. 2A S1A–C). By contrast, mutation of cleavage site-1 had little or no effect on NLRP1B cleavage ( Fig. S1A –C). When Casp1 and Il1b cDNA expression vectors were cotransfected into this same 293T system, only CR2A and CR2B were defective for induction of IL-1β processing into p17 above the basal processing induced by CASP1 and NLRP1B prior to stimulation ( Fig. S1B –C). Thus, while confirming the previous finding that both site-1 and site-2 of mouse NLRP1B can be cleaved by LF [42] , these results suggest that site-2 is the predominant LF target within NLRP1B in cells. 10.1371/journal.ppat.1003452.g002 Figure 2 Mouse NLRP1B cleavage by LF is required for inflammasome activation. A) Both WT and CR2A GFP-HA-NLRP1B were transfected into 293T cells and then treated with LeTx for the indicated times, and cleavage was monitored by immunoblotting with indicated antibodies. B) Immortalized macrophages from a C57BL6 mouse were transduced with both forms of GFP-HA-NLRP1B and then treated with LeTx or LFn-Fla+PA (FlaTox). Pyroptosis was assayed by LDH release and normalized to complete detergent lysis. Error bars represent plus and minus one standard deviation from the mean. We tested the ability of the CR2A NLRP1B mutant to form an inflammasome capable of promoting pyroptosis. In these experiments, we used immortalized macrophages from a C57BL/6 (B6) mouse, because the endogenous B6 allele of NLRP1B is not responsive to LeTx. As expected, immortalized B6 macrophages transduced with a retroviral construct expressing the wild-type 129S1 allele of NLRP1B became sensitive to LeTx and underwent pyroptosis, as assessed by release of cytosolic lactate dehydrogenase (LDH) into the supernatant ( Fig. 2B ). By contrast, transduction of B6 macrophages with the CR2A NLRP1B mutant did not confer any measurable sensitivity to LeTx over the same time period. This difference in responsiveness is not due to differences in expression of the NLRP1B alleles ( Fig. S2A ). B6 cells harbor a functional NAIP5 inflammasome; thus, as a further control, the NLRP1B-transduced cells can be tested for inflammasome responses to the cytosolic presence of flagellin. We therefore delivered flagellin to the cytosol, via the protective antigen translocation channel used by lethal factor, as a fusion to the translocation signal in LF (dubbed 'FlaTox') [46] ( Fig. 2B ). Cells transduced with wild-type and CR2A NLRP1B were equally susceptible to FlaTox, indicating that they expressed functionally equivalent levels of anthrax toxin receptor, CASP1, and downstream effectors required for pyroptosis. These data demonstrate that the ability of mouse NLRP1B to respond to LF correlates with the ability of LF to cleave NLRP1B at its N-terminus. LF, expressed in the cytosol in the absence of PA, is sufficient to activate NLRP1B The ability of LF to cleave and activate NLRP1B has only been tested in the presence of PA, since PA is typically required in order to deliver LF to the cytosol. It is therefore unclear if PA is only necessary for the translocation of LF in to the cytosol, or if it is also required for NLRP1B activation. We decided to test the ability of LF expression to induce pyroptosis and cytokine secretion in B6 and 129 immortalized macrophage-like cell lines with a Tet-On inducible vector. We transduced these cell lines with a lentiviral Tet-On GFP or LF expression vector and then treated the transduced cells with doxycycline to induce GFP or LF expression. LF expression was able to consistently induce pyroptosis in 129 (NLRP1B LeTx-responsive) cells but not B6 (NLRP1B LeTx-nonresponsive) cells ( Fig. S5A ). Further addition of PA had no additional effect on pyroptosis induction. Similar results were obtained when IL-1β production was used to monitor NLRP1B activation ( Fig. S5B ). These results show that the cytosolic presence of LF is sufficient to activate NLRP1B and that additional putative signals provided by PA pore formation are not required. Cleavage of NLRP1B is sufficient for inflammasome activation Together the above results suggest that mouse NLRP1B requires direct cleavage in order to be activated by LF, but it is difficult to rule out the formal possibility that the CR2A mutant is misfolded or is otherwise non-functional for reasons unrelated to its resistance to cleavage by LF. Moreover, the above experiments did not address whether cleavage alone is sufficient for activation of NLRP1B. For example, LF may have other substrates that must be cleaved in addition to NLRP1B, or LF itself could provide a ligand-like signal for the cleaved NLRP1B receptor. To address these possibilities, we replaced the predicted LF cleavage sites-1 and -2 in NLRP1B with a Tobacco Etch Virus (TEV) NIa protease cleavage-site ( Fig. S1A ). TEV protease was selected because it has no known endogenous substrates in mouse or human cells. We transfected 293T cells with plasmids expressing the wild-type and TEV-site forms of NLRP1B, along with plasmids encoding CASP1, pro-IL-1β, and either LF or TEV protease. As expected, wild-type NLRP1B was cleaved only in the presence of LF, and this coincided with the generation of mature IL-1β ( Fig. 3A ). Importantly, NLRP1B harboring a target sequence for TEV protease in place of the LF target sequence at site-2 (TEV-site2 NLRP1B) was cleaved efficiently by TEV protease, and this cleavage was sufficient to promote IL-1β processing. Consistent with the relatively low sequence specificity of LF, the TEV-site2 NLRP1B protein was also cleaved by LF, but this cleavage was inefficient as most of the NLRP1B remained uncleaved, and IL-1β was not efficiently processed. Cleavage of TEV-site1 also was sufficient to induce IL-1β processing, but this occurred upon expression of either LF or TEV proteases. Furthermore, the TEV-induced cleavage at site-1 produced a fragment of NLRP1B that was smaller than the fragment produced by LF expression ( Fig. 3A and S1D ). Consistent with the mutagenesis experiments shown in Fig. 2 , this observation may indicate that LF prefers to cleave at site-2, which is still present in the TEV-site1 NLRP1B protein. Taken together, these results suggests that cleavage of NLRP1B at either site-1 or site-2 is sufficient to induce inflammasome activation independently of other LF-dependent cellular effects. 10.1371/journal.ppat.1003452.g003 Figure 3 Cleavage of NLRP1B is sufficient to promote inflammasome activation. A) 293T cells were transfected with WT, TEV-site2 or TEV-site1 GFP-HA-NLRP1B along with empty vector, TEV expression vector, or a LF expression plasmids. In all conditions cells were also co-transfected with Casp1 and Il1b expression vectors. Cleavage of GFP-HA-NLRP1B and IL-1β was determined 24 h post transfection. B) Immortalized B6 macrophages were transduced with a retrovirus encoding the indicated GFP-HA-NLRP1B form followed by a sequential transduction with a TEV-expression retrovirus co-expressing THY1.1. Percent transduction was determined by measuring expression of the respective retroviral integration markers (GFP and anti-THY1.1-PE-Cy7) by flow cytometry, and are expressed in relative fluorescent units (RFU). The numbers within each quadrant represent the percentage of live cells within the respective quadrant. C) RAW264.7 macrophages were transduced with GFP-HA-NLRP1B and a Tet-On construct expressing the indicated gene. Cells were treated with 5 µg/ml doxycycline for 20 h and supernatants were assayed for LDH release. D) 293T cells were transfected with empty vector, FL-NLRP1B-HA, the truncated NLRP1B-HA, or ΔLRR HA-NLRP1B, along with Casp1 and Il1b and assayed by immunoblotting. We also confirmed that cleavage of NLRP1B is sufficient to induce pyroptosis in macrophages cell lines. We transduced immortalized B6 macrophages with two different retroviral vectors, one expressing GFP-NLRP1B and the other expressing TEV protease with an IRES-Thy1.1 expression marker. If cleavage is sufficient to activate NLRP1B, it is expected that only cells expressing both components would undergo pyroptosis and therefore be underrepresented in the live population of cells. We analyzed the percentage of cells that contained both retroviruses by measuring THY1.1 surface-expression and GFP fluorescence by flow cytometry. As expected, an underrepresentation of the THY1.1 and GFP double-positive population was specifically seen in cells transduced with TEV and TEVsite2-NLRP1B, while the frequency of THY1.1+ cells was similar in cells that where negative for both forms of NLRP1B ( Fig. 3B ). To further confirm that cleavage of NLRP1B is sufficient for inflammasome activation, we transduced RAW 264.7 cells with retroviral vectors encoding various NLRP1B alleles, as well as a lentiviral Tet-On vector that inducibly expresses GFP, TEV-protease or LF after exposure of cells to doxycycline. In this system, TEV expression induced high levels of LDH release only for cells expressing TEVsite2-NLRP1B. As expected, since RAW cells express an endogenous functional allele of NLRP1B, LF induced pyroptotic lysis of cells expressing either wild-type or TEVsite2-NLRP1B ( Fig. 3C ). The percent LDH release was generally consistent with the percentage of cells that expressed both constructs ( Fig. S2B ). These data demonstrate that cleavage of NLRP1B is sufficient to activate this inflammasome in macrophages and cause pyroptosis. No apparent role for the N-terminal NLRP1B cleavage fragment We next tested whether the N-terminal fragment generated by cleavage of NLRP1B by LF at site-2 must be present along with the corresponding C-terminal fragment. We generated a construct to express a 'pre-cleaved' C-terminal fragment by deleting residues 1–44 of full-length NLRP1B and replacing amino acid 45 (leucine) with an initiator methionine. The resulting C-terminal fragment contains all known functional domains of NLRP1B ( Fig. 1D ). In the 293T cell system, high levels of spontaneous IL-1β cleavage was observed upon expression of the precleaved NLRP1B. The activity of pre-cleaved NLRP1B was comparable to that of a ΔLRR mutant, a form of NLRP1B that is known to be constitutively active ( Fig. 3D and S3C ) [47] . When the N-terminal fragment (amino acids 1–44) was coexpressed with the precleaved C-terminal fragment, no change in the amount of IL-1β processing was observed ( Fig. S3B ), suggesting it is neither necessary nor inhibitory when expressed in trans. For all of these experiments, the differences in the amount of IL-1β cleavage were not explained by differences in expression of NLRP1B ( Fig. S3A –C). Proteasome inhibitors and FIIND processing do not affect LF-dependent cleavage NLRP1B inflammasome activation can be blocked by proteasome inhibitors, an effect that is observed with multiple inhibitors and is specific to the NLRP1B inflammasome and not the NAIP/NLRC4 inflammasome [37] , [39] . The mechanism by which proteasome inhibitors affect NLRP1B inflammasome activation is not currently known. Therefore, we tested whether the proteasome inhibitor MG132 blocked NLRP1B cleavage. In the 293T system, an equivalent amount of cleaved of NLRP1B occurred in the presence of MG132 and its vehicle ( Fig. 4A ), suggesting NLRP1B cleavage is not the step at which MG132 interferes with NLRP1B activation ( Fig. S4A ). 10.1371/journal.ppat.1003452.g004 Figure 4 Proteasome inhibition and FIIND-processing do not affect NLRP1B cleavage by LF. A) 293T cells expressing GFP-HA-NLRP1B were co-treated with 1 µg/ml LeTx and 10 µM MG132 (proteasome inhibitor) or the DMSO vehicle and assayed for cleavage. B) Cleavage susceptibility of WT and S984A (FIIND mutant) GFP-HA-NLRP1B was determined in 293T cells at the indicated time points. The FIIND of NLRP1B contains a ZU-5/UPA-like domain that can auto-process, and this auto-processing is required for NLRP1B activation [23] , [24] . We tested if auto-processing at the FIIND region is prerequisite for N-terminal cleavage by LF. We tested the FIIND mutant S984A, which cannot auto-process, and found it to be indistinguishably sensitive to LeTx cleavage as wild-type NLRP1B ( Fig. 4B ). Thus FIIND auto-processing appears to be required for a downstream step in NLRP1B activation ( Fig. S4B ), and is not required for NLRP1B to be sensitive to LeTx cleavage. Mouse NLRP1B is cleaved by LF The N-termini of mouse NLRP1B and rat NLRP1 were recently reported to be cleaved by LF [41] , [42] . Interestingly, these proteins do not exhibit much similarity in the region surrounding the cleavage site ( Fig. 1A ), whereas the rest of the protein is highly conserved between mice and rats (37% amino acid identity from position 1–54 vs. 70% identity from residue 55 to the C-terminus). The N-terminal fragment produced by LF is under 10 kDa and appears to be unstable, making it difficult to detect by conventional western blotting techniques in cell lysates. Thus, to confirm that mouse NLRP1B is cleaved by LF, we augmented the mass of the putative N-terminal fragment by 29 kDa by fusing full-length NLRP1B to enhanced green fluorescent protein (EGFP) and a hemagglutinin (HA) affinity-tag. We transfected this construct into HEK 293T cells and then treated the cells with LeTx. As reported previously, NLRP1B constitutively but only partially auto-processes its FIIND domain in untreated cells, resulting in a loss of 29 kDa from the C-terminus, and producing a doublet of 140 kDa and 169 kDa that we will refer to as the 'processed' and 'unprocessed' forms of NLRP1B, respectively ( Fig. 1B and 1D ) [24] . After LeTx addition, an N-terminal fragment smaller than 37 kDa, but larger than EGFP-HA alone (29 kDa), begins to accumulate inside cells. To distinguish LF-dependent cleavage from auto-processing of the FIIND domain, we will refer to the LF-dependent fragments as 'cleaved' (as opposed to 'processed') NLRP1B ( Fig. 1D ). With kinetics corresponding to the appearance of the cleaved N-terminal fragment, the amount of the detectable uncleaved NLRP1B decreased over time, consistent with removal of the N-terminal tag. The LF-dependent cleavage of NLRP1B is not complete even after 6 hours, and thus occurs much more slowly than the LF-dependent cleavage of the MAP kinase kinase MEK2, a canonical LF substrate, which appears complete within 2 hours ( Fig. 1B ). 10.1371/journal.ppat.1003452.g001 Figure 1 Murine NLRP1B from 129S1 mice is cleaved directly by LeTx. A) Protein sequence alignment of the N-terminal region of murine NLRP1B (129S1 allele) and rat NLRP1 (Fischer/CDF allele) was determined by ClutalW with a BLOSUM series matrix. The LF cleavage motif and cleavage site are identified in the rat allele by the bar and arrow above the rat sequence. B) GFP-HA-NLRP1B was transfected into HEK 293T cells and then treated with 1 µg/ml LeTx over the indicate time points followed by immunoblotting (IB) for HA on non-boiled lysates, and boiled lysates when probed with MEK2 and beta-actin antibodies. The arrow-head refers to the LeTx-dependent N-terminal cleavage fragment. C) HA-NLRP1B expressed in 293T cells, immunoprecipitated (IP) with anti-HA beads, and treated with recombinant LF (rLF) for 2 h, followed by immunoblotting for HA. D) Graphic representation of the GFP-HA-NLRP1B construct and annotated functional domains. The different forms of NLRP1B observed are shaded in gray along with their predicted molecular weights, when immunoblotted with an anti-HA antibody. To test if LF cleaves NLRP1B directly, without the GFP fusion, we expressed an HA-tagged NLRP1B in 293T cells, immunoprecipitated NLRP1B, and then treated the purified protein with recombinant LF in vitro . In the sample treated with LF, a fragment smaller than 10 kDa is produced ( Fig. 1C ), suggesting that LF can cleave mouse NLRP1B directly near the N-terminus, confirming recent findings [42] . Cleavage is required for LF activation of NLRP1B Even though the cleavage site in rat NLRP1 is not well-conserved in mouse NLRP1B ( Fig. 1A ), two sequences can be found in the N-terminus of mouse NLRP1B that partially fit the previously established consensus specificity of LF [42] ( Fig. S1A ). For clarity, we refer to the putative site nearest to the N-terminus (cleavage after K38) as site-1, and the C-terminal site (cleavage after K44) as site-2 ( Fig. 1D and S1A ). These two sites were also identified as putative LF cleavage sites in a recent study [42] , but their functional importance was not addressed. We attempted to generate cleavage resistant (CR) forms of NLRP1B by mutating each site. We made a variety of amino acid substitutions at site-1 (CR1A-D) and site-2 (CR2A-C) ( Fig. S1A ), utilizing residues that have previously been used to render MKK3 and MKK6 cleavage resistant [43] , or residues not found in LF consensus sites [44] , [45] . These mutants were transfected into 293T cells, and cells were then treated with LeTx and assayed for cleavage. Mutation of cleavage site-2 produced a cleavage-resistant form, despite the fact that site-1 is intact in this mutant ( Fig. 2A S1A–C). By contrast, mutation of cleavage site-1 had little or no effect on NLRP1B cleavage ( Fig. S1A –C). When Casp1 and Il1b cDNA expression vectors were cotransfected into this same 293T system, only CR2A and CR2B were defective for induction of IL-1β processing into p17 above the basal processing induced by CASP1 and NLRP1B prior to stimulation ( Fig. S1B –C). Thus, while confirming the previous finding that both site-1 and site-2 of mouse NLRP1B can be cleaved by LF [42] , these results suggest that site-2 is the predominant LF target within NLRP1B in cells. 10.1371/journal.ppat.1003452.g002 Figure 2 Mouse NLRP1B cleavage by LF is required for inflammasome activation. A) Both WT and CR2A GFP-HA-NLRP1B were transfected into 293T cells and then treated with LeTx for the indicated times, and cleavage was monitored by immunoblotting with indicated antibodies. B) Immortalized macrophages from a C57BL6 mouse were transduced with both forms of GFP-HA-NLRP1B and then treated with LeTx or LFn-Fla+PA (FlaTox). Pyroptosis was assayed by LDH release and normalized to complete detergent lysis. Error bars represent plus and minus one standard deviation from the mean. We tested the ability of the CR2A NLRP1B mutant to form an inflammasome capable of promoting pyroptosis. In these experiments, we used immortalized macrophages from a C57BL/6 (B6) mouse, because the endogenous B6 allele of NLRP1B is not responsive to LeTx. As expected, immortalized B6 macrophages transduced with a retroviral construct expressing the wild-type 129S1 allele of NLRP1B became sensitive to LeTx and underwent pyroptosis, as assessed by release of cytosolic lactate dehydrogenase (LDH) into the supernatant ( Fig. 2B ). By contrast, transduction of B6 macrophages with the CR2A NLRP1B mutant did not confer any measurable sensitivity to LeTx over the same time period. This difference in responsiveness is not due to differences in expression of the NLRP1B alleles ( Fig. S2A ). B6 cells harbor a functional NAIP5 inflammasome; thus, as a further control, the NLRP1B-transduced cells can be tested for inflammasome responses to the cytosolic presence of flagellin. We therefore delivered flagellin to the cytosol, via the protective antigen translocation channel used by lethal factor, as a fusion to the translocation signal in LF (dubbed 'FlaTox') [46] ( Fig. 2B ). Cells transduced with wild-type and CR2A NLRP1B were equally susceptible to FlaTox, indicating that they expressed functionally equivalent levels of anthrax toxin receptor, CASP1, and downstream effectors required for pyroptosis. These data demonstrate that the ability of mouse NLRP1B to respond to LF correlates with the ability of LF to cleave NLRP1B at its N-terminus. LF, expressed in the cytosol in the absence of PA, is sufficient to activate NLRP1B The ability of LF to cleave and activate NLRP1B has only been tested in the presence of PA, since PA is typically required in order to deliver LF to the cytosol. It is therefore unclear if PA is only necessary for the translocation of LF in to the cytosol, or if it is also required for NLRP1B activation. We decided to test the ability of LF expression to induce pyroptosis and cytokine secretion in B6 and 129 immortalized macrophage-like cell lines with a Tet-On inducible vector. We transduced these cell lines with a lentiviral Tet-On GFP or LF expression vector and then treated the transduced cells with doxycycline to induce GFP or LF expression. LF expression was able to consistently induce pyroptosis in 129 (NLRP1B LeTx-responsive) cells but not B6 (NLRP1B LeTx-nonresponsive) cells ( Fig. S5A ). Further addition of PA had no additional effect on pyroptosis induction. Similar results were obtained when IL-1β production was used to monitor NLRP1B activation ( Fig. S5B ). These results show that the cytosolic presence of LF is sufficient to activate NLRP1B and that additional putative signals provided by PA pore formation are not required. Cleavage of NLRP1B is sufficient for inflammasome activation Together the above results suggest that mouse NLRP1B requires direct cleavage in order to be activated by LF, but it is difficult to rule out the formal possibility that the CR2A mutant is misfolded or is otherwise non-functional for reasons unrelated to its resistance to cleavage by LF. Moreover, the above experiments did not address whether cleavage alone is sufficient for activation of NLRP1B. For example, LF may have other substrates that must be cleaved in addition to NLRP1B, or LF itself could provide a ligand-like signal for the cleaved NLRP1B receptor. To address these possibilities, we replaced the predicted LF cleavage sites-1 and -2 in NLRP1B with a Tobacco Etch Virus (TEV) NIa protease cleavage-site ( Fig. S1A ). TEV protease was selected because it has no known endogenous substrates in mouse or human cells. We transfected 293T cells with plasmids expressing the wild-type and TEV-site forms of NLRP1B, along with plasmids encoding CASP1, pro-IL-1β, and either LF or TEV protease. As expected, wild-type NLRP1B was cleaved only in the presence of LF, and this coincided with the generation of mature IL-1β ( Fig. 3A ). Importantly, NLRP1B harboring a target sequence for TEV protease in place of the LF target sequence at site-2 (TEV-site2 NLRP1B) was cleaved efficiently by TEV protease, and this cleavage was sufficient to promote IL-1β processing. Consistent with the relatively low sequence specificity of LF, the TEV-site2 NLRP1B protein was also cleaved by LF, but this cleavage was inefficient as most of the NLRP1B remained uncleaved, and IL-1β was not efficiently processed. Cleavage of TEV-site1 also was sufficient to induce IL-1β processing, but this occurred upon expression of either LF or TEV proteases. Furthermore, the TEV-induced cleavage at site-1 produced a fragment of NLRP1B that was smaller than the fragment produced by LF expression ( Fig. 3A and S1D ). Consistent with the mutagenesis experiments shown in Fig. 2 , this observation may indicate that LF prefers to cleave at site-2, which is still present in the TEV-site1 NLRP1B protein. Taken together, these results suggests that cleavage of NLRP1B at either site-1 or site-2 is sufficient to induce inflammasome activation independently of other LF-dependent cellular effects. 10.1371/journal.ppat.1003452.g003 Figure 3 Cleavage of NLRP1B is sufficient to promote inflammasome activation. A) 293T cells were transfected with WT, TEV-site2 or TEV-site1 GFP-HA-NLRP1B along with empty vector, TEV expression vector, or a LF expression plasmids. In all conditions cells were also co-transfected with Casp1 and Il1b expression vectors. Cleavage of GFP-HA-NLRP1B and IL-1β was determined 24 h post transfection. B) Immortalized B6 macrophages were transduced with a retrovirus encoding the indicated GFP-HA-NLRP1B form followed by a sequential transduction with a TEV-expression retrovirus co-expressing THY1.1. Percent transduction was determined by measuring expression of the respective retroviral integration markers (GFP and anti-THY1.1-PE-Cy7) by flow cytometry, and are expressed in relative fluorescent units (RFU). The numbers within each quadrant represent the percentage of live cells within the respective quadrant. C) RAW264.7 macrophages were transduced with GFP-HA-NLRP1B and a Tet-On construct expressing the indicated gene. Cells were treated with 5 µg/ml doxycycline for 20 h and supernatants were assayed for LDH release. D) 293T cells were transfected with empty vector, FL-NLRP1B-HA, the truncated NLRP1B-HA, or ΔLRR HA-NLRP1B, along with Casp1 and Il1b and assayed by immunoblotting. We also confirmed that cleavage of NLRP1B is sufficient to induce pyroptosis in macrophages cell lines. We transduced immortalized B6 macrophages with two different retroviral vectors, one expressing GFP-NLRP1B and the other expressing TEV protease with an IRES-Thy1.1 expression marker. If cleavage is sufficient to activate NLRP1B, it is expected that only cells expressing both components would undergo pyroptosis and therefore be underrepresented in the live population of cells. We analyzed the percentage of cells that contained both retroviruses by measuring THY1.1 surface-expression and GFP fluorescence by flow cytometry. As expected, an underrepresentation of the THY1.1 and GFP double-positive population was specifically seen in cells transduced with TEV and TEVsite2-NLRP1B, while the frequency of THY1.1+ cells was similar in cells that where negative for both forms of NLRP1B ( Fig. 3B ). To further confirm that cleavage of NLRP1B is sufficient for inflammasome activation, we transduced RAW 264.7 cells with retroviral vectors encoding various NLRP1B alleles, as well as a lentiviral Tet-On vector that inducibly expresses GFP, TEV-protease or LF after exposure of cells to doxycycline. In this system, TEV expression induced high levels of LDH release only for cells expressing TEVsite2-NLRP1B. As expected, since RAW cells express an endogenous functional allele of NLRP1B, LF induced pyroptotic lysis of cells expressing either wild-type or TEVsite2-NLRP1B ( Fig. 3C ). The percent LDH release was generally consistent with the percentage of cells that expressed both constructs ( Fig. S2B ). These data demonstrate that cleavage of NLRP1B is sufficient to activate this inflammasome in macrophages and cause pyroptosis. No apparent role for the N-terminal NLRP1B cleavage fragment We next tested whether the N-terminal fragment generated by cleavage of NLRP1B by LF at site-2 must be present along with the corresponding C-terminal fragment. We generated a construct to express a 'pre-cleaved' C-terminal fragment by deleting residues 1–44 of full-length NLRP1B and replacing amino acid 45 (leucine) with an initiator methionine. The resulting C-terminal fragment contains all known functional domains of NLRP1B ( Fig. 1D ). In the 293T cell system, high levels of spontaneous IL-1β cleavage was observed upon expression of the precleaved NLRP1B. The activity of pre-cleaved NLRP1B was comparable to that of a ΔLRR mutant, a form of NLRP1B that is known to be constitutively active ( Fig. 3D and S3C ) [47] . When the N-terminal fragment (amino acids 1–44) was coexpressed with the precleaved C-terminal fragment, no change in the amount of IL-1β processing was observed ( Fig. S3B ), suggesting it is neither necessary nor inhibitory when expressed in trans. For all of these experiments, the differences in the amount of IL-1β cleavage were not explained by differences in expression of NLRP1B ( Fig. S3A –C). Proteasome inhibitors and FIIND processing do not affect LF-dependent cleavage NLRP1B inflammasome activation can be blocked by proteasome inhibitors, an effect that is observed with multiple inhibitors and is specific to the NLRP1B inflammasome and not the NAIP/NLRC4 inflammasome [37] , [39] . The mechanism by which proteasome inhibitors affect NLRP1B inflammasome activation is not currently known. Therefore, we tested whether the proteasome inhibitor MG132 blocked NLRP1B cleavage. In the 293T system, an equivalent amount of cleaved of NLRP1B occurred in the presence of MG132 and its vehicle ( Fig. 4A ), suggesting NLRP1B cleavage is not the step at which MG132 interferes with NLRP1B activation ( Fig. S4A ). 10.1371/journal.ppat.1003452.g004 Figure 4 Proteasome inhibition and FIIND-processing do not affect NLRP1B cleavage by LF. A) 293T cells expressing GFP-HA-NLRP1B were co-treated with 1 µg/ml LeTx and 10 µM MG132 (proteasome inhibitor) or the DMSO vehicle and assayed for cleavage. B) Cleavage susceptibility of WT and S984A (FIIND mutant) GFP-HA-NLRP1B was determined in 293T cells at the indicated time points. The FIIND of NLRP1B contains a ZU-5/UPA-like domain that can auto-process, and this auto-processing is required for NLRP1B activation [23] , [24] . We tested if auto-processing at the FIIND region is prerequisite for N-terminal cleavage by LF. We tested the FIIND mutant S984A, which cannot auto-process, and found it to be indistinguishably sensitive to LeTx cleavage as wild-type NLRP1B ( Fig. 4B ). Thus FIIND auto-processing appears to be required for a downstream step in NLRP1B activation ( Fig. S4B ), and is not required for NLRP1B to be sensitive to LeTx cleavage. Discussion Activation of the NLRP1B inflammasome by LeTx is an important resistance mechanism during Bacillus anthracis infections in mice [17] , [18] . However the question of how NLRP1B senses the protease activity of LF remains unresolved. Here we investigated the molecular mechanism by which the protease activity of B. anthracis lethal toxin could be detected by NLRP1B. Our studies provide a clear molecular mechanism for how a pathogen-encoded activity (or 'pattern of pathogenesis' [3] ) can be sensed by the innate immune system. Two recent studies by Moayeri and colleagues provided a considerable advance in our understanding of NLRP1B activation by LeTx [41] , [42] . These two studies showed that both rat and mouse NLRP1 can be cleaved near the N-terminus by LF, and that mutation of the cleavage site abolished responsiveness of rat NLRP1 to LF. While these findings strongly suggest that direct cleavage of rat NLRP1 could be its mechanism of activation, the functional role of cleavage of mouse NLRP1B was not addressed, and it is also possible that mutation of the cleavage site blocked activation of rat NLRP1 by affecting the folding or assembly of NLRP1. Most significantly, the question of whether cleavage of NLRP1B was sufficient for its activation has not been addressed. This question is especially important to address because LT has been shown to have complex effects on cells, including disruption of MAP kinase signaling [43] , [48] , [49] , that could conceivably play a role in NLRP1B activation. Moreover, several other cellular functions, such as proteasome activity and N-end rule degradation pathways, have been implicated in NLRP1B activation [37] , [39] , [40] . Therefore, to demonstrate that cleavage of NLRP1B is sufficient to induce inflammasome activation, we engineered an allele of NLRP1B that could be activated by the heterologous TEV protease. This protease is not known to have endogenous substrates in mouse or human cells, so is likely to exert its effects solely via direct cleavage of the engineered NLRP1B protein. Indeed, the TEV protease did not activate NLRP1B unless NLRP1B contained a site that could be cleaved by TEV ( Fig. 3A ). Recent data have suggested that mouse NLRP1B can be cleaved at two distinct sites by LF [42] , but our cleavage site mutants and TEV-site forms of the receptor are most consistent with site-2 (cleavage between residues 44–45) being the predominant cleavage site. Interestingly, this site coincides with the same amino acid position as the LF cleavage site in rat NLRP1, even though the sequences of the two sites are not conserved ( Fig. 1A ). The low degree of target sequence specificity exhibited by LF may have allowed the sequence of the cleavage site in NLRP1 to diverge without losing responsiveness to LF. The position within NLRP1 at which LF cleaves may be determined in part by interactions between LF and regions of NLRP1 outside of the cleavage site. Indeed, similar non-cleavage-site interactions appear to control the specificity of LF for its other known substrates, the MAPKKs [9] , [10] . The divergence of the amino acid sequence of the N-terminus of NLRP1B is interesting given the high degree of conservation in the rest of the protein. This divergence may be due to random drift of a structurally unconstrained domain, or alternatively, the divergence may reflect evolutionary pressure for NLRP1 to be recognized by other pathogen-encoded proteases. Notably, our data suggest that cleavage outside of the primary LF target site (e.g., at site-1) can also activate NLRP1B ( Fig. 3A ), although it is unclear if LF can cleave and activate NLRP1B at this position. In addition, our data suggest that cleavage of NLRP1B does not necessarily have to be complete to be sufficient to permit inflammasome assembly and CASP1 activation. Taken together, these observations suggest that NLRP1B could be responsive to proteases from other pathogens even if these proteases cleave NLRP1B at different sites with low efficiency. Indeed, countless pathogens, including bacteria, viruses and parasites, depend on cytosolically-localized proteases for virulence [50] – [53] . Therefore the presence of cytosolic proteases could be considered a 'pattern of pathogenesis' [3] , that could be detected by NLRP1 proteins to allow the innate immune system to discriminate pathogenic and harmless microbes. The divergence of rat and mouse NLRP1 may thus reflect evolution under the selective pressure imposed by distinct sets of pathogens in the two different rodents species. It will be interesting to determine if other proteases can activate rodent NLRP1s. The detection of protease activity by NLRP1B represents a fundamentally distinct mode of pathogen recognition in vertebrates as compared to the classic mode of direct recognition of PAMPs observed with most innate immune receptors of the TLR, NLR and RLR families. The N-terminus of NLRP1B appears to function to detect LF activity in a manner analogous to the 'decoy' model [54] , which has been previously proposed to explain detection of certain pathogen effectors by plant NLRs. The proteolytic mechanism by which NLRP1B is activated represents one of the few examples in mammals in which a molecular mechanism has been established for how an innate immune sensor can respond to a pathogen-encoded activity. It is currently unknown how cleavage of the N-terminus results in structural changes that lead to NLRP1B activation. A simple model is that the N-terminus of NLRP1B mediates an auto-inhibitory intramolecular interaction, perhaps via an interaction with the LRR domain, which is known also to be required for auto-inhibition of NLRP1B ( Fig. 3D ) [47] . An alternative model that is not excluded by our data is that the removal of the original N-terminus allows the neo-N-terminus to provide a positive signal to activate NLRP1B. More complicated models involving interactions with other proteins can also be envisaged. We did not observe an inhibitory role of the N-terminal fragment when expressed in trans ( Fig. S3 ). This suggests that the N-terminus is necessary to maintain NLRP1B in a conformation that is inactive, but can only do so when the N-terminus is covalently attached to the rest of NLRP1B. In addition to proteolytic cleavage by LF, additional layers of NLRP1B regulation appear to exist. For example, FIIND auto-processing is required for NLRP1B activity, for reasons that remain poorly understood [24] . Since we found that FIIND auto-processing mutants are still cleaved by LF, the role of FIIND auto-processing appears not to be to render NLRP1B susceptible to cleavage by LF. Further complexities in NLRP1B activation are also suggested by the observation that proteasome and N-end rule pathway inhibitors appear to specifically prevent NLRP1B-dependent CASP1 activation [37] , [39] , [40] . Even though previous studies have shown that proteasome inhibitors do not block cleavage of MAPKK by LF [37] , [39] , we tested if proteasome inhibition might affect cleavage of NLRP1B, which appears to be a less optimal substrate than the MAPKKs. However, we observed no effect of the proteasome inhibitor MG132 on the ability of LF to cleave NLRP1B. Thus it remains unclear how this inhibitor specifically blocks the NLRP1B inflammasome and not other inflammasomes. Models that attempt to explain the mechanism of NLRP1B are further complicated by other unique features of NLRP1B. For example, ATP binding to the NBD of NLRP1B is not necessary for inflammasome activation, and mutants of NLRP1B that are unable to bind ATP are actually constitutively active [55] . This is contrary to what is known for other mammalian NLRs, where ATP binding appears to be required for oligomerization and downstream signaling [21] . Furthermore, gross truncations of NLRP1B can also lead to constitutively active forms of NLRP1B that contain only the very C-terminal CARD and a portion of the FIIND [47] . Thus, other disturbances, by proteolysis or by other means, to the overall structure of NLRP1B could lead to loss of the conformation that mediates auto-inhibition. In general, the molecular conformational changes that occur in NLRs as they transition from an inactive to an active state are poorly understood. Thus, our studies of NLRP1B provide an important point of comparison that helps us to develop a broader understanding of the NLR class of innate immune sensors and the mechanisms of their activation. Materials and Methods Ethics statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee at the University of California, Berkeley (MAUP #: R301-0313BRC). Plasmids and constructs HA-NLRP1B was amplified from a Nlrp1b (DQ117584.1) cDNA (gift of E. Boyden and B. Dietrich, Harvard Medical School) with primers 1–2 and cloned into pCMSCV-IRES-hCD4 using the XhoI and NotI restriction sites ( Fig. S5 ). A construct for expression a GFP-HA-NLRP1B fusion was created by amplifying HA-NLRP1B with primers 3–4 and cloning the resulting PCR product into MSCV downstream of and in-frame with GFP using NotI and SalI sites. TEV expression constructs were created by amplifying a His6-TEV ORF with the primers 7–9 and 8–9 into pCMSCV-IRES-Thy1.1 and pFG12-rtTA-IRES-Thy1.1, respectively. LF was similarly cloned into the same vectors as TEV, but with the primers 10–11 and 12–13 using a template provided by Bryan Krantz (UC Berkeley). Mutagenesis of Nlrp1b was performed using Quickchange (Stratagene/Agilent), but modified by substituting the Pfu polymerase for PrimeSTAR HS (TAKARA/Clonetech). The primers used are listed in Fig. S6 . Cell culture HEK293T (ATCC) cells were grown in complete media (DMEM, 10%FBS, 100 U/ml Penicillin, 100 µg/ml Streptomycin, and supplemented with 2 mM L-glutamine). RAW 264.7 and immortalized B6 macrophages were grown in complete media (RPMI 1640, 10%FBS, 100 U/ml Penicillin, 100 µg/ml Streptomycin). DNA transient transfections HEK 293T cells were seeded the day prior to transfection at a density of 1.5×10 5 cells/well in a 24-well plate with complete media. DNA complexes were made with Lipofectamine 2000 (Invitrogen) according to manufactures instructions and overlaid on cells for 24–36 hours. Western blots Cells were lysed in RIPA buffer supplemented with 1 mM PMSF and 1×X Complete Protease Inhibitor Cocktail (Roche). Lysates were spun down at max speed at 4C for 20 min and supernatants were mixed with 6× Laemmli sample buffer. To detect full length NLRP1B, lysates were incubated at room temperature prior to SDS-PAGE. To analyze all other proteins, including the N-terminally cleaved form of NLRP1B, samples were boiled for 10 min prior to separation. SDS-PAGE was performed with Novex BisTris gel system according to manufacturer instructions (Invitrogen). Separated proteins were transferred on to Immobilon-FL PVDF membranes. Membranes were blocked with Odyssey blocking buffer (Licor). The following antibodies were used for the following antigens: HA mAB 3F10 (Roche), MEK-2 SC-13115 (Santa Cruz), Beta Actin SC-4778 (Santa Cruz), IL-1B AF-401-NA (R&D systems). Secondary antibodies anti-rat, mouse and goat were all conjugated to Alexa Flour-680 (Invitrogen). Immunoprecipitation and LF in vitro cleavage assay Transfected cells were lysed in a non-denaturing buffer (1% NP-40, 137 mM NaCl, 2 mM EDTA, 20 mM Tris pH 8 supplemented with protease inhibitors). Cleared lysates were bound to EZview Red Anti-HA Affinity Gel (Sigma) washed four times with lysis buffer, once in LF cleavage buffer (10 mM NaCl, 5 uM ZnSO4, 10 mM HEPES pH 7.4), and resuspended back into cleavage buffer. One microgram of recombinant LF was added to immunoprecipitated NLRP1B and incubated at 37°C for 2 hours, and analyzed by western blotting as described above. Cytotoxicity/Pyroptosis assay and IL-1β secretion Macrophages were seeded one day prior to treatment in a 96well plate at 5×10 4 cell/well in RPMI media without phenol red. The next day cells were treated with LeTx 1 µg/ml, FlaTox 1 µg/ml [46] , or doxycycline at 5 µg/ml in ethanol for the indicated time, and spun down at 400× g . For IL-1β release cells were pretreated/cotreated with 1 µg/ml of Pam3CSK4. Supernatants were removed and assayed for LDH and IL-1β release as described previously [56] . Ethics statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee at the University of California, Berkeley (MAUP #: R301-0313BRC). Plasmids and constructs HA-NLRP1B was amplified from a Nlrp1b (DQ117584.1) cDNA (gift of E. Boyden and B. Dietrich, Harvard Medical School) with primers 1–2 and cloned into pCMSCV-IRES-hCD4 using the XhoI and NotI restriction sites ( Fig. S5 ). A construct for expression a GFP-HA-NLRP1B fusion was created by amplifying HA-NLRP1B with primers 3–4 and cloning the resulting PCR product into MSCV downstream of and in-frame with GFP using NotI and SalI sites. TEV expression constructs were created by amplifying a His6-TEV ORF with the primers 7–9 and 8–9 into pCMSCV-IRES-Thy1.1 and pFG12-rtTA-IRES-Thy1.1, respectively. LF was similarly cloned into the same vectors as TEV, but with the primers 10–11 and 12–13 using a template provided by Bryan Krantz (UC Berkeley). Mutagenesis of Nlrp1b was performed using Quickchange (Stratagene/Agilent), but modified by substituting the Pfu polymerase for PrimeSTAR HS (TAKARA/Clonetech). The primers used are listed in Fig. S6 . Cell culture HEK293T (ATCC) cells were grown in complete media (DMEM, 10%FBS, 100 U/ml Penicillin, 100 µg/ml Streptomycin, and supplemented with 2 mM L-glutamine). RAW 264.7 and immortalized B6 macrophages were grown in complete media (RPMI 1640, 10%FBS, 100 U/ml Penicillin, 100 µg/ml Streptomycin). DNA transient transfections HEK 293T cells were seeded the day prior to transfection at a density of 1.5×10 5 cells/well in a 24-well plate with complete media. DNA complexes were made with Lipofectamine 2000 (Invitrogen) according to manufactures instructions and overlaid on cells for 24–36 hours. Western blots Cells were lysed in RIPA buffer supplemented with 1 mM PMSF and 1×X Complete Protease Inhibitor Cocktail (Roche). Lysates were spun down at max speed at 4C for 20 min and supernatants were mixed with 6× Laemmli sample buffer. To detect full length NLRP1B, lysates were incubated at room temperature prior to SDS-PAGE. To analyze all other proteins, including the N-terminally cleaved form of NLRP1B, samples were boiled for 10 min prior to separation. SDS-PAGE was performed with Novex BisTris gel system according to manufacturer instructions (Invitrogen). Separated proteins were transferred on to Immobilon-FL PVDF membranes. Membranes were blocked with Odyssey blocking buffer (Licor). The following antibodies were used for the following antigens: HA mAB 3F10 (Roche), MEK-2 SC-13115 (Santa Cruz), Beta Actin SC-4778 (Santa Cruz), IL-1B AF-401-NA (R&D systems). Secondary antibodies anti-rat, mouse and goat were all conjugated to Alexa Flour-680 (Invitrogen). Immunoprecipitation and LF in vitro cleavage assay Transfected cells were lysed in a non-denaturing buffer (1% NP-40, 137 mM NaCl, 2 mM EDTA, 20 mM Tris pH 8 supplemented with protease inhibitors). Cleared lysates were bound to EZview Red Anti-HA Affinity Gel (Sigma) washed four times with lysis buffer, once in LF cleavage buffer (10 mM NaCl, 5 uM ZnSO4, 10 mM HEPES pH 7.4), and resuspended back into cleavage buffer. One microgram of recombinant LF was added to immunoprecipitated NLRP1B and incubated at 37°C for 2 hours, and analyzed by western blotting as described above. Cytotoxicity/Pyroptosis assay and IL-1β secretion Macrophages were seeded one day prior to treatment in a 96well plate at 5×10 4 cell/well in RPMI media without phenol red. The next day cells were treated with LeTx 1 µg/ml, FlaTox 1 µg/ml [46] , or doxycycline at 5 µg/ml in ethanol for the indicated time, and spun down at 400× g . For IL-1β release cells were pretreated/cotreated with 1 µg/ml of Pam3CSK4. Supernatants were removed and assayed for LDH and IL-1β release as described previously [56] . Supporting Information Figure S1 Predicted LF cleavage-site mutation comparison. A) LF cleavage-site1 predicts cleavage between K38-39 and the motif surrounding this site was progressively mutated away from the consensus motif. Cleavage-site 2 predicts cleavage between K44 and L45, and this site was muted only at residues immediately surrounding the site. TEV cleavage sites were introduced to produce cleavage after residue 38 for site-1, and residue 44 for site-2. B) Cleavage site-1 mutant series (CR1A-D)) and CR2A were transfected into 293T cells and treated with 1 µg/ml LeTx for 4 h, analyzed by western blotting with the indicated antibodies. The bottom blot was done in the presence of Casp1 to determine the extent of IL-1β processing. C) WT, CR1D, CR2A-C GFP-HA-NLRP1B were transfected into 293T cells and treated with 1 µg/ml LeTx for the indicated time points and cleavage and IL-1β processing was assayed by western blotting. D) WT and TEV-site1 GFP-HANLRP1B were cotransfected with TEV or empty expression vector into 293T cells. Thirty-six hours post transfection cells were treated with LeTx for 4 h, then lysed and analyzed by immunoblotting with an anti-HA antibody. (TIF) Click here for additional data file. Figure S2 Transduction efficiency is the same in macrophage cell lines. A) Immortalized B6 macrophages were transduced with WT and CR2A GFP-HA-NLRP1B. Expression and cleavage of each NLRP1B was determined by western blotting. Glycine (5 mM) was added 1 hour post the addition of LeTx to block lysis of cells in the 2 h and 3 h time points. B) Percent transduction of RAW 264.7 macrophages was determined by measuring THY1.1 surface expression for the Tet-On vector, and GFP expression for the NLRP1B vector under non-inducing conditions by flow cytometry. GFP and anti-THY1.1-PE-Cy7 fluorescence are expressed in relative fluorescence units (RFU). The numbers within each quadrant represent the percentage of live cells within the respective quadrant. (EPS) Click here for additional data file. Figure S3 NLRP1B's N-terminal fragment has no role in inflammasome activation when expressed in trans. A) Expression of FL, precleaved and ΔLRR mutants of NLRP1B were determined by anti-HA immunoblotting. B) 293T cells were cotransfected with C-terminally HA tagged WT or precleaved NLRP1B along with Casp1 and Il1b expression constructs. The N-terminal fragment (residues 1–44) fused to GFP-HA was co-transfected with the C-terminal fragments and assayed for IL-1β 24 h post-transfection. C) Processing of IL-1β in 293T cells was determined to be dependent on NLRP1B expression and the catalytic activity of CASP1. (TIF) Click here for additional data file. Figure S4 MG132 blocks NLRP1B activity and FIIND processing is required in 293T cells. A) IL-1β processing was analyzed in 293T cells expressing GFP-HA-NLRP1B, Casp1 , Il1b and treated with MG132 (proteasome inhibitor) and LeTx for the indicated time points. B) The necessity of FIIND domain processing for NLRP1B activation in 293T cells was determined by measuring IL-1β processing in cells expressing GFP-HA-NLRP1B, Casp1 , Il1b , after treatment with LeTx for the indicated time points. (TIF) Click here for additional data file. Figure S5 LF expression is sufficient to induce pyroptosis and IL-1β in 129 macrophages. A,B) Immortalized C57BL/6 (B6) and 129 macrophages were transduced with a Tet-On construct expressing the GFP or LF. Cells were treated with 1 µg/ml Pam3CSK4, 5 µg/ml doxycycline and 1 µg/ml PA for 20 h and supernatants were assayed for LDH release (A) and IL-1β release (B) into the supernatant. Cells were also treated with 1 µg/ml LeTx and 5 µg/ml FlaTox for 4 h prior supernatant collection. Error bars represent plus and minus one standard deviation from the mean. ND stands for not determined. (EPS) Click here for additional data file. Figure S6 PCR primers used for cloning and mutagenesis. (EPS) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10305495/

Network-Based Prediction of Side Effects of Repurposed Antihypertensive Sartans against COVID-19 via Proteome and Drug-Target Interactomes

The potential of targeting the Renin-Angiotensin-Aldosterone System (RAAS) as a treatment for the coronavirus disease 2019 (COVID-19) is currently under investigation. One way to combat this disease involves the repurposing of angiotensin receptor blockers (ARBs), which are antihypertensive drugs, because they bind to angiotensin-converting enzyme 2 (ACE2), which in turn interacts with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. However, there has been no in silico analysis of the potential toxicity risks associated with the use of these drugs for the treatment of COVID-19. To address this, a network-based bioinformatics methodology was used to investigate the potential side effects of known Food and Drug Administration (FDA)-approved antihypertensive drugs, Sartans. This involved identifying the human proteins targeted by these drugs, their first neighbors, and any drugs that bind to them using publicly available experimentally supported data, and subsequently constructing proteomes and protein–drug interactomes. This methodology was also applied to Pfizer's Paxlovid, an antiviral drug approved by the FDA for emergency use in mild-to-moderate COVID-19 treatment. The study compares the results for both drug categories and examines the potential for off-target effects, undesirable involvement in various biological processes and diseases, possible drug interactions, and the potential reduction in drug efficiency resulting from proteoform identification. 1. Introduction The proper function of the human body is achieved by the interaction and perfect coordination of several systems and organs. RAAS is a crucial regulator of various body functions necessary for survival, such as blood volume and pressure regulation, sodium and water reabsorption, potassium secretion, and maintenance of the vascular tone [ 1 , 2 ]. Disturbance of this intricate hormonal regulatory system could potentially lead to acute or chronic conditions, such as various cardiovascular and renal disorders, including congestive heart failure (CHF), acute myocardial infarction (AMI), hypertension, and diabetic kidney disease (DKD) [ 1 , 3 ]. Until now, five main axes of the RAAS regulatory action have been reported, each of which utilizes different enzymes, receptors, key substrates, effector peptides, and/or leads to distinct downstream signaling pathways [ 4 ]. The five main axes are the classic angiotensinogen/renin/angiotensin-converting enzyme (ACE)/angiotensin II (ANG II)/angiotensin type 1 receptor (AT1R)/angiotensin I (ANG I)/ANG II/angiotensin-converting enzyme 2 (ACE2)/angiotensin 1–7 (ANG(1–7))/Mas receptor, the ANG II/alanine and proline-rich secreted protein (APA)/ANG III/angiotensin type 2 receptor (AT2R)/NO/cGMP, the ANG III/3-Arylpropiolonitriles (APN)/angiotensin IV (ANG IV)/insulin-regulated aminopeptidase (IRAP)/polyketide synthase (AT4) receptor, and the prorenin/renin/prorenin receptor (PRR or Atp6ap2)/MAP kinases ERK1/2/V-ATPase [ 4 ]. The dynamic equilibrium between the first two arms of the RAAS, ACE (the classical RAAS) and ACE2, is crucial for its regulation [ 5 ]. In the ACE pathway, ANG-I is formed as a result of the cleavage of angiotensinogen (AGT) (produced in the liver) from renin (produced in the kidney) [ 6 ]. Later on, ANG-I is cleaved by ACE into angiotensin II (ANG-II) (produced in the vascular tissue) that binds to two distinct protein receptors of the G protein-coupled family, angiotensin type 1 and type 2 (AT1R and AT2R, respectively) [ 7 ]. The AT1R pathway promotes vasoconstriction, cell growth, sodium, and water retention, as well as sympathetic activation. In contrast, the binding of ANG-II to the AT2R neutralizes the detrimental effects induced by the AT1R pathway [ 8 ]. Oppositely, ACE2 can produce angiotensin 1–9 (ANG 1–9) from ANG-I and angiotensin 1–7 (ANG 1–7) from ANG-II [ 6 ]. The production of ANG 1–7 limits the available ANG-II, thus reducing the activation of the AT1R while simultaneously binding to the MAS-receptor, resulting in vasodepressor, anti-inflammatory, anti-oxidative, and antiproliferative effects [ 5 ]. ACE inhibitors and ARBs (also named Sartans, Figure 1 ) are first-line drugs for hypertension but are also employed to treat certain cases of heart failure and chronic kidney disease [ 9 ]. On the one hand, ACE inhibitors impede the production of ANG-II, reducing the activity of both AT1R and AT2R [ 9 ]. However, because this enzyme is also a kininase, its inhibition raises the overall level of kinins. One of those, bradykinin, is correlated with a series of side effects such as cough, but on top of that, in rare cases, with inhibitor-induced angioedema, which is a potentially life-threatening emergency [ 9 ]. On the other hand, ARBs were engineered as a substitute for patients who could not endure the adverse events of ACE inhibitors. These drugs specifically block the AT1R while augmenting the activation of the AT2R [ 10 ]. Additionally, ARBs can limit inflammation along with endothelial and epithelial dysfunction in various organs. More specifically, there is clinical evidence suggesting that the integrity of the lung's endothelial barrier, which might be disrupted in the case of a viral infection, can be protected by ARBs [ 11 ]. Recently, ARB repurposing efforts have been made for the treatment of COVID-19 in order to address the urgent need for effective therapy for the disease while minimizing the costs, time, and uncertainty that accompany most drug development strategies [ 12 , 13 ]. The rationale behind this work is the fact that Sartans, as AT1R antagonists, are substantially ANG-II mimic molecules and thereby are expected to bind to the ACE2 enzyme [ 12 ]. Previously, the development and in silico study of biSartans, a novel type of Sartans with two anionic biphenyl tetrazole moieties, were reported [ 12 , 13 ]. These molecules were found to bind stronger than Sartans not only to the AT1R but also to the Receptor Binding Domain (RBD)/ACE2 complex [ 13 ]. It is well known that the spike S-protein of SARS-CoV-2 binds to ACE2 through its RBD, thus initiating membrane fusion between the virion and the cell [ 14 ]. One of the antiviral drugs currently approved by the FDA for emergency use for the treatment of mild-to-moderate COVID-19 in certain adults and pediatric patients is Pfizer's Paxlovid ( Figure 1 ). Paxlovid is a combination of PF-07321332 (Nirmatrelvir), an inhibitor of the crucial for the viral proliferation of 3-chymotrypsin-like protease (3CL pro ), and Ritonavir, which acts as a pharmacokinetic boosting agent and is also used for the treatment of the Human Immunodeficiency Virus (HIV) [ 15 ]. As this drug is a strong cytochrome P450 (CYP) 3A4 inhibitor, co-administration with drugs that are highly dependent on CYP3A4 for clearance or potent CYP3A4 inducers could potentially lead to elevated concentrations of those drugs and greatly reduce Paxlovid plasma concentrations, respectively [ 16 ]. The problem that arises is that many drugs used regularly by a large percentage of the population, such as statins, antiarrhythmics, and antipsychotics, are metabolized by CYP3A4, and thus there is a great chance of drug interaction between those drugs and Paxlovid that could conceivably lead to side effects [ 17 , 18 ]. Most of the ARBs (i.e., losartan, irbesartan, candesartan, azilsartan, and valsartan) are predominantly metabolized by another CYP isomorph, CYP2C9, and to a smaller extent by CYP3A4 [ 19 , 20 , 21 ]. In the case of telmisartan, eprosartan, and Olmesartan, however, the CYP system does not participate in their metabolism [ 21 ]. Figure 1 Chemical structures of Valsartan, Telmisartan, Olmesartan, Candesartan cilexetil, Irbesartan, Eprosartan, Losartan, Azilsartan medoxomil, Ritonavir, and Nirmatrelvir. Note that some of these may be deprotonated at physiological pH [ 13 , 22 , 23 ]. Illustrations were made with MarvinSketch, version 22.22.0 [ 24 ]. In this work, a network-based methodology was developed as an initial prediction of the potential for side effects resulting from protein off-target and/or drug–drug interactions that could occur when a drug is repurposed. It has been suggested that the proteins that directly interact with a drug's protein target (first neighbors) could act as off-targets for the specific drug in question, other drugs that share the same drug target with the drug in question, and drugs that target the drug's first neighbors in the network [ 25 ]. Additionally, the drugs that share a drug target or target neighbor proteins in the human interactome could also participate in drug–drug interactions [ 26 ]. Protein off-target and drug–drug interactions can in some cases be beneficial, for example, in drug repurposing or drug combinations for efficiency enhancement. However, in other cases, they can also lead to side effects. In this study, a network-based bioinformatics approach was used to explore the potential of FDA-approved antihypertensive drugs, Sartans, for repurposing against SARS-CoV-2. The analysis investigated their off-target interactions, effects on biological processes, and potential interactions with other drugs by integrating experimental and publicly available proteome interactome and drug-target data. As a comparison, the same methodology was also applied for Pfizer's Paxlovid, as there is an evident structural similarity of its components, especially Ritonavir, with Sartans. This comparison could provide further insight into what would happen if Sartan was co-administered with Paxlovid. Some steps of the methodology were also applied to Perphenazine, a drug that has nothing to do with ACE2 or COVID-19, as a sort of "negative control test" for the relevance of the obtained results. Perphenazine is an antipsychotic drug that displays comparable effectiveness to Haloperidol and is used for the management of schizophrenia symptoms as well as the control of severe nausea and vomiting in adult patients [ 27 , 28 ]. 2. Materials and Methods The methodology workflow is presented in Figure 2 . Το identify the specific proteins that are targeted by the drug in question, the DrugBank database was used [ 29 ]. DrugBank is a web-enabled database that includes thorough molecular details regarding drugs, their mechanisms of action, interactions, and targets [ 30 , 31 , 32 , 33 , 34 ]. To ascertain the structural similarity between the drugs in question, the Tanimoto index was calculated with the use of the Similarity Workbench of ChemMine Tools [ 35 , 36 ]. Additionally, DrugBank was also used to ascertain whether the human protein targets of the drug in question are also targets of other drugs approved at a certain moment in time in at least one jurisdiction. Next, using the Human Protein Atlas, which has all the human proteins mapped in cells, tissues, and organs through the integration of multi-omics data [ 37 , 38 ], the expression of each drug receptor was identified. Human interactome data was downloaded from the Protein InteraCtion KnowLedgebasE (PICKLE) meta-database at the Universal Protein Resource (UniProt) level [ 39 , 40 ]. PICKLE web-source consists of the direct protein-protein interactome of the human proteome, integrating publicly available experimentally supported protein–protein interactions (PPIs) [ 41 , 42 , 43 , 44 ]. The protein targets for the drugs in question were projected into the human interactome, and binary PPIs between protein targets and their first neighbors (i.e., the proteins with which they physically interact) were extracted. The proteome interactomes and drug-target data were integrated to construct protein–drug interaction networks that include the drugs of interest (Sartans or Paxlovid) and their corresponding receptors, along with any other drugs that may also target these receptors. Additionally, the networks include the first neighbors of the receptors in the human interactome, as well as any drugs that target these first neighbors. The proteome interactomes and drug-target data were integrated for the construction of protein–drug interaction networks that include the drugs of interest (Sartans or Paxlovid) and their corresponding receptors, along with any other drugs that may also target these receptors. Additionally, the networks include the interactors of the receptors in the human interactome, as well as any drugs that target these interactors. The networks were constructed as described in previous works [ 45 ], including the import of mined PPI data from databases, visualization tools to adjust the layout, node size and color, edge thickness and color, and analysis tools using clustering algorithms for calculation of centrality measures in Cytoscape (version 3.9.1) [ 46 ]. After the network construction, the node degree, betweenness centrality, and closeness centrality calculations of the interactors of the drugs in question was also performed in Cytoscape. For the network comparison, the Jaccard index (a similarity coefficient equivalent to the Tanimoto index and correspondent to 1-dJ, where dJ is the well-known Jaccard distance for sparse matrixes [ 47 ]) was used to compute the similarity (a proxy of the probability of interaction) in terms of first neighbors between the protein–drug networks. A plug-in of Cytoscape named Biological Networks Gene Ontology tool (BiNGO) was used for the functional annotation of the drug in question's protein targets. BiNGO is an open-source Java tool for the identification of Gene Ontology (GO) keywords that are notably overrepresented in a given set of genes [ 48 ]. The statistical test implemented for the calculation of the overrepresentation is the hypergeometric test, whereas the False Discovery Rate (FDR) correction is carried out with the Benjamini–Hochberg method. BiNGO uses the term-for-term approach that detects overrepresentation of GO terms individually, and in our work, it is used as a validation of the pathways that the drug targets are involved in. For the functional annotation of the interactors of the drugs' targets, the Protein Analysis THrough Evolutionary Relationships (PANTHER) database was used [ 49 ]. PANTHER is a publicly available knowledge base that stores the outcomes of a lengthy phylogenetic reconstruction containing both computational and manual operations as well as quality control stages [ 50 ]. The statistical test used to determine the over-represented GO terms is the Fisher exact test. The Benjamini–Hochberg False Discovery Rate (FDR) is also computed, and all displayed GO terms have an FDR < 0.05 [ 51 ]. The gene–disease association of the protein targets of the drugs in question and their interactors was performed on the DisGeNET (version 7) platform, which contains an extensive catalog of genes and genomic variants associated with human diseases [ 52 ]. The proteoform identification of protein targets and their first neighbors was performed through the iteration of data mined from UniProt, the Online Mendelian Inheritance in Man (OMIM), and GeneCards. Proteoforms resulting from post-translational modifications (PTMs) were identified from UniProt, while those resulting from allelic variants of genes were extracted from OMIM, a comprehensive catalog of human genes, genetic conditions, and attributes that emphasizes the molecular linkage of genetic variation to phenotypic expression [ 53 , 54 ]. Additionally, proteoforms resulting from alternative splicing were extracted from the GeneCards database, which integrates information from multiple web sources about all annotated and predicted human genes [ 55 , 56 ]. 3. Results 3.1. Retrievement of Drug–Protein Target Associations There is an evident structural similarity between Sartans and Paxlovids that was verified by the calculation of the Tanimoto index. The Tanimoto index, along with the Dice index, cosine coefficient, and Soergel distance, have been proven to be the best (and, in some cases, equivalent) metrics for similarity computations [ 57 ]. In this case, the Tanimoto index of Sartans and Ritonavir ranges between 0.13 and 0.23 and the one of Sartans and Nirmatrelvir ranges between 0.04 and 0.23. Since the index is relatively low but different from zero (the total superposition scoring 1), the two kinds of drugs share some common structural motifs, and thus it is worth comparing them with one another to find out what would happen in a case of co-administration. The complete table of the Tanimoto index for each Sartan and the components of Paxlovid can be found in Table S10 . Based on the DrugBank database, all Sartans bind to the AT1R. These receptors, as mentioned above, are vital effectors of the RAAS and are found in various cell types, as ANG II is a molecule with a wide range of actions [ 57 ]. Two of the approved Sartans (Telmisartan and Irbesartan) bind to one more protein each, which is also a target of many other drugs. Irbesartan binds to c-JUN, which is a transcription factor (TF) AP-1 subunit. c-JUN, as a basic leucine zipper (bZIP) TF, takes part in many different cell functions, including proliferation, apoptosis, survival, cancer, and tissue morphogenesis [ 58 ]. Proliferator-activated receptor γ (PPARγ) is one of the targets of Telmisartan. This protein belongs to the nuclear receptor superfamily of TFs and is a main regulator of the differentiation, maturation, and function of colon, breast, prostate, bladder, fat, and immune system cells [ 59 ]. If Sartans are repurposed for the treatment of COVID-19, they are going to target the ACE2 receptor. ACE2 is a homologue of ACE and mainly contributes to the regulation of ANG II levels but also hydrolyzes some proteins such as bradykinin [ 60 , 61 ]. This zinc metalloenzyme and monocarboxypeptidase is a Type 1 integral membrane glycoprotein [ 62 ] and is located both on the surface of endothelial and epithelial cells of the kidney, heart, uterus, placenta, and retina, as well as other tissues [ 60 ]. However, there is also a secreted form of this enzyme in the blood [ 60 ]. Paxlovid has two drug receptors, one for each of its active components. Nirmatrelvir, which is the antiviral component, binds to 3CL pro , whereas Ritonavir ( Figure 1 ), which is essentially a pharmacokinetic boosting agent, targets the nuclear receptor subfamily 1 group I member 2 (NR1I2), also known as the pregnane X receptor (PXR). The expression of drug-metabolic enzymes and transporters that mediate the responses of mammals to their chemical environment and various endogenous chemicals is regulated by this receptor [ 63 ]. Table 1 shows the protein targets of Sartans and the two components of Paxlovid, along with the number of other drugs that bind to the same targets. The full table can be seen in Table S5 . It should be noted that the proteoforms have nothing to do with drug efficiency, but rather contribute to the reliability of the physical drug-receptor interaction. Additionally, the number of proteoforms is proportional to the number of studies conducted on that specific protein. Consequently, it is no surprise that ACE2 has a great number of proteoforms, as there are an enormous number of studies on that receptor, whereas for the SARS-CoV2 3CLpro, no proteoforms have been identified yet. The full table with all the experimentally supported events for proteoforms can be found in Table S6 . Based on the Human Protein Atlas, the RNA tissue specificity, protein tissue expression, and subcellular location of the drugs' protein targets were retrieved ( Table 2 ). Heart, skin, kidneys, blood vessels, skeletal muscles, brain, liver, lungs, and adrenal glands are among the organs where AT1R is expressed [ 64 ], whereas c-JUN is mostly overexpressed in cancer tissue. In adipose, spleen, adrenal gland, and the big colon tissue, the highest concentrations of PPAR mRNA have been identified (4–7). Multiple lines of research also suggest that PPARG is crucial for controlling adipocyte differentiation and glucose homeostasis [ 65 ]. NR1I2 mRNA concentrations are abundant in the liver, and lower concentrations of it have been found in the small intestine, the colon, the stomach, and skeletal muscles [ 66 ]. PXR's highest levels of expression are encountered in the small intestine, the colon, and the liver [ 67 ]. It has been demonstrated that the ACE2 protein is highly expressed in the brush border of enterocytes of the small intestines, but it has also been spotted in lung and endothelial tissue [ 68 , 69 ]. 3.2. Construction of Protein–Protein and Protein–Drug Interaction Networks To identify all the proteins that physically interact with the protein targets mentioned above, three Sartans and one Paxlovid were projected into the experimentally supported human interactome ( Figure 3 a,b). ACE2, the protein that Sartans will target if they are used in the treatment of COVID-19, was also projected into the human interactome in order to identify its first neighbors ( Figure 3 c). Sartans' receptors interact with a greater number of proteins, and their interactome consists of 335 nodes and 2989 edges. The main receptor of all Sartans, AT1R, interacts with 48 different proteins, whereas c-JUN (Irbesartan's receptor) interacts with 186 other proteins and PPAR-γ (Telmisartan's receptor) with 120 more. The last two receptors also interact with themselves, as they form dimers, and with each other. ACE2, Sartans' suggested target, and some of its 9 first neighbors form dimers, and its interactome consists of 10 nodes and 18 edges. NR1I2 interacts with itself, thus forming a dimer, as well as with 28 other proteins; thus, its interactome consists of 30 nodes and 126 edges. Next, the two protein–drug networks for Paxlovid and Sartans that contained the drugs in question, their targets, and their first neighbors, as well as any drugs that bind to them, were constructed ( Figure 4 and Figure 5 ). Sartans' network consists of 1011 nodes and 4133 edges; its diameter is 6 and its radius is 4. Ritonavir's network consists of 215 nodes and 254 edges; its diameter is 4 and its radius is 2. In order to better visualize and compare the results, Sartans' network was divided into three distinct networks: the network of Irbesartan (that binds to AT1R and c-JUN), the network of Telmisartan (that binds to AT1R and PPARG-γ), and the network for all the other Sartans that only bind to AT1R. In the third network, all six Sartans are grouped into one group node. As seen in Figure 6 , the six Sartans that only bind to AT1R (i.e., Valsartan, Olmesartan, Candesartan cilexetil, Eprosartan, Losartan, and Azilsartan medoxomil) have 49 first neighbors and 159 drugs possibly interacting with them; their networks' diameter is 4 and their radius is 2. Irbesartan, that binds to both the AT1R and c-JUN, has 236 first neighbors and 607 drugs possibly interacting with it; the network's diameter is 6 and its radius is 3. Telmisartan, which binds to both the AT1R and PPARG-γ, has 170 first neighbors and 397 drugs possibly interacting with it; the network's diameter is 6 and its radius is 3. The interactome of Sartans that exclusively target AT1R was compared with the Protein–Drug Network of Paxlovid. To ensure a valid network comparison, ACE2 and its neighboring proteins, along with drugs that bind to them, were added to the network of Sartans that exclusively target AT1R ( Figure 7 ). The resulting network has 272 nodes and 420 edges; its diameter is 5 and its radius is 3. The two targets combined have 107 interactors, and there are 163 drugs that may interact with the Sartans. Finally, the protein–drug network of Perphenazine that contained the drug, its three protein targets (i.e., Dopamine Receptor D1 (DRD1), Dopamine Receptor D2 (DRD2), and Calmodulin 1 (CALM1)), their first neighbors, as well as any drugs that bind to them, was constructed ( Figure 8 ). Perphenazine's network consists of 787 nodes and 1182 edges; its diameter is 6 and its radius is 3. The three protein targets have a total of 151 first neighbors, and there are 633 drugs possibly interacting with Perphenazine. In Table S9 , the synoptic table (closeness centrality, betweenness centrality, and node degree) of the analysis of each protein–drug network can be found. 3.3. Shared First Neighbor Distance Metrics (Jaccard Index) In Table 3 , the Jaccard index and Jaccard distance between the shared first neighbors of the two drugs in question and the third drug (Perphenazine) can be seen. 3.4. Proteoform Identification of Protein Drug Targets and Proteins Involved in Sartans' and Paxlovid's Based Interactomes The term "proteoform", established by Smith et al., is used to describe "all of the different molecular forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts and post-translational modifications" [ 70 ]. In this work, we focused on the proteoforms that result from PTMs, allelic variants of genes, and alternative splicing of the two drugs' protein targets and their interactors. The full tables of the experimentally supported events of PTMs of both the drugs' receptors and their first neighbors can be seen in Table S6 . In Figure 9 , it can be seen that the PTMs of Sartans' protein targets result from modified residues (excluding lipids, glycans, and protein crosslinks) (37.78%), glycosylation (covalently attached glycan group(s)) (24.44%), disulfide bonds (cysteine residues participating in disulfide bonds) (11.11%), chain (extent of a polypeptide chain in the mature protein) (11.11%), cross-link (residues participating in covalent linkage(s) between proteins) (11.11%), lipidation (covalently attached lipid group(s)) (2.22%), and signal (sequence targeting proteins to the secretory pathway or periplasmic space) (2.22%). The PTMs of Sartans' protein targets, if repurposed for the treatment of COVID-19, arise from glycosylation (41.67%), disulfide bonds (20.83%), chain (20.83%), modified residues (8.33%), lipidation (4.17%), and signal (4.17%). According to UniProt, the protein target of Paxlovid does not get modified post-translationally. In Figure 10 , the PTMs of the interactors AT1R, ACE2, and NR1I2 are presented. The PTMs of the first neighbors of AT1R are a result of modified residues (59.36%), chain (11.87%), disulfide bonds (10.27%), glycosylation (7.76%), cross-link (3.65%), lipidation (3.20%), initiator methionine (cleaved initiator methionine) (2.74%), signal (0.91%), and transit peptide (extent of a transit peptide for organelle targeting) (0.23%). The PTMs of the first neighbors of ACE2 arise from modified residues (54.95%), chain (8.11%), peptide (extent of an active peptide in the mature protein) (7.21%), glycosylation (7.21%), cross-link (5.41%), disulfide bond (5.41%), signal (4.50%), lipidation (2.70%), initiator methionine (2.70%), and propeptide (part of a protein that is cleaved during maturation or activation) (1.80%). The PTMs of the interactors of NR1I2 are a result of modified residues (76.70%), cross-link (10.92%), chain (7.52%), initiator methionine (cleaved initiator methionine) (2.91%), lipidation (0.97%), signal (0.49%), and glycosylation (0.49%). The proteoforms arising from allelic variations are 5 for the AT1R receptor gene and 13 for the PPARG-γ receptor gene. The full tables of allelic variations can be seen in Table S1 . Out of Sartans receptor genes, the AT1R gene has 6 transcript variants that result from alternative splicing, PPARG-γ has 16, ACE2 has 6, and Paxlvovid's receptor gene has 3. The full tables of alternative transcripts can be seen in Table S2 . 3.5. Functional and Disease Annotation of Proteins Involved in Sartans' and Paxlovids' Based Interactomes Functional annotation analysis revealed that AT1R, in addition to its angiotensin type I receptor activity, is associated with acetyltransferase activator activity, bradykinin receptor binding, angiotensin type II receptor activity, peptide receptor activity, G-protein-coupled receptor binding, protein heterodimerization activity, and enzyme activator activity. The TF c-JUN is related to R-SMAD binding, Rho and Ras GTPase activator activity, RNA polymerase II transcription factor activity, enhancer binding, double-stranded DNA binding, promoter binding, DNA regulatory region binding, structure-specific DNA binding, and transcription coactivator activity, amongst others. Finally, PPAR-γ is linked with arachidonic acid, eicosatetraenoic acid, and eicosanoid binding, transcription activator binding, prostaglandin, and retinoid X receptor binding, prostanoid receptor activity, retinoic acid receptor binding, fatty acid binding, and more. The molecular function Gene Ontology (GO) terms that are associated with Paxlovid's human receptor, NR1I2, are steroid hormone receptor activity, ligand-dependent nuclear receptor activity, and drug binding. Finally, ACE2 is associated with glycoprotein binding as well as viral receptor, carboxypeptidase, and exopeptidase activity. The full table with all the over-represented GO terms of the drugs' protein targets, along with the GO-IDs and p -values, can be seen in Table S7 . As far as the first neighbors are concerned, the ten molecular function GO terms associated with Sartans' and Paxlovid's receptors that had the best p -values were chosen. As it can be seen below, AT1R's, c-JUN's, and PPAR-γ's first neighbors are related to transcription factor binding, protein domain specific binding, acetyltransferase activity, phosphothreonine residue binding, transferase activity, catalytic activity, acting on DNA, peptide butyryltransferase activity, peptide crotonyltransferase activity, and histone H2B acetyltransferase activity. NR1I2′s first neighbors are linked with transcription factor binding, transcription regulator activity, nuclear steroid receptor activity, DNA binding, nuclear thyroid hormone receptor binding, nuclear estrogen receptor binding, transcription coregulator binding, acetyltransferase activity, DNA-binding transcription factor activity, and STAT family protein binding. If Sartans are repurposed against COVID-19, they are going to have two protein receptors, the AT1R and the ACE2, because only the sartans that originally had one protein target were chosen. The two receptors' first neighbors are related to exogenous protein binding, G protein-coupled receptor binding, adenylate cyclase regulator activity, phosphatase activator activity, protein-containing complex binding, beta-adrenergic receptor kinase activity, titin binding, dopamine receptor binding, molecular function regulator activity, and binding ( Table 4 ). The full table with all the over-represented GO terms of the first neighbors of the drugs' protein targets, along with the GO-IDs, p -values, fold enrichment, and FDR, can be seen in Table S8 . The gene–disease association of the protein targets of the drugs in question is shown in Figure 11 . The AT1R gene (target of Sartans) is mostly associated with neoplasm metastasis, patent ductus arteriosus, aortic aneurysm/abdominal, proteinuria, and hypertensive disease. The JUN gene (target of Sartans) shows a strong association with malignant tumors of the colon, lung, liver, and stomach, as well as osteosarcoma. The PPARG-γ gene (target of Sartans) shows a significant correlation with diabetes mellitus, inflammation, familial partial lipodystrophy type 2, malignant colon tumors, and diabetic nephropathy. The ACE2 gene (target of Sartans if they are repurposed for the treatment of COVID-19) is associated with congestive heart failure, heart failure, myocardial infarction, tubal abortion, and early pregnancy loss. The NRI2 gene (target of Paxlovid) is linked with diabetes mellitus, steatohepatitis, drug-induced liver disease, adenocarcinoma, and obesity. The gene–disease association of the interactors of the protein targets of the drugs in question is shown in Figure 12 . The first neighbors of the targets of AT1R, cJUN, and PPARG-γ are mainly related with malignant neoplasms of the breast, polycythemia vera, Von Hippel–Lindau Syndrome, primary myelofibrosis, and Burkitt lymphoma. The first neighbors of AT1R (Sartans protein target) show a strong association with mast syndrome, leopard syndrome, essential thrombocythemia, uveal melanoma, and acute myelocytic leukemia. The first neighbors of ACE2 (Sartans protein target if they are repurposed for the treatment of COVID-19) are mostly associated with breast carcinoma, melanoma, malignant neoplasms of the breast, hypertensive disease, and ventricular tachycardia. The first neighbors of NR1I2 (target of Paxlovid) are linked with colorectal carcinoma, obesity, Fanconi renotubular syndrome with maturity-onset diabetes of the young, thrombocytopenia, and cardiomyopathy. The full table with all the diseases associated with every target and its first neighbors ranked by disease score can be found in Tables S3 and S4 . 3.6. Connectivity of AT1R, ACE2, and NR1I's Interactors The calculation of the node degree of the first neighbors of Sartans' two receptors, AT1R and ACE2 (when repurposed for COVID-19), as well as Paxlovid's receptor (NR1I2), was performed. In Table 5 , the ten nodes with the highest node degree as well as the mean node degree of all the first neighbors can be seen. The mean node degree for AT1R's first neighbors is 74, and the mean node degree for ACE2's first neighbors is 170. NR1I2's first neighbors mean node degree is 153. The full table with the network analysis (closeness centrality, betweenness centrality, and degree) of the protein–drug networks can be seen in Table S9 . 3.1. Retrievement of Drug–Protein Target Associations There is an evident structural similarity between Sartans and Paxlovids that was verified by the calculation of the Tanimoto index. The Tanimoto index, along with the Dice index, cosine coefficient, and Soergel distance, have been proven to be the best (and, in some cases, equivalent) metrics for similarity computations [ 57 ]. In this case, the Tanimoto index of Sartans and Ritonavir ranges between 0.13 and 0.23 and the one of Sartans and Nirmatrelvir ranges between 0.04 and 0.23. Since the index is relatively low but different from zero (the total superposition scoring 1), the two kinds of drugs share some common structural motifs, and thus it is worth comparing them with one another to find out what would happen in a case of co-administration. The complete table of the Tanimoto index for each Sartan and the components of Paxlovid can be found in Table S10 . Based on the DrugBank database, all Sartans bind to the AT1R. These receptors, as mentioned above, are vital effectors of the RAAS and are found in various cell types, as ANG II is a molecule with a wide range of actions [ 57 ]. Two of the approved Sartans (Telmisartan and Irbesartan) bind to one more protein each, which is also a target of many other drugs. Irbesartan binds to c-JUN, which is a transcription factor (TF) AP-1 subunit. c-JUN, as a basic leucine zipper (bZIP) TF, takes part in many different cell functions, including proliferation, apoptosis, survival, cancer, and tissue morphogenesis [ 58 ]. Proliferator-activated receptor γ (PPARγ) is one of the targets of Telmisartan. This protein belongs to the nuclear receptor superfamily of TFs and is a main regulator of the differentiation, maturation, and function of colon, breast, prostate, bladder, fat, and immune system cells [ 59 ]. If Sartans are repurposed for the treatment of COVID-19, they are going to target the ACE2 receptor. ACE2 is a homologue of ACE and mainly contributes to the regulation of ANG II levels but also hydrolyzes some proteins such as bradykinin [ 60 , 61 ]. This zinc metalloenzyme and monocarboxypeptidase is a Type 1 integral membrane glycoprotein [ 62 ] and is located both on the surface of endothelial and epithelial cells of the kidney, heart, uterus, placenta, and retina, as well as other tissues [ 60 ]. However, there is also a secreted form of this enzyme in the blood [ 60 ]. Paxlovid has two drug receptors, one for each of its active components. Nirmatrelvir, which is the antiviral component, binds to 3CL pro , whereas Ritonavir ( Figure 1 ), which is essentially a pharmacokinetic boosting agent, targets the nuclear receptor subfamily 1 group I member 2 (NR1I2), also known as the pregnane X receptor (PXR). The expression of drug-metabolic enzymes and transporters that mediate the responses of mammals to their chemical environment and various endogenous chemicals is regulated by this receptor [ 63 ]. Table 1 shows the protein targets of Sartans and the two components of Paxlovid, along with the number of other drugs that bind to the same targets. The full table can be seen in Table S5 . It should be noted that the proteoforms have nothing to do with drug efficiency, but rather contribute to the reliability of the physical drug-receptor interaction. Additionally, the number of proteoforms is proportional to the number of studies conducted on that specific protein. Consequently, it is no surprise that ACE2 has a great number of proteoforms, as there are an enormous number of studies on that receptor, whereas for the SARS-CoV2 3CLpro, no proteoforms have been identified yet. The full table with all the experimentally supported events for proteoforms can be found in Table S6 . Based on the Human Protein Atlas, the RNA tissue specificity, protein tissue expression, and subcellular location of the drugs' protein targets were retrieved ( Table 2 ). Heart, skin, kidneys, blood vessels, skeletal muscles, brain, liver, lungs, and adrenal glands are among the organs where AT1R is expressed [ 64 ], whereas c-JUN is mostly overexpressed in cancer tissue. In adipose, spleen, adrenal gland, and the big colon tissue, the highest concentrations of PPAR mRNA have been identified (4–7). Multiple lines of research also suggest that PPARG is crucial for controlling adipocyte differentiation and glucose homeostasis [ 65 ]. NR1I2 mRNA concentrations are abundant in the liver, and lower concentrations of it have been found in the small intestine, the colon, the stomach, and skeletal muscles [ 66 ]. PXR's highest levels of expression are encountered in the small intestine, the colon, and the liver [ 67 ]. It has been demonstrated that the ACE2 protein is highly expressed in the brush border of enterocytes of the small intestines, but it has also been spotted in lung and endothelial tissue [ 68 , 69 ]. 3.2. Construction of Protein–Protein and Protein–Drug Interaction Networks To identify all the proteins that physically interact with the protein targets mentioned above, three Sartans and one Paxlovid were projected into the experimentally supported human interactome ( Figure 3 a,b). ACE2, the protein that Sartans will target if they are used in the treatment of COVID-19, was also projected into the human interactome in order to identify its first neighbors ( Figure 3 c). Sartans' receptors interact with a greater number of proteins, and their interactome consists of 335 nodes and 2989 edges. The main receptor of all Sartans, AT1R, interacts with 48 different proteins, whereas c-JUN (Irbesartan's receptor) interacts with 186 other proteins and PPAR-γ (Telmisartan's receptor) with 120 more. The last two receptors also interact with themselves, as they form dimers, and with each other. ACE2, Sartans' suggested target, and some of its 9 first neighbors form dimers, and its interactome consists of 10 nodes and 18 edges. NR1I2 interacts with itself, thus forming a dimer, as well as with 28 other proteins; thus, its interactome consists of 30 nodes and 126 edges. Next, the two protein–drug networks for Paxlovid and Sartans that contained the drugs in question, their targets, and their first neighbors, as well as any drugs that bind to them, were constructed ( Figure 4 and Figure 5 ). Sartans' network consists of 1011 nodes and 4133 edges; its diameter is 6 and its radius is 4. Ritonavir's network consists of 215 nodes and 254 edges; its diameter is 4 and its radius is 2. In order to better visualize and compare the results, Sartans' network was divided into three distinct networks: the network of Irbesartan (that binds to AT1R and c-JUN), the network of Telmisartan (that binds to AT1R and PPARG-γ), and the network for all the other Sartans that only bind to AT1R. In the third network, all six Sartans are grouped into one group node. As seen in Figure 6 , the six Sartans that only bind to AT1R (i.e., Valsartan, Olmesartan, Candesartan cilexetil, Eprosartan, Losartan, and Azilsartan medoxomil) have 49 first neighbors and 159 drugs possibly interacting with them; their networks' diameter is 4 and their radius is 2. Irbesartan, that binds to both the AT1R and c-JUN, has 236 first neighbors and 607 drugs possibly interacting with it; the network's diameter is 6 and its radius is 3. Telmisartan, which binds to both the AT1R and PPARG-γ, has 170 first neighbors and 397 drugs possibly interacting with it; the network's diameter is 6 and its radius is 3. The interactome of Sartans that exclusively target AT1R was compared with the Protein–Drug Network of Paxlovid. To ensure a valid network comparison, ACE2 and its neighboring proteins, along with drugs that bind to them, were added to the network of Sartans that exclusively target AT1R ( Figure 7 ). The resulting network has 272 nodes and 420 edges; its diameter is 5 and its radius is 3. The two targets combined have 107 interactors, and there are 163 drugs that may interact with the Sartans. Finally, the protein–drug network of Perphenazine that contained the drug, its three protein targets (i.e., Dopamine Receptor D1 (DRD1), Dopamine Receptor D2 (DRD2), and Calmodulin 1 (CALM1)), their first neighbors, as well as any drugs that bind to them, was constructed ( Figure 8 ). Perphenazine's network consists of 787 nodes and 1182 edges; its diameter is 6 and its radius is 3. The three protein targets have a total of 151 first neighbors, and there are 633 drugs possibly interacting with Perphenazine. In Table S9 , the synoptic table (closeness centrality, betweenness centrality, and node degree) of the analysis of each protein–drug network can be found. 3.3. Shared First Neighbor Distance Metrics (Jaccard Index) In Table 3 , the Jaccard index and Jaccard distance between the shared first neighbors of the two drugs in question and the third drug (Perphenazine) can be seen. 3.4. Proteoform Identification of Protein Drug Targets and Proteins Involved in Sartans' and Paxlovid's Based Interactomes The term "proteoform", established by Smith et al., is used to describe "all of the different molecular forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts and post-translational modifications" [ 70 ]. In this work, we focused on the proteoforms that result from PTMs, allelic variants of genes, and alternative splicing of the two drugs' protein targets and their interactors. The full tables of the experimentally supported events of PTMs of both the drugs' receptors and their first neighbors can be seen in Table S6 . In Figure 9 , it can be seen that the PTMs of Sartans' protein targets result from modified residues (excluding lipids, glycans, and protein crosslinks) (37.78%), glycosylation (covalently attached glycan group(s)) (24.44%), disulfide bonds (cysteine residues participating in disulfide bonds) (11.11%), chain (extent of a polypeptide chain in the mature protein) (11.11%), cross-link (residues participating in covalent linkage(s) between proteins) (11.11%), lipidation (covalently attached lipid group(s)) (2.22%), and signal (sequence targeting proteins to the secretory pathway or periplasmic space) (2.22%). The PTMs of Sartans' protein targets, if repurposed for the treatment of COVID-19, arise from glycosylation (41.67%), disulfide bonds (20.83%), chain (20.83%), modified residues (8.33%), lipidation (4.17%), and signal (4.17%). According to UniProt, the protein target of Paxlovid does not get modified post-translationally. In Figure 10 , the PTMs of the interactors AT1R, ACE2, and NR1I2 are presented. The PTMs of the first neighbors of AT1R are a result of modified residues (59.36%), chain (11.87%), disulfide bonds (10.27%), glycosylation (7.76%), cross-link (3.65%), lipidation (3.20%), initiator methionine (cleaved initiator methionine) (2.74%), signal (0.91%), and transit peptide (extent of a transit peptide for organelle targeting) (0.23%). The PTMs of the first neighbors of ACE2 arise from modified residues (54.95%), chain (8.11%), peptide (extent of an active peptide in the mature protein) (7.21%), glycosylation (7.21%), cross-link (5.41%), disulfide bond (5.41%), signal (4.50%), lipidation (2.70%), initiator methionine (2.70%), and propeptide (part of a protein that is cleaved during maturation or activation) (1.80%). The PTMs of the interactors of NR1I2 are a result of modified residues (76.70%), cross-link (10.92%), chain (7.52%), initiator methionine (cleaved initiator methionine) (2.91%), lipidation (0.97%), signal (0.49%), and glycosylation (0.49%). The proteoforms arising from allelic variations are 5 for the AT1R receptor gene and 13 for the PPARG-γ receptor gene. The full tables of allelic variations can be seen in Table S1 . Out of Sartans receptor genes, the AT1R gene has 6 transcript variants that result from alternative splicing, PPARG-γ has 16, ACE2 has 6, and Paxlvovid's receptor gene has 3. The full tables of alternative transcripts can be seen in Table S2 . 3.5. Functional and Disease Annotation of Proteins Involved in Sartans' and Paxlovids' Based Interactomes Functional annotation analysis revealed that AT1R, in addition to its angiotensin type I receptor activity, is associated with acetyltransferase activator activity, bradykinin receptor binding, angiotensin type II receptor activity, peptide receptor activity, G-protein-coupled receptor binding, protein heterodimerization activity, and enzyme activator activity. The TF c-JUN is related to R-SMAD binding, Rho and Ras GTPase activator activity, RNA polymerase II transcription factor activity, enhancer binding, double-stranded DNA binding, promoter binding, DNA regulatory region binding, structure-specific DNA binding, and transcription coactivator activity, amongst others. Finally, PPAR-γ is linked with arachidonic acid, eicosatetraenoic acid, and eicosanoid binding, transcription activator binding, prostaglandin, and retinoid X receptor binding, prostanoid receptor activity, retinoic acid receptor binding, fatty acid binding, and more. The molecular function Gene Ontology (GO) terms that are associated with Paxlovid's human receptor, NR1I2, are steroid hormone receptor activity, ligand-dependent nuclear receptor activity, and drug binding. Finally, ACE2 is associated with glycoprotein binding as well as viral receptor, carboxypeptidase, and exopeptidase activity. The full table with all the over-represented GO terms of the drugs' protein targets, along with the GO-IDs and p -values, can be seen in Table S7 . As far as the first neighbors are concerned, the ten molecular function GO terms associated with Sartans' and Paxlovid's receptors that had the best p -values were chosen. As it can be seen below, AT1R's, c-JUN's, and PPAR-γ's first neighbors are related to transcription factor binding, protein domain specific binding, acetyltransferase activity, phosphothreonine residue binding, transferase activity, catalytic activity, acting on DNA, peptide butyryltransferase activity, peptide crotonyltransferase activity, and histone H2B acetyltransferase activity. NR1I2′s first neighbors are linked with transcription factor binding, transcription regulator activity, nuclear steroid receptor activity, DNA binding, nuclear thyroid hormone receptor binding, nuclear estrogen receptor binding, transcription coregulator binding, acetyltransferase activity, DNA-binding transcription factor activity, and STAT family protein binding. If Sartans are repurposed against COVID-19, they are going to have two protein receptors, the AT1R and the ACE2, because only the sartans that originally had one protein target were chosen. The two receptors' first neighbors are related to exogenous protein binding, G protein-coupled receptor binding, adenylate cyclase regulator activity, phosphatase activator activity, protein-containing complex binding, beta-adrenergic receptor kinase activity, titin binding, dopamine receptor binding, molecular function regulator activity, and binding ( Table 4 ). The full table with all the over-represented GO terms of the first neighbors of the drugs' protein targets, along with the GO-IDs, p -values, fold enrichment, and FDR, can be seen in Table S8 . The gene–disease association of the protein targets of the drugs in question is shown in Figure 11 . The AT1R gene (target of Sartans) is mostly associated with neoplasm metastasis, patent ductus arteriosus, aortic aneurysm/abdominal, proteinuria, and hypertensive disease. The JUN gene (target of Sartans) shows a strong association with malignant tumors of the colon, lung, liver, and stomach, as well as osteosarcoma. The PPARG-γ gene (target of Sartans) shows a significant correlation with diabetes mellitus, inflammation, familial partial lipodystrophy type 2, malignant colon tumors, and diabetic nephropathy. The ACE2 gene (target of Sartans if they are repurposed for the treatment of COVID-19) is associated with congestive heart failure, heart failure, myocardial infarction, tubal abortion, and early pregnancy loss. The NRI2 gene (target of Paxlovid) is linked with diabetes mellitus, steatohepatitis, drug-induced liver disease, adenocarcinoma, and obesity. The gene–disease association of the interactors of the protein targets of the drugs in question is shown in Figure 12 . The first neighbors of the targets of AT1R, cJUN, and PPARG-γ are mainly related with malignant neoplasms of the breast, polycythemia vera, Von Hippel–Lindau Syndrome, primary myelofibrosis, and Burkitt lymphoma. The first neighbors of AT1R (Sartans protein target) show a strong association with mast syndrome, leopard syndrome, essential thrombocythemia, uveal melanoma, and acute myelocytic leukemia. The first neighbors of ACE2 (Sartans protein target if they are repurposed for the treatment of COVID-19) are mostly associated with breast carcinoma, melanoma, malignant neoplasms of the breast, hypertensive disease, and ventricular tachycardia. The first neighbors of NR1I2 (target of Paxlovid) are linked with colorectal carcinoma, obesity, Fanconi renotubular syndrome with maturity-onset diabetes of the young, thrombocytopenia, and cardiomyopathy. The full table with all the diseases associated with every target and its first neighbors ranked by disease score can be found in Tables S3 and S4 . 3.6. Connectivity of AT1R, ACE2, and NR1I's Interactors The calculation of the node degree of the first neighbors of Sartans' two receptors, AT1R and ACE2 (when repurposed for COVID-19), as well as Paxlovid's receptor (NR1I2), was performed. In Table 5 , the ten nodes with the highest node degree as well as the mean node degree of all the first neighbors can be seen. The mean node degree for AT1R's first neighbors is 74, and the mean node degree for ACE2's first neighbors is 170. NR1I2's first neighbors mean node degree is 153. The full table with the network analysis (closeness centrality, betweenness centrality, and degree) of the protein–drug networks can be seen in Table S9 . 4. Discussion Sartans are antihypertensive drugs that act on the RAAS by targeting the AT1R and activating the AT2R [ 10 ]. Apart from lowering blood pressure, ARBs are associated with lung endothelial protection and inflammation reduction [ 11 ]. Lately, in silico studies for drug repurposing for the treatment of COVID-19 have been made, as they constitute a fast and cost-effective plan of action [ 12 , 13 ]. Sartans, as well as the recently developed biSartans, antagonize the AT1R, and therefore it can be assumed that they will also bind to ACE2, the protein that interacts with the S-protein of SARS-CoV-2 and is the entry point of the virus into the cell [ 12 , 13 ]. Pfizer's Paxlovid, a combination of Nirmatrelvir and Ritonavir, is currently approved by the FDA for emergency use in the treatment of mild to moderate cases of COVID-19 in certain adult and pediatric patients. Paxlovid is, however, a potent CYP3A4 inhibitor, and because this isoenzyme is responsible for the metabolism of many common drugs that are prescribed daily, drug interactions with this antiviral drug could occur [ 16 , 17 , 18 ]. In the past few years, efforts have been made to repurpose Sartans and a new generation of angiotensin receptor-blocking drugs with a Sartan scaffold called biSartans. Nevertheless, before the process of the development of novel antiviral agents is initiated, it is of utmost importance to carefully consider the potential for off-target interactions (i.e., interactions occurring between the drug and unintended protein targets), as they are a major contributor to drug toxicity. Examples reported regarding drug off-target interactions include inhibition of cytochrome P450 enzymes, alteration of protein kinase activity, interference with ion channels, disruption of transporter function, and alteration of hormone signaling [ 71 ]. The identification of these off-target interactions is essential for the establishment of drug safety and the minimization of unintended consequences, such as preclinical and clinical toxic events. Hitherto, no in silico study has been performed to predict the toxicity risks of Sartans or a novel class of synthetic antihypertensive drugs referred to as biSartans in the case that they target ACE2 and are repurposed to treat COVID-19. In this work, a network-based bioinformatics approach was applied to investigate the potential of known FDA-approved Sartans for protein off-target interactions, their unwanted involvement in various biological processes, and their potential interactions with other drugs. It has been proposed that the first neighbors of a drug's protein target in the human interactome could act as off-targets either for that specific drug or for other drugs targeting the same protein or proteins at close proximity in the human interactome. Consequently, these drugs have a high probability of participating in a drug–drug interaction event or sharing the same side effects [ 25 , 72 , 73 ]. A comparative analysis of the protein–drug associations for both Sartans and Paxlovid was performed to ascertain the potential for off-target interactions and any possible interactions with other drugs. Additionally, the different proteoforms of the drugs' protein targets and their first neighbors were studied to investigate the possibility of their interference with drug binding. Furthermore, the functional and disease annotation of both drugs' protein targets and their first neighbors was performed so that their undesired participation in a variety of biological processes and their possible implication in disease onset could be pinpointed. On the one hand, Table S1 shows that all three of Sartans' targets are also targeted by 32 additional drugs. Moreover, if Sartans were to be repurposed for COVID-19 treatment, they would target ACE2, which is already the target of 4 drugs. On the other hand, Paxlovid, which only targets NR1I2, is bound by 52 other drugs. The latter further validates the fact that Paxlovid may cause various drug interactions with common drugs, as they share a receptor (i.e., NR1I2). The protein-drug interaction networks demonstrate that Sartans' drug targets have 319 first neighbors and 682 drugs that could interact with them or their first neighbors, while Paxlovid has 185 drugs that could interact with it, and its target is directly interacting with 28 more proteins. More specifically, the six Sartans that only bind to the AT1R (i.e., Valsartan, Olmesartan, Candesartan cilexetil, Eprosartan, Losartan, and Azilsartan medoxomil) have 49 first neighbors and 159 drugs possibly interacting with them. Irbesartan, which binds to both the AT1R and c-JUN, has 236 first neighbors and 607 drugs possibly interacting with it. Telmisartan, which binds to both the AT1R and PPARG-γ, has 170 first neighbors and 397 drugs possibly interacting with it. The interactome of the six Sartans that exclusively target AT1R was compared to Paxlovid's network. For their proper comparison, as mentioned above, ACE2 was added as a protein target, and in the new network, the two protein targets together have 107 first neighbors and 163 drugs that could target them or their first neighbors. From that, it can be seen that even if Sartans target ACE2, the number of drugs that could potentially interact with them through the PPI network is almost the same. Additionally, it can also be observed that the six Sartans that only interact with the AT1R have fewer drugs that could possibly interact with them than Paxlovid, suggesting that if they are indeed used as a treatment for COVID-19, they may have fewer side effects due to drug-drug interactions. Nevertheless, because Sartans are also repurposed for the treatment of diabetic nephropathy, ischemic stroke, ventricular dysfunction, and more, their potential for off-target interactions should be considered before they are repurposed outside the scope of the original medical indication. From the Jaccard index calculation, it can be seen that the network of Sartans, when used as anti-COVID-19 drugs compared to the network of Paxlovid have the smallest similarity in terms of shared first neighbors. The same thing is observed in the comparison of Paxlovid and Perphenazine, which is an anti-psychotic drug totally unrelated to ACE2 binding. The network of Sartans when used as antihypertensive drugs and their network when used as an anti-COVID-19 strategy share the most first neighbors, which validates the results of this method. The protein targets of Sartans, when they are used as antihypertensive drugs, mostly undergo post-translational modifications that result in modified residues, glycosylation, and cross-links. These modified residues are mainly phosphoserine, phosphothreonine, and N6-acetyllysine, whereas the glycans are attached to the N-terminal end of asparagine and the oxygen of threonine. The cross-links on their end result in glycyl lysine isopeptide formation. On the contrary, the most common PTMs of the receptors of Sartans, if they are repurposed for the treatment of COVID-19, are glycosylation, disulfide bond formation, and differences in chain lengths. In this case, the addition of glycans leads to N-linked (GlcNAc) asparagine formation, and disulfide bonds are formed between cysteines in the extracellular region of ACE2. ACE2 can be found in two forms: the cellular, which is membrane-bound, and the circulating, soluble one. For the formation of the soluble form, the full-length ACE2 is processed either by the metalloprotease ADAM Metallopeptidase Domain 12 (ADAM12) or the Transmembrane Serine Protease 2 (TMPRSS2), resulting in two slightly different circulating forms that lack a small fragment of the C-terminus [ 74 ]. The allelic variations of the AT1R gene that result in different receptor isoforms are associated with hypertension and renal tubular dysgenesis, while the allelic variations of the PPARG-γ are linked with severe obesity, type 2 diabetes mellitus and insulin resistance, somatic colon cancer, and type 3 partial familial lipodystrophy. When Sartans are used as antihypertensive drugs, two of their targets (i.e., AT1R and PPARG-γ) are alternatively spliced and result in 22 alternatively spliced transcripts, whereas if they are repurposed for the treatment of COVID-19, they result in 12 alternatively spliced transcripts. Paxlovid's receptor mRNA also undergoes alternative splicing, which leads to the formation of three alternative transcripts. The interactors of the protein targets of Sartans, when the drugs are used as antihypertensive drugs, mostly undergo post-translational modifications that result in modified residues, disulfide bond formation, and differences in chain lengths. These modified residues are mainly phosphoserine, phosphotyrosine, and phosphothreonine. On the contrary, the most common PTMs of interactors of the ACE2 receptor of Sartans, if they are repurposed for the treatment of COVID-19, are modified residues, glycosylation, disulfide bond formation, and cross-links. These modified residues are mainly phosphoserine, phosphotyrosine, and phosphothreonine or are modified on the N6 atom. In the case of ACE2 interactors, the addition of glycans leads to N-linked (GlcNAc) asparagine formation. The interactors of NR1I2 (i.e., Paxlovid's receptor) mostly undergo PTMs that result in modified residues, different chain lengths, and an initiator methionine cleavage. These modified residues are, for the most part, phosphoserine, phosphotyrosine, and phosphothreonine, but they are also residues modified on the N6 atoms and asymmetric dimethylarginines. The cross-links on their ends result in glycyl lysine isopeptide formation. There has been some evidence suggesting that phosphorylation could have an impact on drug binding, affinity, and consequently clinical efficacy, and that impact is expected to be stronger the closer the phosphorylation is to the drug binding pocket [ 75 ]. Furthermore, acetylation may drastically alter protein function via modification of its properties, such as hydrophobicity, solubility, and surface properties [ 76 ]. In the case of N-acetylation, which is one of the most common types, protein-protein interactions could be altered, resulting in a higher binding affinity with substrates, cofactors, and other macromolecules [ 77 ]. Until recently, the disulfide bond, where two cysteines spontaneously form a bond under redox conditions, was the most well-known covalent crosslink [ 78 ]. These bonds contribute to protein stability and are therefore crucial for protein structure and function, as well as the regulation of protein activities [ 79 ]. However, apart from disulfide bonds, in proteins there can be other types of cross-links, such as ester and isopeptide bonds [ 78 ]. The isopeptide bonds, which were originally discovered about 10 years ago, are peptide bonds that are developed outside the main protein chain [ 80 ]. They are intramolecular bonds that form autocatalytically between the side chains of lysine and asparagine/aspartic acid under hydrophobic conditions and provide great stability and structural firmness [ 78 ]. Finally, proteins are frequently stabilized by protein glycosylation, which extends their half-life and protects them from denaturation or proteolytic degradation [ 81 ]. According to some studies, the level of glycosylation is proportional to the stability increase and reduced flexibility of the protein [ 82 ] and is also involved in cell membrane formation and cell–cell adhesion [ 83 ]. Specifically, N -glycosylation affects protein folding, decreases protein dynamics, and most likely leads to increased protein stability [ 84 ], whereas O -glycosylation may alter protein–protein interactions [ 85 ]. Proteoforms can change protein dynamics, protein binding, and metabolism, and this may have an impact on how well drugs work; therefore, they can impair the effectiveness of commonly used medications or render cells resistant to some medications. However, there has not been any experimental data in this situation to suggest that the proteoforms have an effect on the efficacy of either Sartans or Paxlovids. Since it reflects the number of studies that are pertinent to them, the number of proteoforms effectively serves as a verification of the existence of a physical interaction between the medicine in question and its target. From the gene–disease association of the protein targets of Sartans, when used as antihypertensive drugs, it can be concluded that the major diseases they are associated with are cancer and neoplasm metastasis, diabetes mellitus, and inflammation, whereas if they are repurposed for COVID-19, apart from cancer, they are also linked with heart failure and related complications, as well as pregnancy loss. Paxlovid's target appears to be involved in the development of diabetes mellitus, liver disease, and obesity. The proteins associated with Sartans are mainly involved in protein binding and, at a lower percentage, in nucleic acid and DNA binding. PPIs are indispensable for basic cellular functions in living cells, as they carry out a variety of tasks, including altering the kinetics of enzymes, catalyzing metabolic processes, activating or inactivating proteins, modifying their specificity, and regulating the concentrations of transporting molecules [ 49 ]. On top of that, PPIs form a vital part of the signaling events that impact cell growth and transformation and, because of their dynamic nature, provide the versatility that characterizes the mechanisms involved in pathway regulation [ 50 ]. DRBPs, or DNA and RNA-binding proteins, are a large class of molecules that make up a sizable portion of cellular proteins and play crucial roles in cells [ 51 ]. They create complex dynamic multilevel networks that control nucleotide metabolism, gene expression [ 52 ], transcription and translation, DNA repair, splicing, apoptosis, and mediate stress responses [ 51 ], among other cellular processes. The interactors of Sartans targets, when the drugs are used for the treatment of hypertension, are mostly associated with breast cancer, leukemia, lymphoma, and obesity. However, if these drugs are repurposed as a treatment strategy for COVID-19, their targets' interactors are affiliated with breast cancer, leukemia, melanoma, thrombocythemia, hypertensive disease, and heart complications. On the contrary, the proteins associated with Ritonavir (Paxlovid's component) mostly take part in transcription regulation and demonstrate TF and transcriptional cofactor (COF) activities. On the one hand, TFs are protein molecules that bind to DNA-regulatory sequences (promoters, enhancers, and silencers), which are typically found in the 5′ upstream region of genes, to modulate the rate of gene transcription and subsequently stimulate or inhibit gene expression and transcription, as well as protein synthesis [ 53 , 54 ]. These proteins are essential for embryogenesis and development and are usually found in larger multiprotein complexes [ 54 ]. On the other hand, COFs operate as central effectors of transcription activation and gene expression as they transmit regulatory cues from enhancers to promoters [ 55 ]. More specifically, they are scaffold proteins, histone/protein-modifying enzymes, and chromatin remodelers that work in concert to arrange the local chromatin structure and balance the neighboring post-translational modifications, which helps to regulate transcription generally [ 56 ]. Paxlovid's receptor interactors have, for the most part, been linked with diabetes, thrombocythemia, leukemia, and cancer. From the functional annotation analysis, it can be observed that AT1R and ACE2 first neighbors are mainly involved in protein and enzyme binding, whereas NR1I2 first neighbors are mostly involved in transcription regulation. Consequently, if Sartans are repurposed for COVID-19 treatment, they probably will not be involved in different biological processes apart from calcium ion binding. Additionally, Sartans and Paxlovids are going to be involved in different biological processes, which will probably lead to different side effects due to off-target interactions. Additionally, from the gene–disease association analysis, it can be seen that Sartans' protein targets are linked with cancer either when they are used as antihypertensive or anti-COVID-19 drugs. If they are used for COVID-19, however, they are also implicated in heart failure and pregnancy loss. Paxlovid's target is associated with completely different diseases. The first neighbors of Sartans' targets, either when they are used for the treatment of hypertension or COVID-19, are associated with hypertension, breast cancer, and leukemia. Nonetheless, when they are used as a treatment strategy for COVID-19, they are also linked with some different types of cancer (i.e., melanoma) and thrombocythemia. The first neighbors of NR1I2′s targets are also associated with cancer, leukemia, and thrombocythemia, such as the first neighbors of Sartans' targets if used as anti-COVID-19 medications, but they are also linked with diabetes. When Sartans are used for different purposes, they are mostly associated with the same diseases but also some different ones. The first neighbors of both drugs' targets (i.e., Sartans and Paxlovid), when used for the treatment of COVID-19, are for the most part associated with the same diseases. It is evident that both drugs are mainly linked to some type of cancer. This is no surprise because it has been computed that for 15.233 out of the 17.371 human genes, there is at least one paper in PubMed that associated them with some type of cancer [ 86 ], thus this finding can be considered purely contingent. Even if there is not a study that connects one particular gene with cancer, it is most likely that there will be one in the future. As far as the other associated diseases are concerned, the gene–disease association could serve as an indication of the implications that that specific drug could have either on the development and progression of that particular disease or on the effect it could have on a patient already suffering from it. According to the node degree results, the centrality of ACE2 is higher but comparable to the centrality of AT1R, but its neighbors are more central than those of AT1R. According to the analysis, if Sartans are repurposed for the treatment of COVID-19, they might have a higher possibility of side effects that result from off-target interactions than when they are used for the treatment of high blood pressure. This is also consistent with the fact that ARBs (Sartans) were engineered as a substitute for patients who could not endure the adverse events of ACE inhibitors, as mentioned above. As far as Paxlovid is concerned, NR1I2 is less central than the Sartans' drug targets, but its centrality is still comparable to theirs. The centrality of its first neighbors is also comparable to that of ACE2. The RAAS has been the prime target for the design of drugs for the treatment of hypertension and cardiovascular diseases. ARBs have been discovered and developed to specifically block the AT1R [ 87 ]. Pioneering research on RAAS has resulted in the discovery of the first orally active ARB antagonist, Losartan (DUP753), followed by the development of a series of other potent Sartans for the treatment of hypertension [ 88 ]. Since ACE2, which is a part of RAAS and the entry point of SARs-CoV-2 in the cell, Sartans bearing anionic groups, tetrazolate, and carboxylate, were investigated as possible antivirals for the treatment of COVID-19, either by blocking entry or replication of the virus [ 13 , 22 , 23 ]. Extensive clinical studies have shown that ARBs are beneficial in the treatment of hypertensive patients infected by COVID-19 [ 89 , 90 ]. Other studies investigating the mechanism of triggering disease have shown that an imbalance in RAAS in favor of ANG-II deregulates the system, exaggerates the SARS-CoV-2 specific T-cell response, and thus increases COVID-19 severity and mortality [ 91 , 92 ]. One of the main contributors to morbidity is the release of inflammatory cytokines. Consequently, ARBs could be a promising treatment plan not only for COVID-19 but also for autoimmune diseases. ARBs modulate the cytokine production of T helper (Th)1 and Th17 effector cells by converting pathogenic cytokines to regulatory [ 93 , 94 ]. Modulation of cytokine production has also been reported in other studies [ 95 , 96 ]. The conformation of angiotensin, the principal effector of RAAS, led to the development of non-peptide mimetic ARBs and biSartans ( Figure 1 and Figure 13 ) based on structure–activity relationship (SAR) studies, nuclear magnetic resonance (NMR), fluorescence, and molecular modeling techniques [ 97 , 98 , 99 , 100 , 101 , 102 ]. In all these studies, the arginine residues play a catalytic role in the basic cleavage sites (R685–S686 and R815–S816) for the S-protein's cleavage and therefore trigger infection induced by proteases, such as furin and transmembrane serine protease 2 (TMPRSS2) [ 103 , 104 , 105 ]. Furthermore, arginine mutations on RBD enhance the binding of S-protein with ACE2, increasing transmissibility and infectivity [ 106 ]. Based on the above, arginine blockers are also potential therapeutics for treating COVID-19 [ 13 , 22 ]. Proteases such as furin, trypsin, TMPRSS2, and 3CLpro are potential targets for designing novel COVID-19 drugs [ 107 , 108 ]. It is worth mentioning that before the implementation of any of those alternative treatments for COVID-19 or autoimmune disorders, their thorough experimental study is of essence. Data provided by the "COVID-19 Drug Interactions" online database, which is an evidence-based drug-drug interaction resource, indicates that the co-administration of Paxlovid and two Sartans (i.e., Valsartan and Irbesartan) may also need close surveillance. In the case of Valsartan, clinically significant interactions have been reported and, thus, require additional monitoring, such as alteration of drug dosage or timing of administration [ 109 ] (data produced on 2 February 2023). Finally, data provided by DrugBank's "Drug Interaction Checker" indicates that the metabolism of Irbesartan can be decreased when combined with Ritonavir [ 110 , 111 ]. 5. Conclusions Sartans are antihypertensive drugs that target the AT1R and activate the AT2R to lower blood pressure. Recently, efforts have been made to repurpose them for the treatment of COVID-19 as a fast and cost-effective alternative to drug discovery. Pfizer's Paxlovid, a combination of Nirmatrelvir and Ritonavir, is currently approved by the FDA for emergency use in the treatment of mild to moderate COVID-19 in certain adult and child patients, but it is also a potent CYP3A4 inhibitor, which could lead to drug interactions. However, given that off-target interactions (i.e., interactions between the medication and undesired protein targets) are a primary cause of drug toxicity, it is crucial to thoroughly assess their possibility before the process of developing novel antiviral medicines is initiated to ensure the reduction of undesired effects. However, to date, no in silico analysis has been carried out to estimate the potential toxic effects of Sartans or new-generation drugs in this family if they target ACE2 and are used to treat COVID-19. This work used a network-based bioinformatics approach to analyze the potential of known FDA-approved Sartans and Paxlovid for protein off-target interactions, drug–drug interactions, and their potential involvement in numerous biological processes, leveraging experimental support from publicly available proteomic interactome and drug-target data. Network analysis revealed that Sartans' protein targets (i.e., AT1R, c-JUN, PPAR-γ, and ACE2) are also targets for 36 more drugs, whereas Paxlovid's only human drug target (i.e., NR1I2) binds 52 other drugs. Additionally, the protein–drug interaction networks demonstrate that Sartans' drug targets have 319 first neighbors and 682 drugs that could interact with them or their first neighbors, while Paxlovid has 185 drugs that could interact with it, and its target is directly interacting with 28 more proteins. Interestingly, the six Sartans that only bind to the AT1R (i.e., Valsartan, Olmesartan, Candesartan cilexetil, Eprosartan, Losartan, and Azilsartan medoxomil) have 107 first neighbors and 163 drugs possibly interacting with them, suggesting that they may have fewer side effects due to drug–drug interactions than Paxlovid if they are repurposed against COVID-19 and a higher possibility for side effects that result from off-target interactions than when they are used for the treatment of high blood pressure. From the proteoform identification, it is evident that Sartans' protein targets have far more proteoforms than Paxlovid's target, either when they are used for the treatment of hypertension or COVID-19. From the study of the most connected interactors of both drugs' targets, it can be seen that the first neighbors of Paxvlovid's target have fewer proteoforms than the first neighbors of Sartans' targets if they are used for the treatment of COVID-19. The study of proteoforms is necessary as they could impact protein binding, dynamics, and metabolism. In this case, however, there has not been any experimental evidence that could indicate an impact of the proteoforms on the drug effectiveness of either Sartans or Paxlovid. The number of proteoforms is essentially a verification of the existence of a physical interaction between the drug in question and its target, as it reflects the number of studies relevant to them. Furthermore, the functional annotation of both drugs' protein targets and their first neighbors revealed that the proteins associated with Sartans were mainly involved in protein and enzyme binding, whereas the proteins associated with Paxlovid were mostly involved in transcription regulation and demonstrated TF and cofactor activities, so the two drugs will most likely be involved in different biological processes and will probably lead to different side effects due to off-target interactions. Additionally, the gene–disease association showed that Sartans' protein targets (for either use) are mainly involved in cancer, heart complications, and pregnancy loss, whereas Paxlovid's target is linked with obesity, liver disease, and diabetes. The first neighbors of Sartans' targets (for either use) are for the most part linked with the same diseases (i.e., hypertension, breast cancer, and leukemia). Nonetheless, the interactors of Paxlovid's target are involved with the same diseases as the interactors of Sartans' targets if used as a treatment for COVID-19. As a result, the targets of Sartans, whether the drugs are used as antihypertensives or as an anti-COVID-19 strategy, are associated with the same diseases. As far as the two COVID-19 therapies discussed in this work (i.e., Sartans and Paxlovid) are concerned, the interactors of both drugs are mainly linked to the same diseases. Since most of the human genes are linked with cancer in one way or another, the focus should be on the non-cancer associated diseases, as the gene–disease association may provide a hint as to the potential effects that a given drug may have, either on the onset or progression of that disease. Based on the results of this study, no specific problem arises from the combined use of Paxlovid and Sartans. A limitation of this computational study is that, although it is based on experimental data, it cannot fully reproduce the complexity of drug interactions and potential side effects in vivo. Furthermore, while the study provides valuable insights into the potential for off-target interactions and drug–drug interactions, it does not account for other factors that could affect the efficacy and safety of Sartans and Paxlovid in treating COVID-19, such as patient characteristics and dosage. Future studies should aim to integrate in vitro and in vivo data with network-based bioinformatics approaches to provide a more comprehensive analysis of the potential effects of these drugs on treating COVID-19.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3238316/

Regulating Caspase-1 During Infection: Roles of NLRs, AIM2, and ASC

Pathogens are detected by a variety of innate immune sensors in host cells leading to rapid induction of cell autonomous responses. Proinflammatory cytokine secretion and a specialized form of inflammatory cell death called pyroptosis are induced during infection through activation of caspase-1. Pathogen-induced caspase-1 activation is regulated in large part by a vast array of cystosolic sensor proteins, including NLRs and AIM2, and an adaptor protein called ASC. Together, these proteins cooperate in forming caspase-1 activation platforms and, more importantly, direct caspase-1 toward cytokine secretion or cell death. Introduction Caspase-1 is a key mediator of inflammation in response to pathogen-derived molecules and endogenous danger signals. This cysteine protease cleaves a large repertoire of substrates leading to diverse downstream activities, including proinflammatory cytokine activation and secretion [ 1 ], and induction of cell death [ 2 ]. Activation of caspase-1 is regulated by upstream sensor proteins and adaptors, including the nucleotide-binding domain, leucine-rich repeat containing proteins (NLRs), absent in melanoma 2 (AIM2), and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). These proteins are responsible for coordinating caspase-1 activation complexes in response to endogenous and foreign molecules associated with cellular danger. Understanding the regulation of caspase-1activation is of particular importance, as improper activation of caspase-1 in response to endogenous molecules is associated with a number of inflammatory diseases. The hereditary disorders familial Mediterranean fever (FMF), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and chronic infantile cutaneous neurological articular syndrome (CINCA) are each associated with excessive activation of caspase-1-mediated proinflammatory cytokine production. Similarly, diseases such as Type 2 diabetes, gout, atherosclerosis, and Alzheimer's disease are associated with increased caspase-1 activation and inflammation. Recent reviews have discussed in detail the relationship between caspase-1 activation and inflammatory disorders [ 3 , 4 ]. Major advances toward understanding caspase-1 activation and regulation have come from examining the role of caspase-1 during the innate immune response to microbial pathogens. One major class of foreign agonists leading to caspase-1 activation is composed of pathogen-derived molecules. Pathogens including bacteria, viruses, and protozoans have proven to be useful tools in dissecting the regulation of caspase-1 and the upstream proteins involved in activation. This review will discuss the current understanding of caspase-1 activation and highlight studies with pathogens that have uncovered new details in the regulation of caspase-1-associated activities. NLRs, AIM2 and ASC: regulators of caspase-1 Caspase-1 activation is regulated in part by cytosolic sensor proteins comprised of the NLRs and AIM2. NLRs are characterized primarily by their shared domain architecture. The C-terminal region typically contains a leucine-rich repeat domain (LRR), and the region immediately upstream contains a nucleotide-binding domain (NBD). The N-terminal domains of NLRs further subdivide these proteins into distinct subfamily members. Members of the NLRP subfamily contain a N-terminal pyrin domain (PYR), while the NLRC proteins contain a caspase recruitment domain (CARD) at this position. The NAIP subfamily members contain baculovirus inhibitory domains (BIR) at their N-termini. Separate from the NLRs, AIM2 is a member of the PYHIN family of proteins and lacks both an NBD and LRR region. Instead, this protein contains a C-terminal HIN200 domain and an N-terminal PYR domain. The N-terminal domains of NLRs and AIM2 determine their ability to directly or indirectly interact with caspase-1. Proteins with CARD domains, such as NLRC4 and NLRP1, are able to directly interact with caspase-1 [ 5 , 6 ]. In contrast, AIM2 and the NLRP proteins, with the exception of NLRP1, require an adaptor protein in order to interact with caspase-1. This adaptor, known as ASC, is comprised of a PYD and a CARD domain. This bipartite architecture enables ASC to bridge PYD-containing proteins with the CARD of caspase-1. Although lacking PYD or CARD domains, NAIP proteins were initially thought to interact with caspase-1 through their BIR domains, which share homology with BIR domains of inhibitor of apoptosis proteins (IAPs) that bind to apoptotic caspases [ 7 ]. However, recent studies indicate that NAIP proteins signal to caspase-1 indirectly through binding interactions with NLRC4 [ 8 , 9 ]. Through these various routes of association, NLRs, AIM2, and ASC interact with caspase-1, resulting in formation of a caspase-1 activation complex called an inflammasome [ 10 ]. The role of inflammasome formation in caspase-1 activities will be discussed in more detail in a subsequent section. Genetic and biochemical evidence have provided useful insight into the mechanistic details behind NLR activation. The LRR domains of NLRs are thought to act in a manner similar to LRRs found in Toll-like receptors (TLRs) and likely are important for protein-protein or receptor-ligand interactions resulting from exposure to an appropriate agonist. Furthermore, LRRs are thought to be autoinhibitory to NLR function [ 5 , 8 ]. Until sensing of an appropriate signal by the LRRs, it is believed that the LRRs maintain the NLR in a conformational state that prevents downstream activation events. The central NBD found in NLRs has been shown to bind nucleotides and promote oligomerization of the NLR with itself and other components in signaling complexes [ 6 , 8 ]. Upon relief of autoinhibition by the LRRs, it is thought that oligomerization is initiated through the NBD while simultaneously exposing the N-terminal PYD, BIR, or CARD domain in order to recruit downstream molecules such as ASC, caspase-1, or another NLR. Ongoing studies continue to examine the interplay between NLRs, ASC, and caspase-1. One of the most heavily studied proteins involved in caspase-1 activation is NLRP3. The detailed mechanism for activation of this NLR is a major area of research, and progress has been made in recent years. NLRP3 activation requires two distinct signals prior to activation. The first signal, typically a TLR agonist, is known as a priming event and involves transcriptional upregulation of NLRP3 [ 11 ]. The second signal involves stimulation with a NLRP3 agonist. A diverse group of pathogen-derived and endogenous molecules signal through NLRP3 to induce caspase-1 activation, suggesting that NLRP3 is responding to an intermediate signal rather than directly sensing any one of these microbial or host-derived agonists. Studies thus far have led to multiple models, each involving the sensing of a general change in the cellular environment following stimulation with a given agonist. One proposed model for activation involves reactive oxygen species (ROS) production. ROS production is a common event following exposure to several NLRP3 agonists and is required for caspase-1 activation in multiple instances [ 12 - 15 ]. However, recent data suggest that the role of ROS may be further upstream in the signaling pathway than previously thought and may play a role solely for priming, or transcriptional upregulation of NLRP3 [ 16 ]. Furthermore, macrophages derived from mice deficient in NOX2, a major producer of ROS in these cells, are still competent for activation of caspase-1 in response to a subset of NLRP3 agonists, suggesting that ROS production is not the sole player in this pathway [ 17 ]. A second model argues that lysosomal leakage as a result of membrane perturbation results in caspase-1 activation. This model proposes that proteases, which are normally active only in lysosomal compartments, are released into the cytosol following perturbation of the phagosomal membrane during endocytosis. The strongest evidence for this model are data indicating that the lysosomal protease cathepsin B plays an important role in NLRP3-dependent caspase-1 activation in response to several agonists [ 15 , 17 - 19 ]. However, much like the ROS model, the requirement for cathepsin B does not appear to be absolute as macrophages deficient for this protease are still competent for induction of caspase-1 activation in response to a subset of agonists, suggesting that other proteases or activities are sufficient for induction [ 18 , 19 ]. Finally, the most common feature of NLRP3-induced inflammasome activation is the requirement for potassium efflux. Potassium efflux occurs in response to a number of NLRP3 agonists, and many NLRP3-mediated pathways for caspase-1 activation are efficiently inhibited by high levels of extracellular potassium [ 12 , 15 , 20 - 23 ]. Although the connection between potassium efflux and NLRP3-mediated activation of caspase-1 remains unclear, a role for the adaptor ASC has been proposed. Biochemical data suggest that under conditions of low potassium, ASC oligomerizes more efficiently and provides a better scaffold for the recruitment and activation of caspase-1 [ 24 ]. Therefore, in the presence of an NLRP3 agonist, a resulting loss of potassium may render the cytosol more favorable for oligomerization of ASC and promote more robust caspase-1 activation. However, it remains unclear if NLRP3 and sensing of potassium efflux are directly linked since oligomerization of purified recombinant ASC and subsequent caspase-1 cleavage was found to occur in the absence of NLRP3 when potassium levels were reduced in vitro [ 24 ]. In addition, potassium efflux has been shown to be important for NLRP3-independent but ASC-dependent pathways leading to caspase-1 activation, arguing that potassium efflux may be a more general requirement for ASC-dependent pathways [ 25 - 27 ]. Regardless, it cannot be ruled out that each of these hypotheses may contribute at least partially to the overall mechanism of activation for NLRP3. It is possible that these three events ― ROS production, lysosomal leakage, and potassium efflux ― each result in a distinct signal that is capable of signaling to NLRP3 either directly or through an intermediate molecule. In support of this latter possibility, it has been shown that ROS production promotes association of thioredoxin interacting protein (TXNIP) with NLRP3 and that TXNIP is required for activation of caspase-1 in response to NLRP3 agonists [ 28 ]. Future work identifying upstream components involved in sensing of NLRP3 agonists should further clarify the mechanistic details behind activation of this sensor molecule. Caspase-1 activation by pathogens Bacterial, viral, and eukaryotic pathogens have all been shown to induce caspase-1 activation. The mechanisms for activation by these various pathogens share a number of features, suggesting that caspase-1 is activated in response to common activities or products of pathogenic organisms. One key determinant for the activation of caspase-1 is the escape of microbial products into the host cell cytosol and subsequent detection by NLRs and AIM2. Microbial products gain access to host cell cytosols through a number of mechanisms, including pore-forming activities of toxins, specialized protein secretion systems that inject microbial products into host cells, or phagosomal leakage resulting from endocytosis of microbial products. NLRC4 A major implication for bacterial-induced caspase-1 activation came with the elucidation of NLRC4 as a key mediator in the response to bacterial flagellin. NLRC4 has been shown to be involved in the response to flagellin during infection of macrophages by Legionella pneumophila, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes [ 29 - 35 ]. In addition, there exist flagellin-independent pathways leading to caspase-1 activation that require NLRC4. The type III secretion system (T3SS) rod protein is sensed in a manner dependent on NLRC4. Pseudomonas aeruginosa, Shigella flexneri , and S. Typhimurium are examples of organisms capable of inducing caspase-1 activation following detection of the T3SS rod protein [ 36 ]. In either case, flagellin and T3SS rod protein are thought to gain access to host cell cytosols through accidental release by the various secretion mechanisms or membrane disrupting activities required for survival of these organisms during infection. For instance, L. pneumophila deficient in type IV secretion, S. Typhimurium or P. aeruginosa deficient in type III secretion, and L. monocytogenes deficient in listeriolysin O production all fail to induce caspase-1 activation [ 33 , 35 , 37 , 38 ]. Thus, NLRC4 enables host cells to discriminate between organisms by specifically detecting activities associated with virulence. Activation of NLRC4-dependent pathways for caspase-1 activation requires signaling from an upstream NAIP protein. Multiple NAIP proteins in mice and the lone human NAIP protein are able to directly bind to distinct agonists, resulting in formation of high molecular weight oligomers containing NLRC4 [ 8 , 9 ]. These NLRC4-containing oligomers are competent for caspase-1 recruitment and activation [ 8 , 9 ]. These data indicate that NLRC4 serves as an adaptor protein for the various NAIP proteins, rather than acting as a direct receptor for microbial agonists. Instead, it is evident that NAIPs serve as the primary receptors and enable caspase-1 activation through NLRC4 in response to multiple microbial-derived agonists. NLRC4-dependent caspase-1 activation mediated by bacterial flagellin has been shown to require NAIP5 in murine macrophages, while signaling in response to the T3SS rod protein from various bacterial species has been shown to require NAIP2 [ 8 , 9 ]. Interestingly, in contrast to flagellin- and T3SS rod protein-mediated activation observed in murine cells, activation of NLRC4 in human macrophage-like cells by NAIP occurs in response to T3SS needle protein [ 9 ]. Together, these data indicate that NAIP proteins impart a level of specificity for the NLRC4 inflammasome. Future studies should elucidate if other ligands exist for the other NAIP proteins encoded in the mouse genome. NLRP3 In addition to NLRC4, a major factor in the caspase-1-mediated response to bacterial-derived molecules is NLRP3. The group of agonists leading to NLRP3-dependent activation of caspase-1 is extensive and continues to grow. One class of activators contains a number of bacterial-derived pore-forming toxins. Examples of pore-forming toxins that induce NLRP3-dependent caspase-1 activation are streptolysin O ( Streptococcus pyogenes ) [ 39 ], listeriolysin O ( L. monocytogenes ) [ 40 ], toxin A ( Clostridium difficile ) [ 41 ], hemolysins ( Staphylococcus aureus ) [ 42 ], and aerolysin ( Aeromonas hydrophila ) [ 43 ]. Activation by pore-forming toxins is thought to occur as a result of potassium efflux. It has been demonstrated that caspase-1 activation induced by pore-forming toxins can be efficiently blocked by increasing extracellular potassium concentrations [ 40 , 42 , 43 ]. In addition to pore-forming toxins, other bacterial products appear to contribute to NLRP3-dependent caspase-1 activation. S. Typhimurium and Yersinia spp. induce caspase-1 activation through an NLRP3-mediated pathway [ 21 , 44 , 45 ]. The mechanism for NLRP3 activation by S. Typhimurium is unclear. Activation by Y. pestis is thought to occur through effector protein YopJ-mediated inhibition of NF-κB signaling, which has been shown previously to negatively regulate inflammasome activation [ 21 ]. NLRP3 also has been shown to be important for sensing of eukaryotic pathogens. The fungal pathogens Candida albicans and Aspergillus fumigatus are two examples of organisms capable of inducing NLRP3-dependent activation of caspase-1 [ 22 , 46 ]. The mechanism behind activation of NLRP3 in response to these fungal pathogens is relatively unclear. Studies with C. albicans indicate that uptake of the yeast form of this pathogen and subsequent differentiation into the hyphal form is required for efficient activation of caspase-1 [ 46 ]. One hypothesis is that transition into the hyphal form leads to phagosomal disruption or reactive oxygen species production, which in turn stimulates NLRP3 [ 46 ]. In addition to fungal pathogens, the protozoan genus Plasmodium induces NLRP3-dependent activation of caspase-1 [ 15 , 47 , 48 ]. The mode of activation involves a byproduct of hemoglobin metabolism produced by the protozoan known as hemozoin. During the detoxification of heme, which is a normal part of hemoglobin metabolism by Plasmodium parasites, hemozoin is produced as an insoluble waste product [ 49 ]. The mechanism for activation by hemozoin is relatively unclear, although data suggests that activation may take place indirectly through the release of uric acid, a well-studied NLRP3 agonist [ 48 ]. Viruses also are sensed by NLRP3-dependent mechanisms. Typically, the sensing of viruses involves detection of nucleic acid in the host cell cytosol. It has been demonstrated that RNA triggers an NLRP3-dependent inflammasome [ 50 ]. RNA viruses, including influenza and sendai virus, are capable of inducing activation of caspase-1 through NLRP3 [ 50 , 51 ]. Interestingly, in addition to sensing of viral RNA, NLRP3-dependent activation of caspase-1 occurs in response to the influenza virus M2 protein, which acts as a proton-selective ion channel during viral pathogenesis [ 52 ]. It is unclear how M2 leads to NLRP3 activation, although the ability of M2 to influence cellular ionic gradients is likely a factor [ 52 ]. NLRP1 Anthrax lethal toxin is another example of a bacterial toxin capable of inducing caspase-1 activation. This toxin is produced by the bacterium Bacillus anthracis and is capable of entering host macrophages through receptor mediated endocytosis, followed by translocation of the catalytic subunit of this protein complex, known as lethal factor (LF), into the host cell cytosol [ 53 ]. Once in the cytosol, LF is sensed by caspase-1 inflammasomes in a manner dependent on NLRP1 (NLRP1b in mice) [ 54 , 55 ]. LF has been shown previously to possess protease activity against mitogen-activated protein kinase kinases (MEKs) [ 56 ]. Protease activity was found to be critical for activation of caspase-1 by lethal factor in murine macrophages [ 26 ]. However, MEK cleavage alone was not sufficient to induce activation, suggesting that MEK cleavage per se may not be the signal for caspase-1 activation through NLRP1b [ 26 ]. Furthermore, activation of caspase-1 by anthrax lethal toxin was found to require proteasome activity and, similar to NLRP3, potassium efflux [ 26 , 57 ]. The role of the proteasome during caspase-1 activation is unclear. One hypothesis is that NLRP1 is kept in an inactive state by another protein that becomes degraded through a proteasome-dependent mechanism following the sensing of anthrax lethal toxin. In addition to anthrax lethal toxin, a bacterial cell wall component, muramyl dipeptide (MDP), has been shown to activate caspase-1 through human NLRP1 [ 6 , 58 ]. In vitro reconstitution experiments suggest that MDP may be a direct ligand for NLRP1, as no other protein components are required to induce NLRP1 activation in this setting [ 6 ]. However, it remains unclear whether a direct interaction between this bacterial agonist and host sensor protein takes place in vivo . Furthermore, MDP-mediated activation of caspase-1 in murine cells occurs through a NOD2-dependent mechanism, and it remains unclear whether a NLRP1 homologue is required for activation in this system. AIM2 DNA is also sensed in host cell cytosols and can induce caspase-1 activation. AIM2 has been shown to directly bind to DNA and induce inflammasome formation during infection by the intracellular bacterial pathogens, Francisella tularensis and L. monocytogenes [ 59 - 65 ]. In addition to bacterial infections, infections by DNA viruses such as vaccinia virus lead to potent AIM2-dependent activation of caspase-1 [ 60 ]. Similar to the NLRPs, activation of caspase-1 by AIM2 requires the adaptor protein ASC [ 59 - 62 ]. NLRP6 In addition to responding to virulent microorganisms, caspase-1 inflammasomes have been shown recently to be critical for maintaining balance of normal microbiota in the murine gut. Mice deficient for NLRP6 were found to be colonized more heavily relative to wild-type mice by several bacterial species, including members of the bacterial genus Prevotella [ 66 ]. Consequently, NLRP6-deficient mice were more susceptible to dextran sodium sulfate (DSS)-induced colitis [ 66 ]. Resistance to colitis was restored with antibiotic treatment, suggesting a possible link between increased inflammation and the expansion of Prevotella or other bacterial species in the gut [ 66 ]. Mice deficient for caspase-1 or the caspase-1-dependent cytokine IL-18 displayed phenotypes similar to NLRP6-deficient mice, suggesting that NLRP6/caspase-1-mediated production of IL-18 helps to maintain balance of normal gut microbiota [ 66 ]. It is unclear whether NLRP6 is directly sensing and responding to Prevotella or whether the absence of NLRP6-dependent signaling enables Prevotella persistence and expansion. These data suggest that inflammasomes may not only serve as a primary responder to pathogenic organisms, but also as regulators of homeostasis among microbial communities in the host gut. NLRC4 A major implication for bacterial-induced caspase-1 activation came with the elucidation of NLRC4 as a key mediator in the response to bacterial flagellin. NLRC4 has been shown to be involved in the response to flagellin during infection of macrophages by Legionella pneumophila, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes [ 29 - 35 ]. In addition, there exist flagellin-independent pathways leading to caspase-1 activation that require NLRC4. The type III secretion system (T3SS) rod protein is sensed in a manner dependent on NLRC4. Pseudomonas aeruginosa, Shigella flexneri , and S. Typhimurium are examples of organisms capable of inducing caspase-1 activation following detection of the T3SS rod protein [ 36 ]. In either case, flagellin and T3SS rod protein are thought to gain access to host cell cytosols through accidental release by the various secretion mechanisms or membrane disrupting activities required for survival of these organisms during infection. For instance, L. pneumophila deficient in type IV secretion, S. Typhimurium or P. aeruginosa deficient in type III secretion, and L. monocytogenes deficient in listeriolysin O production all fail to induce caspase-1 activation [ 33 , 35 , 37 , 38 ]. Thus, NLRC4 enables host cells to discriminate between organisms by specifically detecting activities associated with virulence. Activation of NLRC4-dependent pathways for caspase-1 activation requires signaling from an upstream NAIP protein. Multiple NAIP proteins in mice and the lone human NAIP protein are able to directly bind to distinct agonists, resulting in formation of high molecular weight oligomers containing NLRC4 [ 8 , 9 ]. These NLRC4-containing oligomers are competent for caspase-1 recruitment and activation [ 8 , 9 ]. These data indicate that NLRC4 serves as an adaptor protein for the various NAIP proteins, rather than acting as a direct receptor for microbial agonists. Instead, it is evident that NAIPs serve as the primary receptors and enable caspase-1 activation through NLRC4 in response to multiple microbial-derived agonists. NLRC4-dependent caspase-1 activation mediated by bacterial flagellin has been shown to require NAIP5 in murine macrophages, while signaling in response to the T3SS rod protein from various bacterial species has been shown to require NAIP2 [ 8 , 9 ]. Interestingly, in contrast to flagellin- and T3SS rod protein-mediated activation observed in murine cells, activation of NLRC4 in human macrophage-like cells by NAIP occurs in response to T3SS needle protein [ 9 ]. Together, these data indicate that NAIP proteins impart a level of specificity for the NLRC4 inflammasome. Future studies should elucidate if other ligands exist for the other NAIP proteins encoded in the mouse genome. NLRP3 In addition to NLRC4, a major factor in the caspase-1-mediated response to bacterial-derived molecules is NLRP3. The group of agonists leading to NLRP3-dependent activation of caspase-1 is extensive and continues to grow. One class of activators contains a number of bacterial-derived pore-forming toxins. Examples of pore-forming toxins that induce NLRP3-dependent caspase-1 activation are streptolysin O ( Streptococcus pyogenes ) [ 39 ], listeriolysin O ( L. monocytogenes ) [ 40 ], toxin A ( Clostridium difficile ) [ 41 ], hemolysins ( Staphylococcus aureus ) [ 42 ], and aerolysin ( Aeromonas hydrophila ) [ 43 ]. Activation by pore-forming toxins is thought to occur as a result of potassium efflux. It has been demonstrated that caspase-1 activation induced by pore-forming toxins can be efficiently blocked by increasing extracellular potassium concentrations [ 40 , 42 , 43 ]. In addition to pore-forming toxins, other bacterial products appear to contribute to NLRP3-dependent caspase-1 activation. S. Typhimurium and Yersinia spp. induce caspase-1 activation through an NLRP3-mediated pathway [ 21 , 44 , 45 ]. The mechanism for NLRP3 activation by S. Typhimurium is unclear. Activation by Y. pestis is thought to occur through effector protein YopJ-mediated inhibition of NF-κB signaling, which has been shown previously to negatively regulate inflammasome activation [ 21 ]. NLRP3 also has been shown to be important for sensing of eukaryotic pathogens. The fungal pathogens Candida albicans and Aspergillus fumigatus are two examples of organisms capable of inducing NLRP3-dependent activation of caspase-1 [ 22 , 46 ]. The mechanism behind activation of NLRP3 in response to these fungal pathogens is relatively unclear. Studies with C. albicans indicate that uptake of the yeast form of this pathogen and subsequent differentiation into the hyphal form is required for efficient activation of caspase-1 [ 46 ]. One hypothesis is that transition into the hyphal form leads to phagosomal disruption or reactive oxygen species production, which in turn stimulates NLRP3 [ 46 ]. In addition to fungal pathogens, the protozoan genus Plasmodium induces NLRP3-dependent activation of caspase-1 [ 15 , 47 , 48 ]. The mode of activation involves a byproduct of hemoglobin metabolism produced by the protozoan known as hemozoin. During the detoxification of heme, which is a normal part of hemoglobin metabolism by Plasmodium parasites, hemozoin is produced as an insoluble waste product [ 49 ]. The mechanism for activation by hemozoin is relatively unclear, although data suggests that activation may take place indirectly through the release of uric acid, a well-studied NLRP3 agonist [ 48 ]. Viruses also are sensed by NLRP3-dependent mechanisms. Typically, the sensing of viruses involves detection of nucleic acid in the host cell cytosol. It has been demonstrated that RNA triggers an NLRP3-dependent inflammasome [ 50 ]. RNA viruses, including influenza and sendai virus, are capable of inducing activation of caspase-1 through NLRP3 [ 50 , 51 ]. Interestingly, in addition to sensing of viral RNA, NLRP3-dependent activation of caspase-1 occurs in response to the influenza virus M2 protein, which acts as a proton-selective ion channel during viral pathogenesis [ 52 ]. It is unclear how M2 leads to NLRP3 activation, although the ability of M2 to influence cellular ionic gradients is likely a factor [ 52 ]. NLRP1 Anthrax lethal toxin is another example of a bacterial toxin capable of inducing caspase-1 activation. This toxin is produced by the bacterium Bacillus anthracis and is capable of entering host macrophages through receptor mediated endocytosis, followed by translocation of the catalytic subunit of this protein complex, known as lethal factor (LF), into the host cell cytosol [ 53 ]. Once in the cytosol, LF is sensed by caspase-1 inflammasomes in a manner dependent on NLRP1 (NLRP1b in mice) [ 54 , 55 ]. LF has been shown previously to possess protease activity against mitogen-activated protein kinase kinases (MEKs) [ 56 ]. Protease activity was found to be critical for activation of caspase-1 by lethal factor in murine macrophages [ 26 ]. However, MEK cleavage alone was not sufficient to induce activation, suggesting that MEK cleavage per se may not be the signal for caspase-1 activation through NLRP1b [ 26 ]. Furthermore, activation of caspase-1 by anthrax lethal toxin was found to require proteasome activity and, similar to NLRP3, potassium efflux [ 26 , 57 ]. The role of the proteasome during caspase-1 activation is unclear. One hypothesis is that NLRP1 is kept in an inactive state by another protein that becomes degraded through a proteasome-dependent mechanism following the sensing of anthrax lethal toxin. In addition to anthrax lethal toxin, a bacterial cell wall component, muramyl dipeptide (MDP), has been shown to activate caspase-1 through human NLRP1 [ 6 , 58 ]. In vitro reconstitution experiments suggest that MDP may be a direct ligand for NLRP1, as no other protein components are required to induce NLRP1 activation in this setting [ 6 ]. However, it remains unclear whether a direct interaction between this bacterial agonist and host sensor protein takes place in vivo . Furthermore, MDP-mediated activation of caspase-1 in murine cells occurs through a NOD2-dependent mechanism, and it remains unclear whether a NLRP1 homologue is required for activation in this system. AIM2 DNA is also sensed in host cell cytosols and can induce caspase-1 activation. AIM2 has been shown to directly bind to DNA and induce inflammasome formation during infection by the intracellular bacterial pathogens, Francisella tularensis and L. monocytogenes [ 59 - 65 ]. In addition to bacterial infections, infections by DNA viruses such as vaccinia virus lead to potent AIM2-dependent activation of caspase-1 [ 60 ]. Similar to the NLRPs, activation of caspase-1 by AIM2 requires the adaptor protein ASC [ 59 - 62 ]. NLRP6 In addition to responding to virulent microorganisms, caspase-1 inflammasomes have been shown recently to be critical for maintaining balance of normal microbiota in the murine gut. Mice deficient for NLRP6 were found to be colonized more heavily relative to wild-type mice by several bacterial species, including members of the bacterial genus Prevotella [ 66 ]. Consequently, NLRP6-deficient mice were more susceptible to dextran sodium sulfate (DSS)-induced colitis [ 66 ]. Resistance to colitis was restored with antibiotic treatment, suggesting a possible link between increased inflammation and the expansion of Prevotella or other bacterial species in the gut [ 66 ]. Mice deficient for caspase-1 or the caspase-1-dependent cytokine IL-18 displayed phenotypes similar to NLRP6-deficient mice, suggesting that NLRP6/caspase-1-mediated production of IL-18 helps to maintain balance of normal gut microbiota [ 66 ]. It is unclear whether NLRP6 is directly sensing and responding to Prevotella or whether the absence of NLRP6-dependent signaling enables Prevotella persistence and expansion. These data suggest that inflammasomes may not only serve as a primary responder to pathogenic organisms, but also as regulators of homeostasis among microbial communities in the host gut. Regulation of inflammasome formation, cytokine processing and cell death Microbe-induced signaling through NLRs or AIM2 ultimately leads to recruitment and activation of caspase-1 via inflammasome formation, followed by the action of this protease on its downstream targets. Two heavily studied activities of caspase-1 include cleavage of the pro-inflammatory cytokines IL-1β and IL-18 and the induction of a cell death pathway termed pyroptosis. IL-1β and IL-18 are synthesized as inactive precursor proteins (pro-IL-1β and pro-IL-18). Caspase-1-mediated cleavage of these proteins results in production of the bioactive form of the cytokines, which are then secreted through an unconventional protein secretion pathway [ 1 , 67 ]. The targets of caspase-1 that lead to cell death are less clear. Following caspase-1 activation, pores are formed in host cell membranes that disrupt ion fluxes, resulting in osmotic lysis and death of the cell [ 68 ]. In murine macrophages, large inflammasome complexes have been observed in response to agonists of NLRC4, NLRP3, NLRP1b, and AIM2 [ 26 , 34 , 45 , 69 ], suggesting that these structures are a common feature of caspase-1 activation. These complexes are organized by the adaptor protein ASC, which as discussed previously undergoes oligomerization during caspase-1 activation. NLRs and AIM2 may participate in complex formation by directly associating with ASC and caspase-1. Recent evidence using pathogen models have indicated that caspase-1-mediated cytokine processing and cell death induction are influenced by the adaptor ASC. The intracellular pathogens S. Typhimurium and L. pneumophila induce NLRC4- and ASC-dependent pathways for caspase-1 activation in murine macrophages [ 25 , 45 ]. During infection with these bacteria, complex formation mediated by NLRC4 and ASC is critical for efficient processing of caspase-1 into its active subunits [ 69 , 70 ]. However, in the absence of ASC-dependent complex formation, caspase-1-mediated cell death via NLRC4 occurs normally [ 25 , 38 , 70 , 71 ]. These data suggest that while NLRs and AIM2 cooperate with ASC in inflammasome formation to induce cleavage of caspase-1, NLRC4 also can function independently of ASC and direct distinct inflammasome formation. Recent studies have provided evidence in support of this hypothesis. Reconstitution studies using 293T cells suggest that NLRC4 is able to form large molecular weight complexes with NAIP5 or NAIP2 in response to flagellin or T3SS rod protein, respectively, and that formation of these complexes are sufficient for induction of cell death [ 8 ]. This complex formation presumably is independent of ASC, as 293T cells do not express detectable levels of this protein [ 72 ]. This suggests that NLRC4-mediated pathways of caspase-1 activation involve complex formation distinct from ASC-dependent pathways. Furthermore, formation of these distinct complexes significantly influences downstream caspase-1-associated activities. Although the ability of caspase-1 to induce cell death in the absence of ASC requires the catalytic activity of the protease, cleavage of caspase-1 is not required [ 70 ]. In fact, a variant of caspase-1 that is unable to undergo proteolytic processing is still able to induce pyroptosis. In contrast, this variant protein is unable to efficiently cleave pro-IL-1β into its active form, even in the presence of ASC [ 70 ]. These data suggest that inflammasome formation through ASC-dependent mechanisms leads to caspase-1 autoproteolysis and that this form of the active protease is critical for cytokine cleavage. In the absence of ASC, caspase-1 activation occurs without autoproteolysis, and this form of the protease targets a distinct subset of substrates critical for the induction of pyroptosis [ 70 ]. Together, these recent findings indicate that formation of distinct inflammasomes control the ability of caspase-1 to target specific substrates. It remains unclear how proteins lacking a CARD domain, such as NLRP3 and AIM2, induce cell death through ASC. One possibility is that the main ASC complex formed in response to all caspase-1 agonists also promotes cell death. It is possible that within the ASC complex, a pool of caspase-1 is processed and acts on cytokines to promote their cleavage and secretion, and an alternate pool is activated but not processed which then targets substrates leading to pore formation and cell death. Alternatively, it cannot be ruled out that processed and non-processed caspase-1 target the same substrates leading to cell death and that processed caspase-1 may simply be more efficient at cleavage of substrates than non-processed caspase-1. In this scenario, inefficient caspase-1 activity may have a more drastic consequence on the levels of cytokines, which are direct targets of caspase-1. In contrast, it is possible that caspase-1-mediated cell death may involve a signal amplification cascade that requires very little caspase-1 activity in order to initiate the pathway. Future studies examining the substrates of processed and non-processed caspase-1 will provide more insight into the ability of caspase-1 to differentially regulate cytokine processing and cell death. Conclusions Studies examining macrophage responses to microbial pathogens and their associated molecules have yielded significant mechanistic insight into caspase-1 activation and regulation. A diverse class of cytosolic sensor proteins comprised of the NLRs and AIM2 enables host cells to detect a range of microbial products that gain access to host cell cytosols. Sensing of microbial products by NLRs and AIM2 may occur through direct binding of an agonist to a sensor protein, production of intermediate signals that bind to a sensor protein, or detection of an overall change in the cellular environment. Ultimately, different signals are able to converge on caspase-1 through the adaptor ASC, which organizes specialized platforms leading to caspase-1 processing and cytokine cleavage. Independent of ASC-mediated complex formation, caspase-1 can be directly recruited and activated by NLRC4 and possibly other sensors without processing, leading to induction of cell death. Studies thus far have uncovered mechanisms for regulating caspase-1 specificity with respect to cytokine secretion and induction of cell death. Future work in this area will likely identify roles for previously uncharacterized NLRs and further elucidate mechanistic details behind NLR activation and regulation of caspase-1-mediated activities.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3579574/

A virus-like particle vaccine platform elicits heightened and hastened local lung mucosal antibody production after a single dose

Highlights ► We conjugated OVA to a sHsp nanoparticle, and show its utility for vaccination. ► The immune response to OVA–sHsp was quick and intense with a single i.n. dose. ► Delivery of OVA–sHsp resulted in the same immune response to OVA as sHsp itself. ► sHsp acted as an adjuvant in the lungs, and induced the production of mucosal IgA. ► Pre-treatment with sHsp provided protective ab and cellular responses to influenza. 1 Introduction Respiratory infections are one of the most prominent afflictions in individuals of all ages and immune statuses, signifying a significant global health concern. Unfortunately, we are currently unable to provide vaccines against many clinically relevant lower respiratory tract pathogens, nor are we able to fully predict the identity of future outbreaks. This global vulnerability has been clearly illustrated by several epidemics in recent memory, including the newly emerged coronavirus, SARS-CoV outbreak of 2002, various influenza strain reassortments (H5N1 "bird flu" and H1N1pandemic), and bioterrorism events involving pathogen aerosolization. Thus, there is an urgent and crucial need for the development of broad-spectrum, rapidly acting vaccination strategies. Unlike most other mucosal sites in the body, which are protected and immunologically shaped by their commensal microbial communities, the lung is more or less sterile. Therefore, the lung relies on an intricate network of sentinel dendritic cells, antimicrobial secretions, and resident macrophages for defense. Importantly, immune responses in the lung must be tightly regulated to promote immunity, while avoiding tissue damage associated with either the pathogen or the host response. As such, pulmonary immune responses are quite unique, and as suggested by others, the individual history of specific pathogen exposures to the lungs may contribute to shaping the appropriate ensuing immune responses to subsequent challenges [1] , [2] , [3] , [4] . Here (and elsewhere [5] ), we further demonstrate that virus-like particles (VLPs), which are unrelated to the antigen of subsequent challenge, can similarly impact the lung microenvironment, without the associated pathology, thereby shaping future immune responses. Many groups have previously suggested that the lung may provide an important route of delivery for mucosal vaccination [6] , [7] , [8] . However, the potential for utilizing localized mucosal vaccination strategies in the lower respiratory tract have historically been overlooked, and approaches which elicit tissue-specific immune responses are just beginning to be developed. An FDA-approved tribute to the realization of this strategy is the highly effective Flumist vaccine, which is delivered intranasally, and provides better comprehensive local immunity than injectable versions [9] , [10] , [11] . Additionally, the application of nanomaterials to biomedicine is one of the most exciting and potentially revolutionary applications of nanotechnology. Here, we describe a mechanism by which we can enhance primary local immune responses to antigens without the necessity of specific antigen priming. We achieved this result by intranasally delivering empty virus-like particles (VLPs), which act to modulate the lung microenvironment, and harness and focus immune responses. Recent exploration in the utilization of nanoparticles [12] , [13] , [14] , virus-like particles [15] , [16] , [17] , [18] , [19] , [20] , [21] , and viruses which have no mammalian cell tropism, has shown that these platforms are naturally immunostimulatory, and likely utilize evolutionarily conserved cell surface receptors, thereby safely engaging immune signaling pathways without replicating [22] , [23] , [24] , [25] , [26] . Furthermore, many of these strategies are currently on the market or undergoing clinical trials, and have already had broad global impact in safely preventing disease [21] , [27] , [28] , [29] . Importantly, virus-like particles can be produced in large quantities, provide a stable product, often are amenable to lyophilization or freeze-drying, and are fiscally economical. These features are especially important for less industrialized nations. In the following studies we utilize two empty, non-pathogenic virus-like particles – a small heat shock protein cage nanoparticle (sHsp) and the P22 phage-derived virus-like particle (P22) as immunomodulatory antigens in both a non-specific pre-priming scenario, and as a platform for the delivery of specific antigen to the lung. The small heat shock protein 16.5 (sHsp) from Methanocaldococcus jannaschii (a hyperthermophilic archaeon) is comprised of 24 repeating subunits [30] , [31] . These subunits self-assemble to produce an empty cage-like structure, comparable to that of a virus capsid by virtue of the high symmetry and quaternary structure [5] , [32] , [33] , [34] . We have previously shown that sHsp can be genetically engineered to incorporate cysteine residues, thereby providing attachment sites for bioconjugation [33] , [35] , [36] , which we exploit here for the display of a foreign protein, similarly to described strategies [37] , [38] . P22 is a bacteriophage capsid which infects Salmonella typhimurium (when intact tail fibers are present) [39] , [40] . The P22 used here is devoid of both genetic material and tail fibers, and is therefore composed of only the non-infectious empty viral capsid. We would like to note here that previously we have referred to the sHsp nanoparticles as "protein cage nanoparticles" or PCN [5] . However, we now find it to be more descriptive to classify both the sHsp and P22 as virus-like particles, given their structural architecture and immunological parallels to other particles described in the literature. Importantly, neither sHsp nor P22 target known mammalian pattern-recognition receptors, nor do they infect mammalian cells. And while the immunomodulatory potential in exploiting non-pathogenic viruses is still in its infancy, others have begun to describe similar successful strategies [24] , [25] , [26] . P22 and sHsp are herein used as immunomodulatory agents alone in a lung priming strategy to achieve heightened heterologous immunity to distinct antigens, such as OVA and influenza virus; or, in the case of sHsp, as a vaccine delivery platform for a model antigen, OVA, which we have conjugated (in its entirety) to the exterior surface of the sHsp cage. This immunization strategy is advantageous because it accelerates and intensifies the primary immune response, after only a single dose. We further show that conjugating a model antigen (OVA) to sHsp elicits an immune response to OVA which mirrors the response to the sHsp itself. sHsp conjugation also acts to adjuvant OVA, and the local delivery of sHsp and antigen complexes induce potent local IgA secretion in sHsp pretreated mice. Therefore, we show that VLPs (sHsp and P22) can be used as both immunomodulatory agents by pretreating the lung and/or as a carrier of antigens, allowing local immunization of the lower respiratory tract against a pathogen of interest, with a single dose. This platform could be useful for both pre- and post-exposure prophylaxis. 2 Materials and methods 2.1 Production of small heat-shock protein cage nanoparticle (sHsp) The small heat-shock protein (sHsp 16.5) was initially purified as described previously [33] . In a slight modification of the previously described protocol, the supernatants from 2 L of cell culture were combined and concentrated to 10 mL with a 100k MWCO amicon filter (Millipore, Billerica, MA) prior to size exclusion chromatography on a Superose 6 column (GE Healthcare, Piscataway, NJ) so that purification of large amounts of sHsp could be accomplished efficiently. In order to remove residual protein contaminants, additional steps were added to the previous protocol. The fractions containing sHsp from size exclusion chromatography were re-concentrated to 10 mL and purified by anion exchange on a Sepharose Q column (GE Healthcare) using a linear gradient from 300 mM to 800 mM NaCl in 25 mM HEPES Buffer at pH 7.3. Following this, the fractions containing sHsp were dialyzed overnight into 50 mM phosphate, 100 mM NaCl, and 5 mM EDTA, at pH 7.3, concentrated a third time to 10 mL, and re-subjected to size exclusion chromatography using a Superose 6 column (GE Healthcare) with the same buffer. For the S121C sHsp variant protein, tris(2-carboxyethyl)phosphine (Pierce, Rockford, IL) was added to a final concentration of 2 mM during each amicon concentration step prior to the FPLC runs to inhibit disulfide formation between external thiols on sHsp. The sHsp protein concentration was determined via UV–visible spectroscopy using an extinction coefficient of A280 = 0.565 (mg/mL) −1 as previously documented [33] . 2.2 OVA Preparation Larger aggregates from commercially available ovalbumin (OVA) (Sigma–Aldrich, Saint Louis, MO, A5503) were removed by size exclusion chromatography using a Superose 6 column (GE Healthcare). Fifty millimolar phosphate, 100 mM NaCl, and 5 mM EDTA at pH 7.3 was used as an elution buffer. Protein concentrations were determined by UV–visible spectroscopy using a previously documented extinction coefficient of A280 = 0.789 (mg/mL) −1 for OVA [41] . 2.3 P22 preparation The P22 K118C coat and scaffold protein were expressed in Escherichia coli , purified, and assembled in vitro as described previously [39] . The purified P22 particles were heated for 20 min at 65 °C to remove the scaffold protein, purified on a sephacryl 500 column (Amersham, Piscataway, NJ), and concentrated to 2 mg/mL, and dialyzed extensively into 50 mM phosphate, 100 mM NaCl, and at pH 7.2. The P22 concentration was calculated using an extinction coefficient A280 = 1.4 (mg/mL) −1 . 2.4 Optimization of OVA–sHsp conjugation To determine the initial conditions for conjugation, a matrix of linking conditions were tested. All reactions were carried out in 50 mM phosphate, 100 mM NaCL, 5 mM EDTA, pH 7.3. OVA (80 μL, 7.72 mg/mL) was labeled with a 2-fold molar excess of each SM(PEG) n linker (SM(PEG) 2 , SM(PEG) 6 , or SM(PEG) 12 ) using a stock solution of 25 mM linker in DMSO respectively. These samples were incubated at room temperature for 50 min, and un-conjugated linker was immediately removed using Micro Bio-Spin Columns P30 (Biorad). The labeled OVA samples were combined with either sHsp S121C or sHsp E102C cages using a 5:1 molar ratio. The proteins were combined, vortexed, and reacted 1 h at room temperature and overnight at 4 °C. The final sHsp concentration was 2.34 mg/mL and the OVA concentration was 11.72 mg/mL in a total volume of 45 μL for each reaction. These reactions were repeated using a 20-fold excess of each SM(PEG) n linker (SM(PEG) 2 , SM(PEG) 6 , or SM(PEG) 12 ) from a stock solution of 250 mM to label the OVA. Prior to conjugation, lysines on sHsp G41C were reacted with 50-fold molar excess of SPDP (Thermo Scientific) per sHsp subunit by the addition of 500 mM SPDP in DMSO to 2 mL of protein at 2.61 mg/mL. This reaction was stirred for 90 min at room temperature, and the labeled sHsp was purified by Superose 6, and stored at 4 °C overnight. For these reactions, a seven-fold molar excess of TCEP (Invitrogen) was added to the sHsp-SPDP (above) to reduce the SPDP, and this reaction was left for 30 min at room temperature to reduce the disulfides on the linker. UV–visible spectroscopy was used to monitor the completeness of the reaction as indicated by increasing absorbance at 324 nm corresponding to the thiopyridone product [42] . This sulfhydryl functionalized sHsp was reacted with OVA (80 μL, 7.72 mg/mL) samples labeled with a 2-fold or 20-fold molar excess of each SM(PEG) n linker (SM(PEG) 2 , SM(PEG) 6 , or SM(PEG) 12 ) using the protocol described above for the S121C and E102C reactions. The linking conditions for the sHsp G41C mutant combined with the OVA contained a final concentration of 30 mM TCEP. The final sHsp G41C and OVA concentrations were normalized to the conditions used with the other mutants: 2.34 mg/mL and 11.72 mg/mL respectively, in a total volume of 45 μL. 2.5 Synthesis and purification of the OVA–sHsp conjugate A 250 mM stock solution of the commercially labeled cross-linking reagent SM(PEG) 6 (Thermo Scientific, Waltham, MA) was made in DMSO. The OVA was concentrated to 9.75 mg/mL, and 2.0 mL was reacted with a 2-fold molar excess of the linker, added dropwise to a vigorously stirring solution. The reaction was stirred for 40 min at room temperature, followed by immediate purification of the maleimide functionalized OVA from un-conjugated small molecule linker by Superose 6 size exclusion chromatography (GE Healthcare). Immediately after elution, the maleimide functionalized OVA was concentrated to approximately 13.4 mg/mL (using E = 0.789 (mg/mL) −1 ) with a 10k MWCO Microcon filter (Millipore), and was mixed with sHsp S121C (2.62 mg/mL) for 1 h at room temperature followed by overnight at 4 °C. A molar ratio of 5:1 OVA to sHsp was used to give final concentrations of 1.34 mg/mL OVA and 2.4 mg/mL sHsp within the linking reactions. Control reactions containing identical concentrations of sHsp or linker-labeled OVA alone were run in parallel. Free sulfhydryls on the proteins were then capped by reaction with 20-fold excess N-ethyl maleimide (Pierce) per sHsp subunit for 2 h at room temperature. The above procedure was repeated six times to obtain sufficient yield of the conjugated construct. Similar samples from these reactions were combined, spun down (5 min × 17,000 × g ) to remove precipitate, and dialyzed into 25 mM triethanolamine (USB Corp, Santa Clara, CA) at pH 7.3. The samples were purified by anion exchange chromatography on a MonoQ column (Amersham Pharmacia) using a linear gradient of 0 M to 500 mM NaCl in 25 mM triethanolamine at pH 7.3 ( Fig. S1 ). Fractions corresponding to conjugated sample, sHsp and maleimide functionalized OVA were combined based on SDS-PAGE analysis of the fractions. The sHsp and maleimide functionalized OVA were mixed in a 5:1 molar ratio to obtain the mixed sample used for the in vivo experiments. All the samples were extensively dialyzed into 50 mM phosphate, 100 mM NaCl, and at pH 7.2 prior to in vivo experiments. The admixture of OVA and sHsp was comprised of equal microgram amounts of both sHsp (100 μg) and OVA (57 μg) (Sigma–Aldrich) as the conjugated form, and was suspended in sterile PBS. 2.6 Limulus amebocyte lysate assay Prior to administration in vivo the endotoxin contamination for each protein preparation was determined using a limulus amebocyte lysate (LAL) assay (Associates of Cape Cod, Inc.; East Falmouth, MA). We determined that sHsp alone contained 1.3 μg LPS per dose, the OVA–sHsp conjugate contained 2.4 ng LPS per dose, the admixture of sHsp and OVA contained 3.4 ng LPS per dose, the P22 preparation contained 3 ng LPS per dose, and OVA alone contained 619 ng LPS per dose. A second batch of P22 was used for only Fig. 3 B, with the following LAL results: The "LPS-high" P22 contained 8 μg LPS per dose, while the "LPS-low" P22 contained 14 ng per dose. 2.7 Confirmatory analysis The size distribution of the VLPs was determined using dynamic light scattering (Brookhaven 90Plus particle size analyzer). For the Bradford assay, a series of protein standards was made using BSA (Sigma, A7906). One hundred microliters of Bradford Reagent (Amresco) was combined with 5 μL of each protein solution, and samples were incubated for 20 min, and the absorbance was measured at 315 and 605 nm. Samples were also run on 1 mm SDS-PAGE reducing gels (15% acrylamide for the running gel, 4% acrylamide for the stacking gel). The AlphaEaseFC software (Alpha Innotech) was used to identify bands and calculate the migration distances of species on the gels. 2.8 Western Blots Proteins were transferred from the SDS-PAGE gels to Hybond C nitrocellulose membranes for 2 h at 200 mA. Membranes were blocked overnight with 5% milk and 0.01% Tween-20 in Tris-buffered saline, incubated with a 1:10,000 dilution of rabbit anti-OVA polyclonal antibody or rabbit anti-sHsp antibody (Millipore) for 3 h. The anti-sHsp antibody was purified from rabbit serum by ammonium sulfate precipitation. The membranes were incubated with a ratio of 1:5000 anti-rabbit antibody HRPO conjugate to blocking solution for 30 min, and blots were detected using an Opti-4CN kit (Biorad). Densitometry analysis was done using AlphaEaseFC software (Alpha Innotech). 2.9 Influenza virus The influenza virus A/PR8/8/34 was produced at the Trudeau Institute, Saranac Lake, NY. Briefly, 10-day-old embryonated chicken eggs were infected for 72 h, and resultant allantoic fluid was recovered and stored at −80 °C until used. 2.10 Mice, pretreatment, and challenges BALB/c, C57BL/6 or TLR4−/− mice were bred in-house at Montana State University, Bozeman, MT. At 6–8 weeks of age, male or female mice were enrolled in described experiments ( n = 5 per group). In experiments utilizing intranasal pretreatments, 100 μg of sHsp (sHspG41C), or P22 were delivered in 50 μL volumes while the mice were lightly anesthetized under inhaled 5% isofluorane. Five pretreatment doses were delivered either daily for five days, or spaced evenly over the course of 2 weeks (both schedules produced equivalent results). Importantly, we have extensively explored potential adverse side-effects on pulmonary function due to repeated sHsp administration, and have found none [5] , [34] . Pretreated mice were rested for 72 h, then challenged. In some experiments mice were challenged with the OVA–sHsp conjugate (sHspS121C), the OVA and sHsp (sHspS121C) admixture, OVA alone, or sHsp alone (sHspS121C), delivered i.n. in 100 μL volumes, again under light anesthesia. The OVA concentration (0.57 mg/mL), as determined by UV–visible spectroscopy, was held constant throughout groups, regardless of conjugation. For subcutaneous (s.c.) studies, no pretreatment was utilized. Instead, one s.c. dose of 100 μL OVA–sHsp, OVA with sHsp admixture, OVA with alum (10% AlkSO 4 ) admixture, alum alone (10% AlkSO 4 ), or OVA alone (0.57 mg/mL) were injected and antibody titers were measured over time. For influenza challenge studies, 1500 plaque forming units (pfu) A/PR8/8/34 influenza virus were delivered in 50 μL i.n. In experiments to determine the impacts of residual LPS, mice were similarly pretreated with "LPS-high" P22, "LPS-low" P22, an equal amount of LPS as in the "LPS-high" P22, or sterile pyrogen-free PBS in 50 μL i.n. All mice were then rested, and subsequently challenged with 100 μg sHspG41C in 50 μL i.n. Mice were bled at relevant timepoints, and serum was separated from whole blood by centrifugation in separation tubes (Sarstedt; Germany). At indicated timepoints per experiment, mice were euthanized by intraperitoneal injection of sodium pentobarbital (90 mg/kg) and exsanguinated after no pedal response could be elicited. Mice were then lavaged with sterile PBS with 3 mM EDTA. BALF and sera were used to determine antibody titers by ELISA. In some experiments, lungs and tracheobronchial lymph nodes (TBLNs) were additionally collected and either homogenized through a wire mesh screen, or digested with agitation in 0.2% collagenase (Worthington Biochemical Corporation; Lakewood, NJ) with DNase (Sigma) at 37 °C for 1 h. Red blood cells were lysed from the lung homogenates using ACK lysis buffer, washed, resuspended in FcR block (clone 93), and stained for flow cytometry. Total cells from each tissue (BAL, lungs and TBLNs) were counted by hemocytometer. In some cases, whole lungs were instilled with OCT (SakuraFinetek; Torrance, CA), excised, and snap frozen in liquid nitrogen for histology. All animal procedures were pre-approved by Montana State University's IACUC. Experimental results were confirmed by at least two independent repetitions of similar design. 2.11 Immunostaining and flow cytometry Frozen blocks were cut into 5 μM sections by cryostat (Leica Microsystems; Buffalo Grove, IL) and resultant lung sections were stained for expression of the polymeric Ig receptor (pIgR) with biotinylated goat anti-mouse pIgR (R&D Systems, Minneapolis, MN) followed by AF488-streptavidin (Invitrogen, Carlsbad, CA). Control sections were utilized to determine staining specificity. Images were acquired on a Nikon Eclipse E800 microscope (Nikon Instruments, Melville, NY) using Nikon NIS-Elements Imaging software. Antibodies used for FACS staining of lung and TBLN homogenates included CD4 (GK1.5), B220 (RA3-6B2), Fas (Jo2), CXCR5 (2G8), CD138 (281-2), and streptavidin from BD Pharmingen; San Diego, CA; GL7 (GL7), and CXCR4 (2B11) from eBioscience; San Diego, CA; and ICOS (C398.4A) from Biolegend, San Diego, CA. FACS data was collected on a FACSCanto (BD) and FACS analysis was completed using FlowJo Software (Treestar, Ashland, OR). Briefly, forward and side scatter plots were gated on lymphocytes, as determined by size and granularity. Lymphocyte populations were then further analyzed for the expression of appropriate combinations of surface antigens, based off negative staining controls. Total cell numbers were then calculated based off total hemocytometer cell counts for each tissue. 2.12 ELISA Serum and BALF antibodies levels were determined by ELISA. Briefly, high-binding polystyrene plates (Corning; Corning, NY) were coated and incubated with antigen (OVA, sHsp, or influenza virus membrane preparation) at 37 °C for 3 h, then moved to 4 °C overnight. Plates were washed with PBS with 0.05% tween, and blocked with nonfat dry milk. Serum samples were diluted at 1:100, and BALF samples were plated neat (from a 2 mL lavage) in duplicate. For influenza-specific ELISAs, samples were diluted in 2-fold dilutions to endpoint titer. All ELISAs were then incubated at 37 °C for 2 h. Plates were again washed and appropriate HRP-conjugated secondary antibodies (whole IgG, IgG1, IgG2a, IgG2b, IgG2c, IgG3, and IgA from SouthernBiotech, Birmingham, AL) were added and incubated for an additional 2 h at 37 °C. Finally, plates were again washed, developed using TMB substrate (Sigma–Aldrich), stopped with 1 M H 3 PO 4 , and read on a SpectraMax Plus plate reader (Molecular Devices; Sunnyvale, CA) at 450 nm. In some results, OVA-specific IgG was quantified using the mouse monoclonal antibody to OVA (Abcam, Cambridge, MA, ab17292). 2.13 Statistics Statistical significance was determined by one-way ANOVA with a Bonferroni post-test of multiple comparisons, or in some cases an unpaired t -test was used. Significance was indicated by * p 62 kDa) in Fig. 1 Biii represented a polydisperse species of conjugated OVA–sHsp, and were later combined in total for use as the OVA–sHsp conjugate in vivo (further described below). To determine if both OVA and sHsp were present in the upper bands of the sample in Fig. 1 Biii, we performed Western Blot analysis using anti-sHsp ( Fig. 1 C) and anti-OVA ( Fig. 1 D) antibodies. Reactivity to both sHsp and OVA antibodies within the upper bands of the conjugated sample alone ( Fig. 1 Ciii and Diii) indicated the conjugation of OVA to sHsp. A semi-quantitative Western Blot ( Fig. S4 ) suggested an average ratio of 2.6 OVA per sHsp cage within the conjugated sample. The total protein concentration of the conjugated (0.99 mg/mL), admixed (0.82 mg/mL), sHsp (0.61 mg/mL), and OVA (0.66 mg/mL) samples used for in vivo experiments were confirmed via Bradford assay. The molecular weights of the bands corresponding to the OVA–sHsp conjugates were determined based upon their migration distance on SDS-PAGE gels ( Figs. 1B and S5 ), and densitometry analysis (not shown). Protein bands corresponding to covalent conjugation between OVA and sHsp subunits on SDS-PAGE gel were identified by their presence within the conjugated sample ( Fig. 1 Biii) and absence within the sHsp ( Fig. 1 Bi), maleimide functionalized OVA ( Fig. 1 Bii), and the admixture ( Fig. 1 Biv) controls. We detected bands with calculated molecular weights of 58 and 62 kDa Fig. 1 Biii and Fig. S5 that corresponded to the conjugation of a single sHsp subunit (16.5 kDa) to one cleaved OVA (40.1 kDa) or full length OVA (45 kDa), respectively. The upper bands detected between 67 and 84 kDa likely resulted from various forms of conjugation of OVA to sHsp ( Fig. S5 ), and the smear of sample corresponding to molecular weights 85–150 kDa likely represented the conjugation of OVA to multiple sHsp subunits, as this entire smear reacted to both anti-OVA and anti-sHsp antibodies on a Western Blot. Thus, the resultant conjugated OVA–sHsp sample used for immunization was the culmination of a polydisperse species of OVA–sHsp, with varying degrees of multivalent array architecture. We utilized size exclusion chromatography and dynamic light scattering measurements to probe the native state and size distribution of the particles within the samples used for immunization. The OVA–sHsp conjugate contained particles that were larger in size than those in the admixture, sHsp, or OVA. Size exclusion chromatography indicated that a distribution of larger species was present within the OVA–sHsp sample ( Fig. S6 ) and by dynamic light scattering, the OVA–sHsp conjugate showed a larger average diameter than the maleimide functionalized OVA, sHsp S121C, or the admixture. The range of average diameters based on the intensity measurements on the samples repeated seven times were 6.0–9.9 nm for OVA, 15.1–17.3 nm for the admixture, 15.6–18.1 nm for sHsp S121C, and 29.9–41.0 nm for the OVA–sHsp conjugated sample ( Fig. S7 ). 3.3 The immune response to OVA–sHsp is quick and intense after only a single intranasal dose In our first in vivo studies we determined the potential of sHsp to serve as a novel vaccine delivery platform by its ability to facilitate the generation of antigen-specific immunity to OVA. Simultaneously, we determined how pretreatment of the lung with a heterologous VLP, P22, affects the subsequent response to antigen challenge. BALB/c mice were pretreated with either P22 or vehicle (PBS) intranasally (i.n.) in five doses and then allowed to rest for 72 h. Mice were then challenged with OVA conjugated to sHsp (OVA–sHsp), OVA and sHsp separately in solution (OVA–sHsp admixture), OVA alone, or sHsp alone. Serum was then collected at a range of timepoints post-antigen challenge for kinetic analysis. We found that mice which had received the P22 pretreatment, followed by a single dose of the OVA–sHsp conjugate, produced high amounts of OVA-specific serum IgG as early as 4 days post-challenge ( Fig. 2 A). This combination far outperformed any of the other treatment scenarios in both rapidity of antibody production and amount. Interestingly, the next highest antibody producing group, at early timepoints, was those mice that had also been challenged with the single dose OVA–sHsp conjugate, but had been pretreated with vehicle (PBS) only. This group also produced significantly higher antibody titers than the remaining combinations, although the response was delayed by about one day, as compared to those mice which had received P22 pretreatment (P22/OVA–sHsp conjugate). The remaining groups however, did not achieve the same peak titers until day 14 post-challenge, if at all, and equal early responses were delayed by 3–4 days (P22/OVA–sHsp admixture) or more (PBS/OVA–sHsp admixture), as compared to the P22/OVA–sHsp conjugate group. This indicated that the OVA–sHsp conjugate is immunologically recognized differently than is the admixture, and further that this resulted in accelerated antibody production. Fig. 2 The immune response to sHsp is accelerated and intensified after only a single intranasal dose. Mice were pretreated with P22 or vehicle (PBS), then challenged with the OVA–sHsp conjugate, the OVA and sHsp admixture, OVA alone, or sHsp alone. At indicated timepoints post-challenge, serum was collected and total OVA-specific IgG (A) or IgG1 (B) were determined by ELISA, and expressed as either O.D., or concentration (ng/mL). Results in this figure were compiled from two independent experiments with alternate days of serum collection. Statistics : In (A) (*) corresponds to the P22/OVA–sHsp conjugate group, (#) corresponds to the PBS/OVA–sHsp conjugate group, ($) corresponds to the P22/OVA–sHsp admixture group, and (&) corresponds to the top 4 traces as compared to the bottom four traces. In (B) the P22/OVA–sHsp conjugate group was evaluated against each other group and significance is indicated by (*). We also determined the absolute concentration of the OVA-specific IgG1 response to the same pre- and post-treatment combinations ( Fig. 2 B). Consistent with our other results, mice that were pretreated with P22, then challenged with the OVA–sHsp conjugate, produced OVA-specific serum IgG1 in significant levels by day 5, and the quantities increased over the next nine days. Second best at producing high quantities of antibodies were both those mice which had received the control (PBS) pretreatment, but had been challenged with the OVA–sHsp conjugate, and those mice which had been pretreated with P22, then challenged with the sHsp and OVA admixture, which were measurable by day 7 post-challenge. Again, by day 14 post-challenge, the amounts of OVA-specific IgG1 were converging in only those groups in which the sHsp was delivered with OVA. Importantly, two additional conditions further heightened and accelerated the initiation of antibody production—heterologous VLP pretreatment with P22, and the physical conjugation of OVA to sHsp. While we will further discuss the impacts of pretreatment on subsequent challenge, we have demonstrated here that we can elicit an accelerated and intensified immune response to a weak antigen, in a single dose, by covalently conjugating it to sHsp. 3.4 The conjugation of an antigen to sHsp results in the generation of the same immune response to that antigen as sHsp itself We have previously demonstrated that the priming of the lung with sHsp elicits an enhanced immune response to a subsequent pathogen challenge [5] . However, we had yet to define the immune response to sHsp itself. Therefore to determine the rate and intensity of the sHsp-specific antibody response we again pretreated mice intranasally with P22, or vehicle. Mice were then rested for 72 h, and challenged with the OVA–sHsp conjugate, the OVA and sHsp admixture, OVA alone, or sHsp alone. We then evaluated the sHsp-specific serum antibody response over 14 days post-challenge ( Fig. 3 A). We found that after only one challenge dose, all mice exposed to sHsp generated strong sHsp-specific IgG1 responses, which peaked as early as day 5 post-challenge in mice which had been pretreated with P22. Mice that did not receive P22 pretreatment were again two days delayed in the production of similar amounts of sHsp-specific antibody. However by day 7 post-challenge, all groups (except control OVA-only challenged mice) had generated similar levels of systemic sHsp-specific IgG1 in response to a single intranasal dose of sHsp antigen. Thus, the above-described OVA-specific response ( Fig. 2 ) mirrors the accelerated kinetics of the sHsp-specific response, indicating that sHsp facilitates a carrier effect that results in an immune response to OVA that is similar to the response to the sHsp in both onset of antibody production and quantities produced. Fig. 3 The conjugation of an antigen to sHsp results in the generation of the same immune response to that antigen as sHsp itself. In (A) mice were pretreated with P22 or vehicle (PBS) i.n. All mice were then challenged with either the OVA–sHsp conjugate, OVA and sHsp admixture, sHsp alone, or OVA alone. At indicated times, serum was collected and sHsp-specific IgG1 was measured by ELISA (A). In (B) mice were pretreated with "LPS-high" P22 (8 μg LPS per dose), "LPS-low" P22 (14 ng LPS per dose), an equivalent amount of LPS alone (8 μg per dose), or sterile pyrogen-free PBS in five doses i.n. All mice were then challenged with 100 μg sHsp and whole serum IgG to sHsp was determined over 8 days (B). Statistics : In (A), at day 3, the P22/OVA–sHsp conjugate group (*) had significantly higher levels of antibody than the PBS/sHsp group only. At day 5, the P22/OVA–sHsp conjugate group had significantly higher levels of antibody than both the PBS/OVA–sHsp conjugate (**) and PBS/OVA–sHsp admixture (*) groups. Additionally, the P22/OVA–sHsp admixture and P22/sHsp groups had significantly higher levels of antibody than the PBS/OVA–sHsp conjugate group (## and #, respectively). In (B) at day 4 all groups had produced significantly more antibody than the LPS pretreated group where both P22 groups ("LPS-high" and "LPS-low") were *** p 62 kDa) in Fig. 1 Biii represented a polydisperse species of conjugated OVA–sHsp, and were later combined in total for use as the OVA–sHsp conjugate in vivo (further described below). To determine if both OVA and sHsp were present in the upper bands of the sample in Fig. 1 Biii, we performed Western Blot analysis using anti-sHsp ( Fig. 1 C) and anti-OVA ( Fig. 1 D) antibodies. Reactivity to both sHsp and OVA antibodies within the upper bands of the conjugated sample alone ( Fig. 1 Ciii and Diii) indicated the conjugation of OVA to sHsp. A semi-quantitative Western Blot ( Fig. S4 ) suggested an average ratio of 2.6 OVA per sHsp cage within the conjugated sample. The total protein concentration of the conjugated (0.99 mg/mL), admixed (0.82 mg/mL), sHsp (0.61 mg/mL), and OVA (0.66 mg/mL) samples used for in vivo experiments were confirmed via Bradford assay. The molecular weights of the bands corresponding to the OVA–sHsp conjugates were determined based upon their migration distance on SDS-PAGE gels ( Figs. 1B and S5 ), and densitometry analysis (not shown). Protein bands corresponding to covalent conjugation between OVA and sHsp subunits on SDS-PAGE gel were identified by their presence within the conjugated sample ( Fig. 1 Biii) and absence within the sHsp ( Fig. 1 Bi), maleimide functionalized OVA ( Fig. 1 Bii), and the admixture ( Fig. 1 Biv) controls. We detected bands with calculated molecular weights of 58 and 62 kDa Fig. 1 Biii and Fig. S5 that corresponded to the conjugation of a single sHsp subunit (16.5 kDa) to one cleaved OVA (40.1 kDa) or full length OVA (45 kDa), respectively. The upper bands detected between 67 and 84 kDa likely resulted from various forms of conjugation of OVA to sHsp ( Fig. S5 ), and the smear of sample corresponding to molecular weights 85–150 kDa likely represented the conjugation of OVA to multiple sHsp subunits, as this entire smear reacted to both anti-OVA and anti-sHsp antibodies on a Western Blot. Thus, the resultant conjugated OVA–sHsp sample used for immunization was the culmination of a polydisperse species of OVA–sHsp, with varying degrees of multivalent array architecture. We utilized size exclusion chromatography and dynamic light scattering measurements to probe the native state and size distribution of the particles within the samples used for immunization. The OVA–sHsp conjugate contained particles that were larger in size than those in the admixture, sHsp, or OVA. Size exclusion chromatography indicated that a distribution of larger species was present within the OVA–sHsp sample ( Fig. S6 ) and by dynamic light scattering, the OVA–sHsp conjugate showed a larger average diameter than the maleimide functionalized OVA, sHsp S121C, or the admixture. The range of average diameters based on the intensity measurements on the samples repeated seven times were 6.0–9.9 nm for OVA, 15.1–17.3 nm for the admixture, 15.6–18.1 nm for sHsp S121C, and 29.9–41.0 nm for the OVA–sHsp conjugated sample ( Fig. S7 ). 3.3 The immune response to OVA–sHsp is quick and intense after only a single intranasal dose In our first in vivo studies we determined the potential of sHsp to serve as a novel vaccine delivery platform by its ability to facilitate the generation of antigen-specific immunity to OVA. Simultaneously, we determined how pretreatment of the lung with a heterologous VLP, P22, affects the subsequent response to antigen challenge. BALB/c mice were pretreated with either P22 or vehicle (PBS) intranasally (i.n.) in five doses and then allowed to rest for 72 h. Mice were then challenged with OVA conjugated to sHsp (OVA–sHsp), OVA and sHsp separately in solution (OVA–sHsp admixture), OVA alone, or sHsp alone. Serum was then collected at a range of timepoints post-antigen challenge for kinetic analysis. We found that mice which had received the P22 pretreatment, followed by a single dose of the OVA–sHsp conjugate, produced high amounts of OVA-specific serum IgG as early as 4 days post-challenge ( Fig. 2 A). This combination far outperformed any of the other treatment scenarios in both rapidity of antibody production and amount. Interestingly, the next highest antibody producing group, at early timepoints, was those mice that had also been challenged with the single dose OVA–sHsp conjugate, but had been pretreated with vehicle (PBS) only. This group also produced significantly higher antibody titers than the remaining combinations, although the response was delayed by about one day, as compared to those mice which had received P22 pretreatment (P22/OVA–sHsp conjugate). The remaining groups however, did not achieve the same peak titers until day 14 post-challenge, if at all, and equal early responses were delayed by 3–4 days (P22/OVA–sHsp admixture) or more (PBS/OVA–sHsp admixture), as compared to the P22/OVA–sHsp conjugate group. This indicated that the OVA–sHsp conjugate is immunologically recognized differently than is the admixture, and further that this resulted in accelerated antibody production. Fig. 2 The immune response to sHsp is accelerated and intensified after only a single intranasal dose. Mice were pretreated with P22 or vehicle (PBS), then challenged with the OVA–sHsp conjugate, the OVA and sHsp admixture, OVA alone, or sHsp alone. At indicated timepoints post-challenge, serum was collected and total OVA-specific IgG (A) or IgG1 (B) were determined by ELISA, and expressed as either O.D., or concentration (ng/mL). Results in this figure were compiled from two independent experiments with alternate days of serum collection. Statistics : In (A) (*) corresponds to the P22/OVA–sHsp conjugate group, (#) corresponds to the PBS/OVA–sHsp conjugate group, ($) corresponds to the P22/OVA–sHsp admixture group, and (&) corresponds to the top 4 traces as compared to the bottom four traces. In (B) the P22/OVA–sHsp conjugate group was evaluated against each other group and significance is indicated by (*). We also determined the absolute concentration of the OVA-specific IgG1 response to the same pre- and post-treatment combinations ( Fig. 2 B). Consistent with our other results, mice that were pretreated with P22, then challenged with the OVA–sHsp conjugate, produced OVA-specific serum IgG1 in significant levels by day 5, and the quantities increased over the next nine days. Second best at producing high quantities of antibodies were both those mice which had received the control (PBS) pretreatment, but had been challenged with the OVA–sHsp conjugate, and those mice which had been pretreated with P22, then challenged with the sHsp and OVA admixture, which were measurable by day 7 post-challenge. Again, by day 14 post-challenge, the amounts of OVA-specific IgG1 were converging in only those groups in which the sHsp was delivered with OVA. Importantly, two additional conditions further heightened and accelerated the initiation of antibody production—heterologous VLP pretreatment with P22, and the physical conjugation of OVA to sHsp. While we will further discuss the impacts of pretreatment on subsequent challenge, we have demonstrated here that we can elicit an accelerated and intensified immune response to a weak antigen, in a single dose, by covalently conjugating it to sHsp. 3.4 The conjugation of an antigen to sHsp results in the generation of the same immune response to that antigen as sHsp itself We have previously demonstrated that the priming of the lung with sHsp elicits an enhanced immune response to a subsequent pathogen challenge [5] . However, we had yet to define the immune response to sHsp itself. Therefore to determine the rate and intensity of the sHsp-specific antibody response we again pretreated mice intranasally with P22, or vehicle. Mice were then rested for 72 h, and challenged with the OVA–sHsp conjugate, the OVA and sHsp admixture, OVA alone, or sHsp alone. We then evaluated the sHsp-specific serum antibody response over 14 days post-challenge ( Fig. 3 A). We found that after only one challenge dose, all mice exposed to sHsp generated strong sHsp-specific IgG1 responses, which peaked as early as day 5 post-challenge in mice which had been pretreated with P22. Mice that did not receive P22 pretreatment were again two days delayed in the production of similar amounts of sHsp-specific antibody. However by day 7 post-challenge, all groups (except control OVA-only challenged mice) had generated similar levels of systemic sHsp-specific IgG1 in response to a single intranasal dose of sHsp antigen. Thus, the above-described OVA-specific response ( Fig. 2 ) mirrors the accelerated kinetics of the sHsp-specific response, indicating that sHsp facilitates a carrier effect that results in an immune response to OVA that is similar to the response to the sHsp in both onset of antibody production and quantities produced. Fig. 3 The conjugation of an antigen to sHsp results in the generation of the same immune response to that antigen as sHsp itself. In (A) mice were pretreated with P22 or vehicle (PBS) i.n. All mice were then challenged with either the OVA–sHsp conjugate, OVA and sHsp admixture, sHsp alone, or OVA alone. At indicated times, serum was collected and sHsp-specific IgG1 was measured by ELISA (A). In (B) mice were pretreated with "LPS-high" P22 (8 μg LPS per dose), "LPS-low" P22 (14 ng LPS per dose), an equivalent amount of LPS alone (8 μg per dose), or sterile pyrogen-free PBS in five doses i.n. All mice were then challenged with 100 μg sHsp and whole serum IgG to sHsp was determined over 8 days (B). Statistics : In (A), at day 3, the P22/OVA–sHsp conjugate group (*) had significantly higher levels of antibody than the PBS/sHsp group only. At day 5, the P22/OVA–sHsp conjugate group had significantly higher levels of antibody than both the PBS/OVA–sHsp conjugate (**) and PBS/OVA–sHsp admixture (*) groups. Additionally, the P22/OVA–sHsp admixture and P22/sHsp groups had significantly higher levels of antibody than the PBS/OVA–sHsp conjugate group (## and #, respectively). In (B) at day 4 all groups had produced significantly more antibody than the LPS pretreated group where both P22 groups ("LPS-high" and "LPS-low") were *** p < .001, and PBS was * p < .05 comparatively to the LPS group. Because our samples were purified from an E. coli expression system, we examined whether or not the heightened antibody responses observed were amplified by the residual LPS contained in our VLPs. We pretreated mice with either "LPS-low" P22, "LPS-high" P22, an equivalent amount of LPS as contained in the "LPS-high" P22 preparation, or sterile pyrogen-free PBS in five daily intranasal doses, as above. Mice were then challenged with sHsp alone, and serum was collected over 8 days to evaluate total anti-sHsp IgG. Importantly, we found that both "LPS-low" and "LPS-high" P22 elicited an equally enhanced early antibody response to the heterologous VLP challenge, while mice which were pretreated with LPS did not ( Fig. 3 B). In addition, we pretreated TRL4−/− or C57BL/6 mice with sHsp or PBS and challenged all mice with high-dose influenza virus. Importantly, only those mice that had been pretreated with sHsp were protected from influenza-induced body weight loss, regardless of their ability to respond to LPS ( Fig. S2 ). These results indicated that the observed heightened immunity in VLP pretreated mice was not dependent on contaminating LPS. 3.5 sHsp can act as an adjuvant in the lungs Next, to determine how the addition of sHsp, either delivered as a conjugate or an admixture with OVA, could act as an adjuvant for the immune response in the lungs, we pretreated mice with PBS only, and subsequently challenged them with the OVA–sHsp conjugate, admixture, or OVA alone, as described above. At day 7 post-challenge, we determined the level of OVA-specific serum IgG subclasses, and found that both the conjugated and admixture preparations of OVA elicited high-titer serum antibody O.Ds., while no such response to OVA alone was seen ( Fig. 4 A). Interestingly however, the OVA–sHsp conjugate promoted accelerated class-switching to IgG2a, IgG2b and IgG3 while only IgG1 was significantly detected for the admixture. To further determine the early events of isotype switching, we pretreated mice with either sHsp or vehicle, and challenged with the OVA–sHsp conjugate, or admixture. At days 0, 4, and 6, serum was collected and the OVA-specific IgG subclasses were determined by ELISA. We found that OVA-specific total IgG and IgG1 production was quicker by about two days in mice that had been pretreated with sHsp and challenged with the conjugated OVA–sHsp ( Fig. 4 B and C) as compared to the other treatments. The early IgG1 response in this group then translated into an enhanced production of class-switched IgG2a and IgG2b at day 6 post-challenge ( Fig. 4 D and E). This extent of isotype switching was not fully realized in any other group. Interestingly however, the enhanced class-switching capacity appeared to be more dependent upon the structure of the challenge antigen (OVA–sHsp conjugate vs. admixture) than the pretreatment with sHsp. Mice which had received only PBS pretreatment and had been challenged with the OVA–sHsp conjugate generated significantly higher IgG1, 2a, 2b, and IgG3 titers at day 6 post-challenge than those which were challenged with the admixture of sHsp and OVA ( Fig. 4 C–E and G). Very little IgG2c was produced at any timepoint ( Fig. 4 F). Thus, sHsp acts as an adjuvant when mixed with OVA and acts as a carrier when conjugated to OVA, resulting in unexpectedly potent antibody titers as early as 4 days after a single immunization in naïve mice. Fig. 4 sHsp can act as an adjuvant in the lungs. In (A), all mice were pretreated with PBS i.n., and then challenged with the OVA–sHsp conjugate, OVA and sHsp admixture, or OVA alone. Serum IgG subclass titers were then determined by ELISA at day 7 post-challenge. In (B)–(G), mice were pretreated with either sHsp or vehicle (PBS) i.n., then challenged with the OVA–sHsp conjugate, or the OVA and sHsp admixture. At days 0, 4, and 6 serum was collected and total OVA-specific IgG subclasses were determined by ELISA. In (H), mice were not pretreated, but were challenged s.c. with the OVA–sHsp conjugate, OVA and sHsp admixture, OVA and alum, OVA alone, or alum alone. Serum was then collected over 28 days, and OVA-specific serum IgG was determined by ELISA. Statistics : For (A) (*) denote the PBS/OVA–sHsp conjugate group as compared to the PBS/OVA–sHsp admixture group and OVA alone and (&) denotes the PBS/OVA–sHsp admixture group as compared to OVA alone. For (B)–(C) symbols are used per line to indicate significance over all other groups. In (D) (∧) represents that the sHsp/OVA–sHsp conjugate group is significant as compared to the PBS/OVA–sHsp admixture group, but not the PBS/OVA–sHsp conjugate group at day 6. And in (E), at days 4 and 6 (∧) indicates that the sHsp/OVA–sHsp conjugate group was significant over the PBS/OVA–sHsp admixture group, and was also significant over the PBS/OVA–sHsp conjugate group (*) at day 6 only. For (H) at day 5 (*) denotes that the OVA–sHsp conjugate group was significant as compared to the OVA–alum admixture. At day 7, both the OVA–sHsp conjugate group (*) and the OVA–alum admixture group (&) were significant as compared to the remaining groups. At day 14 both the OVA–sHsp conjugate group (*) and the OVA–alum admixture group (&) were significant as compared to the OVA alone group. Significance was not denoted for differences against alum alone. Because we found that sHsp is a strong mucosal adjuvant, we next determined if sHsp would elicit a similar response when delivered to a non-mucosal site. We subcutaneously (s.c.) injected naïve mice with either the OVA–sHsp conjugate, the OVA and sHsp admixture, OVA alone, or OVA with its classical adjuvant–alum. We then determined the resultant serum antibody responses and found that when conjugated to antigen, sHsp acts as a strong adjuvant to OVA to produce an accelerated OVA-specific antibody response as early as day 5 post-challenge, while OVA–alum responses are delayed by two days comparatively ( Fig. 4 H). Again, the OVA and sHsp admixture elicited high titer antibody responses, as did the OVA and alum admixture, however, these combinations required more time to produce similar results. Taken together, sHsp conjugated to antigen acts as a carrier and elicits an accelerated antibody response to that antigen when delivered to several immunologically distinct sites. 3.6 sHsp-treatment induces local IgA responses Growing recognition of the importance of site-specific immunity at mucosal surfaces [7] , as well as tailoring immune responses per tissue [44] , [45] , [46] , [47] led us to determine whether pretreatment with sHsp or the conjugation of sHsp to OVA affects local IgA production. After pre-treatment with sHsp or PBS, and challenge with either the OVA–sHsp conjugate or the admixture, lung lavage fluids contained high levels of mucosal IgA and IgG in only those mice pretreated with sHsp ( Fig. 5 A and B). Local IgG production was further enhanced by the conjugation of sHsp to OVA ( Fig. 5 B). Notably, while many of our results demonstrate that the admixed OVA and sHsp preparation does not elicit the same enhancement as the conjugate, here, the admixture is equally potent in initiating mucosal IgA responses ( Fig. 5 A). sHsp thus serves a dual role—first as an immunomodulatory agent during pretreatment, and second, as an adjuvant for specific antigens. Furthermore, we found that the pretreatment of the lung with sHsp caused an upregulation of the polymeric Ig receptor (pIgR) on lung epithelial cell surfaces prior to antigen challenge ( Fig. 5 C). Thus, the capacity for the release of local secretory IgA into the airway lumen is enhanced by sHsp pretreatment. Fig. 5 sHsp treatment induces local IgA responses. Mice were pretreated with sHsp or vehicle control (PBS) i.n., then challenged with the OVA–sHsp conjugate, or the OVA and sHsp admixture. In (A) and (B), BALF OVA-specific IgA and IgG were measured at day 7 post-challenge by ELISA. In (C), frozen lungs were sectioned and stained for the presence of the polymeric Ig receptor in either sHsp- (left) or vehicle-primed (right) mice. Statistics : For (A) the sHsp/OVA–sHsp conjugate was significant as compared to both PBS pretreated groups. In (B) the sHsp/OVA–sHsp conjugate was significant as compared to all the below groups, as was the sHsp/OVA–sHsp admixture, and the PBS/OVA–sHsp conjugate. 3.7 sHsp pretreatment of the lungs enhances influenza-specific antibody responses and changes the lung environment, making it more conducive to local antibody responses We have previously shown that sHsp pretreatment of mice subsequently infected with influenza, accelerates the onset and intensity of an influenza-specific IgG response similar to how sHsp pretreatment enhances the antibody response to OVA–sHsp [5] . We then determined whether sHsp pretreatment also affected the kinetics of antibody class switching after influenza infection as it does with OVA–sHsp. In mice pretreated with sHsp or control, then challenged with 1500 pfu mouse-adapted PR8 (H1N1) influenza virus, we determined the corresponding endpoint-dilution titer of influenza-specific IgG subclasses in the local BALF, and found that, as had been expected due to the OVA results, influenza-specific BALF IgG was enhanced both in titer and in the rate of class-switching in mice which had been exposed to sHsp prior to infection ( Fig. 6 A–F). Thus, sHsp pretreatment similarly affects antibody responses to a model antigen and a pathogen-associated antigen. Fig. 6 sHsp pretreatment accelerates the onset of influenza-specific IgG in BALF. Mice were pretreated with either sHsp or vehicle (PBS), and challenged with 1500 pfu PR8 influenza i.n. At indicated times post-challenge, mice were sacrificed and lavaged. Influenza-specific whole IgG (A), IgG1 (B), IgG2a (C), IgG2b (D), IgG2c (E), and IgG3 (F) in the BALF were determined to endpoint titer in 2-fold dilutions by ELISA. We next determined how sHsp pretreatment modulates the lung environment. We pretreated mice with sHsp, then challenged with influenza. We found that sHsp pretreatment stimulated the formation of germinal centers (GC) in the lung and enlarged GC B cell areas in the tracheobronchial lymph node (TBLN) ( Fig. 7 A and B). In both tissues, GC B cells were more prevalent by at least one log in sHsp-primed mice before infection, indicating that the microenvironment within the lung and local lymph node had been stimulated to adjust to the current antigenic exposure. We also found that T follicular helper (T FH ) cells were more abundant in both the lungs and TBLNs of sHsp-primed mice ( Fig. 7 C and D). Thus, germinal center reactions were more easily facilitated within the lungs and local lymph nodes of sHsp-primed mice. Over the course of infection, a clear advantage in GC organization and establishment was observed as both GC B cells and T FH cells retained significantly higher numbers in sHsp-primed mice as opposed to controls. Thus, highly active GC reactions, complete with T FH cell help, were likely facilitating the enhanced antibody isotype switching and elevated antibody production. Fig. 7 sHsp pretreatment of the lungs leads to an even faster response to subsequent antigens. Mice were pretreated with sHsp or vehicle (PBS) i.n., infected with 1500 pfu PR8, and then sacrificed at indicated times post-infection. Lungs and tracheobronchial lymph nodes (TBLNs) were homogenized and stained for germinal center (GC) B cells (A and B), T follicular helper cells (TFH) (C and D), or plasma cells (E and F) and total cell numbers were quantified by FACS and cell counts. Given that GC's are enhanced, we also stained for the presence of plasma cells, as defined by their lymphocyte size and morphology, loss of B220 and CD19, with or without CD138 (syndecan) expression, and the upregulation of CXCR4 ( Fig. 7 E and F). Interestingly, we found that in the lung, plasma cells at day 0, assumedly secreting antibody against sHsp, were markedly more abundant in primed mice. However, the number of cells present rapidly declined over the course of the influenza infection, which may represent profound plasticity in the specificity of the inhabitants of GC areas of the lung (as has been described by others) [48] . In the TBLN, plasma cells were again more numerous before infection in the sHsp-primed mice. However, here, they remained elevated as compared to control mice until the resolution of infection, at which point the plasma cell numbers in the TBLNs of both groups converged. Unlike the lung however, similar trends were followed by both groups in the TBLN. We surmise from this data that sHsp-specific plasma cells in the lung are decreasing, while simultaneously, influenza-specific plasma cells are increasing. Therefore, due to the likely variable rates of influx and efflux, we may not have exclusively quantified the rate of increase in influenza-specific plasma cells due to sHsp priming. What remains puzzling however is how sHsp-specific germinal centers and plasma cells become influenza- or OVA-specific at an accelerated rate when compared to control mice. Given our experimental data, we surmise, as have others [2] , that the pulmonary microenvironment adjusts in the context of innate, humoral, and cellular immune responses in reaction to the menagerie of antigenic exposures encountered, which are unique to that individual. Thus, sHsp pretreatment significantly enhances the efficiency of future immune responses. 4 Discussion We have shown previously that sHsp pretreatment induces pulmonary changes that protect against a subsequent challenge with a variety of pathogens, and additionally sHsp pretreatment decreases tissue damage, while accelerating viral clearance [5] . We demonstrate here that both sHsp pretreatment, and the conjugation of sHsp to an antigen of interest, provided enhanced immunity, by likely distinct immunological mechanisms. Pretreatment with sHsp elicited the organization of GCs in the lungs and TBLNs, which then functioned to accelerate and intensify the onset of antibody production. Most importantly, pre-priming of the lung with sHsp elicited an enhanced mucosal IgA response, and the upregulation of epithelial pIgR. This response is not dependent upon the physical conjugation of OVA to sHsp, as the OVA and sHsp admixture elicited IgA production equally well. For local BALF IgG production, however while sHsp pretreatment seemed to be the factor majorly responsible for the enhanced antibody titers, the conjugation of sHsp to OVA also contributed. Finally, heterologous VLP pretreatment was equally efficient at eliciting heightened immunity, as demonstrated with P22 pretreatment followed by OVA–sHsp challenge, and sHsp pretreatment followed by influenza challenge. While heterologous immunity has been extensively described in both the context of cross-protective immunity, and inappropriate or harmful skewing, the underlying mechanisms are still incompletely defined [1] , [2] , [3] , [48] , [49] , [50] , [51] . We demonstrate here that pretreatment of mice with non-pathogenic VLPs stimulated immunity through some type of priming, which resulted in a subsequent enhanced response to OVA, and furthermore, facilitated pathogen clearance and decreased damage in response to viral challenge ( Fig. 5 , Fig. 6 and Ref. [5] ). In addition to pretreatment, we also demonstrate the utility of sHsp as a novel vaccine platform through the direct conjugation to an antigen of interest. In contrast to the conjugation of antigenic peptides to a multivalent scaffold, we present the conjugation of an entire protein antigen to the protein cage surface for in vivo application. Through this approach, it is possible for the conjugated antigen to be seen by the immune system as part of the VLP, and elicits a response similar to that the VLP elicits. As previous work has demonstrated that chemical conjugation efficiency of large unrelated proteins to a viral capsid is inhibited as the size of the protein is increased [38] , conditions for the chemical conjugation of the 45 kDa OVA to the sHsp cage were optimized for these proteins. We observed that the attachment position on the sHsp cage, the length of the chemical linker, and the stoichiometry of linker labeling affected the conjugation efficiency. When delivered as an OVA–sHsp conjugate, sHsp acted to enhance OVA-specific early antibody production. Thus, sHsp acted as a carrier, causing the OVA-specific response to mirror the sHsp-specific response in onset of antibody production and quantity produced. Interestingly, while we have utilized sHsp in two scenarios, as a pretreatment, or a vaccine delivery platform, and these scenarios likely are working through distinct immunostimulatory mechanisms, the activities of each are not mutually exclusive, and can in fact, synergize—as the antibody responses to the conjugated OVA–sHsp are enhanced by the pretreatment with either sHsp or P22. While we have yet to define the exact cellular receptors responsible for signal transduction to initiate the immune response to the VLPs, we hypothesize that the repeating subunits of sHsp or P22 allow for the ability to crosslink or otherwise engage single or multiple cellular receptor domains. And as it has been suggested by others, cell surface integrins on antigen-presenting cells likely play a role in recognizing viral capsids [22] , [23] , [25] . Targeting evolutionarily conserved domains therefore facilitates plasticity in this delivery system, allowing for the utilization of sHsp conjugation with other antigens—especially those which have complicated or precluded the design and manufacture of potent vaccines. Additionally, the complex geometry of the OVA–sHsp conjugate may be impacting the resultant immune responses observed. It has been well demonstrated that the specific geometric display of multivalent antigens significantly heightens immune responses [20] , [52] , [53] , and especially B cell recognition [52] , [54] , [55] , [56] , and further, that such displays may even provide enough co-stimulation to break tolerance to self-antigens without the need for adjuvants [55] , [57] . Thus the arrangement of the OVA–sHsp conjugate may explain the enhanced class-switching which we observed only in those mice which received the conjugated form of OVA–sHsp ( Fig. 4 A–G). We therefore demonstrate the utility in displaying a poorly immunogenic antigen as a multivalent array through the conjugation to a VLP platform to elicit enhanced specific immunity. Furthermore, the implications for a single-dose vaccine that accelerates the antibody response to antigens, producing high titer antibody within 4–7 days of primary immunization holds significant clinical potential. In this regard, new strategies which employ post-exposure prophylaxis represent an arguably greater demand than pretreatment, as we continually lose antibiotic options due to the emergence of resistant strains of bacteria, and are no better at predicting future viral outbreaks. Thus, we may be able to exploit sHsp, conjugated to an antigen of choice, to elicit early IgA responses. Importantly, few adjuvants currently on the market can be safely delivered to the lung, nor do they produce high titer, site-specific mucosal IgA [58] . We have presented a novel vaccine platform that is amenable to easy manipulation, facilitating flexibility and broad implications for vaccination strategies against many types of pathogens. This type of platform is especially noteworthy, as it may be utilized as an immunomodulatory agent alone (as in the case of pretreatments), for modifying the current state of immune homeostasis, or as a carrier and adjuvant for a defined antigen of interest. This phenomenon of immune homeostasis and skewing has been extensively described in terms of an individual's history of pathogen and allergen exposure [3] , [51] . However, to date few attempts to harness the potential of immune priming, with or without involving pathogen-specific epitopes or proteins, have been described [4] . Thus, we propose that we have identified an approach which utilizes strategies for priming against undefined broad-spectrum antigens, and also for delivering a highly efficacious single-dose vaccine against a defined antigen. Importantly, we have additionally shown that the route of delivery (directly to the lung by intranasal instillation) results in the creation of an immune response that is specifically tailored to that tissue, and its individual requirements. While concern in purposely eliciting an immune response in the lung is justified, we and others [52] indicate here (and elsewhere [5] ) that by doing so with VLPs, the power of tissue-specific immunity can be harnessed to provide a safe and appropriate response in the context of both the pathogen (sterilizing immunity), and the protection of lung function. Furthermore, the onset of immunity when the lung is pre-exposed to sHsp is accelerated, again indicating that the pretreatment with sHsp impacts the future, unpredictable pathogen challenge. In addition, we have previously shown that the deposition of VLPs in mouse lungs has no adverse effects and even attenuates lung hypersensitivity [5] . Virus-like particle and nanoparticle vaccination strategies are gaining recognition as the next class of safe and effective platforms. Notably, while many vaccines are expensive to produce, and require refrigeration, many types of VLPs and nanoparticles are stable, and easily preserved through freeze-drying, creating opportunities for distribution that may be otherwise precluded due to cost or logistics. Therefore, we have herein described a novel mucosal vaccination strategy that accounts for tissue-specificity, and exploits natural immunity to provide accelerated and enhanced antibody and cellular immune responses to primary antigen challenge. Appendix A Supplementary data

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968091/

Therapeutic monoclonal antibodies for respiratory diseases: Current challenges and perspectives, March 31 – April 1, 2016, Tours, France

ABSTRACT Monoclonal antibody (mAb) therapeutics have tremendous potential to benefit patients with lung diseases, for which there remains substantial unmet medical need. To capture the current state of mAb research and development in the area of respiratory diseases, the Research Center of Respiratory Diseases (CEPR-INSERM U1100), the Laboratory of Excellence "MAbImprove," the GDR 3260 "Antibodies and therapeutic targeting," and the Grant Research program ARD2020 "Biotherapeutics" invited speakers from industry, academic and government organizations to present their recent research results at the Therapeutic Monoclonal Antibodies for Respiratory Diseases: Current challenges and perspectives congress held March 31 – April 1, 2016 in Tours, France. Abbreviations [18F]FES 16α-[18F]Fluoro-17β-estradiol ADC antibody-drug conjugate ADCC antibody-dependent cell-mediated cytotoxicity AhR aryl hydrocarbon receptor BAFF B-cell activating factor BAL bronchial-alveolar lavages CARD9 caspase activation and recruitment domains 9 CCL16 Club cell protein-16 CDC complement-dependant cytotoxicity CODV cross-over dual variable COPD chronic obstructive pulmonary disease CS cigarette smoke CT computed tomography CTLA-4 cytotoxic T lymphocyte-associated antigen 4 Da Dalton DVD dual-variable-domain EGFR epidermal growth factor receptor EPR enhanced permeability and retention ER estrogen receptor FcRn neonatal Fc receptor HER2 human epidermal growth factor receptor 2 Hrsv human respiratory syncytial virus HRV human rhinovirus IL interleukin IPF idiopathic pulmonary fibrosis IV intravenous kDa Kilodaltons LDH lactate dehydrogenase mAb monoclonal antibody MMP-9 matrix metalloproteinase-9 MRI magnetic resonance imaging NHP non-human primate NIRF near-infrared fluorescence NLRP3 pyrin domain containing 3 NSCLC non-small cell lung cancer ORR objective response rate OS overall survival PEG polyethylene glycol PD pharmacodynamics PD-1 programmed death 1 PD-L1 PD-1 ligand PET positron emission tomography PFS progression-free survival PK pharmacokinetics PMN polymorphonuclear leucocytes RSV respiratory syncytial virus scFv single-chain variable fragment SCID severe combined immunodeficiency SPECT radioisotopic single photon emission CT ST2 interleukin 1 receptor-like 1 TBTi tetravalent bispecific tandem immunoglobulin Th T-helper TNFα tumor necrosis factor α TMARD Monoclonal Antibodies for Respiratory Diseases: Current challenges and perspectives UK United Kingdom US United States of America VEGF vascular endothelial growth factor On March 31, 2016, the meeting Monoclonal Antibodies for Respiratory Diseases (TMARD, http://tmard.sciencesconf.org/ ): Current challenges and perspectives was opened by Hervé Watier (Co-Director of LabEx MAbImprove), Nathalie Heuzé-Vourc'h (President of TMARD scientific committee), Patrice Diot (Dean of Tours School of Medicine) and Pierre Commandeur (Vice-president of Center-Val de Loire region), who welcomed participants and thanked the organizers and both institutional and industrial sponsors. The first speaker, Janice M. Reichert (The Antibody Society; Reichert Biotechnology Consulting LLC), discussed monoclonal antibody (mAb) therapeutics in development for respiratory disorders, as well as neurological, infectious, cardiovascular / hemostasis diseases. 1 While antibody therapeutics for cancer and common immune-mediated disorders, e.g. , rheumatoid arthritis, psoriasis, Crohn's disease, are discussed frequently, development of mAbs for these other therapeutic areas are not often the focus of attention. To provide context, Dr. Reichert noted that global antibody therapeutics research and development by the biopharmaceutical industry has undergone remarkable expansion in the past ∼5 years. Resulting from a substantial dedication of effort and resources, over 100 novel antibodies entered first clinical studies in 2015, and the overall clinical pipeline now includes ∼480 antibodies. Importantly for patients, these molecules are progressing through the phases of clinical testing, and being approved for marketing. To date, over 50 antibody therapeutics for a variety of diseases are in Phase 3 studies. 1 A record number, i.e. , 9 new products, were granted first marketing approvals in the United States or European Union in 2015, and the evidence suggests that a similar number may be approved in 2016. As examples, Dr. Reichert provided details for obiltoxaximab, ixekizumab and reslizumab, 3 mAb products already approved by the Food and Drug Administration in 2016. Obiltoxaximab (Anthim®), targeting the protective antigen of Bacillus anthracis exotoxin, is indicated in adult and pediatric patients for treatment of inhalational anthrax due to Bacillus anthracis in combination with appropriate antibacterial drugs, and for prophylaxis of inhalational anthrax when alternative therapies are not available or appropriate. Ixekizumab (Taltz®), targeting interleukin IL17A, is indicated for treatment of adults with moderate-to-severe plaque psoriasis, whereas reslizumab (Cinqair®), targeting IL5, is indicated for severe asthma in adults. Dr. Reichert then focused on antibodies developed for respiratory, neurological, infectious or cardiovascular / hemostasis diseases, which, excluding cancer and common immune-mediated disorders, are the therapeutic areas that include the most mAbs in development. Neurological disorders represent the largest area (27 mAbs; 11 in Phase 2/3 or Phase 3 studies), followed by cardiovascular / hemostasis (25 mAbs; 6 in Phase 3 studies), infectious disease (24 mAbs; 3 in Phase 3 studies) and respiratory disease (22 mAbs, 4 in Phase 3 studies). Most (∼90%) mAbs in development for these therapeutic areas are canonical IgG 1 , IgG 2 , IgG 4 or IgM that may have been Fc- or glyco-engineered. In contrast, relatively few, i.e. , less than 20 mAbs, have non-canonical formats, e.g ., bispecific, antibody fragments (domain, nanobody, single-chain variable fragment (scFv), Fab). In the respiratory disorder area, Dr. Reichert discussed 4 mAbs that are undergoing evaluation in Phase 3 studies for asthmatic patients: anti-IL5 receptor benralizumab, anti-IL4 receptor α dupilumab, anti-IL13 lebrikizumab, and anti-IL13 tralokinumab. Benralizumab is also undergoing evaluation in Phase 3 studies of patients with chronic obstructive pulmonary disease (COPD). Thus, Dr. Reichert noted that this latter is an "antibody to watch" in 2016, because its results for 4 Phase 3 studies of asthma patients are expected in 2016, and, if they are positive, marketing application submissions may occur later in the year. In concluding her talk, Dr. Reichert emphasized that the recent increase in the number of mAbs in clinical studies is expected to drive a trend toward first approvals for ∼6–8 new mAbs per year (or more), including mAbs for respiratory disorders. She cautioned that the sustainability of this trend depends on the verification of expected increases in potency of engineered antibodies and bispecific antibodies, and the validity of the novel targets for mAbs in development. However, if the recent past reflects the near future, unmet medical need should be reduced and patient choices for antibody therapeutics should increase in the next ∼8 years, which is the average time for mAb clinical development. Session 1: Anti-infectious monoclonal antibodies Matthew Sleeman (MedImmune) opened the session dedicated to anti-infectious mAbs with a talk entitled 'Targeting Pathogens'. Increasingly, mAbs targeting different cytokines, including IL13 and IL5, are being considered as therapeutic options for the treatment of severe respiratory conditions such as asthma, idiopathic pulmonary fibrosis (IPF) and COPD. While many of these anti-cytokine approaches demonstrated promise, the majority of hospitalizations in these diseases are caused by common pathogens triggering exacerbations of their conditions. Therefore, therapies directly targeting pathogens may provide significant benefit. He showed first that, due to antigenic diversity, direct targeting of pathogens has been challenging and requires a detailed understanding of the stable proteins on a pathogen surface that can be accessed by mAbs. One such example is the antibody palivizumab (Synagis®), which binds to the key target fusion-protein of respiratory syncytial virus (RSV). It has been approved for the prevention of infections in premature infants. In addition to this, anti-RSV vaccines have been generated that, if successful, could provide longer term protection for at risk individuals. MAb therapies, such as palivizumab, are unfortunately the exception rather than the rule in the prevention or treatment of infectious disease. Thereafter, Dr. Sleeman discussed an alternative approach, which is the design of antibodies to the host co-receptor to prevent viral entry and infection. To illustrate his talk, he presented the human rhinovirus (HRV), which is responsible for the common cold and virally-driven respiratory exacerbations in asthma and COPD. HRV is made up of 3 distinct clades: HRV-A, HRV-B and HRV-C consisting of greater than 167 distinct serotypes. With such diversity, the ability to design a mAb that would neutralize all serotypes is extremely challenging; a different option could be to target one of the co-receptors: intercellular adhesion molecule 1 (ICAM-1), low-density lipoprotein receptor (LDLR) or cadherin-related family member 3 (CDHR3). Using mouse models of HRV infection, Dr. Sleeman showed that ICAM-1 was elevated on the bronchial epithelium of the lung, and that an ICAM1 neutralizing antibody (14C11) could prevent HRV driven lung inflammation, viral infection and cytokine production whether the antibody was administered directly to the airways or was given systemically. 2 In addition, he also showed published data that soluble-ICAM1 given to healthy human volunteers pre-inoculation with HRV significantly reduced daily symptoms scores compared with placebo. 3 While these data support the hypothesis of using the co-receptors as a target to prevent viral infections, significant questions remain, especially in the context of a respiratory exacerbations, such as which viral serotypes triggers the exacerbations, could non-HRV viruses, bacteria or fungal pathogens be the cause of some exacerbations or would one need to target all 3 co-receptors for maximal impact? Finally, Dr. Sleeman considered targeting the downstream pro-inflammatory molecules produced as a consequence of pathogen invasion in the lungs. One of these key targets gaining substantial interest is the cytokine IL33, which is highly expressed in the epithelium of the lung and rapidly released upon lung infection with, for example, influenza-A, RSV or HRV. Using a mouse model of COPD, he also went on to show that chronic cigarette smoke (CS) exposure caused an accumulation of IL33 in the epithelium, and that this was rapidly released following an influenza infection, causing chronic inflammation. 4 Furthermore, he showed that this response was significantly blunted in mice deficient in either IL33 or interleukin 1 receptor-like 1 (ST2). The association with this cytokine and respiratory exacerbations has also been shown in samples from severe asthmatics. 5 In conclusion, targeting respiratory pathogens with mAbs is a distinct possibility and has been clearly demonstrated with molecules such as palivizumab; however, significant but not insurmountable challenges remain. Firstly, isolating neutralizing antibodies to many pathogens is complex and challenging; secondly, targeting co-receptors is a distinct possibility, but there could be significant redundancy where a pathogen may use different or multiple co-receptors; or thirdly targeting downstream pathways of infection could provide benefit, but may require a personalized healthcare approach to identify the key pathways that predominate in any one individual. Thomas Secher (INSERM U1220-IRSD) presented results on panobacumab, a human IgM against Pseudomonas aeruginosa serotype O11 lipopolysaccharides. 6-8 Panobacumab is able to reduce P. aeruginosa burden in lungs by enhancing neutrophil recruitment and reducing the host-derived production of pro-inflammatory mediators, and thereby reduces lung injury in a murine model of lung infection, whatever the immune status. Regarding its additional effects when given in association with meropenem, panobacumab may be combined with standard antibiotic therapy. These encouraging preclinical data have been recently confirmed in a short-scale Phase 2 clinical trial in which the full treatment of panobacumab was shown to induce a complete resolution in patients presenting with nosocomial P. aeruginosa pneumonia compared to untreated individuals. 9 Session 2: Anti-cancer monoclonal antibodies Karen L. Reckamp (City of Hope Comprehensive Cancer Center) started the second session of the congress by reminding attendees how clinical trials using mAbs in lung cancer have recently improved patient outcomes. She began her talk with a discussion of the anti-vascular endothelial growth factor (VEGF) bevacizumab (Avastin®), which was approved for treatment of non-small cell lung cancer (NSCLC) a decade ago. 10 The initial Phase 3 trial, which compared carboplatin and paclitaxel with and without bevacizumab in patients with advanced NSCLC with non-squamous histology, demonstrated a statistically significant improvement in objective response rate (ORR) and progression-free survival (PFS) with the addition of bevacizumab. It was also the first study to report a median overall survival (OS) greater than 12 months in such a population. A subsequent 3-arm Phase 3 trial investigated cisplatin and gemcitabine with placebo or bevacizumab at 2 dose levels in patients with similar NSCLC; 11 patients assigned to the bevacizumab arms had statistically superior ORR and PFS. Dr. Reckamp then showed results of the Phase 3 trial in which docetaxel with placebo or ramucirumab (Cyramza®), a mAb against the extra-cellular domain of VEGF-receptor 2, was administered to patients who had experienced disease progression after platinum-based therapy. 12 Statistically significant higher ORR, longer PFS and longer OS were observed with ramucirumab. In all, predictive biomarkers for anti-angiogenesis antibodies have not been identified. Afterwards, Dr. Reckamp reported the multiple clinical trials that have evaluated anti-epidermal growth factor receptor (EGFR) mAbs in combination with chemotherapy to enhance the efficacy of cytotoxic therapy. For instance, a Phase 3 trial investigated cisplatin and gemcitabine with and without necitumumab (Portrazza®), a mAb against the extra-cellular domain of the EGFR in patients with advanced NSCLC with squamous histology. 13 Those who received necitumumab had a similar response rate, but a statistically significant longer PFS and OS, while a similar trial in patients with advanced non-squamous NSCLC failed to demonstrate improved efficacy. 14 Thereafter, Dr. Reckamp showed how anti-programmed death 1 (PD-1) mAbs, like nivolumab (Opdivo®), have improved survival as monotherapy for patients with NSCLC as second-line therapy compared to docetaxel. 15-17 A Phase 3 study of nivolumab versus docetaxel in squamous cell histology showed an improvement in OS of 9.2 months vs. 6 months, respectively. Nonetheless, in these subjects, PD-1 ligand (PD-L1) expression did not appear to be prognostic or predictive of patient outcome at any level. In addition, the Phase 3 trial in patients with previously treated non-squamous histology showed that median OS was significantly improved at 12.2 months for nivolumab and 9.4 months for docetaxel. In contrast with the previous trial, in this study PD-L1 expression was predictive of clinical benefit, although the OS was also similar in both arms when PD-L1 was not expressed. Then, Dr. Reckamp provided details on pembrolizumab (Keytruda®), a highly selective humanized IgG 4 -kappa mAb against PD-1: a single arm trial enrolled 495 NSCLC patients and correlated PD-L1 expression with response to treatment. 18 In a Phase 2/3 randomized trial assessing pembrolizumab compared to docetaxel in patients with previously treated advanced NSCLC who were PD-L1 positive, the median OS was significantly longer for both doses of pembrolizumab. 17 The OS was substantially longer in those who had at least 50% PD-L1 expression in tumor cells. Atezolizumab is another mAb that prevents the binding of PD-L1 to PD-1 and CD80, in addition to preserving the interaction between PD-L2 and PD-1. It demonstrated efficacy when compared to docetaxel in NSCLC in a randomized Phase 2 study. 19 To note, activation of the immune system to produce antitumor responses leads to distinct toxicities related to immune stimulation. Dr. Reckamp underlined the numerous immune-related adverse events in clinical trials and practice, which are greater with cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibition compared to anti-PD-1 and anti-PD-L1 agents, although all these mAbs may result in life-threatening toxicities. She concluded that antibody-based therapy for lung cancer is well-established, and new treatments with antibodies have demonstrated improved efficacy, confirming the success of this modality. Antibody-drug conjugates (ADC) are emerging agents in clinical trials that may lead to new therapeutic options in the future. Rita De Santis (Sigma Tau SpA) drew the attention of the audience with her talk about AvidinOX –conjugated biotinylated-antibody to treat cancer. AvidinOX is an oxidized version of avidin that forms Schiff's bases with tissue proteins, thus constituting an artificial stable receptor for biotinylated therapeutics. 20 Biotinylated anti-cetuximab and anti-panitumumab antibodies, which both target EGFR, showed anti-tumor efficacy at a dose lower than 1/25,000 the intravenous (IV) effective dose, when delivered after AvidinOX through the airways, in an experimental model of lung cancer in severe combined immunodeficiency (SCID) mice. She explained the effect was due to the inability of EGFR to dimerize, and subsequent inhibition of signaling. 21 She also emphasized the excellent tolerability of AvidinOX in animals. Dr. De Santis concluded that AvidinOX offers a great opportunity to treat lung tumors using aerosol delivery. Session 3: Monoclonal antibodies in asthma Opening the third session, Bernhard Ryffel ( INEM - UMR7355; CNRS) reviewed clinical asthma and recent progress with therapeutic antibodies for severe and steroid resistant asthma, highlighting bi- and multi-specific antibodies. Novel mechanisms of allergic asthma focusing on Th2 and Th17 polarized immune responses were discussed, together with recent discoveries that pyrin domain containing 3 (NLRP3) may act as a Th2 transcription factor enhancing Th2 dependent asthma in mice. 22 By contrast, the NLRP3 inflammasome complex is required for IL1/IL17-dependent asthma. 23 The IL1 family cytokine IL33 is involved in the Th2 response, since antibody neutralization reduces asthma, while the NLRP3 inflammasome is a negative regulator dampening allergic asthma. 24 In addition, RORγc-dependent production of IL17A/F and IL22 by ILC3 and T-cells drives severe neutrophilic asthma, representing additional novel therapeutic targets. With the present insights of the interplay of microbiota, nutrition, infection and the immune response, new preventive/therapeutic options are likely to emerge. Selected food, probiotics, recombinant bacilli, and microbial metabolites such as short chain fatty acids may have anti-inflammatory effects and could be used as complementary prevention or therapy. Reduced production of tryptophan metabolites by microbiota from caspase activation and recruitment domains 9 (CARD9)-deficient mice and patients with Crohn's disease diminish aryl hydrocarbon receptor (AhR) activation and increase inflammation. 25 Therefore, the administration of AhR agonists inducing an AhR-dependent protective IL22 response may be beneficial in asthma. Dr. Ryffel also underlined the use of germfree and mono-colonization with protective bacterial species, which may provide new insights on the protective effect of Clostridia controlling inflammatory Bacteriodetes species. However, investigation by bacterial depletion with antibiotics is problematic due to selection of resistant strains. Dr. Ryffel then reminded the attendees that environmental factors, including air pollution, chemicals, tobacco smoke, ozone and respiratory infections, are other factors influencing the allergic respiratory responses. Experimental studies in mice exposed to ozone develop severe, neutrophilic, steroid resistant airway hyper-reactivity and inflammation representing a major therapeutic challenge. Preliminary data using ozone exposure suggest that this type of severe asthma is sensitive to M3 muscarinic antagonists, but IL17 targeting might also be considered. 26 In addition to classical immune mediators, the targeting of neural reflexes, central nervous system mediators/neuropeptides could represent another exciting and promising approach. The preclinical investigation of novel human targeted therapeutics is required prior to use in patients. Human mAbs, but also single chain antibodies, dominant negative inhibitors, nanofitines, fusion proteins and lipocalins 27 targeting specific human proteins may be tested in non-human primates, if there is sufficient cross-reactivity. The recent development of humanized immune system mice and human knock-in mice, in which mouse protein is replaced by the human analog of, for example, tumor necrosis factor (TNF), CD20, CD64, B-cell activating factor (BAFF), or neonatal Fc receptor (FcRn), are available and may be helpful. These novel humanized mice will enable in vivo investigations of efficacy, pharmacokinetics and, importantly, novel routes of administration such as inhalation. In conclusion, Dr. Ryffel added that basic research leading to new therapeutics that neutralize key-mediators is emerging, which may improve the control of allergic asthma and reduce steroids. Harshad P. Patil (Université Catholique de Louvain) began his talk by reminding attendees of the deleterious role of IL17 in asthma. He showed the benefit of an anti-IL17 Fab' antibody associated with high molecular-weight polyethylene glycol (PEG, 20–40 KDa) and delivered locally into the lungs to achieve local targeted activity against inflammation. The PEGylated anti-IL17 Fab' substantially reduced neutrophils recruitment, lactate dehydrogenase (LDH) and TNF in bronchial-alveolar lavages (BAL) of asthmatic mice. Moreover, weekly pulmonary delivery of 20 µg PEGylated anti-IL17 Fab' was as effective as 10 times-higher doses of non-PEGylated anti-IL17 Fab' given by subcutaneous route. Dr Patil also revealed that PEGylation prolonged residency of anti-IL17 Fab' in the luminal side of the lungs, through different mechanisms like enhanced proteolytic resistance, attachment to the mucus and escape from alveolar macrophages. 28 These findings are promising for the future development of anti-asthma strategies. Session 4: Antibody format Opening session 4, Jörg P. Adamczewski (Sanofi R&D) gave an overview on bi- and multi-specific antibodies from a drug developer's perspective. He raised 2 key questions that were discussed on the basis of current examples from multiple therapeutic areas: 1) how to exploit the biological effects of a multi-specific antibody to achieve a therapeutic effect, and 2) what antibody format to choose for a desired outcome. Bispecific antibodies have 2 biological effects: they bind to 2 targets simultaneously and bring them into spatial proximity. Therefore, which of these mechanisms is exploited to achieve the desired therapeutic effect defines the 2 basic classes of such antibodies. However, even if the spatial action is not the aim, it needs to be considered as a potential source of undesired biological outcomes, especially if neither target is a soluble protein. Dr. Adamczewski reminded attendees that multi-targeting antibodies typically block 2 or more receptors or ligands to inhibit parallel pathways leading to converging biological or pathophysiological processes. However, they can also be used to target an escape pathway at the same time as the principal target, or to achieve deep inhibition by blocking the same pathway at 2 points. As specific example, SAR156597 is a tetravalent bispecific antibody that targets the 2 Th2 cytokines IL4 and IL13. Both cytokines have been implicated in pathophysiology of IPF, a progressive fibrotic disease of the lungs in which 3 key cell types play a possible role in the disease pathogenesis, airway epithelial cells, lung fibroblasts and lung macrophages, making them an attractive target for parallel pathway blockage. He pointed out that SAR156597 is currently the only multi-specific antibody in clinical development for non-infectious respiratory disease, with a Phase 2 proof-of-concept trial in IPF (clinicaltrials.gov/ct2/show/NCT02345070) ongoing. Dr. Adamczewski then highlighted the interest in using bispecific antibodies to bring 2 targets into physical proximity. Spatial use is the most common approach for anti-cancer immune cell engagers, in which the antibody activates an immune effector cell, typically a cytotoxic T-cell targeted on CD3, and homes it to a surface antigen on a tumor cell, resulting in cell death. The approval of blinatumomab (Blincyto®), targeting both CD3 and the B-lineage antigen CD19, for relapsed/refractory acute lymphoblastic leukemia demonstrated the power of such T-cell engagers. 29 An equivalent approach can be used to force the association of 2 proteins, as illustrated by emicizumab, which induces binding of factor IXa to factor X in the absence of sufficient factor VIII, thus restoring blood coagulation. A Phase 3 trial in hemophilia A patients is ongoing (clinicaltrials.gov/ct2/show/NCT02622321). The speaker explained that optimization for different biological properties, but also parallel historical developments, led to over 50 bispecific formats, allowing fine-tuning for the desired biological effect, e.g ., pharmacokinetics (PK), including tissue penetration, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependant cytotoxicity (CDC). Stable symmetric, IgG-like formats such as the dual-variable-domain (DVD) format class or tetravalent bispecific tandem immunoglobulin (TBTi) including SAR156597, combine longer PK, the option of effector functions and greater ease of process development over smaller constructs based on antibody fragments, which may have superior tissue penetration. Dr. Adamczewski indicated that Sanofi's third-generation crossover dual variable Ig-like proteins (CODV) bispecific format allows greater versatility and maintains full binding specificity of both parental antibody sequences. 30 He concluded that multi-specific antibodies are thus a versatile tool to achieve powerful therapeutic effects that currently seems underutilized in respiratory diseases, and may provide additional opportunities for patient benefit in the future. Next, Jean-François Gestin (Inserm U892 – CNRS 6299) gave an overview of the radiolabelled companion diagnostic tests that he introduced as non-invasive "imaging biopsy-like" tools. For instance, he explained that radiolabelled vector 16α-[18F]Fluoro-17β-estradiol ([18F]FES) has proven to be a valuable tracer for the studies of the estrogen receptor (ER) status of primary and metastatic breast cancer. Dr. Gestin then focused on other impressive examples related to radiolabelled companion diagnostic tests, like anti-human epidermal growth factor receptor 2 (HER2) antibody ( 89 Zr-trastuzumab or 64 Cu-DOTA-trastuzumab) to determine the HER2 status in primary breast tumor, but also PK and accessibility to adapt the treatment in case of relapse and metastasis. 31,32 He concluded that radiolabeled companion diagnostics, like radiolabeled mAbs, are supporting tools to aid therapeutic decisions, ensuring effective selection of patient eligible for treatment and giving individuals more effective and safer treatments. They may be of interest for lung cancer patients. Session 5: Inhaled antibodies Theresa Sweeney (Nektar Therapeutics) opened the session focused on use of the pulmonary route to deliver mAbs for respiratory diseases. She noted that mAb therapy is well accepted for the treatment of cancer and auto-immune disease, but few mAbs are approved for the treatment of respiratory disease and none are delivered by the pulmonary route. In theory, inhalation delivery offers the advantage over intravenous delivery of a high dose delivered directly to the target organ, i.e. , lungs, while limiting system exposure and potential side effects. Yet until recently, the delivery efficiency of inhalation devices and the added cost of therapy have limited the development of inhaled biologics for pulmonary disease. Dr. Sweeney reviewed the history of inhaled mAbs delivery by describing the experience with anti-IgE (omalizumab, Xolair®) for the treatment of asthma. She described the rationale for inhalation delivery of anti-IgE and the data that supported the targeting of lung IgE over systemic IgE. 33 Anti-IgE was not successful in ameliorating the broncho-constrictive response in mild asthmatics when delivered by inhalation despite a high lung dose, 34 suggesting that both lung and systemic IgE neutralization was required for efficacy. Full-length mAbs have low bioavailability, 35 and smaller forms could offer advantages because they may better penetrate tissues to reach target receptors. Dr. Sweeney suggested that recent advances in the pathogenesis of asthma and COPD may provide better understanding of therapeutic targets, and that the development of new technologies may offer improvements over full-length mAbs. Lower molecular weight and more potent forms of antibodies are being evaluated for topical delivery, including an anti-IL13 Fab for the treatment of asthma 36 and a highly potent nanobody™ to treat RSV infection. 37 Dr. Sweeney concluded that improved knowledge of therapeutic targets, antibody fragments, potent molecules, and more efficient delivery systems are important for the success of inhalation delivery for the treatment of pulmonary disease. Next, Rita Vanbever (Université catholique de Louvain) unraveled the fate of pulmonary-delivered mAbs. She noted that a major drawback of inhalation is the short residence time of antibodies in the lungs as they are mostly cleared from the lungs within one to 2 d. 28,35 In contrast, plasma half-lives of full-length antibodies after injection reach 3 weeks and more. 38 Accordingly, most antibodies developed for the treatment of respiratory diseases are delivered by injection, 39 except for an inhaled single-domain antibody (Nanobody ® ) specific for RSV fusion protein. Because injection presents limitations, including high delivered doses, low antibody penetration in the lungs and potential systemic side effects, she explained that inhalation of antibody therapeutics deserves further investigation. Based on that, Dr. Vanbever presented her results on antibody formats that would allow the longest retention within the lungs. Compared with full-length antibodies, engineered antibody fragments present advantages, such as enhanced tissue penetration, binding to cryptic epitopes, multi-specific actions and economical production in bacteria. 40 A F(ab') 2 and a Fab' antibody fragments were shown to be as quickly cleared from the lungs as a full-length antibody. 28 Therefore, full-length antibodies do not provide any apparent lung pharmacokinetic advantage over these antibody fragments. Interestingly, she showed that conjugation of antibody fragments to one large molecular weight PEG chain sustained the residence time of the proteins within the lungs. 28 High levels of PEGylated antibody fragments persisted in the lungs for more than 2 d post-delivery. All antibody constructs were principally located within the airway lumen rather than the lung parenchyma. In addition, PEG increased pulmonary retention of antibody fragments through muco-adhesion and escape from alveolar macrophages, rather than by increased hydrodynamic size or improved enzymatic stability. The deeper the deposition site of the antibody constructs within the lungs, the longer the molecules were retained. A two-armed 40 kDa PEG better prolonged residence time than a linear 20 kDa PEG, although the effect of PEG molecular weight was slight. Dr. Vanbever concluded by showing that the increased pulmonary residency of antibody fragments through PEGylation was confirmed in 3 small animal species. Finally, Renaud Respaud (CEPR-INSERM U1100) showed evidence to support that delivery of full-length mAbs through the airways is feasible and relevant in the treatment of respiratory diseases. 35,41-43 When they are delivered through the pulmonary route, mAbs achieved a therapeutic response, in some experimental conditions, greater than when they were delivered systemically. Although the fate of mAbs within the lungs remained unclear after airway delivery, they passed slowly and poorly into the systemic circulation. Dr. Respaud also highlighted the importance of mAb stability during aerosolization, and showed that mesh nebulizers prevented better mAbs degradation and concentration. Addition of surfactant was critical to maintain mAb molecular integrity and pharmacological activity during vibrating-mesh nebulization. Concluding remarks Patrice Diot (Dean of Tours School of Medicine; CEPR-INSERM U1100) concluded the first day of the TMARD symposium by reiterating the basics and challenges related to respiratory medicine. He started by summarizing the challenges in medicine, from the beginning of its history to the current times, i.e. , 1) to take into account the epidemiology of the diseases, and to establish priorities to set up public health programs; 2) to understand their pathophysiology, which has eventually allowed over time to simplify the concepts and to make bridges between various types of disorders; 3) to establish the accurate diagnosis; 4) to prescribe the efficient treatment; and 5) eventually to establish and communicate the prognosis, at the level of individuals and of communities. He underlined that respiratory medicine is special because it mainly concerns diseases which, in terms of epidemiology, are at the 2 extremes, either very frequent, or on the opposite very rare or even orphan, and which, in terms of treatment, can often not be cured nowadays. As examples of frequent, severe and non-curable respiratory diseases worldwide, COPD is projected to be the third leading cause of death by 2020, while lung cancer is currently the first cause of death by cancer, and 16% of children are suffering from asthma. In contrast, cystic fibrosis and IPF seem rarer, extremely severe, and non-curable diseases. In terms of their pathophysiology, Pr. Diot said that: "respiratory diseases, as diseases of any other system in fact, are very simple, as they basically correspond to genetic, infectious, inflammatory or cancer disorders." He pointed out that the concept of the accuracy of a treatment, in medicine in general, is a mixture of its efficiency, side effects, convenience, and price. There have been several periods in the history of medicine with regards to the development of therapeutics: the chemical period, with for example the major advances represented by the discovery of aspirin, penicillin, or streptomycin; then the robotic advent which allowed major advances in surgery; and thereafter the digital period. More recently, personalized therapies, like those based on mAbs, have arisen, especially in respiratory diseases. Pr. Diot concluded that several challenges with regards to the proliferation of mAbs in therapeutics, e.g., the target(s), the physico-chemical characteristics, the route of administration, the safety and the price, and acknowledged that all these aspects have been addressed during the various sessions of the symposium. Session 6: Animal models in respiratory diseases On the second day, Caroline Owen (Brigham and Women's Hospital; Harvard Medical School) started by outlining the healthcare burden associated with COPD. She pointed out that, despite over 50 y of research on COPD, we still lack any disease-modifying medical therapies. She described animal models of COPD that have been used to test hypotheses on pathogenesis or the efficacy of novel therapies for this disease: the current gold standard model for COPD is to expose mice to CS for 6 months. 44 Dr. Owen described the advantages of CS-exposed mice as a model system, including the fact that: 1) they develop some features of the human disease (pulmonary inflammation, small airway fibrosis and airspace enlargement); 44 2) their genome can be manipulated to delete or over-express single genes; 3) they breed rapidly; 4) they are relatively inexpensive to house and study; and 5) tools to study their pathways are generally commercially-available. Dr. Owen then enumerated some disadvantages of CS-exposed mice as a model system for COPD, including the major differences in the anatomy and immunology between humans and mice, the fact that mice develop minimal COPD-like airway disease, they do not develop mucus hyper-secretion like in chronic bronchitis, and the fact that longitudinal blood and lung sampling is challenging or not feasible in mice. Dr. Owen also highlighted that therapies that had efficacy in limiting the progression of COPD-like lung disease in CS-exposed mice were subsequently shown not to have efficacy in limiting disease progression in human COPD patients in randomized clinical trials. 45,46 Dr. Owen then discussed the anatomical and immunological relevance of non-human primates (NHP), and thus how they could have potential as a new animal model of COPD. Thus, she went on to describe a novel NHP larger animal model of COPD-like airway disease that she and her colleagues have recently characterized. 47 After presenting the methodology, she showed that NHPs exposed to CS for 12 weeks did not lose weight, but developed robust COPD-like airway disease, including airway epithelial mucus cell metaplasia, submucosal gland hypertrophy and hyperplasia, had higher airway leukocyte counts, i.e. , macrophages, polymorphonuclear (PMN) leucocytes, and lymphocytes, small airway fibrosis, increases in peri-bronchial lymphoid aggregates, and robust reductions in airway Club cell protein-16 (CCL16) immunostaining similar to that occurring in human COPD airways. 48 Although CS-exposed NHPs did not develop emphysema or increases in lung compliance by 12 weeks, they exhibited changes that contribute to emphysema development including increases in: 1) BAL fluid levels of matrix metalloproteinase-9 (MMP-9), interleukin-8 and CCL2; 2) alveolar macrophage MMP-12 levels; 3) parenchymal macrophage, PMN, and lymphoid aggregate counts; 4) lung oxidative stress levels; and 5) alveolar septal cell apoptosis. CS-exposed NHPs also had a strong trend toward reduction in forced expiratory volume in 0.1 second, which may reflect the small airway disease that develops after 12 weeks of CS exposure. 47 Dr. Owen concluded that CS-exposed NHPs have considerable potential as a model of airway disease phenotypes occurring in COPD patients, especially chronic bronchitis and small airway fibrosis. Unlike mice, NHPs can safely undergo longitudinal sampling in both blood and lung compartments, which could be useful for validating novel biomarkers for COPD, and performing PK and pharmacodynamics (PD) studies of novel therapeutics for COPD-like airway disease. Next, Frédéric Ducancel (iMETI; DRF; CEA; U1184; IDMIT) presented the IDMIT facility for "Infectious Disease Models and Innovative Therapies" (IDMIT, http://www.idmitcenter.fr ) dedicated to preclinical studies using NHP models. He emphasized that these latter are under development in the field of respiratory medicine to test different prophylactic and/or therapeutic candidates for human influenza, whooping-cough, tuberculosis and anthrax. For instance, a model of flu infection is being carried out in collaboration with the Baylor Institute for Immunology Research and Tulane University to study vaccine proteins engineered to specifically target skin and mucosal dendritic cells. The goals are to characterize the early molecular and cellular mechanisms at the skin level following vaccination by in vivo imaging to evaluate immune-induced to vaccine-candidates, and to compare/establish the protective efficacy of vaccine versus challenging with various flu virus strains. Session 7: Relevance of animal models Clive Page (King's College of London) gave an engaging lecture, picking up on Pr. Diot's points and enlightening attendees on the strengths and limitations of animal models to support drug development for respiratory diseases and predict translation to humans. First, he mentioned that the mouse is increasingly being utilized as a model to investigate the pathogenesis of asthma and COPD, and to help in the search for novel treatments. However, there is growing concern about how predictable murine models are in selecting new treatments because there has been a catalog of failures of potential new drugs based on work in the mouse. He explained that a major problem is that much of our understanding of allergic asthma has heavily relied on allergic models in the mouse but there is very little consistency between laboratories concerning the protocols used for sensitization and challenge. The superficial attractiveness of the mouse is the availability of tools and reagents, particularly to investigate the immunological basis of allergic inflammation. However, as stated before by Caroline Owen through the example of COPD, the lung physiology of the mouse is quite different to that of man, and due to their size they are not the most convenient species to reliably measure lung function, particularly using the same animal as its own control to investigate longitudinal changes in lung function or bronchial responsiveness. Furthermore, most models of allergic asthma have been acute and have evaluated drugs on allergen-induced eosinophilia rather than evaluating chronic changes or measurements of lung function. Pr. Page then underlined that one of the major phenotypic characteristic of asthma is bronchial hyperresponsiveness, which is not normalized by current therapies, even following treatment with glucocorticosteroids for a decade. 49 This suggests that mechanisms other than airways inflammation contribute to this phenomenon. 50 Accordingly, animal models that allow longitudinal measurement of lung function in the same animal investigated chronically are required to understand this process in more details. Furthermore, such a model must exhibit the spectrum of airway responsiveness to both direct and indirect acting provocation stimuli as seen in patients with clinical asthma. 51 Then, Pr. Page showed how his group used rabbits and guinea-pigs, rather than just relying on mice, over several decades to investigate the mechanisms of bronchial hyperresponsiveness. 52-54 He first pointed the value of the rabbit in this context as it is of a size that allows minimally invasive lung function recordings to be made in the same animals over time. 52,53 More recently, Pr. Page's group used the guinea pig to aid in the development of a novel class of inhaled bifunctional drug for the treatment of asthma and COPD, the dual phosphodiesterase (PDE) 3/4 inhibitor RPL554, 55 where animal studies predicted the clinical efficacy of this drug in man. RPL554 is now in Phase 2b clinical trials. 56 He concluded on the importance of choosing the right species for drug development, in particular for mAbs. Next, Stéphanie Blanc (Cynbiose) presented a cynomolgus macaque model of mild infection with human RSV (hRSV), 57,58 a common respiratory infection in premature infants and children, associated with a high mortality when developing with other chronic diseases. She showed that age and repeated infections affected virological, clinical and immunological parameters. Even in infant macaques, intranasal hRSV infection induced both local and systemic immune responses to efficiently control the virus. Then, Dr. Blanc illustrated that this model was pharmacologically validated using a reference topical treatment in humans based on palivizumab (Synagis®) administered intranasally, allowing a significant reduction in virus replication. Overall, this model of hRSV is relevant, and therefore, may be used for the testing of new therapies against RSV and different routes of administration. Antoine Guillon (CEPR-INSERM U1100) described a novel method to determine lung PK. First, he reminded the audience that PK studies are required to characterize the kinetics of tissue/fluid deposition, transformation and clearance of an inhaled drug for pulmonary disease. Classically, PK parameters are estimated by monitoring drug concentrations in the systemic circulation, then computed in mathematical compartmental models to predict the behavior of both local and systemically acting drugs. Dr. Guillon emphasized that mAbs do not diffuse passively into organ/tissue compartments; thus, indirect estimation of lung concentrations by modeling from plasma drug profiles is limited and sometimes biased. He provided details on a new method to quantify the time-course exposure of inhaled mAb by direct sampling in the lung parenchyma, using lung microdialysis. Lung microdialysis was already established and validated, but not for large molecules. 59,60 In vitro , the recovery of mAbs with a 1,000 KDa cut-off semi-permeable membrane allowed 34% drug recovery and sampling rate every 180 min with a flow rate of 0.3 µL/min. In vivo , lung microdialysis was set up in NHPs, a relevant animal model for both biotherapeutics and aerosols therapy to attempt the dynamic quantification of mAbs in the interstitial lung space. He explained that a microdialysis probe was implanted in the lung by thoracic surgery immediately after delivery of mAb aerosol in conscious NHPs, and animals were thereafter maintained under prolonged anesthesia and mechanical ventilation for at least 55 hours. Microdialysate and blood samples were collected at time intervals for the determination of mAbs and endogenous/control markers to control the permeability of the probe and determine in vivo mAb recovery. He concluded that the conditions are now established for lung microdialysis of inhaled mAbs targeting soluble-antigens, although this technique remains challenging. Invited lecture on imaging modalities in animals to explore the lungs To complete the "animal models" sessions, Alain Le Pape and Stéphanie Lerondel (Center for Small Animal Imaging, Phenomin-TAAM CNRS) gave an overview of imaging technology to explore the lungs. They described the imaging modalities available for lung exploration: X-ray computed tomography (CT), radioisotopic single photon emission CT (SPECT) or positron emission tomography (PET) imaging and magnetic resonance imaging (MRI), which directly result from medical imaging. Thanks to technological advances, the detectors had been improved for both resolution and sensitivity, making it possible to use these devices in rats and mice to perform lung explorations with sub-millimetric resolution. During the last decade, development of optical approaches such as bioluminescence and near-infrared fluorescence (NIRF) imaging have revolutionized the use of molecular imaging in mice for upstream translational research. Imaging can provide anatomical or functional information, sometimes at a molecular level, that can be combined by multimodality approaches. Pr. Le Pape and Dr. Lerondel discussed the opportunities and applications for lung in vivo imaging, from mice to primates and men, giving examples from lung cancer, asthma, emphysema and illustrating aerosol-based therapies delivery. They highlighted the advantages and limitations of the different techniques depending on their physical basis and the size of the specimen submitted to examination. In particular, they provided details on: 1) the requirement for synchronization of images during acquisition to prevent blurring in imaging at millimetric resolution due to respiratory movements; 2) the dosimetry delivered to the tumor for CT and nuclear bimodalities SPECT/CT and PET/CT for onco-pharmacology studies in mice models to avoid bias; 3) the limitations for accurate quantitative imaging in bioluminescence when the tumor becomes hypoxic; and 4) the potential of NIRF imaging with a variety of probes to explore biomarkers for cancer, inflammation and infection. Drs. Le Pape and Lerondel also described the advanced imaging strategies to assess interaction of mAbs or bioactive molecules with their targets. First, they explained that direct quantitation of the amount of a conjugated molecules concentrated into a lesion is not an accurate quantitation of the specific recognition/interaction because the expanded space of diffusion associated with inflammation, compartments inside the lesion and its environment, enhanced permeability and retention (EPR) effect, Fc interactions with mAbs and many other parameters may contribute to non-specific uptake. Quantitation of interaction requires that imaging data be corrected using those obtained with a representative but non-active molecule, like mutant protein or isotype mAb for example. They also presented some results of high resolution CT and Krypton 81 m ventilation scintigraphy imaging in animal models of asthma and emphysema. They went on to explain the rationale for the choice of the imaging modalities (tomoscintigraphy vs. NIRF imaging) as illustrated with a biodistribution study of cetuximab in a mouse model of lung cancer. Finally, the potential of imaging for translational research was illustrated with aerosolized therapies, e.g. , cetuximab or gemcitabine in lung cancer, papillomavirus vaccine, which provided key information on efficacy, biodistribution and safety assessments in mice, rats, primates and humans. Conclusion Up to 2015, a limited number of mAbs for respiratory disease were in the market, but several molecules were recently approved and numerous are in late phase development. This demonstrates the potential of mAbs to benefit patients with respiratory diseases that still represent unmet medical need. This meeting aimed at giving an overview of the molecules approved or in development for major respiratory diseases, discussing the format and route of administration to improve efficacy and safety of those drugs. As mentioned by Pr. Diot, one should keep in mind that the accuracy of a treatment is a subtle balance between its efficiency, side effects, convenience, and price. Major respiratory diseases are still non-curable, with drugs addressing mainly the symptoms or limiting disease progression rather than reversing it. This symposium also raised concerns regarding relevant animal models for preclinical studies. Although animal models are critical in the development of mAbs, the choice of the species depends on the scientific question to be addressed, and the model that might mimic human disease best. Funding, transparency declarations and disclosures This symposium has been funded with support from the French Higher Education and Research ministry under the program "Investissements d'avenir" Grant Agreement: LabEx MAbImprove ANR-10-LABX-53-01. ARD 2020, GDR ACCITH. Several companies also sponsored this meeting: Astrazeneca, GlaxoSmithKline, Sanofi Genzyme, and Terali. No funder has played any decision-making role in this meeting report. NHV declares receiving financial support by Sanofi Genzyme for study on mAbs inhalation. MH is employed by MedImmune. JPA is an employee and shareholder of Sanofi. TDS is employed by Nektar Therapeutics. The other authors declare neither conflict of interest nor ongoing financial support. All authors declare to have given their agreement for the submission. A professional medical writer, Karen D. Mittleman (Sanofi), was involved in the editing of the manuscript. The editors have our entire permission to reproduce any content of the article, after potential acceptation. Disclosure of potential conflicts of interest NHV declares receivingnancial support by Sanofi Genzyme for study on mAbs inhalation. MH is employed by MedImmune. JPA is an employee and shareholder of Sanofi. TDS is employed by Nektar Therapeutics. The other authors declare neither conflict of interest nor ongoing financial support. Acknowledgments The authors wish to thank all the speakers who participated in the congress and provided a PDF of their presentations, thereby contributing to the report in this way. The authors are grateful to Hervé Watier, Annie Gauvineau, Marc Bonnemaison, Guillaume Parrot, Florence Dambrine, Isabelle Thurmel, and the Scientific Committee for their precious help in organizing the congress. They were responsible for a large part of its success.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2227706/

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores ▿

A single-chain Fv (scFv) antibody was developed and applied for efficient and specific detection of Bacillus anthracis spores. The antibody was isolated from a phage display library prepared from spleens of mice immunized with a water-soluble extract of the outer membrane of the B. anthracis spore (exosporium). The library (7 × 10 6 PFU) was biopanned against live, native B. anthracis ATCC Δ14185 spores suspended in solution, resulting in the isolation of a unique soluble scFv antibody. The antibody was affinity purified and its affinity constant (3 × 10 8 ± 1 × 10 8 M −1 ) determined via flow cytometry (FCM). Preliminary characterization of scFv specificity indicated that the scFv antibody does not cross-react with representatives of some phylogenetically related Bacillus spores. The potential use of scFv antibodies in detection platforms was demonstrated by the successful application of the soluble purified scFv antibody in enzyme-linked immunosorbent assays, immunofluorescence assays, and FCM.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10470788/

Crystal structure and induced stability of trimeric BxpB: implications for the assembly of BxpB-BclA complexes in the exosporium of Bacillus anthracis

ABSTRACT The outermost exosporium layer of Bacillus anthracis spores, the causative agents of anthrax, is comprised of a basal layer and an external hair-like nap. The nap includes filaments composed of trimers of the collagen-like glycoprotein BclA. Essentially all BclA trimers are attached to the spore in a process in which part of the 38-residue amino-terminal domain (NTD) of BclA forms an extremely stable interaction with the basal layer protein BxpB. Evidence indicates that the BclA-BxpB interaction is direct and requires trimeric BxpB. To further investigate the nature of the BclA-BxpB interaction, we determined the crystal structure of BxpB. The structure was trimeric with each monomer consisting of 11 β strands with connecting loops. The structure did not include apparently disordered amino acids 1–19, which contain the only two cysteine residues of the 167-residue BxpB. The orientation of the structure reveals regions of BxpB that could be involved in interacting with the BclA NTD and with adjacent cysteine-rich proteins in the basal layer. Furthermore, the BxpB structure closely resembles that of the 134-residue carboxyl-terminal domain of BclA, which forms trimers that are highly resistant to heat and detergent. We demonstrated that BxpB trimers do not share this resistance. However, when BxpB trimers are mixed with a peptide containing residues 20–38 of BclA, they form a complex that is as stable as BclA-BxpB complexes extracted from spores. Together, our results provide new insights into the mechanism of BclA-BxpB attachment and incorporation into the exosporium. IMPORTANCE The B. anthracis exosporium plays major roles in spore survival and infectivity, but the complex mechanism of its assembly is poorly understood. Key steps in this process are the stable attachment of collagen-like BclA filaments to the major basal layer structural protein BxpB and the insertion of BxpB into an underlying basal layer scaffold. The goal of this study is to further elucidate these interactions thereby advancing our understanding of exosporium assembly, a process shared by many spore-forming bacteria including important human pathogens. IMPORTANCE The B. anthracis exosporium plays major roles in spore survival and infectivity, but the complex mechanism of its assembly is poorly understood. Key steps in this process are the stable attachment of collagen-like BclA filaments to the major basal layer structural protein BxpB and the insertion of BxpB into an underlying basal layer scaffold. The goal of this study is to further elucidate these interactions thereby advancing our understanding of exosporium assembly, a process shared by many spore-forming bacteria including important human pathogens. INTRODUCTION The Gram-positive, aerobic soil bacterium Bacillus anthracis forms spores when starved for nutrients and contact with these spores can cause the potentially lethal disease anthrax in animals and humans. Sporulation starts with an asymmetric septation that divides the vegetative cell into two genome-containing compartments called the mother cell and forespore. The mother cell then engulfs the smaller forespore and surrounds it with three protective layers: a peptidoglycan-containing cortex, a closely apposed proteinaceous coat, and a loosely fitting exosporium ( 1 ). Subsequent lysis of the mother cell releases a dormant spore capable of surviving under harsh conditions for many years. Upon encountering a nutrient-rich aqueous environment, spores can rapidly germinate and grow as vegetative cells. The outermost exosporium layer of B. anthracis spores plays key roles in spore viability ( 2 , 3 ) and apparently in the progression of disease within an infected host ( 4 , 5 ). It also serves as the source of molecular markers used to detect B. anthracis spores ( 6 , 7 ), a preferred weapon of bioterrorism and biological warfare. The exosporium is a prominent bipartite structure comprised of a paracrystalline basal layer and an external hair-like nap ( 8 ). Each filament of the nap is formed solely by a trimer of the collagen-like glycoprotein BclA ( 9 - 11 ). BclA is composed of three domains: a 38-residue amino-terminal domain (NTD), a central collagen-like region containing a strain-specific number of triplet amino-acid repeats, and a 134-residue carboxy-terminal domain (CTD) that promotes trimer formation ( 10 , 12 , 13 ). The collagen-like region and CTD are glycosylated ( 14 , 15 ). In contrast to the nap, the basal layer of the exosporium contains approximately 25 different proteins ( 16 ). One of these proteins is BxpB (also called ExsFA), which is required for the attachment of approximately 98% of the BclA present in the exosporium ( 17 , 18 ). In this process, each filament of the nap is attached to a basal layer surface protrusion that appears to be formed by a trimer of BxpB ( 9 ). Basal layer attachment of BclA occurs through, and requires only, its NTD ( 12 , 19 , 20 ). Efficient attachment requires proteolytic cleavage between BclA residues 19 and 20 ( 19 ), which occurs only after the NTD is bound to the developing forespore ( 20 ). In mature wild-type spores, BclA is included in high molecular mass (>250-kDa) complexes that also include BxpB and in some cases other exosporium proteins, such as ExsY and its homolog CotY ( 17 , 21 ). These complexes are resistant to heat, detergents, and reducing agents, conditions designed to dissociate non-covalently bound protein complexes and to reduce disulfide bonds. These results suggested that BclA and BxpB are attached through a non-disulfide covalent bond ( 16 , 17 ), although attempts to identify such a bond have been unsuccessful. To further investigate the nature of the BclA-BxpB attachment, we determined the crystal structure of BxpB. The resulting trimeric structure revealed surfaces that could physically interact with the NTD of BclA and with other basal layer proteins. The structure of the BxpB monomer closely resembles that of the BclA CTD, which forms extremely stable trimers ( 12 ). BxpB trimers do not share this stability; however, when mixed with a segment of the BclA NTD, they form complexes as stable as BclA-BxpA complexes found in spores. We discuss all these results in terms of mechanisms of BclA-BxpB complex formation and insertion into the exosporium. RESULTS Crystal structure of a BxpB trimer An amino-terminally His 6 -tagged version of the BxpB protein of B. anthracis was expressed in Escherichia coli , affinity purified, and cleaved with Factor Xa protease to precisely remove the His 6 tag and adjacent Xa cleavage site. The His 6 tag and Factor Xa were removed by affinity capture. The resulting highly purified 167-residue BxpB, in Factor Xa cleavage buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM CaCl 2 , hereafter Xa buffer) containing 20 mM dithiothreitol (DTT), was used for crystallization. X-ray diffraction data were collected from a single frozen crystal (see Materials and Methods and Table S1 ). The structure of BxpB was solved by molecular replacement using a homology model and refined to 1.4 à resolution (PDB ID: 8D02). Although it was confirmed by mass spectrometry that the protein used for crystallization contained all 167 residues (data not shown), the structure included only residues 20–167. The crystal structure displayed a trimeric assembly of BxpB monomers ( Fig. 1A and B ). Each monomer folds in a jelly roll-like structure composed of two antiparallel β sheets, labeled A and B, containing six and five strands, respectively ( Fig. 1A ; Fig. S1 ). β sheet B includes two β hairpin motifs. A novel feature of the BxpB crystal structure is a bound calcium ion hexa-coordinated by oxygen atoms of residues Asp87, Ser89, Glu94, and Thr153 located on neighboring β turns ( Fig. S2 ). The side view in Fig. 1A shows that the BxpB termini represented by residues Thr20 and Ser167 are close together at the bottom of the image. The top view of the trimeric structure shows that the β sheets of each monomer form a tight core within the trimeric structure ( Fig. 1C ). Fig 1 Crystal structures of BxpB. ( A ) Diagram showing the structure of the BxpB monomer. N-terminal 19 residues could not be modeled presumably because of disorder. BxpB residues 20 to 167 fold into a jelly roll-like structure consisting of two antiparallel β sheets, A and B, composed of six (cyan) and five (dark blue) β strands, respectively. One Ca 2+ ion (yellow sphere) is hexa-coordinated by amino acids located on two β turns connecting the β sheets. β sheet B exhibits two β hairpins (residues 61–63, 68–70 and 95–102, 114–117) as indicated by analysis of the structure using PROMOTIF ( 22 ). The N- and C-terminal residues of BxpB are labeled. In the crystal structure, residue 20 was modeled as Ala (instead of Thr) as there was no density for the sidechain. Also see topology diagram in Fig. S1 . ( B ) Diagram showing the BxpB trimer formed by threefold symmetry related BxpB monomers in the crystal structure. Monomers are colored cyan, orange, and light pink. N and C termini are labeled. The Ca 2+ ion of each monomer is shown as a sphere, with the yellow sphere labeled. ( C ) Diagram of the BxpB trimer rotated forward 90° relative to panel B to provide a top view of the structure. BxpB is a trimer in solution To test if the trimeric assembly of BxpB (17.3 kDa) in the crystal structure is an artifact of crystallization, we confirmed the oligomeric state of BxpB in solution. A sample of BxpB (0.26 mg/mL) in Xa buffer containing 2 mM Tris(2-carboxyethyl)phosphine (TCEP) was analyzed by size exclusion chromatography with multi-angle light scattering (SEC-MALS). This technique uses UV absorbance, differential refractive index, and multi-angle light scattering to provide an absolute measurement of the molecular mass of a protein or protein complex ( 23 ). The results indicate that essentially all protein in the sample (as judged by the UV scan) migrates as a single species with a molecular mass of 47 kDa ± 2% ( Fig. 2 ). This mass is nearly the same as that expected for a BxpB trimer, which is 52 kDa, indicating that the trimer is a stable and preferred oligomeric state of BxpB. The only other protein species exhibiting a strong light scattering signal appeared to be large aggregates of BxpB with a molecular mass of 1.8 × 10 6 Da. Judged by the UV scan, the amount of protein in this peak was extremely small. The UV scan also indicated the presence of a small amount of protein smaller (slower eluting) than the BxpB trimer; however, the molecular mass of this material could not be determined. Finally, the large differential refractive index peak near the end of the chromatogram is not due to protein but to a system peak resulting from compressed gas in the sample or small differences between sample and system buffers. Fig 2 SEC-MALS analysis of BxpB in Xa buffer. BxpB was separated (eluted with Xa buffer containing 2 mM TCEP) and analyzed using an analytical SEC column. The chromatogram displays UV at 280 nm (green), light scattering (red), differential refractive index (blue), and calculated molar mass (black). Nearly all BxpB (i.e., UV-absorbing material) elutes at 8.5 min with a calculated mass of 47 kDa ± 2, close to that expected for a BxpB trimer. The artifactual system peak is labeled. We also used SEC-MALS to examine a sample of BxpB (0.48 mg/mL) in a different buffer, namely, PBS containing 2 mM TCEP. BxpB trimers were again detected, although they appeared somewhat less stable than those formed in Xa buffer ( Fig. S3 ). The calculated molecular mass for BxpB trimers in PBS was 49 kDa ± 2%, slightly closer to the predicted mass than that observed in Xa buffer. Comparing structures of BxpB and the BclA CTD A previously reported I-TASSER prediction of monomeric BxpB structure indicated that it was homologous to the determined crystal structure of the CTD of BclA ( 9 ). The 134-residue BclA CTD folds into an all-β structure with a jelly roll topology ( 24 ). The structure includes 13 β strands arranged in three antiparallel β sheets. The BclA CTD crystallizes as a tight, globular trimer with the buried core formed by the β strands from each monomeric unit. The N and C termini come together on the side of the trimer that faces the basal layer ( 12 ). No cations (e.g., calcium) were found bound to the protein in the crystal structure. The results of the current study now allow a comparison of experimentally determined crystal structures of BxpB and the BclA CTD. An overlay of monomeric structures of BxpB and the BclA CTD showed that overall structures including the orientation of β strands, loops, and the termini are similar ( Fig. 3 ). For both proteins, the β sheets of each monomer pack to form the trimer (as in Fig. 1C ). However, the root-mean-square deviation measured for the two monomeric structures is 6.84, indicating significant variations in local structures. Fig 3 Overlay of BxpB (purple) and BclA CTD (pink) monomeric crystal structures. The Ca 2+ ion bound to BxpB (yellow sphere) is shown, and the N and C termini of each protein are at the bottom of the image. Comparing stabilities of trimers of BxpB and the CTD of BclA A hallmark of trimers formed by the CTD of BclA is extreme stability. When heated for 8 min in PBS or in sample buffer containing 2% sodium dodecyl sulfate (SDS) and then analyzed by SDS-PAGE, the T m values for CTD trimers were 95°C and 84°C, respectively ( 12 ). In sharp contrast, when BxpB trimers (2 mg/mL) in either Xa buffer or PBS were mixed with 0.25 vol of 5X sample buffer (final SDS and BxpB concentrations of 2% and 1.6 mg/mL, respectively) and immediately analyzed by SDS-PAGE without heating, virtually all BxpB ran as a 17-kDa monomer ( Fig. 4 ). For direct comparison, a sample of BclA CTD trimers (2 mg/mL) in Xa buffer was mixed with 0.25 vol of 5X sample buffer, portions were heated from 22 to 100°C for 8 min, and samples were cooled to room temperature and immediately analyzed by SDS-PAGE ( Fig. 4 ). The results show a T m of approximately 85°C, like that previously reported for BclA trimers examined under similar conditions. Overall, under the conditions examined, BxpB trimers are much less stable than trimers of the BclA CTD. Fig 4 Comparing the stabilities of BxpB trimers and trimers of the BclA CTD. Samples of BxpB trimers in either Xa buffer or PBS and BclA CTD trimers in Xa buffer were made 1× in sample buffer. Portions of the BclA CTD trimer sample were heated at the indicated temperatures for 8 min. A fraction of each sample containing approximately the same amount of protein was analyzed by SDS-PAGE; the Coomassie-stained gel is shown. Filled and open arrowheads indicate the positions of trimeric and monomeric BclA, respectively. Trimers of the BclA CTD migrate faster than predicted from their mass, as previously described ( 12 ). The molecular masses of protein standards are indicated. BxpB trimers and a peptide containing residues 20–38 of the BclA NTD form stable complexes in vitro The NTD of BclA, specifically residues 20–38, forms an extremely stable complex with BxpB during sporulation of B. anthracis ( 12 , 19 , 20 ). To determine if this complex could be recapitulated in vitro , we mixed equal volumes of a 0.2 mM BxpB solution (in Xa buffer) and a 1 mM solution of a peptide containing BclA residues 20–38 (in water) and incubated the sample at room temperature (22°C). After 30 min and 5 h of incubation, a portion of the reaction mixture was removed and mixed with 0.25 vol of 5X sample buffer. For each time point, this sample was divided into two aliquots with one heated at 100°C for 8 min. As a control, we prepared a 0.1 mM solution of BxpB (in 0.5X Xa buffer), which was mixed with sample buffer as above and divided into two aliquots, with one heated at 100°C for 8 min. Equal portions of all samples were analyzed by SDS-PAGE and western blotting with an anti-BxpB mAb ( Fig. 5 ). Fig 5 Formation of stable complexes between BxpB trimers and a peptide containing BclA NTD residues 20–38 in vitro . A reaction mixture containing trimeric BxpB and a 5× molar excess of a peptide containing BclA residues 20–38 was incubated at room temperature for the indicated time (i.e., 30 min and 5 h), made 1× in sample buffer, and a portion heated as described in the text. Same-sized portions of each unheated and heated mixture of BxpB and peptide (+P) and of unheated and heated BxpB alone ( B ) were analyzed by western blotting with an anti-BxpB mAb. The molecular masses of protein standards are indicated. The positions of BxpB monomers ( M ), dimers ( D ), and trimers ( T ) and of the heat-stable BxpB-peptide complexes ( C ) are also marked. The results show that in the unheated samples containing the BclA peptide, a large fraction of BxpB is present in apparently multimeric forms. These include a distinct band with an apparent molecular mass of 52 kDa, the mass expected for a BxpB trimer. Immediately above the putative trimer band, a wide and pronounced band, with a molecular mass from approximately 55 to 65 kDa, was detected. A gel slice containing this material was excised from the gel, treated in situ with chymotrypsin, and analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS), which showed the presence of the BclA NTD peptide. Most likely, the 55 to 65-kDa band represents a complex between BxpB trimers and one or more (probably three) molecules of the BclA peptide. These complexes could be the source of the putative BxpB trimers. Additionally, bands of BxpB-containing material were detected with apparent molecular masses above 75 kDa. These bands were much more abundant in the 5 h sample, and their diffuse shapes and apparent masses suggest that they are larger aggregates of BxpB and the BclA peptide. Based on their apparent masses, some of these larger aggregates could be multimers of the putative BxpB trimer-BclA peptide complex. Inspection of the heated samples showed that the effect of heat (in the presence of detergent) on the various forms of multimeric BxpB was different. The putative (52 kDa) BxpB trimers and >75-kDa species were almost completely destabilized. A similar effect was seen on dimeric BxpB, which can be seen in both the BxpB only and BxpB plus BclA peptide lanes. In contrast, the putative (55 to 65-kDa) BxpB trimer-BclA peptide complexes appeared to be unaffected by the heat treatment. This stability mimics that of BxpB-BclA complexes isolated from B. anthracis spores ( 17 ). A gel slice containing the heat-treated 55 to 65-kDa band was also analyzed by LC-MS/MS as above, which confirmed the presence of the BclA NTD peptide and identified peptides that covered the entire BxpB amino acid sequence. BxpB trimers and a peptide containing residues 20–38 of the BclA NTD form stable complexes in vivo To demonstrate that the same stable BxpB-BclA peptide complexes formed in vitro could be formed in sporulating cells, we constructed a mutant version of the B. anthracis Sterne strain (designated CLT406) in which a stop codon was introduced after codon 38 of the chromosomal bclA gene. Instead of full-length BclA, this strain produces a peptide containing BclA residues 1–38, which should be normally cleaved between residues 19 and 20 during spore formation. Purified spores of this strain were produced and mixed with 1× sample buffer, and exosporium proteins were extracted by heating at 100°C for 8 min. As controls, we extracted exosporium proteins from spores of the Sterne stain and from a ΔbclA variant unable to produce BclA. In addition, we prepared more heat-treated BxpB-BclA peptide complexes in vitro as described above (except incubation was for 1 h). All samples were analyzed by SDS-PAGE and western blotting with an anti-BxpB mAb ( Fig. 6 ). This mAb does not detect the BxpB paralog ExsFB under the conditions employed ( Fig. S4 ). Fig 6 Formation of BxpB trimers and BxpB trimer-BclA NTD peptide complexes in vivo . A heat-treated reaction mixture containing BxpB plus a peptide containing BclA residues 20–38 (+P) and exosporium proteins extracted from an equal number of spores of the CLT406, CLT306 (Δ bclA ), and wild-type Sterne (Strn) strains of B. anthracis were analyzed by SDS-PAGE and western blotting with an anti-BxpB mAb (see text for additional details). Both the Coomassie-stained gel and a western blot are shown. The molecular masses of protein standards are indicated. The positions of BxpB monomers ( M ), dimers ( D ), and putative trimers ( T ) and of the apparent heat-stable BxpB-BclA NTD peptide complexes ( C ) are also marked. The western blot showed striking similarities between the bands from the CLT406 and in vitro samples. Most importantly, the CLT406 lane contained a series of distinct bands with the same apparent aggregate mass as that of the wide band of putative BxpB trimer-BclA peptide complexes formed in vitro . The CLT406 sample also contained a major band at approximately 52 kDa that was presumed to be BxpB trimers. This band was as pronounced as the BclA trimer band detected in an unheated in vitro reaction mixture ( Fig. 5 ). Distinct bands of the putative BxpB trimer-BclA peptide complexes and BxpB trimer were not detected in the ΔbclA and Sterne samples, indicating the dependence of these species on the BclA NTD peptide. Other BxpB-containing material, including monomeric and dimeric BxpB and uncharacterized species larger than BxpB trimer-BclA peptide complexes, was present in similar amounts in the CLT406 and ΔbclA samples. In the case of Sterne spores, except for a small amount of monomer, BxpB was restricted to >250-kDa complexes previously shown to contain BxpB and BclA ( 17 ). Taken together, the results above confirm that similar heat-stable BxpB-BclA NTD peptide complexes are formed in vitro with purified components and in sporulating cells. Crystal structure of a BxpB trimer An amino-terminally His 6 -tagged version of the BxpB protein of B. anthracis was expressed in Escherichia coli , affinity purified, and cleaved with Factor Xa protease to precisely remove the His 6 tag and adjacent Xa cleavage site. The His 6 tag and Factor Xa were removed by affinity capture. The resulting highly purified 167-residue BxpB, in Factor Xa cleavage buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM CaCl 2 , hereafter Xa buffer) containing 20 mM dithiothreitol (DTT), was used for crystallization. X-ray diffraction data were collected from a single frozen crystal (see Materials and Methods and Table S1 ). The structure of BxpB was solved by molecular replacement using a homology model and refined to 1.4 à resolution (PDB ID: 8D02). Although it was confirmed by mass spectrometry that the protein used for crystallization contained all 167 residues (data not shown), the structure included only residues 20–167. The crystal structure displayed a trimeric assembly of BxpB monomers ( Fig. 1A and B ). Each monomer folds in a jelly roll-like structure composed of two antiparallel β sheets, labeled A and B, containing six and five strands, respectively ( Fig. 1A ; Fig. S1 ). β sheet B includes two β hairpin motifs. A novel feature of the BxpB crystal structure is a bound calcium ion hexa-coordinated by oxygen atoms of residues Asp87, Ser89, Glu94, and Thr153 located on neighboring β turns ( Fig. S2 ). The side view in Fig. 1A shows that the BxpB termini represented by residues Thr20 and Ser167 are close together at the bottom of the image. The top view of the trimeric structure shows that the β sheets of each monomer form a tight core within the trimeric structure ( Fig. 1C ). Fig 1 Crystal structures of BxpB. ( A ) Diagram showing the structure of the BxpB monomer. N-terminal 19 residues could not be modeled presumably because of disorder. BxpB residues 20 to 167 fold into a jelly roll-like structure consisting of two antiparallel β sheets, A and B, composed of six (cyan) and five (dark blue) β strands, respectively. One Ca 2+ ion (yellow sphere) is hexa-coordinated by amino acids located on two β turns connecting the β sheets. β sheet B exhibits two β hairpins (residues 61–63, 68–70 and 95–102, 114–117) as indicated by analysis of the structure using PROMOTIF ( 22 ). The N- and C-terminal residues of BxpB are labeled. In the crystal structure, residue 20 was modeled as Ala (instead of Thr) as there was no density for the sidechain. Also see topology diagram in Fig. S1 . ( B ) Diagram showing the BxpB trimer formed by threefold symmetry related BxpB monomers in the crystal structure. Monomers are colored cyan, orange, and light pink. N and C termini are labeled. The Ca 2+ ion of each monomer is shown as a sphere, with the yellow sphere labeled. ( C ) Diagram of the BxpB trimer rotated forward 90° relative to panel B to provide a top view of the structure. BxpB is a trimer in solution To test if the trimeric assembly of BxpB (17.3 kDa) in the crystal structure is an artifact of crystallization, we confirmed the oligomeric state of BxpB in solution. A sample of BxpB (0.26 mg/mL) in Xa buffer containing 2 mM Tris(2-carboxyethyl)phosphine (TCEP) was analyzed by size exclusion chromatography with multi-angle light scattering (SEC-MALS). This technique uses UV absorbance, differential refractive index, and multi-angle light scattering to provide an absolute measurement of the molecular mass of a protein or protein complex ( 23 ). The results indicate that essentially all protein in the sample (as judged by the UV scan) migrates as a single species with a molecular mass of 47 kDa ± 2% ( Fig. 2 ). This mass is nearly the same as that expected for a BxpB trimer, which is 52 kDa, indicating that the trimer is a stable and preferred oligomeric state of BxpB. The only other protein species exhibiting a strong light scattering signal appeared to be large aggregates of BxpB with a molecular mass of 1.8 × 10 6 Da. Judged by the UV scan, the amount of protein in this peak was extremely small. The UV scan also indicated the presence of a small amount of protein smaller (slower eluting) than the BxpB trimer; however, the molecular mass of this material could not be determined. Finally, the large differential refractive index peak near the end of the chromatogram is not due to protein but to a system peak resulting from compressed gas in the sample or small differences between sample and system buffers. Fig 2 SEC-MALS analysis of BxpB in Xa buffer. BxpB was separated (eluted with Xa buffer containing 2 mM TCEP) and analyzed using an analytical SEC column. The chromatogram displays UV at 280 nm (green), light scattering (red), differential refractive index (blue), and calculated molar mass (black). Nearly all BxpB (i.e., UV-absorbing material) elutes at 8.5 min with a calculated mass of 47 kDa ± 2, close to that expected for a BxpB trimer. The artifactual system peak is labeled. We also used SEC-MALS to examine a sample of BxpB (0.48 mg/mL) in a different buffer, namely, PBS containing 2 mM TCEP. BxpB trimers were again detected, although they appeared somewhat less stable than those formed in Xa buffer ( Fig. S3 ). The calculated molecular mass for BxpB trimers in PBS was 49 kDa ± 2%, slightly closer to the predicted mass than that observed in Xa buffer. Comparing structures of BxpB and the BclA CTD A previously reported I-TASSER prediction of monomeric BxpB structure indicated that it was homologous to the determined crystal structure of the CTD of BclA ( 9 ). The 134-residue BclA CTD folds into an all-β structure with a jelly roll topology ( 24 ). The structure includes 13 β strands arranged in three antiparallel β sheets. The BclA CTD crystallizes as a tight, globular trimer with the buried core formed by the β strands from each monomeric unit. The N and C termini come together on the side of the trimer that faces the basal layer ( 12 ). No cations (e.g., calcium) were found bound to the protein in the crystal structure. The results of the current study now allow a comparison of experimentally determined crystal structures of BxpB and the BclA CTD. An overlay of monomeric structures of BxpB and the BclA CTD showed that overall structures including the orientation of β strands, loops, and the termini are similar ( Fig. 3 ). For both proteins, the β sheets of each monomer pack to form the trimer (as in Fig. 1C ). However, the root-mean-square deviation measured for the two monomeric structures is 6.84, indicating significant variations in local structures. Fig 3 Overlay of BxpB (purple) and BclA CTD (pink) monomeric crystal structures. The Ca 2+ ion bound to BxpB (yellow sphere) is shown, and the N and C termini of each protein are at the bottom of the image. Comparing stabilities of trimers of BxpB and the CTD of BclA A hallmark of trimers formed by the CTD of BclA is extreme stability. When heated for 8 min in PBS or in sample buffer containing 2% sodium dodecyl sulfate (SDS) and then analyzed by SDS-PAGE, the T m values for CTD trimers were 95°C and 84°C, respectively ( 12 ). In sharp contrast, when BxpB trimers (2 mg/mL) in either Xa buffer or PBS were mixed with 0.25 vol of 5X sample buffer (final SDS and BxpB concentrations of 2% and 1.6 mg/mL, respectively) and immediately analyzed by SDS-PAGE without heating, virtually all BxpB ran as a 17-kDa monomer ( Fig. 4 ). For direct comparison, a sample of BclA CTD trimers (2 mg/mL) in Xa buffer was mixed with 0.25 vol of 5X sample buffer, portions were heated from 22 to 100°C for 8 min, and samples were cooled to room temperature and immediately analyzed by SDS-PAGE ( Fig. 4 ). The results show a T m of approximately 85°C, like that previously reported for BclA trimers examined under similar conditions. Overall, under the conditions examined, BxpB trimers are much less stable than trimers of the BclA CTD. Fig 4 Comparing the stabilities of BxpB trimers and trimers of the BclA CTD. Samples of BxpB trimers in either Xa buffer or PBS and BclA CTD trimers in Xa buffer were made 1× in sample buffer. Portions of the BclA CTD trimer sample were heated at the indicated temperatures for 8 min. A fraction of each sample containing approximately the same amount of protein was analyzed by SDS-PAGE; the Coomassie-stained gel is shown. Filled and open arrowheads indicate the positions of trimeric and monomeric BclA, respectively. Trimers of the BclA CTD migrate faster than predicted from their mass, as previously described ( 12 ). The molecular masses of protein standards are indicated. BxpB trimers and a peptide containing residues 20–38 of the BclA NTD form stable complexes in vitro The NTD of BclA, specifically residues 20–38, forms an extremely stable complex with BxpB during sporulation of B. anthracis ( 12 , 19 , 20 ). To determine if this complex could be recapitulated in vitro , we mixed equal volumes of a 0.2 mM BxpB solution (in Xa buffer) and a 1 mM solution of a peptide containing BclA residues 20–38 (in water) and incubated the sample at room temperature (22°C). After 30 min and 5 h of incubation, a portion of the reaction mixture was removed and mixed with 0.25 vol of 5X sample buffer. For each time point, this sample was divided into two aliquots with one heated at 100°C for 8 min. As a control, we prepared a 0.1 mM solution of BxpB (in 0.5X Xa buffer), which was mixed with sample buffer as above and divided into two aliquots, with one heated at 100°C for 8 min. Equal portions of all samples were analyzed by SDS-PAGE and western blotting with an anti-BxpB mAb ( Fig. 5 ). Fig 5 Formation of stable complexes between BxpB trimers and a peptide containing BclA NTD residues 20–38 in vitro . A reaction mixture containing trimeric BxpB and a 5× molar excess of a peptide containing BclA residues 20–38 was incubated at room temperature for the indicated time (i.e., 30 min and 5 h), made 1× in sample buffer, and a portion heated as described in the text. Same-sized portions of each unheated and heated mixture of BxpB and peptide (+P) and of unheated and heated BxpB alone ( B ) were analyzed by western blotting with an anti-BxpB mAb. The molecular masses of protein standards are indicated. The positions of BxpB monomers ( M ), dimers ( D ), and trimers ( T ) and of the heat-stable BxpB-peptide complexes ( C ) are also marked. The results show that in the unheated samples containing the BclA peptide, a large fraction of BxpB is present in apparently multimeric forms. These include a distinct band with an apparent molecular mass of 52 kDa, the mass expected for a BxpB trimer. Immediately above the putative trimer band, a wide and pronounced band, with a molecular mass from approximately 55 to 65 kDa, was detected. A gel slice containing this material was excised from the gel, treated in situ with chymotrypsin, and analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS), which showed the presence of the BclA NTD peptide. Most likely, the 55 to 65-kDa band represents a complex between BxpB trimers and one or more (probably three) molecules of the BclA peptide. These complexes could be the source of the putative BxpB trimers. Additionally, bands of BxpB-containing material were detected with apparent molecular masses above 75 kDa. These bands were much more abundant in the 5 h sample, and their diffuse shapes and apparent masses suggest that they are larger aggregates of BxpB and the BclA peptide. Based on their apparent masses, some of these larger aggregates could be multimers of the putative BxpB trimer-BclA peptide complex. Inspection of the heated samples showed that the effect of heat (in the presence of detergent) on the various forms of multimeric BxpB was different. The putative (52 kDa) BxpB trimers and >75-kDa species were almost completely destabilized. A similar effect was seen on dimeric BxpB, which can be seen in both the BxpB only and BxpB plus BclA peptide lanes. In contrast, the putative (55 to 65-kDa) BxpB trimer-BclA peptide complexes appeared to be unaffected by the heat treatment. This stability mimics that of BxpB-BclA complexes isolated from B. anthracis spores ( 17 ). A gel slice containing the heat-treated 55 to 65-kDa band was also analyzed by LC-MS/MS as above, which confirmed the presence of the BclA NTD peptide and identified peptides that covered the entire BxpB amino acid sequence. BxpB trimers and a peptide containing residues 20–38 of the BclA NTD form stable complexes in vivo To demonstrate that the same stable BxpB-BclA peptide complexes formed in vitro could be formed in sporulating cells, we constructed a mutant version of the B. anthracis Sterne strain (designated CLT406) in which a stop codon was introduced after codon 38 of the chromosomal bclA gene. Instead of full-length BclA, this strain produces a peptide containing BclA residues 1–38, which should be normally cleaved between residues 19 and 20 during spore formation. Purified spores of this strain were produced and mixed with 1× sample buffer, and exosporium proteins were extracted by heating at 100°C for 8 min. As controls, we extracted exosporium proteins from spores of the Sterne stain and from a ΔbclA variant unable to produce BclA. In addition, we prepared more heat-treated BxpB-BclA peptide complexes in vitro as described above (except incubation was for 1 h). All samples were analyzed by SDS-PAGE and western blotting with an anti-BxpB mAb ( Fig. 6 ). This mAb does not detect the BxpB paralog ExsFB under the conditions employed ( Fig. S4 ). Fig 6 Formation of BxpB trimers and BxpB trimer-BclA NTD peptide complexes in vivo . A heat-treated reaction mixture containing BxpB plus a peptide containing BclA residues 20–38 (+P) and exosporium proteins extracted from an equal number of spores of the CLT406, CLT306 (Δ bclA ), and wild-type Sterne (Strn) strains of B. anthracis were analyzed by SDS-PAGE and western blotting with an anti-BxpB mAb (see text for additional details). Both the Coomassie-stained gel and a western blot are shown. The molecular masses of protein standards are indicated. The positions of BxpB monomers ( M ), dimers ( D ), and putative trimers ( T ) and of the apparent heat-stable BxpB-BclA NTD peptide complexes ( C ) are also marked. The western blot showed striking similarities between the bands from the CLT406 and in vitro samples. Most importantly, the CLT406 lane contained a series of distinct bands with the same apparent aggregate mass as that of the wide band of putative BxpB trimer-BclA peptide complexes formed in vitro . The CLT406 sample also contained a major band at approximately 52 kDa that was presumed to be BxpB trimers. This band was as pronounced as the BclA trimer band detected in an unheated in vitro reaction mixture ( Fig. 5 ). Distinct bands of the putative BxpB trimer-BclA peptide complexes and BxpB trimer were not detected in the ΔbclA and Sterne samples, indicating the dependence of these species on the BclA NTD peptide. Other BxpB-containing material, including monomeric and dimeric BxpB and uncharacterized species larger than BxpB trimer-BclA peptide complexes, was present in similar amounts in the CLT406 and ΔbclA samples. In the case of Sterne spores, except for a small amount of monomer, BxpB was restricted to >250-kDa complexes previously shown to contain BxpB and BclA ( 17 ). Taken together, the results above confirm that similar heat-stable BxpB-BclA NTD peptide complexes are formed in vitro with purified components and in sporulating cells. DISCUSSION The crystal structure of BxpB provides important new insight into its functioning in exosporium formation. The structure provided the first direct evidence that BxpB forms trimers, which we confirmed as a preferred oligomeric state in solution by SEC-MALS analysis. A trimeric structure for BxpB implies a one-to-one correspondence with attached trimeric BclA filaments. A trimeric structure for BxpB was also predicted from cryo-electron microscopic analyses of exosporia from B. anthracis and closely related Bacillus species (9 , 25 , 26) . Analysis of exosporia from wild-type and mutant ( ΔbclA and ΔbxpB ) B. anthracis spores identified the aforementioned basal layer protrusions to which BclA filaments are attached. Evidence that these protrusions were BxpB trimers was that they were present in wild-type and ΔbclA exosporia but not in the ΔbxpB exosporium and their volume was equal to that calculated for a BxpB trimer ( 9 ). BxpB trimers were also proposed to occupy positions of threefold symmetry within a two-dimensional array of hexagonal subunits that comprise the predominant scaffold of the basal layer ( 9 , 25 , 26 ). The hexagonal subunits of this scaffold appear to be formed by self-assembly of the cysteine-rich protein ExsY and stabilized by disulfide bonding ( 26 ). The positioning of BxpB trimers within the basal layer appears strategic in that, in the absence of BxpB, the major basal layer scaffold is disordered ( 9 ) and multiple exosporium proteins are aberrantly localized ( 16 , 27 , 28 ). It was proposed that BxpB trimers act as a glue that links ExsY hexameric rings together allowing proper assembly of other exosporium proteins ( 9 , 26 ). With respect to the role of BxpB trimers in the assembly of the exosporium overall, it should be noted that the ExsY/BxpB scaffold described above appears to be the major structural element of most but not all of the developing exosporium. The exosporium is divided into two distinct domains: a cap, which is comprised of the first approximately 25% of the exosporium formed and covers one end of the forespore, and a larger noncap region ( 29 ). The ExsY/BxpB scaffold appears to be restricted primarily to the noncap region of the wild-type exosporium. The cap appears to employ an analogous scaffold formed by paralogs of ExsY and BxpB, namely, CotY and ExsFB, respectively ( 16 , 26 , 30 ). ExsFB is required for the attachment of the small amount of BclA that does not involve BxpB ( 17 , 18 ). The crystal structure of BxpB also suggests an orientation for BxpB trimers within the basal layer and a mechanism for BxpB attachment to ExsY. Although the first 19 residues of BxpB are presumably disordered and thus not visible in the crystal structure, the position of residue Thr20 suggests that these disordered residues are most likely positioned at the bottom of the structure shown in Fig. 1B . These residues are particularly noteworthy because they include the longest stretch of nonidentical amino acids when the sequences of BxpB and ExsFB are compared. These 167-residue proteins exhibit 78% sequence identity, but only six of the first 18 residues (and three between positions 5 and 18) are identical. It seems reasonable to suspect that the amino-terminal residues of BxpB and ExsFB (i.e., residues 1–18) are responsible, at least in part, for the differential localization of the two proteins within the exosporium: BxpB binding to ExsY in the noncap region and ExsFB binding to CotY in the cap. Furthermore, the only cysteine residues in BxpB are located within the amino-terminal region at positions 6 and 13. Twelve of the 152 amino acids of ExsY are cysteine residues, and disulfide bond formation involving at least some of these cysteines is important for stabilizing the self-assembled ExsY scaffold ( 26 ). Perhaps, other ExsY cysteines could participate in disulfide bond formation with one or both cysteines of BxpB, allowing covalent attachment of the two proteins. A similar situation could occur with ExsFB, which contains one cysteine residue at position 13, and CotY, in which 14 of its 156 residues are cysteines. Taken together, the observations above suggest that the bottom of the BxpB trimer structure shown in Fig. 1B directly contacts an underlying ExsY scaffold and that this contact could be stabilized by disulfide bond formation between BxpB and ExsY. According to the proposed orientation for BxpB trimers in the basal layer, the top of the structure shown in Fig. 1B would contact the amino-terminal region of an attached BclA trimer. This interaction could be between individual monomers of BclA and BxpB or something more complex. In either event, the region of BxpB proposed to interface with the BclA NTD is comprised of three loops, two of which are involved in binding Ca 2+ ( Fig. 1A and B ). The importance of Ca 2+ in BxpB structure and function is unknown, but Ca 2+ is abundant in spores ( 31 ). Although the crystal structure of the BxpB trimer resembles that of a trimer of the CTD of BclA in several ways, BxpB trimers are much less stable than BclA CTD trimers when treated with heat and detergent. On the other hand, when BxpB trimers are incubated with a peptide containing residues 20–38 of the NTD of BclA, they form a complex with an apparent molecular mass of 55–65 kDa that is as stable to heat and detergent as either BclA CTD trimers or BxpB-BclA complexes extracted from spores. This result provides, for the first time, unambiguous evidence for a direct and highly stable interaction between BxpB and the NTD of BclA. In addition, upon analysis by SDS-PAGE, it appears that 52 kDa BxpB trimers dissociate from the putative BxpB trimer-BclA NTD peptide complexes and that these trimers are much more stable than those formed by purified BxpB. Apparently, binding of the BclA NTD peptide to a BxpB trimer induces a change in the structure of the trimer to a more stable form capable of entrapping the NTD of BclA, and this stable structure persists even after dissociation of the peptide. The structure of BxpB within the BxpB trimer-BclA NTD peptide complex is presently being investigated. We were also able to recapitulate the formation of BxpB trimer-BclA NTD peptide complexes in vivo using a B. anthracis variant (CLT406) that produces the NTD of BclA instead of the full-length protein. These complexes were similar in size and exhibited similar stabilities to those formed in vitro . The heterogeneity in the complex bands, formed both in vitro and in vivo , indicate variability in the stoichiometry of complex components (e.g., number of peptides) and/or the shape of the complexes. We also observed high levels of 52- kDa trimeric BxpB with strain CLT406, which was not observed with wild-type and ΔbclA strains. These results indicate that the interactions between BxpB and the BclA NTD and NTD-induced changes in BxpB trimer structure are the same with purified proteins in a test tube as they are during spore development. The extreme stability of BxpB-BclA complexes extracted from B. anthracis spores and the virtual absence of free BclA in this extract raised the possibility that the connection between the two proteins was covalent ( 17 ). It was also speculated that such a covalent connection was linked to the proteolytic cleavage of the BclA NTD between residues 19 and 20 ( 17 ). However, the formation of BxpB-BclA complexes and their insertion into the basal layer were subsequently shown to occur prior to cleavage of the BclA NTD ( 20 ). Furthermore, we have searched for evidence of a covalent linkage between BxpB and BclA by proteolytically digesting BxpB-BclA complexes extracted from spores and exhaustively analyzing the resulting peptides by LC-MS/MS. No cross-linked peptides containing fragments of both BxpB and BclA were detected. Thus, the case for a covalent linkage between BxpB and BclA is weak. On the other hand, the results presented in this paper make a strong argument for a highly stable but noncovalent interaction between BxpB and BclA. The structural change in BxpB trimers induced by the BclA NTD, which results in a highly stable trimer, could also entrap the BclA NTD in a similarly stable complex. Such entrapment could involve the multiple loops at the top of BxpB as well as other exposed regions of the protein. In addition, BxpB trimers appear to dissociate from BxpB trimer-BclA NTD peptide complexes, indicating that their association with the BclA NTD is noncovalent. In contrast, BclA does not readily dissociate from BxpB-BclA complexes. This difference suggests that trimerization of BclA enhances the stability of BxpB-BclA complexes, an effect that appears to be independent of BclA glycosylation ( 14 ). Furthermore, stable BxpB trimer-BclA NTD peptide complexes form in vitro in the absence of other protein factors and an obvious source of energy that would be required for covalent attachment. The involvement of proteolytic cleavage of the BclA NTD in this process is precluded by the use of a BclA NTD peptide containing only residues 20–38. If the interaction between BxpB and BclA is indeed noncovalent, elucidating the exact nature of the linkage will require additional structural analysis of the BxpB-BclA complex. A detailed understanding of BxpB-BclA attachment will be generally useful as many spore-forming bacteria, including important pathogens, appear to use the same or analogous mechanisms to attach BclA and BclA-like filamentous trimers to the basal layer of the exosporium ( 26 , 32 ). Finally, it is necessary to discuss a recent publication by Durand-Heredia et al. ( 33 ) that suggests a fundamentally different role for BxpB in exosporium assembly than that described in this paper. The critical difference can be summarized by a single statement in Durand-Heredia et al.: "BclA may be stably attached to an exosporium basal layer protein, but that protein is not BxpB." Essentially, two arguments led to this conclusion, and each will be discussed separately. First, according to Durand-Heredia et al., the component of the BxpB-BclA complex that directs its insertion into the basal layer is BclA, not BxpB. In fact, in the absence of BclA, incorporation of BxpB into the basal layer is very inefficient, a co-dependence model first proposed in 2011 ( 34 ). There is clear evidence that contradicts the co-dependence model. This model was first refuted in 2014 in a paper that included the cryo-electron microscopic analysis of exosporia of wild-type, ΔbclA , and ΔbxpB spores of B. anthracis mentioned in the preceding text. The relevant point here is that the structures of wild-type and Δ bclA exosporia are indistinguishable, except for the presence of BclA-containing filaments in the wild-type exosporium. In both cases, the exosporium was highly ordered, and the basal layer exhibited the same level of putative BxpB-containing projections to which filaments are attached in the wild-type exosporium. In sharp contrast, the absence of BxpB resulted in a disordered basal layer that lacked projections, a readily detectable phenotype. Accordingly, the absence of this phenotype in the ΔbclA exosporium indicates a full complement of BxpB without the aid of BclA. Additionally, previous studies have shown that comparable levels of BxpB-containing material are extracted from wild-type and ΔbclA spores ( 17 ). This same result can be seen in Fig. 6 of this paper. Taken together, these data indicate that BxpB does not require BclA for efficient (wild-type level) incorporation into the basal layer. Second, Durand-Heredia et al. state that the actual tight-binding partner for BclA is an unidentified basal layer protein with a mass of 28–32 kDa, hereafter called P30. The most definitive evidence offered is that, in vivo , a His 12 -tagged version of the BclA NTD tightly associates with P30, a complex detected with antiserum against the His tag. This experiment is essentially the same as that with strain CLT406 shown in Fig. 6 , where abundant BxpB trimer-BclA NTD peptide complexes (and BxpB trimers) were detected. However, the BclA NTD-containing complexes in the two experiments appear to be different with apparent molecular masses of 30–40 kDa and 55–65 kDa. It does not appear that Durand-Heredia et al. probed the 30 to 40-kDa complex with anti-BxpB antibodies to exclude the possibility that the complex contained BxpB. At this point, an assessment of the role of P30 in BclA attachment must await its identification. It is possible that the BclA NTD stably interacts with P30 after the insertion of BxpB-BclA complexes into the basal layer, perhaps associated with BclA NTD cleavage. However, extensive published genetic, biochemical, and structural data and the data presented in our paper make an extremely strong case that BxpB, and to a lesser extent ExsFB, are the only basal layer proteins involved in the primary attachment of BclA to the basal layer. MATERIALS AND METHODS Bacterial strains The Sterne 34F2 avirulent veterinary vaccine strain of B. anthracis was used as the wild-type strain and as the parent in strain constructions. Two mutant variants of the Sterne strain were constructed by an allelic exchange on the chromosome essentially as previously described ( 14 , 17 ). These constructs were cured of intermediate plasmids vehicles used to introduce changes into the bclA locus. For strain CLT306 ( ΔbclA ), the entire bclA gene was deleted and replaced with a spectinomycin resistance cassette. For strain CLT406, a TAA stop codon was inserted into the bclA gene immediately after codon 38. This construction also included a spectinomycin resistance cassette inserted after the bclA transcription terminator. All constructions were confirmed by PCR amplification and DNA sequencing of the relevant regions of the chromosome. Strain CLT307 ( ΔbxpB ), another Sterne variant in which codons 1–163 of the bxpB gene were replaced with a spectinomycin resistance cassette, was described previously ( 17 ). Preparation of BxpB and the BclA CTD BxpB was expressed and purified essentially as previously described ( 17 ). Briefly, the bxpB gene of the Sterne strain was expressed in E. coli strain RY3041 (BL21(DE3) slyD ::Tn10) from a modified version of expression vector pET15B (pCLT1733) producing a recombinant BxpB with a His 6 -tag and a Factor Xa cleavage site immediately preceding the BxpB initiating methionine. Recombinant BxpB was purified under native (and reducing) conditions by immobilized metal affinity chromatography (QIAGEN or Cube Biotech), dialyzed into Xa buffer, and cleaved by Factor Xa (New England BioLabs). Passage through a second affinity column removed the His-tag-containing amino-terminal polypeptide, and Factor Xa was removed using a Factor Xa capture kit (Novagen). Purified BxpB was concentrated using an Amicon Ultra-4 centrifugal filter. A His 6 -tagged version of the BclA CTD was expressed in pCLT1218/RY3041 and purified by immobilized metal affinity chromatography essentially as previously described ( 12 ). The resulting recombinant protein, which was dialyzed into Xa buffer, contains the amino-terminal sequence MGSSHHHHHHSSGLVPRGSHNIEGR fused to the 134 amino acids of the CTD of BclA. The concentrations of purified BxpB and His 6 -tagged BclA CTD were measured spectroscopically at 280 nM using calculated molar extinction coefficients (ExPASy). All genetic constructions encoding proteins described above were confirmed by DNA sequence analysis. BclA NTD peptide A ≥98% pure preparation of a peptide containing BclA residues 20–38 was purchased from GenScript. The sequence was confirmed by mass spectrometry. Crystallization, X-ray diffraction data collection, and structure determination of BxpB Purified BxpB was concentrated by ultrafiltration to a final concentration of 28 mg/mL (1.6 mM) and crystallized using the sitting drop vapor diffusion technique. The reservoir solution containing 30% Jeffamine M-600, pH 7.0, and protein solution were mixed in a 1:1 ratio and equilibrated at 22°C. For X-ray data collection, a single crystal was frozen in liquid nitrogen without cryo-preservation. Diffraction data extending to 1.4 à were collected at APS SER-CAT ID synchrotron beam line.22ID on Dectris Eiger X 16M detector at 100K. The crystal structure was solved by a molecular replacement method using Phaser ( 35 ) and a homology model generated using RoseTTAFold ( 36 ). Phenix version 1.19.2 ( 37 ) and Coot ( 38 ) were used for refinement and model building. The crystal structure has been refined to 1.4 à resolution. The final R and R free values are 0.1763 and 0.1894. Details of data collection and refinement statistics are listed in Table S1 . SEC-MALS A 100-µL sample of BxpB was injected onto a WTC-MP015N5 column, 4.6 by 300 mm I.D; particle size, 5 mm; pore size, 150 à (Wyatt Technology, Santa Barbara, CA) on a Shimadzu Prominence HPLC System (Shimadzu Corp., Kyoto, Japan) with an isocratic run at 0.3 mL/min for 17 min, and a solution of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM CaCl2, and 2 mM TCEP was used as the mobile phase. UV absorbance was set at 280 nm using Shimadzu HPLC Explorer software (Shimadzu Corp., Kyoto, Japan). A DAWN 8 MALS detector (Wyatt Technology, Santa Barbara, CA), set at 659 nm, and an Optilab refractometer (Wyatt Technology, Santa Barbara, CA) were used in tandem for detection. Bovine serum albumin (Wyatt Technology, Santa Barbara, CA) was used to normalize the static light scattering detector. The delay volume, band broadening parameters, and the light scattering and differential refractive index measurements were analyzed using Astra 8 software (Wyatt Technology, Santa Barbara, CA). Gel electrophoresis and immunoblotting Purified and exosporium proteins in sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 100 mM DTT, 0.012% bromophenol blue, and 10% (v/v) glycerol were separated by SDS-PAGE in a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and visualized by staining with Coomassie brilliant blue. Before loading samples of extracted exosporium proteins, insoluble material was removed by centrifugation for 1 min at 18,000 × g . For immunoblotting, proteins were electrophoretically transferred from an unstained polyacrylamide gel to a nitrocellulose membrane and treated as described in the manual for the Bio-Rad Immun-Blot HRP Assay kit. The primary antibody used for immunoblotting was the mouse anti-BxpB mAb 10-44-1, prepared as previously described ( 6 , 19 ). Preparation of spores Spores were prepared by growing B. anthracis strains at 37°C on LB agar plates until sporulation was complete, typically 3 d. Spores were washed from plates with cold (4°C) sterile water, collected by centrifugation, washed twice with cold water, and stored in water at 4°C for 12–24 h. Spores were then purified by sedimentation through a two-step gradient of 20% and 45% Isovue-300 (Bracco Diagnostics) and washed three times with cold water. Spores were stored at −20°C and quantitated microscopically with a Petroff-Hausser counting chamber. Mass spectrometry For protein analysis by mass spectrometry, a Coomassie stained protein band was excised from a polyacrylamide gel and digested with sequencing grade chymotrypsin (Promega, Madison, WI) ( 39 ). Proteolytic fragments were analyzed by LC-MS/MS with electrospray ionization using a Thermofisher Hypersil Gold 80 à reverse-phase column (Torrance, CA) and Exion UHPLC linked to a SCIEX 5600 Triple-Tof mass spectrometer (SCIEX, Toronto, Canada). The MS/MS data were processed to provide protein identifications using an in-house Protein Pilot 5.0 search engine (Sciex, Toronto, Canada) using the B. anthracis UniProt protein database and a chymotrypsin plus missed cleavage digestion parameter. Sequences identified in the software were verified by manual de novo sequencing for authenticity against the known sequences of BclA and/or BxpB proteins. Bacterial strains The Sterne 34F2 avirulent veterinary vaccine strain of B. anthracis was used as the wild-type strain and as the parent in strain constructions. Two mutant variants of the Sterne strain were constructed by an allelic exchange on the chromosome essentially as previously described ( 14 , 17 ). These constructs were cured of intermediate plasmids vehicles used to introduce changes into the bclA locus. For strain CLT306 ( ΔbclA ), the entire bclA gene was deleted and replaced with a spectinomycin resistance cassette. For strain CLT406, a TAA stop codon was inserted into the bclA gene immediately after codon 38. This construction also included a spectinomycin resistance cassette inserted after the bclA transcription terminator. All constructions were confirmed by PCR amplification and DNA sequencing of the relevant regions of the chromosome. Strain CLT307 ( ΔbxpB ), another Sterne variant in which codons 1–163 of the bxpB gene were replaced with a spectinomycin resistance cassette, was described previously ( 17 ). Preparation of BxpB and the BclA CTD BxpB was expressed and purified essentially as previously described ( 17 ). Briefly, the bxpB gene of the Sterne strain was expressed in E. coli strain RY3041 (BL21(DE3) slyD ::Tn10) from a modified version of expression vector pET15B (pCLT1733) producing a recombinant BxpB with a His 6 -tag and a Factor Xa cleavage site immediately preceding the BxpB initiating methionine. Recombinant BxpB was purified under native (and reducing) conditions by immobilized metal affinity chromatography (QIAGEN or Cube Biotech), dialyzed into Xa buffer, and cleaved by Factor Xa (New England BioLabs). Passage through a second affinity column removed the His-tag-containing amino-terminal polypeptide, and Factor Xa was removed using a Factor Xa capture kit (Novagen). Purified BxpB was concentrated using an Amicon Ultra-4 centrifugal filter. A His 6 -tagged version of the BclA CTD was expressed in pCLT1218/RY3041 and purified by immobilized metal affinity chromatography essentially as previously described ( 12 ). The resulting recombinant protein, which was dialyzed into Xa buffer, contains the amino-terminal sequence MGSSHHHHHHSSGLVPRGSHNIEGR fused to the 134 amino acids of the CTD of BclA. The concentrations of purified BxpB and His 6 -tagged BclA CTD were measured spectroscopically at 280 nM using calculated molar extinction coefficients (ExPASy). All genetic constructions encoding proteins described above were confirmed by DNA sequence analysis. BclA NTD peptide A ≥98% pure preparation of a peptide containing BclA residues 20–38 was purchased from GenScript. The sequence was confirmed by mass spectrometry. Crystallization, X-ray diffraction data collection, and structure determination of BxpB Purified BxpB was concentrated by ultrafiltration to a final concentration of 28 mg/mL (1.6 mM) and crystallized using the sitting drop vapor diffusion technique. The reservoir solution containing 30% Jeffamine M-600, pH 7.0, and protein solution were mixed in a 1:1 ratio and equilibrated at 22°C. For X-ray data collection, a single crystal was frozen in liquid nitrogen without cryo-preservation. Diffraction data extending to 1.4 à were collected at APS SER-CAT ID synchrotron beam line.22ID on Dectris Eiger X 16M detector at 100K. The crystal structure was solved by a molecular replacement method using Phaser ( 35 ) and a homology model generated using RoseTTAFold ( 36 ). Phenix version 1.19.2 ( 37 ) and Coot ( 38 ) were used for refinement and model building. The crystal structure has been refined to 1.4 à resolution. The final R and R free values are 0.1763 and 0.1894. Details of data collection and refinement statistics are listed in Table S1 . SEC-MALS A 100-µL sample of BxpB was injected onto a WTC-MP015N5 column, 4.6 by 300 mm I.D; particle size, 5 mm; pore size, 150 à (Wyatt Technology, Santa Barbara, CA) on a Shimadzu Prominence HPLC System (Shimadzu Corp., Kyoto, Japan) with an isocratic run at 0.3 mL/min for 17 min, and a solution of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM CaCl2, and 2 mM TCEP was used as the mobile phase. UV absorbance was set at 280 nm using Shimadzu HPLC Explorer software (Shimadzu Corp., Kyoto, Japan). A DAWN 8 MALS detector (Wyatt Technology, Santa Barbara, CA), set at 659 nm, and an Optilab refractometer (Wyatt Technology, Santa Barbara, CA) were used in tandem for detection. Bovine serum albumin (Wyatt Technology, Santa Barbara, CA) was used to normalize the static light scattering detector. The delay volume, band broadening parameters, and the light scattering and differential refractive index measurements were analyzed using Astra 8 software (Wyatt Technology, Santa Barbara, CA). Gel electrophoresis and immunoblotting Purified and exosporium proteins in sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 100 mM DTT, 0.012% bromophenol blue, and 10% (v/v) glycerol were separated by SDS-PAGE in a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and visualized by staining with Coomassie brilliant blue. Before loading samples of extracted exosporium proteins, insoluble material was removed by centrifugation for 1 min at 18,000 × g . For immunoblotting, proteins were electrophoretically transferred from an unstained polyacrylamide gel to a nitrocellulose membrane and treated as described in the manual for the Bio-Rad Immun-Blot HRP Assay kit. The primary antibody used for immunoblotting was the mouse anti-BxpB mAb 10-44-1, prepared as previously described ( 6 , 19 ). Preparation of spores Spores were prepared by growing B. anthracis strains at 37°C on LB agar plates until sporulation was complete, typically 3 d. Spores were washed from plates with cold (4°C) sterile water, collected by centrifugation, washed twice with cold water, and stored in water at 4°C for 12–24 h. Spores were then purified by sedimentation through a two-step gradient of 20% and 45% Isovue-300 (Bracco Diagnostics) and washed three times with cold water. Spores were stored at −20°C and quantitated microscopically with a Petroff-Hausser counting chamber. Mass spectrometry For protein analysis by mass spectrometry, a Coomassie stained protein band was excised from a polyacrylamide gel and digested with sequencing grade chymotrypsin (Promega, Madison, WI) ( 39 ). Proteolytic fragments were analyzed by LC-MS/MS with electrospray ionization using a Thermofisher Hypersil Gold 80 à reverse-phase column (Torrance, CA) and Exion UHPLC linked to a SCIEX 5600 Triple-Tof mass spectrometer (SCIEX, Toronto, Canada). The MS/MS data were processed to provide protein identifications using an in-house Protein Pilot 5.0 search engine (Sciex, Toronto, Canada) using the B. anthracis UniProt protein database and a chymotrypsin plus missed cleavage digestion parameter. Sequences identified in the software were verified by manual de novo sequencing for authenticity against the known sequences of BclA and/or BxpB proteins. DATA AVAILABILITY Atomic coordinates and structure factors for BxpB have been deposited in the Protein Data Bank: PDB ID: 3D02 and Deposition ID D_1000263664. DIRECT CONTRIBUTION This article is a direct contribution from Charles L. Turnbough, Jr., a Fellow of the American Academy of Microbiology, who arranged for and secured reviews by Charles Moran, Jr., Emory University, and Tina Henkin, Ohio State University. SUPPLEMENTAL MATERIAL The following material is available online at https://doi.org/10.1128/mbio.01172-23 . 10.1128/mbio.01172-23.SuF1 Fig. S1 mbio.01172-23-s0001.tif Topology diagram of BxpB generated by using PDBsum. Click here for additional data file. 10.1128/mbio.01172-23.SuF2 Fig. S2 mbio.01172-23-s0002.tiff Ca 2+ coordination within the BxpB crystal structure. Click here for additional data file. 10.1128/mbio.01172-23.SuF3 Fig. S3 mbio.01172-23-s0003.tif SEC-MALS analysis of BxpB in PBS. Click here for additional data file. 10.1128/mbio.01172-23.SuF4 Fig. S4 mbio.01172-23-s0004.tif Examining the cross-reactivity of anti-BxpB mAb 10-44-1. Click here for additional data file. 10.1128/mbio.01172-23.SuF5 Table S1 mbio.01172-23-s0005.docx X-ray data collection and refinement statistics. Click here for additional data file. ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2243339/

Value of ICD-9 coded chief complaints for detection of epidemics.

To assess the value of ICD-9 coded chief complaints for early detection of epidemics, we measured sensitivity, positive predictive value, and timeliness of Influenza detection using a respiratory set (RS) of ICD-9 codes and an Influenza set (IS). We also measured inherent timeliness of these data using the cross-correlation function. We found that, for a one-year period, the detectors had sensitivity of 100% (1/1 epidemic) and positive predictive values of 50% (1/2) for RS and 25% (1/4) for IS. The timeliness of detection using ICD-9 coded chief complaints was one week earlier than the detection using Pneumonia and Influenza deaths (the gold standard). The inherent timeliness of ICD-9 data measured by the cross-correlation function was two weeks earlier than the gold standard.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9408887/

Development of Hydroxamic Acid Compounds for Inhibition of Metallo-β-Lactamase from Bacillus anthracis

The emergence of resistant bacteria takes place, endangering the effectiveness of antibiotics. A reason for antibiotic resistance is the presence of lactamases that catalyze the hydrolysis of β-lactam antibiotics. An inhibitor of serine-β-lactamases such as clavulanic acid binds to the active site of the enzymes, thus solving the resistance problem. A pressing issue, however, is that the reaction mechanism of metallo-β-lactamases (MBLs) hydrolyzing β-lactam antibiotics differs from that of serine-β-lactamases due to the existence of zinc ions in the active site of MBLs. Thus, the development of potential inhibitors for MBLs remains urgent. Here, the ability to inhibit MBL from Bacillus anthracis (Bla2) was investigated in silico and in vitro using compounds possessing two hydroxamate functional groups such as 3-chloro-N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)benzamide (Compound 4 ) and N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)-3-methoxybenzamide (Compound 6 ). In silico docking and molecular dynamics simulations revealed that both Compounds 4 and 6 were coordinated with zinc ions in the active site, suggesting that the hydroxamate group attached to the aromatic ring of the compound plays a crucial role in the coordination to the zinc ions. In vitro kinetic analysis demonstrated that the mode of inhibitions for Compounds 4 and 6 were a competitive inhibition with K i values of 6.4 ± 1.7 and 4.7 ± 1.4 kcal/mol, respectively. The agreement between in silico and in vitro investigations indicates that compounds containing dihyroxamate moieties may offer a new avenue to overcome antibiotic resistance to bacteria. 1. Introduction Anthrax is an infectious disease caused by the Gram-positive bacteria Bacillus anthracis . This infection is commonly found in mammals; however, it could be fatal if the inhalation infection cases are not treated soon after exposure [ 1 , 2 ]. An infection of B. anthracis used to be treated with a class of β-lactam antibiotics, but the increased occurrence of antibiotic-resistant strains has made treating such infections increasingly difficult. This emergence of antibiotic resistance has been partially attributed to the production of β-lactamase enzymes capable of hydrolyzing the β-lactam ring within many types of these common antibiotics, and thereby rendering them ineffective in fighting off infection. Today the different variants of β-lactamases discovered can be categorized into the four groups A, B, C, and D [ 3 ]. Groups A, C, and D make up a class of β-lactamases called serine-β-lactamases (SBLs) [ 3 ]. This class relies on an active site serine residue that functions as the nucleophile in hydrolysis of the β-lactam substrate [ 3 ]. The group B β-lactamases, commonly referred to as metallo-β-lactamases (MBLs), require the presence of Zn(II) ions within the active site to carry out catalytic activity [ 3 ]. This group of MBLs can be further divided into three subgroups (B1, B2, B3) that vary primarily in substrate preference and the number of Zn(II) ions they require [ 4 ]. The B1 subgroup can require one or two Zn(II) ions, referred to as Zn 1 and Zn 2 . The B2 subgroup generally prefers only one Zn(II) ion, and the B3 subgroup often requires two Zn(II) ions where Zn 2 is stabilized by an additional His residue in place of a Cys [ 4 ]. The Sterne strain of B. anthracis produces an MBL of the B1 subtype, referred to as Bla2 [ 5 , 6 ]. The Bla2 active site can contain both the tightly bound Zn 1 and loosely bound Zn 2 , with the presence or absence of Zn 2 having no significant impact on catalytic activity [ 3 ]. Some clinically available β-lactamase inhibitors, such as clavulanic acid and tazobactam, are used to inhibit a wide array of SBLs but remain ineffective against MBLs today [ 3 ]. This is due in part to the broad substrate profile that MBLs show in hydrolyzing β-lactam antibiotics [ 7 ]. Given that MBLs rely on the presence of Zn(II) ions within their active sites, a promising approach to developing broad-spectrum MBL inhibitors could be chelating of the active site Zn(II) ions. It has been shown that metalloproteinases can be effectively inhibited by compounds containing a hydroxamic acid group that binds the catalytically crucial zinc ions, and thereby inactivating the enzyme [ 8 ]. The use of hydroxamic acid compounds was further investigated in the context of inhibiting Aeromonas hydrophila , FEZ-1, and B. anthracis MBLs, all with promising results [ 5 , 9 , 10 ]. Here we seek to further explore the potential for two newly-developed hydroxamic acid compounds to inhibit Bla2 activity by way of binding active site Zn(II) ions. The ability of each compound to effectively bind the Zn(II) ions within the active site was studied by methods of in silico and in vitro analysis. We performed a series of molecular dynamics (MD) simulations of Bla2 in complex with the newly developed hydroxamic acid inhibitors and the common hydroxamic acid compound suberoylanilide hydroxamic acid (SAHA). Here we report the results of three simulations in which enzyme-inhibitor stability and key interactions are compared, and later kinetic analysis that was used to experimentally test the ability of each compound to inhibit Bla2. 2. Results 2.1. Synthesis of Compound 4 and 6 We previously synthesized Compound 2 without any substituents as zinc-dependent enzymes including inhibitors for MBLs [ 5 ]. Syntheses of Compound 4 with meta-chloro and Compound 6 with meta-methoxy on benzohydramate moiety were similar to its Compound 2 ( Scheme 1 ) [ 5 ]. Subsequent reaction of either 3 or 5 with aqueous hydroxylamine (3 eq) and potassium hydroxide (4 eq) in the presence of methanol yielded di-hydroxamic acid derivatives 4 and 6 in good yields (72 and 66%, respectively). These compounds were synthesized to learn the influence of enzymatic activities and selectivity at the active site of Bla2 due to their different sizes and polarities. 2.2. Molecular Docking To obtain models of the inhibitors Compound 4 , Compound 6 , and SAHA in complex with Bla2 the molecular docking simulations were carried out. The docking result of lowest energy was selected from each complex and used for further dynamics study ( Table 1 ). In reviewing the conformation for the complex chosen for 3-chloro-N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)benzamide (Compound 4 ) and N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)-3-methoxybenzamide (Compound 6 ), a hydroxamate group attached to the aromatic end (referred to as the aromatic hydroxamate group) can be found binding both Zn(II) ions. The hydrophobic nature of the active site residues F63 and V68 seem to form a more nonpolar environment favoring the aromatic end of the compounds. In addition, several potential hydrogen bonds were identified between the compounds (Compound 4 and Compound 6 ) and the active site residues, with N209 forming a potential hydrogen bond with the aromatic hydroxamate group, and K200, S201, and H239, forming a potential hydrogen bond with a hydroxamate group attached to the aliphatic end, referred to as the aliphatic hydroxamate group ( Figure 1 A,B). The side chain of residue N209 is also within range to form a hydrogen bond with the methoxy of Compound 6 ( Figure 1 B). For the SAHA conformation, the aliphatic hydroxamate group is bound to the Zn(II) ions and also could potentially form hydrogen bonds with N209. The amide group toward the aromatic end of SAHA also forms a potential hydrogen bond with K200. In reviewing all of the intermolecular interactions, most of the stability of SAHA within the Bla2 active site comes from the hydrophobic interactions between the aliphatic backbone of SAHA and the nonpolar active site residues F63, W88, and V68 ( Figure 1 C). 2.3. Molecular Dynamics A comparison of the Bla2 backbone RMSDs between the simulations demonstrates that Bla2 stability is achieved around 0.17 nm in relation to the energy minimized conformation, indicating that the systems are sufficiently equilibrated within at most 5 ns ( Figure 2 A). The Bla2 backbone RMSDs for all three systems were relatively similar ( Figure 2 A). In comparing the RMSDs of the Compounds themselves ( Figure 2 B), one can see that the Bla2:Inhibitor complex for each system is maintained throughout the simulation. It should be mentioned here that the RMSD of SAHA appears to be rather different from the cases of Compounds 4 and 6 . The possible explanation would be that the aliphatic hydroxamate of SAHA binds to the zinc ion whereas the binding hydroxamate of Compounds 4 and 6 is opposite. In addition, we performed a 70 ns simulation of Bla2 alone to further investigate the stability of the protein. Given the radius of gyration of the Bla2 backbone structure in the 70 ns simulation, the structure remains stable throughout the simulation process, indicating no significant unfolding of the protein structure; furthermore, the RMSD of the Bla2 structure in the 70 ns simulation revealed that the stability of the protein backbone is maintained around 0.175 nm ( Supplemental Figure S5 ). Both Compounds 4 and 6 deviate only within a range of 0.15–0.3 nm in relation to the protein active site. In comparing this analysis to the RMSD of SAHA, minimal difference in complex stability is seen. To develop a more detailed understanding of the interactions taking place between Bla2 and the inhibitors, the decomposition data ( Supplemental Figures S3 and S4 ) for each system were analyzed. In reviewing the dynamics for Compound 4 , the aromatic hydroxamate group remains bound to both Zn(II) ions. Throughout the simulation, a potential parallel π-stacking interaction between the aromatic ring of Compound 4 and the side chain of H239 is sustained ( Figure 3 A). This π-stacking interaction and the nonpolar environment provided by active site residues, are the likely reason for the stability and lack of torsion throughout the simulation for the aromatic end of Compound 4 . The aliphatic hydroxamic group continued to form frequent hydrogen bonds with the side chain of K200, backbone of H239, and the side chain of S201 ( Figure 3 A). The most dominant residues in stabilizing Compound 4 were H239 with the π-stacking interactions and hydrogen bonding, in addition to the hydrogen bonding of the K200 side chain ( Supplemental Figure S4 ). The same π-stacking interaction of H239, and hydrophobic surrounding of F63 and V68, can be found stabilizing the aromatic ring of Compound 6 ( Figure 3 B). Similar to Compound 4 , the aromatic hydroxamate group of Compound 6 remained bound to both Zn(II) ions throughout the simulation. The aliphatic hydroxamic group of Compound 6 manages to sustain a noticeably stable network of hydrogens bonds between the K200, S201, and H239 ( Figure 3 B). In reviewing the dynamics for SAHA, the aliphatic hydroxamic group does remain bound to both Zn(II) ions. The aromatic end of SAHA associates mostly with peripheral residues of the Bla2 active site ( Supplemental Figure S1 ). The stability of SAHA within the Bla2 active site seems to be sustained primarily by way of hydrophobic interactions with the F63 and V68 residues. Although it does form occasional hydrogen bonds with H239 and K200, these interactions with Bla2 do not appear to be as stable as they are for Compounds 4 and 6 . In calculating the end-state free energy of each simulation, the free energy change of the Compound 6 :Bla2 complex was the most stable ( Table 1 ), while the free energy changes calculated for the Compound 4 :Bla2 and SAHA:Bla2 complexes were higher in value and relatively similar to one another ( Table 1 ). Looking further into the free energy components for each system, Compounds 4 and 6 had electrostatic energy contributions of −32.50 ± 1.05 kcal/mol and −38.96 ± 1.18 kcal/mol, respectively. This is in comparison to the electrostatic energy contribution of SAHA at −0.16 ± 0.98 kcal/mol. 2.4. Inhibition Tests To explore the possibility of inhibition of Bla2 activity by Compound 4 and 6 , IC 50 values were determined. As a control experiment, SAHA was used due to the presence of a hydroxamate functional group in the compound. Concentrations of Compound 4 and 6 were used from a range of 0.5 μM to 100 mM to gain IC 50 values. All the data obtained were fit to a concentration-response plot with the equation v i / v o = 1/(1 + ([I]/IC 50 ) h ), where I is an inhibitor and h is the Hill coefficient (the Hill coefficients used were between 0.5 and 1). As shown in Figure 4 , the data points for compound 4 were well fit to the semilog concentration-response plot, and the IC 50 was able to be calculated from the plot at 50% inhibition: that is, 20.0 ± 5.0 μM. Compound 6 behaves similarly to Compound 4 with the IC 50 value of 14.9 ± 9.8 μM ( Figure 4 ). However, the IC 50 value for SAHA was higher than 100 μM, which is too high to be considered as a drug candidate ( Supplemental Figure S2 ). Attempts were made to explore the mode of inhibition, inhibitory enzyme assays were carried out using various concentrations of the substrate nitrocefin as well as Compound 4 and 6 . Figure 5 shows Lineweaver–Burk plots with a nest of lines that intersect at the y -axis for both Compound 4 and 6 . K i values were determined on the basis of the plots with a K i value of 6.4 ± 1.7 μM for Compound 4 and a K i value of 4.7 ± 1.4 μM for compound 6 , where K i values were calculated from the slope based on K m and apparent K m values were obtained from Figure 5 . The IC 50 and K i values for Compound 4 and 6 and SAHA are listed in Table 2 . 2.1. Synthesis of Compound 4 and 6 We previously synthesized Compound 2 without any substituents as zinc-dependent enzymes including inhibitors for MBLs [ 5 ]. Syntheses of Compound 4 with meta-chloro and Compound 6 with meta-methoxy on benzohydramate moiety were similar to its Compound 2 ( Scheme 1 ) [ 5 ]. Subsequent reaction of either 3 or 5 with aqueous hydroxylamine (3 eq) and potassium hydroxide (4 eq) in the presence of methanol yielded di-hydroxamic acid derivatives 4 and 6 in good yields (72 and 66%, respectively). These compounds were synthesized to learn the influence of enzymatic activities and selectivity at the active site of Bla2 due to their different sizes and polarities. 2.2. Molecular Docking To obtain models of the inhibitors Compound 4 , Compound 6 , and SAHA in complex with Bla2 the molecular docking simulations were carried out. The docking result of lowest energy was selected from each complex and used for further dynamics study ( Table 1 ). In reviewing the conformation for the complex chosen for 3-chloro-N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)benzamide (Compound 4 ) and N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)-3-methoxybenzamide (Compound 6 ), a hydroxamate group attached to the aromatic end (referred to as the aromatic hydroxamate group) can be found binding both Zn(II) ions. The hydrophobic nature of the active site residues F63 and V68 seem to form a more nonpolar environment favoring the aromatic end of the compounds. In addition, several potential hydrogen bonds were identified between the compounds (Compound 4 and Compound 6 ) and the active site residues, with N209 forming a potential hydrogen bond with the aromatic hydroxamate group, and K200, S201, and H239, forming a potential hydrogen bond with a hydroxamate group attached to the aliphatic end, referred to as the aliphatic hydroxamate group ( Figure 1 A,B). The side chain of residue N209 is also within range to form a hydrogen bond with the methoxy of Compound 6 ( Figure 1 B). For the SAHA conformation, the aliphatic hydroxamate group is bound to the Zn(II) ions and also could potentially form hydrogen bonds with N209. The amide group toward the aromatic end of SAHA also forms a potential hydrogen bond with K200. In reviewing all of the intermolecular interactions, most of the stability of SAHA within the Bla2 active site comes from the hydrophobic interactions between the aliphatic backbone of SAHA and the nonpolar active site residues F63, W88, and V68 ( Figure 1 C). 2.3. Molecular Dynamics A comparison of the Bla2 backbone RMSDs between the simulations demonstrates that Bla2 stability is achieved around 0.17 nm in relation to the energy minimized conformation, indicating that the systems are sufficiently equilibrated within at most 5 ns ( Figure 2 A). The Bla2 backbone RMSDs for all three systems were relatively similar ( Figure 2 A). In comparing the RMSDs of the Compounds themselves ( Figure 2 B), one can see that the Bla2:Inhibitor complex for each system is maintained throughout the simulation. It should be mentioned here that the RMSD of SAHA appears to be rather different from the cases of Compounds 4 and 6 . The possible explanation would be that the aliphatic hydroxamate of SAHA binds to the zinc ion whereas the binding hydroxamate of Compounds 4 and 6 is opposite. In addition, we performed a 70 ns simulation of Bla2 alone to further investigate the stability of the protein. Given the radius of gyration of the Bla2 backbone structure in the 70 ns simulation, the structure remains stable throughout the simulation process, indicating no significant unfolding of the protein structure; furthermore, the RMSD of the Bla2 structure in the 70 ns simulation revealed that the stability of the protein backbone is maintained around 0.175 nm ( Supplemental Figure S5 ). Both Compounds 4 and 6 deviate only within a range of 0.15–0.3 nm in relation to the protein active site. In comparing this analysis to the RMSD of SAHA, minimal difference in complex stability is seen. To develop a more detailed understanding of the interactions taking place between Bla2 and the inhibitors, the decomposition data ( Supplemental Figures S3 and S4 ) for each system were analyzed. In reviewing the dynamics for Compound 4 , the aromatic hydroxamate group remains bound to both Zn(II) ions. Throughout the simulation, a potential parallel π-stacking interaction between the aromatic ring of Compound 4 and the side chain of H239 is sustained ( Figure 3 A). This π-stacking interaction and the nonpolar environment provided by active site residues, are the likely reason for the stability and lack of torsion throughout the simulation for the aromatic end of Compound 4 . The aliphatic hydroxamic group continued to form frequent hydrogen bonds with the side chain of K200, backbone of H239, and the side chain of S201 ( Figure 3 A). The most dominant residues in stabilizing Compound 4 were H239 with the π-stacking interactions and hydrogen bonding, in addition to the hydrogen bonding of the K200 side chain ( Supplemental Figure S4 ). The same π-stacking interaction of H239, and hydrophobic surrounding of F63 and V68, can be found stabilizing the aromatic ring of Compound 6 ( Figure 3 B). Similar to Compound 4 , the aromatic hydroxamate group of Compound 6 remained bound to both Zn(II) ions throughout the simulation. The aliphatic hydroxamic group of Compound 6 manages to sustain a noticeably stable network of hydrogens bonds between the K200, S201, and H239 ( Figure 3 B). In reviewing the dynamics for SAHA, the aliphatic hydroxamic group does remain bound to both Zn(II) ions. The aromatic end of SAHA associates mostly with peripheral residues of the Bla2 active site ( Supplemental Figure S1 ). The stability of SAHA within the Bla2 active site seems to be sustained primarily by way of hydrophobic interactions with the F63 and V68 residues. Although it does form occasional hydrogen bonds with H239 and K200, these interactions with Bla2 do not appear to be as stable as they are for Compounds 4 and 6 . In calculating the end-state free energy of each simulation, the free energy change of the Compound 6 :Bla2 complex was the most stable ( Table 1 ), while the free energy changes calculated for the Compound 4 :Bla2 and SAHA:Bla2 complexes were higher in value and relatively similar to one another ( Table 1 ). Looking further into the free energy components for each system, Compounds 4 and 6 had electrostatic energy contributions of −32.50 ± 1.05 kcal/mol and −38.96 ± 1.18 kcal/mol, respectively. This is in comparison to the electrostatic energy contribution of SAHA at −0.16 ± 0.98 kcal/mol. 2.4. Inhibition Tests To explore the possibility of inhibition of Bla2 activity by Compound 4 and 6 , IC 50 values were determined. As a control experiment, SAHA was used due to the presence of a hydroxamate functional group in the compound. Concentrations of Compound 4 and 6 were used from a range of 0.5 μM to 100 mM to gain IC 50 values. All the data obtained were fit to a concentration-response plot with the equation v i / v o = 1/(1 + ([I]/IC 50 ) h ), where I is an inhibitor and h is the Hill coefficient (the Hill coefficients used were between 0.5 and 1). As shown in Figure 4 , the data points for compound 4 were well fit to the semilog concentration-response plot, and the IC 50 was able to be calculated from the plot at 50% inhibition: that is, 20.0 ± 5.0 μM. Compound 6 behaves similarly to Compound 4 with the IC 50 value of 14.9 ± 9.8 μM ( Figure 4 ). However, the IC 50 value for SAHA was higher than 100 μM, which is too high to be considered as a drug candidate ( Supplemental Figure S2 ). Attempts were made to explore the mode of inhibition, inhibitory enzyme assays were carried out using various concentrations of the substrate nitrocefin as well as Compound 4 and 6 . Figure 5 shows Lineweaver–Burk plots with a nest of lines that intersect at the y -axis for both Compound 4 and 6 . K i values were determined on the basis of the plots with a K i value of 6.4 ± 1.7 μM for Compound 4 and a K i value of 4.7 ± 1.4 μM for compound 6 , where K i values were calculated from the slope based on K m and apparent K m values were obtained from Figure 5 . The IC 50 and K i values for Compound 4 and 6 and SAHA are listed in Table 2 . 3. Discussion We have demonstrated the potential for inhibition of Bla2 by two hydroxamate compounds that have shown the ability to bind both of the active site zinc ions. The effectiveness of the zinc ion binding was investigated by way of in silico and in vitro analyses. The MD simulations of Bla2 in complex with the newly developed hydroxamate compounds and the control compound SAHA further revealed the details of the binding interactions. In addition, kinetic analyses were performed to examine the inhibitory potential of each compound. The performance of the MD simulations for both the Bla2:Compound 4 complex and Bla2:Compound 6 complex, clearly showed the ability of the aromatic hydroxamate group to remain bound to the two active site zinc ions. The binding affinities obtained from Autodock Vina for both Compounds 4 and 6 are comparable at −7.5 and −7.7 kcal/mol, respectively. The free energy values obtained from the MD simulations would show a similar trend with only a slight improvement for Compound 6 as compared to Compound 4 and SAHA. This difference could be attributed to the structures of each compound, concerning the meta-oriented chlorine of Compound 4 , and methoxy of Compound 6. In analyzing the MD simulations, and per-residue decomposition data of each compound, the methoxy of Compound 6 does seem to provide it an exclusive advantage in interacting with the V68 side chain in a variety of ways. This is an interaction that the chlorine of Compound 4 does not allow to occur too frequently. In comparing the results of Compounds 4 and 6 to SAHA, the lack of an electrostatic contribution in the SAHA simulation is certainly more characteristic of its hydrophobic structure and behavior found in the MD simulation, as compared to the more polar structures of Compounds 4 and 6 . This can be seen in the per-residue decomposition data ( Supplemental Figures S3 and S4 ) as polar interactions are not as dominant for SAHA, as they were for Compounds 4 and 6 . The hydrophobic interactions surrounding the aromatic end of Compound 6 also played an important role in stabilizing the compound within the Bla2 active site, as reflected in the per-residue decomposition data. The in silico data obtained for the SAHA simulation showing inhibitory potential comparable to Compounds 4 and 6 clearly is not reflective of the results obtained from in vitro analysis of SAHA. This is likely due to the active site focused grid box used to carry out the docking protocol. Had the grid box been set up to encompass the entire Bla2 structure, SAHA could have potentially shown a preference for a binding conformation beyond the active site. Thus, the current docking protocol may have forced SAHA into a conformation within the Bla2 active site. The inhibition studies by experimental and in silico analyses confirmed that the mode of inhibition is highly likely to be competitive. As the MD simulations showed that these compounds strongly bind the active site zinc ions, reaffirming their potential as competitive inhibitors. The IC 50 values for the compounds range from 15 to 20 μM. Given the overlap in calculated error, these values can be considered comparable. In addition to the IC 50 values, the results of the measured binding interaction strength ( K i values) only range from 4.7 to 6.4 (kcal/mol). Previously, structurally similar compounds containing hydroxamate functional groups were investigated in our laboratory, where 3-(heptyloxy)-N-hydroxybenzamide displayed no inhibition but N-hydroxy-3-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide showed significant inhibition [ 5 ]. It should be noted that 3-(heptyloxy)-N-hydroxybenzamide has only one hydroxamate located at the distal end of the aliphatic group and N-hydroxy-3-((6-(hydroxyamino)-6-oxohexyl)oxy) contains two hydroxamate groups similar to Compounds 4 and 6 . No or little inhibition by 3-(heptyloxy)-N-hydroxybenzamide and SAHA supports the notion that the hydroxamate attached to the aromatic group in the compounds plays a pivotal role in the inhibition and the binding interactions. Although some IC 50 values in the low micromolar to nanomolar range were found in mercaptoacetic acid thiol ester compounds for various MBLs and a disubstitued succinic acid compound with various hydrophobic substituents for IMP-1 from P. aeruginosa [ 11 , 12 , 13 ]. The dihydroxamate-containing compounds (Compounds 4 and 6 ) should be added to the repertoire of promising inhibitory compounds for MBLs. In all consideration, Compounds 4 and 6 may be useful drug candidates as well as lead compounds for further development. To further investigate the development of better drug candidates, some structural modifications would be needed with structure-activity relationship analysis. It should also be mentioned here that these compounds should be examined further for pharmacokinetics, cytotoxicity and specificity prior to clinical use. 4. Materials and Methods 4.1. General Procedures All chemicals were obtained through Sigma or other quality manufacturers. The enzyme Bla2 was purified as previously described [ 4 , 14 ]. The purity of the product through Ni 2+ chromatography was determined by 10% SDS-PAGE. If necessary, gel-filtration chromatography was performed for further purification. Higher than 95% pure enzyme was stored at −20 °C in 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.0), 0.050 mM ZnSO 4 , and 30% ( v / v ) glycerol for further experiments. 3-chloro-N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)benzamide (Compound 4 ) and N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)-3-methoxybenzamide (Compound 6 ) were prepared by dissolving them in dimethyl sulfoxide (DMSO), which was then diluted to various concentrations to assay enzyme activities. The hydroxamate compounds were prepared as previously reported [ 5 ]. 4.2. Molecular Docking Simulations Given that there is no determined crystal structure of Bla2, homology-modelling was used with the SWISS-MODEL program to build a structure file of the enzyme [ 15 ]. The crystal structure of metallo-beta-lactamase BcII (4C09) was used as the template for modelling Bla2, with a final GMQE score of 0.87, and QMEAN score of 1.82 [ 16 ]. The structure files of both Compounds 4 and 6 , and SAHA, were generated using the Avogadro program with the General Amber Force Fields for energy minimization [ 17 ]. A molecular docking simulation was then performed for each ligand using the program Autodock Vina with complete ligand flexibility and a rigid receptor [ 18 ]. A grid box with parameters of (60 à × 52 à × 52 à ) was centered around the active site of Bla2 with the x-y-z coordinates (12.134, 6.796, 24.713). The docking results of lowest energy for each ligand were then prepared for further analysis through molecular dynamics. The Protein-Ligand Interaction Profiler (PLIP) online server was used to observe the interactions from the Autodock Results, and the ending molecular dynamics conformations [ 19 ]. 4.3. Molecular Dynamics Simulations All molecular dynamics simulations were carried out using the Gromacs program version 2021.4. The force field used for these simulations was the AMBER99SB-ILDN force field modified with recently published parameters for zinc(II)-binding residues [ 20 , 21 ]. These modifications are important for accurate coordination of the active site zinc ions, helping to improve the stability of the metalloprotein during the dynamics simulation. The water model used was the SPC/E water model. To generate the ligand topologies for Gromacs, the program Acpype was used [ 22 ]. The first stage of each dynamics simulation was solvating the system, and then neutralizing it with the addition of three chlorine ions. Next, an energy minimization was carried out for 50,000 steps using the steepest descent algorithm. The first phase of equilibration was carried out in a 100 ps run using the NVT ensemble with the leap-frog integrator. This run stabilized the temperature around 300 K. The second phase of equilibration was carried out over a 100 ps run using the NPT ensemble with the leap-frog integrator as well. This run stabilized the pressure of the system around 1 bar. Position restraints were applied on both the receptor and the ligand for each equilibration run. The temperature was regulated using the Berendsen V-rescale thermostat, and the pressure was regulated using the Parrinello-Rahman barostat. The final production MD simulation was run for 20 ns, during which the position restraints were removed. Two replicate simulations were carried out for each Bla2:Inhibitor complex system with different initial velocities. The replicates were then compared to help identify behavior consistent of each system, and from which the most fit replicate was used for further analysis and comparison of inhibitors. The root mean square deviation (RMSD) of each inhibitor trajectory in relation to the protein backbone was calculated using the RMSD module of Gromacs. In addition, a calculation of the protein backbone RMSD relative to the energy minimized conformation was performed for each system, to provide insight into the protein stability throughout the simulations ( Figure 2 ). 4.4. Free Energy Calculations To calculate the average end-state free energy of each simulation, the molecular mechanics generalized Born and surface area model (MM-GBSA) was used with the program gmx_MMPBSA [ 23 ]. For this calculation, 100 snapshots from each simulation were used. The binding free energy calculation used for this simulation can be summarized as: (1) Δ G binding = Δ G complex − ( Δ G receptor + Δ G ligand ) The Δ G complex is the free energy change of the Bla2-Inhibitor complex, Δ G receptor is the free energy change of Bla2 alone, and Δ G ligand is the free energy change of the ligand alone. The variables composing this calculation for the MM-GBSA model can be further understood as so: (2) Δ G binding = Δ E gas + Δ G solv The Δ E gas component is the vacuum interaction energy, derived from the summation of the bonded and non-bonded interactions. The bonded interactions are composed of the bond, angle, and dihedral potentials. The non-bonded interactions are composed of the van der Waals and electrostatic contributions. (3) Δ E gas = Δ E bonded + Δ E non − bonded = [ Δ E bond + Δ E angle + Δ E dihed ] + [ Δ E vdw + Δ E ele ] The Δ G solv component is the solvated free energy made up of the polar ( Δ E gb ) and nonpolar ( Δ E surf ) solvation components. (4) Δ G solv = Δ E gb + Δ E surf In addition to the free energy calculation, gmx_MMPBSA was also used to carry out a per-residue decomposition of the free energy, to help analyze the energy contributions of key active site residues within 4 (à ) of the ligand. 4.5. Inhibition Tests The purified Bla2 was assayed for substrate activity by adding enzyme at a final concentration of 0.13 μg/mL to solutions ranging from 10 to 70 μM nitrocefin in 50 mM MOPS (pH 7.0) to a final volume of 1 mL in quartz cuvettes at room temperature. Reactions were monitored by observing the increase in absorbance at 485 nm due to the hydrolysis of nitrocefin (ε = 15,900 M −1 ·cm −1 ) and were performed in triplicate [ 24 ]. The assay for IC 50 , the concentration of inhibitor necessary to inhibit 50% of the enzymatic activity, was performed under the aforementioned enzyme assay conditions after incubating the reaction mixture for 1 min with varying concentrations of Compounds 4 and 6 before initiating the reaction with a fixed concentration of nitrocefin (0.4 mM). The enzyme concentration used was 0.13 μg/mL (4.8 nM). A separate series of assays for each inhibitor was run at fixed inhibitor concentrations (0–0.030 mM for compounds 9 and 10 ) in which, at each inhibitor concentration, the concentration of the substrate nitrocefin was varied from 0.1 to 0.4 mM. The resulting data were analyzed for mode of inhibition by Lineweaver–Burk plots and were also fit using non-linear regression through SigmaPlot version 11.0 (Sigma, St. Louis, MO, USA), using the competitive inhibition equations: ν = V max ·S/[K m ·(1 + 1/K i ) + S]. All experiments were carried out in triplicate. 4.1. General Procedures All chemicals were obtained through Sigma or other quality manufacturers. The enzyme Bla2 was purified as previously described [ 4 , 14 ]. The purity of the product through Ni 2+ chromatography was determined by 10% SDS-PAGE. If necessary, gel-filtration chromatography was performed for further purification. Higher than 95% pure enzyme was stored at −20 °C in 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.0), 0.050 mM ZnSO 4 , and 30% ( v / v ) glycerol for further experiments. 3-chloro-N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)benzamide (Compound 4 ) and N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)-3-methoxybenzamide (Compound 6 ) were prepared by dissolving them in dimethyl sulfoxide (DMSO), which was then diluted to various concentrations to assay enzyme activities. The hydroxamate compounds were prepared as previously reported [ 5 ]. 4.2. Molecular Docking Simulations Given that there is no determined crystal structure of Bla2, homology-modelling was used with the SWISS-MODEL program to build a structure file of the enzyme [ 15 ]. The crystal structure of metallo-beta-lactamase BcII (4C09) was used as the template for modelling Bla2, with a final GMQE score of 0.87, and QMEAN score of 1.82 [ 16 ]. The structure files of both Compounds 4 and 6 , and SAHA, were generated using the Avogadro program with the General Amber Force Fields for energy minimization [ 17 ]. A molecular docking simulation was then performed for each ligand using the program Autodock Vina with complete ligand flexibility and a rigid receptor [ 18 ]. A grid box with parameters of (60 à × 52 à × 52 à ) was centered around the active site of Bla2 with the x-y-z coordinates (12.134, 6.796, 24.713). The docking results of lowest energy for each ligand were then prepared for further analysis through molecular dynamics. The Protein-Ligand Interaction Profiler (PLIP) online server was used to observe the interactions from the Autodock Results, and the ending molecular dynamics conformations [ 19 ]. 4.3. Molecular Dynamics Simulations All molecular dynamics simulations were carried out using the Gromacs program version 2021.4. The force field used for these simulations was the AMBER99SB-ILDN force field modified with recently published parameters for zinc(II)-binding residues [ 20 , 21 ]. These modifications are important for accurate coordination of the active site zinc ions, helping to improve the stability of the metalloprotein during the dynamics simulation. The water model used was the SPC/E water model. To generate the ligand topologies for Gromacs, the program Acpype was used [ 22 ]. The first stage of each dynamics simulation was solvating the system, and then neutralizing it with the addition of three chlorine ions. Next, an energy minimization was carried out for 50,000 steps using the steepest descent algorithm. The first phase of equilibration was carried out in a 100 ps run using the NVT ensemble with the leap-frog integrator. This run stabilized the temperature around 300 K. The second phase of equilibration was carried out over a 100 ps run using the NPT ensemble with the leap-frog integrator as well. This run stabilized the pressure of the system around 1 bar. Position restraints were applied on both the receptor and the ligand for each equilibration run. The temperature was regulated using the Berendsen V-rescale thermostat, and the pressure was regulated using the Parrinello-Rahman barostat. The final production MD simulation was run for 20 ns, during which the position restraints were removed. Two replicate simulations were carried out for each Bla2:Inhibitor complex system with different initial velocities. The replicates were then compared to help identify behavior consistent of each system, and from which the most fit replicate was used for further analysis and comparison of inhibitors. The root mean square deviation (RMSD) of each inhibitor trajectory in relation to the protein backbone was calculated using the RMSD module of Gromacs. In addition, a calculation of the protein backbone RMSD relative to the energy minimized conformation was performed for each system, to provide insight into the protein stability throughout the simulations ( Figure 2 ). 4.4. Free Energy Calculations To calculate the average end-state free energy of each simulation, the molecular mechanics generalized Born and surface area model (MM-GBSA) was used with the program gmx_MMPBSA [ 23 ]. For this calculation, 100 snapshots from each simulation were used. The binding free energy calculation used for this simulation can be summarized as: (1) Δ G binding = Δ G complex − ( Δ G receptor + Δ G ligand ) The Δ G complex is the free energy change of the Bla2-Inhibitor complex, Δ G receptor is the free energy change of Bla2 alone, and Δ G ligand is the free energy change of the ligand alone. The variables composing this calculation for the MM-GBSA model can be further understood as so: (2) Δ G binding = Δ E gas + Δ G solv The Δ E gas component is the vacuum interaction energy, derived from the summation of the bonded and non-bonded interactions. The bonded interactions are composed of the bond, angle, and dihedral potentials. The non-bonded interactions are composed of the van der Waals and electrostatic contributions. (3) Δ E gas = Δ E bonded + Δ E non − bonded = [ Δ E bond + Δ E angle + Δ E dihed ] + [ Δ E vdw + Δ E ele ] The Δ G solv component is the solvated free energy made up of the polar ( Δ E gb ) and nonpolar ( Δ E surf ) solvation components. (4) Δ G solv = Δ E gb + Δ E surf In addition to the free energy calculation, gmx_MMPBSA was also used to carry out a per-residue decomposition of the free energy, to help analyze the energy contributions of key active site residues within 4 (à ) of the ligand. 4.5. Inhibition Tests The purified Bla2 was assayed for substrate activity by adding enzyme at a final concentration of 0.13 μg/mL to solutions ranging from 10 to 70 μM nitrocefin in 50 mM MOPS (pH 7.0) to a final volume of 1 mL in quartz cuvettes at room temperature. Reactions were monitored by observing the increase in absorbance at 485 nm due to the hydrolysis of nitrocefin (ε = 15,900 M −1 ·cm −1 ) and were performed in triplicate [ 24 ]. The assay for IC 50 , the concentration of inhibitor necessary to inhibit 50% of the enzymatic activity, was performed under the aforementioned enzyme assay conditions after incubating the reaction mixture for 1 min with varying concentrations of Compounds 4 and 6 before initiating the reaction with a fixed concentration of nitrocefin (0.4 mM). The enzyme concentration used was 0.13 μg/mL (4.8 nM). A separate series of assays for each inhibitor was run at fixed inhibitor concentrations (0–0.030 mM for compounds 9 and 10 ) in which, at each inhibitor concentration, the concentration of the substrate nitrocefin was varied from 0.1 to 0.4 mM. The resulting data were analyzed for mode of inhibition by Lineweaver–Burk plots and were also fit using non-linear regression through SigmaPlot version 11.0 (Sigma, St. Louis, MO, USA), using the competitive inhibition equations: ν = V max ·S/[K m ·(1 + 1/K i ) + S]. All experiments were carried out in triplicate. 5. Conclusions We reported the development of novel di-hydroxamate-containing compounds with high affinity for Bla2, which is a major factor of β-lactam antibiotic resistance. The compounds showed great potential by inhibiting Bla2 with IC 50 values in the micromolar range and by binding strength with K i values in the low micromolar range. MD simulations detail some specific amino acids are strongly engaged in the complex formation of the compounds with the enzyme. In addition, the MD simulations provide insight into the subtle differences that can be found in the interactions between the inhibitors and Bla2. The experimental inhibitory studies support that the mode of inhibition is competitive, which is well consistent with the data obtained from in silico analysis. It is strongly suggested that the hydroxamate group attached to the aromatic ring of the compounds is crucial in the interaction with the zinc ions, enhancing the effectiveness of inhibition. It is expected that the compounds protect β-lactam antibiotics from damage by MBLs. Figures, Scheme and Tables ijms-23-09163-sch001_Scheme 1 Scheme 1 Synthesis of meta-substituents' dihydroxamate derivatives. The synthesis condition for Compound 4 and 6 is NH 2 OH (3 eq)/KOH (4 eq) in MeOH. The hydroxamic acid group is colored in red; methoxy group in blue; chlorine in cyan. Figure 1 Autodock binding conformations selected for dynamics. ( A ) Compound 4 in cyan, ( B ) Compound 6 in pink, and ( C ) SAHA in yellow. Figure 2 The RMSDs for the complexes and the compounds. ( A ) Bla2 backbone RMSD for the Bla2-Compound 4 complex in black, and Bla2-Compound 6 complex in red, and Bla2-SAHA complex in yellow. ( B ) RMSD of Compound 4 in black, Compound 6 in red, and SAHA in yellow, in relation to the protein backbone. Plotted as running averages for visual clarity. Figure 3 Average atomic coordinates of the root mean square fluctuation for both Compound 4 in cyan ( A ) and Compound 6 in purple ( B ). Some key amino acids were shown for both Compounds. Figure 4 Concentration-response plots for Bla2 inhibition with hydroxamate compounds: Compound 4 ( A ) and Compound 6 ( B ). All data points are based on average values from triplicate analysis. Figure 5 Lineweaver–Burk plots of inhibition of Bla2 by Compound 4 and 6 . Assays were performed in 50 mM MOPS, pH 7.0. Kinetic constants were determined by fitting of the data to the equation v = V max ·S/(K m ·(1 + 1/K i ) + S) which indicates competitive inhibition. ( A ) The concentrations of compound 4 are 0 (blue), 6 (orange), and 30 μM (gray); ( B ) The concentrations of compound 6 are 0 (blue), 10 (orange), and 30 μM (gray). All data points are based on average values from triplicate analysis. ijms-23-09163-t001_Table 1 Table 1 The Autodock binding affinities for each selected conformation, and end-state free energies of the molecular dynamics simulations. Autodock Binding Affinity (kcal/mol) Average End-State Free Energy (kcal/mol) Compound 4 −7.5 −27.18 ± 0.65 Compound 6 −7.7 −30.08 ± 1.12 SAHA −6.7 −27.82 ± 0.51 ijms-23-09163-t002_Table 2 Table 2 The inhibition effectiveness of Compound 4 and 6 by inhibitory potency (IC 50 ) and affinity strength ( K i ) of Bla2. IC 50 (μM) K i (μM) Compound 4 20.0 ± 5.0 6.4 ± 1.7 Compound 6 14.9 ± 9.8 4.7 ± 1.4 SAHA >100 N.D. * * Data were not determined due to the high IC 50 value.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792709/

A Structural View of Xenophagy, a Battle between Host and Microbes

The cytoplasm in mammalian cells is a battlefield between the host and invading microbes. Both the living organisms have evolved unique strategies for their survival. The host utilizes a specialized autophagy system, xenophagy, for the clearance of invading pathogens, whereas bacteria secrete proteins to defend and escape from the host xenophagy. Several molecules have been identified and their structural investigation has enabled the comprehension of these mechanisms at the molecular level. In this review, we focus on one example of host autophagy and the other of bacterial defense: the autophagy receptor, NDP52, in conjunction with the sugar receptor, galectin-8, plays a critical role in targeting the autophagy machinery against Salmonella ; and the cysteine protease, RavZ secreted by Legionella pneumophila cleaves the LC3-PE on the phagophore membrane. The structure-function relationships of these two examples and the directions of future research will be discussed. INTRODUCTION Autophagy is an evolutionarily conserved cellular pathway that delivers cytoplasmic proteins and organelles to the lysosome for degradation in eukaryotic cells ( Huang and Klionsky, 2007 ; Levine and Klionsky, 2017 ; Nah et al., 2015 ; Nakatogawa et al., 2009 ; Wen and Klionsky, 2016 ). In contrast to the ubiquitin-proteasome system, autophagy can degrade the comparatively large substrates, including protein aggregates, cellular organelles, and invading pathogens ( Levine et al., 2011 ; Mizushima, 2011 ). Initially, autophagy was thought to be a nonselective pathway for the degradation of cytoplasmic components to provide energy and aid survival in nutrient-deprived conditions ( Mizushima et al., 1998 ). However, many recent studies of selective autophagy have been reported ( Boyle and Randow, 2013 ; Farre and Subramani, 2016 ; Kim et al., 2013 ; 2015 ; 2016 ; Kim and Song, 2015 ; Kwon et al., 2017b ; Liu and Du, 2015 ; Svenning and Johansen, 2013 ; Zaffagnini and Martens, 2016 ). Selective autophagy requires specific autophagy receptors and is referred to by different terms based on the cargo molecules: aggrephagy (protein aggregates), lysophagy (lysosomes), mitophagy (mitochondria), pexophagy (peroxisomes), and xenophagy (invading pathogens). The substrates for selective autophagy are recognized either directly or indirectly by phagophores. In mitophagy, phagophore-conjugated LC3-family proteins directly recognize the mitochondrial proteins NIX, BNIP3, and FUNDC1 ( Liu et al., 2014 ). In contrast, other selective autophagy receptors, such as p62/SQSTM1, NDP52 (also known as CALCOCO2), and optineurin (OPTN), simultaneously recognize membrane-conjugated LC3-family proteins and autophagy substrates ( Boyle and Randow, 2013 ). As indicated, the autophagic process utilized for the degradation of bacteria or viruses is termed 'xenophagy' ( Levine, 2005 ). Infectious bacteria are cleared from human cells by host autophagy in combination with other upstream cellular components, such as autophagy receptors, ubiquitin (Ub), diacylglycerol, NOD proteins, galectin-8 (GAL8), and Ub E3-ligases ( Sorbara and Girardin, 2015 ). Furthermore, LC3-associated phagocytosis has also been known as a novel form of non-canonical autophagy where LC3 is conjugated to single membrane phagosomes using a portion of the canonical autophagic molecules ( Heckmann et al., 2017 ). However, as a survival mechanism, many bacteria have also evolved the ability to manipulate the host autophagy pathway through the inhibition of the function of autophagic molecules. Therefore, our cellular environment is essentially a battlefield with microbes and protein molecules are the armor and weapons ( Fig. 1 ). In this review, we discuss the interplay between bacteria and host autophagy from a structural biology perspective, including how the autophagic process targets bacteria for clearance and how bacteria block this process for survival. HOST DEFENSE USING AUTOPHAGY There are many different types of molecules involved in xenophagy. Among them, autophagy receptors (also called cargo receptors or autophagy adaptors) are particularly important for selective autophagy ( Kim et al., 2016 ; Svenning and Johansen, 2013 ; Zaffagnini and Martens, 2016 ). Autophagy receptors commonly possess LIR (LC3-Interacting Region) and Ub-interacting domains (UBA, UBZ, and ZnF), which suggests that ubiquitylation and autophagy are closely linked ( Ji and Kwon, 2017 ; Kim et al., 2016 ; Rahighi and Dikic, 2012 ). When bacteria invade mammalian cells, they are usually restricted within vacuoles; however, some of this bacterial population can escape from the vacuoles and enter the cytoplasm, and the vacuoles themselves can be ruptured by the bacterial growth ( Fig. 1 ). The autophagic proteins of the host target the bacteria through multiple steps. Ubiquitin-coating of bacteria Bacteria are marked as degradation targets by the attachment of Ub molecules in cells ( Levine et al., 2011 ; Thurston et al., 2009 ). Therefore, it is natural that autophagy receptors bridge ubiquitylated cargos, such as Ub-coated bacteria, to the autophagy pathway by using their LIR motif and Ub-binding domains. Although this phenomenon was discovered some time ago ( Perrin et al., 2004 ), the molecular mechanism involved remains unclear. The E3-ligase that attaches Ub to bacteria has only recently been reported ( Celli, 2012 ; Huett et al., 2012 ). Through the genome-wide analysis of leucine-rich repeat (LRR)-containing proteins, LRSAM1 (leucine-rich repeat and sterile alpha motif containing protein 1) was identified as a component of the antibacterial autophagic response ( Ng and Xavier, 2011 ; Ng et al., 2011 ), and later, LRSAM1 was identified as the E3 ligase responsible for bacteria-associated ubiquitylation prior to autophagy ( Huett et al., 2012 ). The LRR domain in LRSAM1 has been shown to be critical for bacterial targeting, whereas the C-terminal RING domain is important for the ubiquitylation of invading pathogens ( Huett et al., 2012 ). Furthermore, LRSAM1 also binds the autophagy receptor NDP52, which subsequently binds the Ub chains and autophagic LC3 protein via UBZ and LIR, respectively. Interestingly, the ubiquitylation of Mycobacterium tuberculosis by Parkin, a well-known E3 Ub-ligase involved in mitophagy, has been also reported, which implied the existence of a functional link between mitophagy and xenophagy ( Manzanillo et al., 2013 ). Very recently, it was reported that the E3-ligase LUBAC (Linear Ub Chain Assembly Complex) generated M1-linked linear poly-Ub patches in the bacteria, which serve as antibacterial and pro-inflammatory signaling platforms ( Noad et al., 2017 ). This showed the coordination of two different defense pathways: xenophagy and NF-kB signaling. Ubiquitylation is an early step of xenophagy ( Fig. 1 ); once bacteria are captured in an autophagosome, the subsequent steps are essentially the same as those enacted in the regular autophagy process. Collaboration between autophagy receptor NDP52 and sugar receptor galectin-8 in xenophagy As noted, the Ub-binding domain of autophagy receptors is particularly important in the recognition of Ub-coated bacteria. At present, at least five selective autophagy receptors have been well-studied (p62/SQSTM1, NBR1, NDP52, OPTN, and TAX1BP1). These autophagy receptors have hetero- or homo-oligomerization domains (p62 and NBR1: PB1 domain; NDP52, OPTN, and TAX1BP1: coiled-coil (CC) domain), which maximize the effect of cargo recognition or strongly interact with phagophore membranes ( Behrends and Fulda, 2012 ). Further, receptor-specific domains allow the proteins to participate in diverse cellular signaling. In particular, NDP52 participates in xenophagic processes related to infectious pathogens, including Salmonella enterica serovar Typhimurium (hereafter, S. typhimurium ), Listeria monocytogenes , Mycobacterium tuberculosis , and Shigella . NDP52 consists of a SKICH domain, LIR motif, CC domain, GALBI (galectin-8 binding) region, and UBZ domain ( Fig. 2A ). The unique GALBI region plays a critical role in the interaction with sugar receptor, GAL8, to clear invading Salmonella ( Thurston et al., 2012 ). Carbohydrates located on mammalian cell surfaces are not exposed to the cytoplasm. Therefore, it has been proposed that these carbohydrates may represent a type of danger signal that is recognized by the danger receptors in the cells ( Fig. 1 ). Randow and colleagues identified several sugar receptors, the galectins, that recognize the carbohydrates when they are exposed through the rupture of Salmonella -containing vacuoles (SCV); in particular, GAL8 binds to the carbohydrates and specifically recruits the autophagy receptor ( Thurston et al., 2012 ). GAL8 belongs to the class of galectins composed of tandem-repeat carbohydrate-recognition domains (CRDs). Both N- and C-terminal CRDs (N-CRD and C-CRD) bind to carbohydrates with different specificities; interestingly, only C-CRD binds to the GALBI region (residues 372–380) of NDP52. Previous structural studies have elucidated the atomic details of how these two receptors interact with each other ( Kim et al., 2013 ; Li et al., 2013 ). Although the full-length structure of NDP52 remains unknown, the structures of each domain in complex with the interacting molecules, LC3C, GAL8, and Ub, have been determined ( Fig. 2B ), except for the central homodimeric CC domain. There are two possibilities for the orientation of CC, parallel and antiparallel, and ACCORD, an assessment tool to determine the orientation of CC has been applied and the NDP52 was determined to have a parallel CC ( Kim et al., 2017 ). The combination of all structural information has allowed a working model for the Salmonella targeting to phagophores to be constructed ( Fig. 2C ). Ubiquitin-coating of bacteria Bacteria are marked as degradation targets by the attachment of Ub molecules in cells ( Levine et al., 2011 ; Thurston et al., 2009 ). Therefore, it is natural that autophagy receptors bridge ubiquitylated cargos, such as Ub-coated bacteria, to the autophagy pathway by using their LIR motif and Ub-binding domains. Although this phenomenon was discovered some time ago ( Perrin et al., 2004 ), the molecular mechanism involved remains unclear. The E3-ligase that attaches Ub to bacteria has only recently been reported ( Celli, 2012 ; Huett et al., 2012 ). Through the genome-wide analysis of leucine-rich repeat (LRR)-containing proteins, LRSAM1 (leucine-rich repeat and sterile alpha motif containing protein 1) was identified as a component of the antibacterial autophagic response ( Ng and Xavier, 2011 ; Ng et al., 2011 ), and later, LRSAM1 was identified as the E3 ligase responsible for bacteria-associated ubiquitylation prior to autophagy ( Huett et al., 2012 ). The LRR domain in LRSAM1 has been shown to be critical for bacterial targeting, whereas the C-terminal RING domain is important for the ubiquitylation of invading pathogens ( Huett et al., 2012 ). Furthermore, LRSAM1 also binds the autophagy receptor NDP52, which subsequently binds the Ub chains and autophagic LC3 protein via UBZ and LIR, respectively. Interestingly, the ubiquitylation of Mycobacterium tuberculosis by Parkin, a well-known E3 Ub-ligase involved in mitophagy, has been also reported, which implied the existence of a functional link between mitophagy and xenophagy ( Manzanillo et al., 2013 ). Very recently, it was reported that the E3-ligase LUBAC (Linear Ub Chain Assembly Complex) generated M1-linked linear poly-Ub patches in the bacteria, which serve as antibacterial and pro-inflammatory signaling platforms ( Noad et al., 2017 ). This showed the coordination of two different defense pathways: xenophagy and NF-kB signaling. Ubiquitylation is an early step of xenophagy ( Fig. 1 ); once bacteria are captured in an autophagosome, the subsequent steps are essentially the same as those enacted in the regular autophagy process. Collaboration between autophagy receptor NDP52 and sugar receptor galectin-8 in xenophagy As noted, the Ub-binding domain of autophagy receptors is particularly important in the recognition of Ub-coated bacteria. At present, at least five selective autophagy receptors have been well-studied (p62/SQSTM1, NBR1, NDP52, OPTN, and TAX1BP1). These autophagy receptors have hetero- or homo-oligomerization domains (p62 and NBR1: PB1 domain; NDP52, OPTN, and TAX1BP1: coiled-coil (CC) domain), which maximize the effect of cargo recognition or strongly interact with phagophore membranes ( Behrends and Fulda, 2012 ). Further, receptor-specific domains allow the proteins to participate in diverse cellular signaling. In particular, NDP52 participates in xenophagic processes related to infectious pathogens, including Salmonella enterica serovar Typhimurium (hereafter, S. typhimurium ), Listeria monocytogenes , Mycobacterium tuberculosis , and Shigella . NDP52 consists of a SKICH domain, LIR motif, CC domain, GALBI (galectin-8 binding) region, and UBZ domain ( Fig. 2A ). The unique GALBI region plays a critical role in the interaction with sugar receptor, GAL8, to clear invading Salmonella ( Thurston et al., 2012 ). Carbohydrates located on mammalian cell surfaces are not exposed to the cytoplasm. Therefore, it has been proposed that these carbohydrates may represent a type of danger signal that is recognized by the danger receptors in the cells ( Fig. 1 ). Randow and colleagues identified several sugar receptors, the galectins, that recognize the carbohydrates when they are exposed through the rupture of Salmonella -containing vacuoles (SCV); in particular, GAL8 binds to the carbohydrates and specifically recruits the autophagy receptor ( Thurston et al., 2012 ). GAL8 belongs to the class of galectins composed of tandem-repeat carbohydrate-recognition domains (CRDs). Both N- and C-terminal CRDs (N-CRD and C-CRD) bind to carbohydrates with different specificities; interestingly, only C-CRD binds to the GALBI region (residues 372–380) of NDP52. Previous structural studies have elucidated the atomic details of how these two receptors interact with each other ( Kim et al., 2013 ; Li et al., 2013 ). Although the full-length structure of NDP52 remains unknown, the structures of each domain in complex with the interacting molecules, LC3C, GAL8, and Ub, have been determined ( Fig. 2B ), except for the central homodimeric CC domain. There are two possibilities for the orientation of CC, parallel and antiparallel, and ACCORD, an assessment tool to determine the orientation of CC has been applied and the NDP52 was determined to have a parallel CC ( Kim et al., 2017 ). The combination of all structural information has allowed a working model for the Salmonella targeting to phagophores to be constructed ( Fig. 2C ). MANIPULATION OF AUTOPHAGY BY MICROBES Although some bacteria are targeted and eliminated by xenophagy, other bacteria have evolved mechanisms to counter or avoid this host defense system. Different bacterial species utilize their unique mechanisms to escape host autophagy, although the autophagosome in host can encapsulate many different intracellular bacteria through the xenophagic process. Therefore, diverse molecules from different bacteria are involved in this blockage of autophagy and these molecules are usually not conserved in the bacterial kingdom. However, they are classified into two main mechanisms: autophagy disarming and camouflage ( Sorbara and Girardin, 2015 ). Unique strategies by different bacteria for inhibition of host autophagy Certain bacteria can inhibit autophagy induction signaling upstream of the autophagosome maturation ( Shin et al., 2010 ; Tattoli et al., 2012 ), evade autophagy recognition by masking the bacterial surface ( Ogawa et al., 2005 ), interfere with the formation of the autophagosome ( Choy et al., 2012 ; Kwon et al., 2017b ), and hijack autophagy for bacterial replication ( Sorbara and Girardin, 2015 ) ( Fig. 1 ). Bacteria secrete their own factors for the modulation of host systems: Eis, anthrax toxin edema factor, and cholera toxin to inhibit the induction of autophagy; IcsB, ActA, and InlK to block the recognition of bacteria by the host autophagy system; RavZ and VirA to directly inhibit the autophagy components; and ESAT-6 and VacA to block the fusion step between the autophagosome and the lysosome ( Huang and Brumell, 2014 ). Functional and structural studies of these molecules are currently in progress to enable the comprehension of their survival mechanisms and the subsequent development of novel antibiotics. Their structures have been reported as follows: Eis from Mycobacterium tuberculosis ( Chen et al., 2011 ; Kim et al., 2012 ; 2014 ), edema factor toxin from Bacillus anthracis ( Santelli et al., 2004 ; Shen et al., 2004 ), cholera toxin from Vibrio cholerae ( Fan et al., 2004 ; Holmner et al., 2004 ; Merritt et al., 1994 ; Zhang et al., 1995 ), InlK from Listeria monocytogenes ( Neves et al., 2013 ), RavZ from Legionella pneumophila ( Horenkamp et al., 2015 ; Kwon et al., 2017a ; 2017b ; Yang et al., 2017 ), VirA from Shigella flexneri ( Davis et al., 2008 ; Germane et al., 2008 ), ESAT-6 from Mycobacterium tuberculosis ( Renshaw et al., 2005 ), and VacA from Helicobacter pylori ( Gangwer et al., 2007 ). Recently, we and other research groups determined the structures of RavZ from Legionella pneumophila ( Horenkamp et al., 2015 ; Kwon et al., 2017a ; 2017b ; Yang et al., 2017 ) ( Fig. 3A ) and independently proposed its mode of action; however, the mechanism for LC3 deconjugation is controversial. Therefore, the current perspectives on RavZ molecule will be included in this review. LC3 deconjugating enzyme, RavZ, from Legionella pneumophila LC3B was originally identified as microtubule-associated proteins 1A/1B light chain 3B encoded by the MAP1LC3B gene ( He et al., 2003 ). It possesses a shared folding pattern with Ub, and the phosphatidylethanolamine (PE) conjugation to LC3 is catalyzed by sequential steps of the autophagic E1-, E2-, and E3-enzymes, which is quite similar to ubiquitylation ( Hong et al., 2011 ; 2012 ; Klionsky and Schulman, 2014 ; Maruyama and Noda, 2018 ; Mizushima et al., 1998 ). PE-conjugated LC3 is the most widely used marker of autophagosomes and plays a central role in the autophagy pathway ( Klionsky et al., 2016 ; Yoshii and Mizushima, 2017 ). Legionella pneumophila elegantly targets this molecule to inhibit host autophagy ( Choy et al., 2012 ): it secretes a cysteine protease RavZ to cleave a specific peptide bond between phenylalanine and the C-terminal glycine of LC3-PE ( Fig. 3B ). The C-terminal segment of LC3 precursor is removed to expose the C-terminal glycine by the host cysteine protease ATG4B in the LC3 maturation step; furthermore, ATG4B also cleaves the bond between C-terminal glycine and PE to recycle LC3 in the cells ( Maruyama and Noda, 2018 ). After RavZ has cleaved LC3-PE from the phagophore membrane, the product cannot be reconjugated to PE as it lacks the C-terminal glycine residue ( Choy et al., 2012 ). Therefore, Legionella RavZ competes with host ATG4B, although there is a difference in specificity. The structure of ATG4B in complex with LC3 has been determined and the enzymatic mechanism has been proposed ( Satoo et al., 2009 ). The primary sequence comparison showed that RavZ was quite different in length and, unexpectedly, there were two LIR motifs at the N-terminal region (N-LIR1/2) and one LIR motif at the C-terminal region (C-LIR) in RavZ ( Kwon et al., 2017b ). Based on the missing electron density map, the regions are very flexible; conversely, they are versatile to seize the LC3-PE molecules on the membrane. In addition to these regions, there are two independent domains, catalytic (CAT) and membrane targeting (MT). The CAT domain shows a similar folding pattern to Ub-like proteases and contains the catalytic triad residues His176-Asp197-Cys258 for the hydrolysis of the peptide bond ( Horenkamp et al., 2015 ; Kwon et al., 2017b ). The function of the MT domain is characterized by a higher preference for phosphatidylinositol 3-phosphate, which is likely to be abundant in the autophagosomal membrane ( Horenkamp et al., 2015 ). Based on this structural information, a working model for RavZ has been proposed ( Fig. 3C ). Both N-LIR1/2 and C-LIR tether the LC3-PE molecules on the membrane and RavZ cuts the third (or one of the tethered) LC3-PE molecule by using its CAT domain. Here, we propose a 'Tethering and Cut' model for further explanation. SAXS (small-angle X-ray scattering) data have shown a 1:2 RavZ-(LC3) 2 complex model, which supports this model, although the resolution is relatively low ( Kwon et al., 2017b ). Later, another model, 'Lift and Cut', was proposed independently ( Pantoom et al., 2017 ; Yang et al., 2017 ) ( Fig. 3D ). This model is based on the conformational change of the hydrophobic α3-helix, which may point towards the membrane, and the structurally similar region in the CAT domain with lipid-binding protein yeast Sec14 homolog (Shf1) ( Yang et al., 2017 ). The α3-helix picks out the LC3-PE from the membrane and the lipid PE moiety is then recognized and cut by RavZ. However, both models are incomplete owing to the absence of the structure of the complex with LC3 bound to the active site of RavZ. Another controversy is the exact role of the two N-LIR1/2 motifs; it is possible that the reported complex structure between RavZ and LC3 might be a crystallization artifact and the only second LIR2 is proposed to be critical ( Yang et al., 2017 ). However, another report showed that deletions, and even point mutations, on any of the LIR motifs resulted in quite significant functional defects in cell-based assays ( Kwon et al., 2017b ). Furthermore, the structure of the complex between LC3 and a longer peptide comprising tandem N-LIR1/2 showed that the first LIR (LIR1) was a major contributory factor for LC3 binding and the tandem LIR motifs formed a characteristic β-sheet conformation to augment the binding affinity ( Kwon et al., 2017a ). Unique strategies by different bacteria for inhibition of host autophagy Certain bacteria can inhibit autophagy induction signaling upstream of the autophagosome maturation ( Shin et al., 2010 ; Tattoli et al., 2012 ), evade autophagy recognition by masking the bacterial surface ( Ogawa et al., 2005 ), interfere with the formation of the autophagosome ( Choy et al., 2012 ; Kwon et al., 2017b ), and hijack autophagy for bacterial replication ( Sorbara and Girardin, 2015 ) ( Fig. 1 ). Bacteria secrete their own factors for the modulation of host systems: Eis, anthrax toxin edema factor, and cholera toxin to inhibit the induction of autophagy; IcsB, ActA, and InlK to block the recognition of bacteria by the host autophagy system; RavZ and VirA to directly inhibit the autophagy components; and ESAT-6 and VacA to block the fusion step between the autophagosome and the lysosome ( Huang and Brumell, 2014 ). Functional and structural studies of these molecules are currently in progress to enable the comprehension of their survival mechanisms and the subsequent development of novel antibiotics. Their structures have been reported as follows: Eis from Mycobacterium tuberculosis ( Chen et al., 2011 ; Kim et al., 2012 ; 2014 ), edema factor toxin from Bacillus anthracis ( Santelli et al., 2004 ; Shen et al., 2004 ), cholera toxin from Vibrio cholerae ( Fan et al., 2004 ; Holmner et al., 2004 ; Merritt et al., 1994 ; Zhang et al., 1995 ), InlK from Listeria monocytogenes ( Neves et al., 2013 ), RavZ from Legionella pneumophila ( Horenkamp et al., 2015 ; Kwon et al., 2017a ; 2017b ; Yang et al., 2017 ), VirA from Shigella flexneri ( Davis et al., 2008 ; Germane et al., 2008 ), ESAT-6 from Mycobacterium tuberculosis ( Renshaw et al., 2005 ), and VacA from Helicobacter pylori ( Gangwer et al., 2007 ). Recently, we and other research groups determined the structures of RavZ from Legionella pneumophila ( Horenkamp et al., 2015 ; Kwon et al., 2017a ; 2017b ; Yang et al., 2017 ) ( Fig. 3A ) and independently proposed its mode of action; however, the mechanism for LC3 deconjugation is controversial. Therefore, the current perspectives on RavZ molecule will be included in this review. LC3 deconjugating enzyme, RavZ, from Legionella pneumophila LC3B was originally identified as microtubule-associated proteins 1A/1B light chain 3B encoded by the MAP1LC3B gene ( He et al., 2003 ). It possesses a shared folding pattern with Ub, and the phosphatidylethanolamine (PE) conjugation to LC3 is catalyzed by sequential steps of the autophagic E1-, E2-, and E3-enzymes, which is quite similar to ubiquitylation ( Hong et al., 2011 ; 2012 ; Klionsky and Schulman, 2014 ; Maruyama and Noda, 2018 ; Mizushima et al., 1998 ). PE-conjugated LC3 is the most widely used marker of autophagosomes and plays a central role in the autophagy pathway ( Klionsky et al., 2016 ; Yoshii and Mizushima, 2017 ). Legionella pneumophila elegantly targets this molecule to inhibit host autophagy ( Choy et al., 2012 ): it secretes a cysteine protease RavZ to cleave a specific peptide bond between phenylalanine and the C-terminal glycine of LC3-PE ( Fig. 3B ). The C-terminal segment of LC3 precursor is removed to expose the C-terminal glycine by the host cysteine protease ATG4B in the LC3 maturation step; furthermore, ATG4B also cleaves the bond between C-terminal glycine and PE to recycle LC3 in the cells ( Maruyama and Noda, 2018 ). After RavZ has cleaved LC3-PE from the phagophore membrane, the product cannot be reconjugated to PE as it lacks the C-terminal glycine residue ( Choy et al., 2012 ). Therefore, Legionella RavZ competes with host ATG4B, although there is a difference in specificity. The structure of ATG4B in complex with LC3 has been determined and the enzymatic mechanism has been proposed ( Satoo et al., 2009 ). The primary sequence comparison showed that RavZ was quite different in length and, unexpectedly, there were two LIR motifs at the N-terminal region (N-LIR1/2) and one LIR motif at the C-terminal region (C-LIR) in RavZ ( Kwon et al., 2017b ). Based on the missing electron density map, the regions are very flexible; conversely, they are versatile to seize the LC3-PE molecules on the membrane. In addition to these regions, there are two independent domains, catalytic (CAT) and membrane targeting (MT). The CAT domain shows a similar folding pattern to Ub-like proteases and contains the catalytic triad residues His176-Asp197-Cys258 for the hydrolysis of the peptide bond ( Horenkamp et al., 2015 ; Kwon et al., 2017b ). The function of the MT domain is characterized by a higher preference for phosphatidylinositol 3-phosphate, which is likely to be abundant in the autophagosomal membrane ( Horenkamp et al., 2015 ). Based on this structural information, a working model for RavZ has been proposed ( Fig. 3C ). Both N-LIR1/2 and C-LIR tether the LC3-PE molecules on the membrane and RavZ cuts the third (or one of the tethered) LC3-PE molecule by using its CAT domain. Here, we propose a 'Tethering and Cut' model for further explanation. SAXS (small-angle X-ray scattering) data have shown a 1:2 RavZ-(LC3) 2 complex model, which supports this model, although the resolution is relatively low ( Kwon et al., 2017b ). Later, another model, 'Lift and Cut', was proposed independently ( Pantoom et al., 2017 ; Yang et al., 2017 ) ( Fig. 3D ). This model is based on the conformational change of the hydrophobic α3-helix, which may point towards the membrane, and the structurally similar region in the CAT domain with lipid-binding protein yeast Sec14 homolog (Shf1) ( Yang et al., 2017 ). The α3-helix picks out the LC3-PE from the membrane and the lipid PE moiety is then recognized and cut by RavZ. However, both models are incomplete owing to the absence of the structure of the complex with LC3 bound to the active site of RavZ. Another controversy is the exact role of the two N-LIR1/2 motifs; it is possible that the reported complex structure between RavZ and LC3 might be a crystallization artifact and the only second LIR2 is proposed to be critical ( Yang et al., 2017 ). However, another report showed that deletions, and even point mutations, on any of the LIR motifs resulted in quite significant functional defects in cell-based assays ( Kwon et al., 2017b ). Furthermore, the structure of the complex between LC3 and a longer peptide comprising tandem N-LIR1/2 showed that the first LIR (LIR1) was a major contributory factor for LC3 binding and the tandem LIR motifs formed a characteristic β-sheet conformation to augment the binding affinity ( Kwon et al., 2017a ). CONCLUSION To fully understand the function of proteins involved in host xenophagy and in the manipulation of host autophagy, the combination of three-dimensional structures with biochemistry and cell biology data is necessary. Novel findings and mechanisms are continuously proposed, which makes the explanation of the structure-function relationships of all molecules challenging. Therefore, we focused on two examples, the NDP52-GAL8 interaction in the host and the RavZ-LC3 interaction in the bacteria Legionella pneumophila . The structural details of the complex between NDP52 and GAL8 have previously been determined ( Kim et al., 2013 ; Li et al., 2013 ) and explain how the sugar receptor is involved in this autophagy pathway. The autophagy receptor NDP52 is targeted to the phagophore via LC3 interaction. We have generated a plausible model for this event ( Fig. 2C ); however, the missing link is bacterial ubiquitylation. Most probably, the Ub-coated bacteria are recognized by the UBZ domain of NDP52, but it is still unclear which molecules of the bacterial surface are ubiquitylated by the host E3-ligases, despite the identification of three E3 Ub-ligases ( Huett et al., 2012 ; Manzanillo et al., 2013 ; Noad et al., 2017 ). The identification of the ubiquitylation target(s) in bacteria is one of the key research directions to understand xenophagy. As noted, each bacterium utilizes unique molecules to manipulate host autophagy. Legionella pneumophila secrete RavZ, an LC3 deconjugating enzyme, for their survival. Structural information on RavZ has been competitively reported by several research groups, although some controversies remain. Two proposed models for RavZ action in the cells should be further verified biochemically and structurally. However, this is not straightforward because the RavZ works in the vicinity of the membrane and thus, all experimental conditions are required to mimic these environments. The regions containing LIR motifs are very flexible and there are many, which complicates the study of the RavZ-LC3 interaction. To provide greater understanding of the RavZ action, we await the structural information of RavZ in complex with LC3-PE at the active site, which will be helpful to develop therapies against Legionnaires' disease.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5618224/

Shiga Toxin Therapeutics: Beyond Neutralization

Ribotoxic Shiga toxins are the primary cause of hemolytic uremic syndrome (HUS) in patients infected with Shiga toxin-producing enterohemorrhagic Escherichia coli (STEC), a pathogen class responsible for epidemic outbreaks of gastrointestinal disease around the globe. HUS is a leading cause of pediatric renal failure in otherwise healthy children, resulting in a mortality rate of 10% and a chronic morbidity rate near 25%. There are currently no available therapeutics to prevent or treat HUS in STEC patients despite decades of work elucidating the mechanisms of Shiga toxicity in sensitive cells. The preclinical development of toxin-targeted HUS therapies has been hindered by the sporadic, geographically dispersed nature of STEC outbreaks with HUS cases and the limited financial incentive for the commercial development of therapies for an acute disease with an inconsistent patient population. The following review considers potential therapeutic targeting of the downstream cellular impacts of Shiga toxicity, which include the unfolded protein response (UPR) and the ribotoxic stress response (RSR). Outcomes of the UPR and RSR are relevant to other diseases with large global incidence and prevalence rates, thus reducing barriers to the development of commercial drugs that could improve STEC and HUS patient outcomes. 1. Introduction Shiga toxins are ribotoxic proteins produced by several species of bacteria responsible for epidemic outbreaks of human gastrointestinal disease [ 1 , 2 ]. The prototypical toxin of this group is Shiga toxin produced by Shigella dysenteriae Type 1, an etiologic cause of bacterial dysentery associated with contaminated water supplies [ 3 , 4 ]. The related proteins Shiga-like toxin 1 (STX1) and Shiga-like toxin 2 (STX2) are produced by various pathogenic strains of Shiga toxin-producing Escherichia coli (STEC) responsible for food-borne illnesses globally, including numerous outbreaks in the United States, Europe, South America, and Japan [ 5 , 6 , 7 ]. STX1 and STX2 are encoded within the genome of lysogenized bacteriophages that can be transferred between related bacteria, creating a diverse array of bacterial strains secreting one or more toxin subtypes [ 1 , 8 ]. Shiga toxins are the etiologic cause of post-diarrheal hemolytic uremic syndrome (HUS), a thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure following a course of bacterially induced hemorrhagic diarrhea [ 9 , 10 , 11 , 12 ]. Neurologic disease is a frequent complication of STEC infection via imprecisely defined mechanistic causes [ 12 , 13 , 14 ]. Approximately 5–30% of patients suffer long term morbidity from chronic renal insufficiency, hypertension, or neurological deficits following the resolution of active HUS [ 15 ]. Children younger than 2 years of age are particularly susceptible to Shiga toxin-induced HUS, and the overall HUS rates vary between 5–15% of confirmed STEC cases depending on the infecting bacterial strain. The recent European outbreak involving an atypical STEC O104:H4 strain showed substantially higher rates of adult HUS in part due to its enteroaggregative properties, and future emerging Shiga toxin-producing pathogens may have variant epidemiological profiles [ 6 , 16 , 17 ]. STEC strains are susceptible to antibiotics, but antibiotic therapy is generally contraindicated due to an association of antibiotic treatment with increased toxin production and risk of HUS development [ 18 , 19 ]. However, antibiotic treatment appeared to be effective during the European O104:H4 outbreak, and this was later confirmed by in vitro evaluation of patient isolates [ 20 ]. This highlights a need for rapid and specific clinical laboratory serotyping coupled with toxin detection, a technology that is not yet available commercially. As a result, the standard of care remains supportive and avoids antibiotics. The clinical management of STEC cases is complicated further by the lack of validated clinical biomarkers capable of predicting HUS onset prior to the development of thrombocytopenia and renal damage. There are no commercially approved therapeutics that specifically treat or prevent HUS caused by Shiga toxin-producing pathogens, and supportive care with careful fluid management is the recommended treatment following diagnosis [ 21 ]. Plasmapheresis and treatment with the C5 complement inhibitor Eculizumab ® have not shown consistent clinical benefits in human patients [ 22 , 23 ]. Due to the diversity of E. coli serotypes capable of causing Shiga toxin-mediated disease and the potential of new emerging Shiga toxin-producing pathogens, treatments that target the activity of the toxin are currently being sought to prevent the development of HUS and to improve HUS patient outcomes. The focus of therapeutic development for Shiga toxicosis and HUS has been the blockade of toxin activity or intracellular trafficking. Thus far, no Shiga toxin-specific therapeutic has advanced past Phase II clinical trials in the United States, partially due to the difficulties in drug development for a sporadic acute disease [ 24 ]. In this review, an alternate strategy of therapeutic development is explored that proposes to target the downstream signaling and outcomes of Shiga toxin activity. The overlap of Shiga toxin-induced stress pathways with common diseases may lead to a more rapid development and approval of commercially available therapeutics to improve patient outcomes compared to the direct targeting of the toxin itself. 2. Shiga Toxin Structure and Activity Shiga toxins are AB5 toxins composed of a single A subunit and a pentameric B subunit [ 2 , 25 ]. Shiga toxins bind to the cell membrane glycolipid globotriaocylceramide (Gb3) via three binding sites on the B subunit to initiate endocytosis and gain cellular entry [ 26 , 27 ]. Retrograde trafficking machinery shuttle the toxin from the early endosome through the Golgi and endoplasmic reticulum (ER) [ 28 ]. During this process, furin-like proteases cleave a target site within the A subunit to create a catalytically active A subunit [ 29 , 30 ]. The active toxin is then transported from the ER into the cytosol to reach its target, the 28S rRNA of ribosomes [ 31 , 32 ]. Shiga toxins depurinate the conserved adenine residue 2260 within the sarcin–ricin loop of 28S eukaryotic rRNA via N-glycosidase activity to inhibit binding by the elongation factor EIF2a, thus terminating peptide elongation [ 31 , 33 ]. Other ribosome-inactivating toxins act on the sarcin–ricin loop of 28S eukaryotic rRNA, and the specific mechanism of toxicity via depurination is shared between Shiga toxins and the plant-derived toxin ricin [ 32 ]. Shiga toxins are highly toxic to Gb3-positive cells in culture, though their sensitivity is known to vary widely between cell lines and toxin subtypes [ 34 , 35 , 36 ]. Shiga toxicity in rodents, pigs, rabbits, and non-human primates differs in the distribution of tissues affected, presumably due to species differences in cellular Gb3 expression and localization. Rodents and rabbits develop gastrointestinal and renal tubular epithelial lesions when challenged with toxin, but fail to develop the glomerular endothelial damage and clinical HUS seen in intoxicated human patients and non-human primates [ 37 , 38 , 39 , 40 ]. Toxin-induced alterations in ribosomal structure and activity initiate cell stress pathways known as the unfolded protein response (UPR) and the ribotoxic stress response (RSR), which activate a variety of pro-inflammatory and pro-apoptotic cellular effector proteins that contribute to cellular dysfunction and disease [ 41 , 42 , 43 , 44 , 45 , 46 ]. The outcomes of these stress responses are inflammatory cytokine secretion, cellular apoptosis, and endothelial dysfunction, all of which are potential contributors to Shiga toxin-induced disease in vivo. 3. Current State of Shiga Toxin-Targeted Therapeutic Development The field of Shiga toxin therapeutic development has focused on inhibiting the toxins at various points in the pathway between entry into the cell and ribosomal depurination by activated toxin A subunits ( Figure 1 ). The therapeutic targeting of the toxin is conceptually justified due to a direct causal link between Shiga toxin and the development of HUS [ 10 , 12 ]. While this review will briefly summarize the current state of toxin-directed therapeutics, a more complete review was recently provided by Melton-Celsa and O'Brien [ 24 ]. Toxin-neutralizing therapeutics and vaccines for other toxin-based diseases, such as tetanus and anthrax, have yielded excellent clinical outcomes, thus providing inspiration for Shiga-like toxin (STX)-neutralizing therapeutic strategies [ 47 , 48 ] ( Table 1 ). Trials using polyclonal anti-sera or monoclonal anti-Shiga toxin antibodies in animal models of Shiga toxicosis successfully rescue from mortality if treatment is given within 48 h of toxin exposure [ 49 , 50 , 51 , 52 ]. Monoclonal humanized murine anti-Shiga toxin antibodies have reached Phase II clinical trials within the U.S., but none have completed Phase III trials [ 53 , 54 ]. Camelid heavy-chain-only antibody constructs containing heavy-chain oligomers specific for the toxin have been developed and are effective in murine and porcine models, but have not been used in humans to date [ 55 ]. The heavy-chain antibody constructs have been administered as a bolus injection or introduced via a replication-incompetent adenoviral construct, with both routes of administration conferring protection against STXs [ 56 ]. One of the challenges facing antibody-based Shiga toxin neutralizers is the variety of toxin subtypes present in various STEC strains. A monoclonal antibody must neutralize STX1 and multiple STX2 subtypes to be universally effective in STEC patients, since each STEC strain can secrete one or more subtypes. Specific multivalent binding of the Shiga toxin B subunit to Gb3 in all STX subtypes relevant to the human disease has led to the generation of synthetic Gb3 analog constructs capable of inhibitory toxin binding. Synsorb-Pk ® is a silicon dioxide backbone containing bound Gb3 capable of neutralizing Shiga toxins in vitro [ 57 ]. A Phase II trial in pediatric HUS patients failed to show any clinical improvement following treatment with Synsorb-Pk ® , possibly due to the inability of the orally administered drug to adsorb toxin produced at the mucosal surface or injected directly into epithelial cells by STEC [ 58 ]. The Nishikawa group has generated metallic backbone tetravalent peptides capable of binding to the Gb3 binding sites of STX1 and STX2 B subunits [ 59 , 60 ]. Variations of the construct have been found to be protective when administered systemically in murine and non-human primate models of Shiga toxicosis, but no human trials have been performed [ 61 , 62 ]. It remains to be seen if immunologic or Gb3-analog toxin neutralizers can be effective in the clinical environment where STEC-infected patients have likely been exposed to Shiga toxins for several days prior to presentation, or if preventing further toxin internalization will improve patient outcomes. The blockade or alteration of the retrograde trafficking system is an alternate strategy for the inhibition of Shiga toxin activity. Stechmann et al. characterized two small molecule re-localizers of the vesicular transport SNARE protein Syntaxin 5 that were capable of rescuing protein synthesis in cells incubated with ricin and Shiga toxins for 4 h [ 63 ]. They named the compounds Retro-1 and 2, and found that the drugs did not impact the localization of several other cargo transport proteins in vitro. Retro-2 was found to be partially protective in a murine model of STEC infection when given orally prior to bacterial toxin induction with mitomycin C and completely protective against nasal ricin challenge if given prophylactically [ 63 , 64 ]. The metallic cofactor manganese has also been found to protect mice from STX1 toxicity by stimulating the degradation of the endosome-to-Golgi transport protein Gpp130, but doses were given prophylactically every 24 h for 5 days prior to a single bolus injection of toxin [ 65 ]. Manganese failed to protect mice from STX2 toxicity due to STX2 transport through proteins other than Gpp130, limiting its usefulness clinically [ 66 ]. To date, there have been no published accounts of reduced clinical toxicity when retrograde trafficking inhibitors are given after toxin exposure in vivo, a condition that more closely replicates the likely clinical scenario in diagnosed STEC patients. 4. Beyond Toxin Neutralization While many toxin neutralizers and inhibitors have shown promise in pre-clinical settings and limited Phase II clinical trials, a direct inhibitor of Shiga toxins has not been successfully brought to market to prevent or ameliorate HUS in clinical STEC patients. Commercial barriers to treatments that specifically target sporadic acute epidemic diseases include difficulties in distribution and storage of the therapeutic as well as a reduced financial incentive to develop and produce therapeutics for an inconstant patient cohort. In addition, the usual HUS rate during an outbreak makes the statistics and finances for a clinical trial almost insurmountable. A simple design of two study groups (STEC versus STEC + HUS; α = 0.05, power 80%) and a 5% HUS rate will need over 600 patients to show effective drug prevention of HUS, which has been the criteria required by the FDA. Most outbreaks are less than 100 patients [ 5 , 72 ]. It is with these barriers in mind that an alternate strategy for STEC and HUS therapy should be considered that does not directly inhibit toxin activity but instead attempts to modulate the downstream stress responses to the catalytic activity of the toxin. Shiga toxins induce cellular stress pathways in sensitive cells following ribotoxicity and translational inhibition. Unfolded or incomplete proteins are detected in the ER by sensor proteins to initiate signaling cascades termed the unfolded protein response (UPR), and changes in ribosome conformation caused by depurination of the sarcin–ricin loop initiate separate signaling pathways termed the ribotoxic stress response (RSR). The UPR and RSR are relevant to other diseases, thus increasing the number of commercially viable drug candidates for the treatment of STEC and HUS patients. It is also possible that by the time of diagnosis, Shiga toxin internalization, processing, and ribosomal inactivation have already progressed to the point where toxin neutralizers and inhibitors alone will not be the most effective way of reducing cellular damage and HUS development. Targeting the UPR and RSR in STEC patients may be successful clinically, either alone or in combination with anti-toxin therapy. 5. The Unfolded Protein Response (UPR) The UPR is a eukaryotic cellular stress response triggered by the accumulation of unfolded or improperly folded peptides within the lumen of ER, also known as ER stress. The UPR is initiated by several proteins located within the ER lumen that sense unfolded proteins and are then activated to initiate signaling cascades that attempt to restore protein homeostasis. The best characterized sensors of ER stress are protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring protein 1α (IRE1α), and activating transcription factor 6α (ATF6α) [ 73 ]. IRE1α is an ER membrane-bound protein that activates its endoribonuclease activity via the dissociation of the inhibitory chaperone protein GRP78 during ER stress. IRE1α cleaves a 26 nucleotide segment from xbox binding protein 1 (XBP1) mRNA. The frameshift created by this cleavage leads to the translation of the active form of XBP1, which acts as a transcription factor for protein folding, protein transport, protein degradation, and mRNA degradation-associated genes. Concurrent with IRE1α activation, the ER stress sensor protein ATF6α moves from the ER to the Golgi for processing into its active form. Active ATF6α travels to the nucleus to upregulate the transcription of chaperone proteins. The combination of ATF6α and IRE1α activation leads to an upregulation of protein-folding and degradation machinery to restore protein homeostasis [ 73 , 74 ]. The ER membrane-bound kinase PERK is activated to initiate a separate signaling pathway during ER stress via phosphorylation and the inactivation of eukaryotic translation initiation factor 2a (eIF2a). The inactivation of eIF2a inhibits translation to allow cellular machinery time to properly fold or degrade misfolded proteins. Phosphorylated eIF2a simultaneously upregulates several transcripts coding for proteins involved in protein degradation, mRNA degradation, chaperone proteins to aid in peptide folding, and protein transporters. A key known transcriptional target of phosphorylated eIF2a is activating transcription factor 4 (ATF4). ATF4 is a transcription factor for antioxidant and amino acid biosynthesis genes as well as CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP). CHOP forms heterodimers with ATF4 to upregulate the transcription of additional UPR targets, including growth arrest and DNA-damage-inducible protein 34 (GADD34). GADD34 is a phosphatase that dephosphorylates eIF2a to reinitiate ribosomal translation [ 73 , 74 , 75 ]. The activation of ATF6α and IRE1α occurs transiently during ER stress, but the activation of PERK persists until ER stress is resolved [ 76 ]. While a short term activation of the UPR promotes survival of the cell, chronic UPR activation eventually leads to cellular apoptosis. The transcription factor CHOP concurrently suppresses anti-apoptotic Bcl-2 expression and enhances pro-apoptotic factor expression if chronically present in the cell. CHOP activates ER oxidase 1α (ERO1α), a protein that facilitates disulfide bond formation in the ER [ 77 ]. In the context of unresolved ER stress, the reactive oxygen species generated by disulfide bond formation lead to increased Ca +2 efflux from the ER, which in turn causes mitochondrial stress. Resuming ribosomal translation via GADD34 phosphatase activity on eIF2a in the context of unresolved ER stress leads to further generation of reactive oxygen species and ATP depletion [ 75 ]. CHOP appears to be the key factor in the progression of the UPR to apoptosis, as the knockout of CHOP in cultured cells and mice protects from apoptosis in various models of chronic UPR [ 77 , 78 ]. A chronic upregulation of CHOP leads to an upregulation of Bcl-2 interacting mediator of cell death (BIM) protein expression with a concurrent downregulation of the anti-apoptotic protein Bcl-2. BIM activates caspase 3, which in turn activates the caspase 8 executioner complex to initiate apoptosis [ 73 ]. 6. The UPR in Health, Disease, and Shiga Toxicosis Although the UPR is considered a stress response, it is a critical process involved in the normal function of eukaryotic organisms during development, cellular differentiation, and during times of intense cellular metabolism. During the differentiation of cells, large scale shifts in protein synthesis occur that can transiently lead to excess amounts of unfolded proteins. B cell development is a known example where abrogation of the UPR hinders cellular functional differentiation [ 79 , 80 , 81 ]. Cells that acutely produce and secrete proteins in response to stimulus also rely on the UPR to function normally and avoid cell death. Insulin-producing beta cells of the pancreas and activated inflammatory cells secreting cytokine and chemokine proteins are known to activate the UPR during stimulation [ 82 , 83 , 84 ]. Induction of the UPR by Shiga toxins has been documented in various susceptible cell types in culture as well as the renal tissue of in vivo mouse models of Shiga intoxication. While apoptosis occurs in susceptible cells exposed to Shiga toxins, it remains unclear if the UPR is the key driver of cellular apoptosis or if other cell stress response pathways, such as the RSR and TNFα death signaling, are involved [ 85 , 86 ]. STXs are known to associate with the ER chaperone proteins HEDJ and BiP during retrograde transport, possibly contributing to initiation of the UPR in addition to ribosomal inactivation [ 87 , 88 ]. Human monocytic leukemia cells upregulate IRE1α, p-PERK, and CHOP when exposed to STX1, and subsequently generate active forms of XBP1 and caspase 8 [ 44 ]. The knockdown of CHOP in THP-1 cells prevents apoptosis and caspase 8 activation [ 89 ]. Human brain microvascular endothelial cells showed a similar upregulation of CHOP and generation of caspase 8 when exposed to STX2 [ 90 ]. Mouse models injected with purified STX2 or infected with the intestinal pathogen Citrobacter rodentium carrying a STX2-containing plasmid showed increased renal expression of CHOP and spliced XBP1 transcripts as well as reduced Bcl-2 transcripts [ 46 ]. The UPR is also induced in a variety of diseases associated with chronically increased cellular protein production and/or secretion ( Table 2 ). Insulin resistance and diabetes chronically induce the UPR in insulin-producing beta cells, leading to beta cell loss over time [ 84 , 91 ]. In diabetes-prone mice, the deletion of CHOP and other late-UPR factors rescues mice from the loss of beta cells. Non-alcoholic fatty liver disease leads to chronic UPR in hepatocytes due to increased fatty acid metabolic pathway activity, eventually contributing to intracellular lipid accumulation and hepatocyte dysfunction [ 92 ]. Neurons upregulate the UPR during neurodegenerative diseases associated with protein aggregates, such as Parkinson's disease and Alzheimer's disease [ 93 ]. Defective UPR responses in gastrointestinal Paneth cells have been suggested to contribute to inflammatory bowel disease, with unresolved ER stress leading to inflammation and a loss of microbiome homeostasis [ 94 , 95 ]. ER stress responses have also been found to contribute to cardiac dysfunction following hypoxia, and are associated with aberrant angiogenesis during vascular retinopathies and neoplastic growth [ 96 , 97 , 98 , 99 , 100 ]. Therapeutic development targeting the UPR is relevant for common chronic diseases, and could also be promising for the treatment of Shiga-intoxicated patients, thus reducing economic barriers for the development of treatments for STEC and HUS. 7. Targeting the UPR The most promising approach to the treatment of UPR-related diseases is to target the downstream effectors of chronic CHOP–ATF6 activity leading to the upregulation of apoptotic factors, the downregulation of anti-apoptotic factors, and the eventual activation of caspase 3 and caspase 8 to initiate apoptosis ( Figure 2 ). A high-throughput therapeutic screening technique validated this theory in a variety of bacterial toxins including ricin, anthrax toxin, and diphtheria toxin by identifying a universally protective compound called biothionol. Biothionol is a small molecule caspase 3, 6, and 7 inhibitor found to prevent cytotoxicity by ricin in RAW264.7 and C32 cells in the face of ribosomal inactivation, and did not show signs of toxicity in mice [ 101 ]. Bithionol has not been used in STX models to date. Ouabain is a cardiotonic steroid that decreases Ca +2 fluxes intracellularly and increases Bcl-XL expression via the activation of Nf-KB p65 subunits, thus blunting the outcomes of chronic UPR activation. Treatment of rat proximal renal tubular cells exposed to STX2 with Ouabain in vitro prevented caspase 3 activation and cellular apoptosis. Ouabain was also found to protect human renal tubular epithelial cells and human glomerular endothelial cells from STX2-induced apoptosis in vitro [ 101 ]. Treatment of mice with continuous subcutaneous infusion of Ouabain prevented renal tubular epithelial apoptosis and a loss of podocytes 48 h after injection with STX2. An alternate protective strategy is to increase the cellular capacity to resolve the UPR in an attempt to avoid chronic ER stress ( Figure 2 ). Small molecule protein chaperones that improve protein folding in cells undergoing ER stress improve outcomes in mouse models of diabetes and inflammatory bowel disease, and have shown positive results in human patients with insulin resistance or cirrhosis [ 102 , 103 , 104 , 105 ]. Extendin-4 is an agonist of glucagon-like peptide agonist 1 that increases the expression of ATF4 in pancreatic beta cells to increase the protective effects of the early UPR [ 106 ]. Extendin-4 has been approved for use in human diabetics in the United States and Europe. To date, therapeutics that enhance UPR resolution have not been studied in the context of Shiga toxin-mediated disease in vitro or in vivo. 8. The Ribotoxic Response In addition to the UPR, damage to domains V or VI of the ribosomal 28S rRNA has been found to initiate a stress response signaling cascade termed the ribotoxic stress response (RSR) [ 107 ]. The activity of ribotoxins such as ricin, sarcin, anisomycin, and deoxynivalenol (DON) leads to the activation of double-stranded-RNA-activated kinase R (PKR), likely through the homodimerization and transphosphorylation of ribosome-associated PKR [ 108 ]. PKR phosphorylates the translation factor eIF2a to inactivate it and initiate a signaling cascade leading to variable activation of classical p38 MAP kinases, ERK, and JNK depending on the cell type. It should be noted that both the UPR and the ribotoxic response involve the phosphorylation of eIF2a, but in cell culture experiments the ribotoxic stress response could be elicited at low levels of translational inhibition by ribotoxins. Furthermore, ribotoxins, such as emetine and pactamycin, that act on ribosomal regions other than domain V or VI fail to elicit the activation of PKR with downstream kinase activation despite their ability to inhibit protein synthesis [ 107 ]. These findings suggest that the RSR is an independent stress pathway that is distinct from the UPR and is not reliant on translational inhibition. Hematopoetic cell kinase (Hck) was found to associate with PKR and the 40S ribosome in human monocytic U937 cells, and the pharmacological inhibition of Hck activity prevented p38 MAPK phosphorylation and IL-8 secretion in response to DON, suggesting that Hck is also a key sensor of ribosomal damage necessary for downstream effector kinase signaling [ 109 ]. While the details of the signaling pathway between PKR and downstream effector kinase activity remain unclear, the zipper sterile alpha motif kinase (ZAK) has been identified as a critical component to signaling via pharmacological and siRNA knockdown studies in vitro. An abrogation of ZAK activity prevents the activation of p38 MAPK, JNK, and ERK in Hct-8 and Vero cells incubated with STX1 [ 81 ]. The ribotoxic stress response may also lead to cell death if chronic stimulation occurs, with NLRP3 inflammasome activation and pyroptosis via caspase 1 activation documented in THP-1 cells incubated with active STX1 in a concentration-dependent manner [ 41 ]. The in vitro impact of the RSR varies depending on the cell type, but is generally characterized by an upregulation of inflammatory cytokine and chemokine transcription and translation. Differentiated macrophage-like THP-1 cells secrete TNFα, IL-1β, IL-8, and MIP-1α in response to stimulation with STXs, and the response could be blunted through an inhibition of JNK, p38 MAPK, and ERK with variance in effect based on the kinase inhibited [ 43 , 110 , 111 , 112 ]. Human intestinal epithelial Hct-8 cells secreted the chemokine IL-8 when incubated with STX1 or the ribotoxic antibiotic anisomycin, and concurrent incubation with E. coli flagella and STX2 led to a superinduction of IL-8 secretion compared to flagella alone [ 113 , 114 ]. Interleukin 8 secretion was reduced through the inhibition of ERK, p38 MAPK, or JNK signaling or through use of a ZAK inhibitor. The amplification of MAPK, JNK, and ERK phosphorylation has also been documented in human dendritic cells co-stimulated with lipopolysaccharide (LPS) and anisomycin, with increased TNFα and IL12 secretion compared to stimulation with LPS alone [ 115 ]. Human brain endothelial cells secreted IL-6 and IL-8 in response to STX1, and the amount of protein secreted was amplified by costimulation with TNFα and STX1 concurrently [ 116 , 117 ]. Human microvascular endothelial cells were found to upregulate the chemokines IL-8, CXCL4, CXCR7, and CXCL12 in response to challenge with STX holotoxins, with delayed degradation of chemokine transcripts [ 118 ]. In vivo experiments in mice and non-human primates (NHPs) have documented acute tissue inflammation in the kidneys as well as increased circulating acute inflammatory proteins following exposure to Shiga toxins via injection or gastrointestinal infection with STEC [ 40 , 52 , 119 ]. In both mice and NHPs, increased circulating and renal TNFα, IL-6, IL-1β, and CXC chemokines are present following injection with purified STX1 and STX2. Mice lacking the gene encoding ZAK were protected from gastrointestinal ricin toxicity, with reduced CXCL1 production following depurination of the sarcin–ricin loop [ 120 ]. The treatment of rabbits with the ZAK kinase inhibitor imatinib reduced the number of neutrophils infiltrating colonic tissue infected with STEC [ 121 ]. In human HUS patients, serum and urine IL-6 levels correlated with severity of disease, and HUS patients suffering from neurologic complications had detectable increases in brain IL-1β content [ 122 , 123 , 124 ]. 9. Targeting the RSR and Inflammation during Shiga Toxicosis Limited research has been performed to evaluate the impact of targeted anti-inflammatory therapy on susceptibility to STX-mediated tissue injury, morbidity, and mortality. Alves-Rosa et al. hypothesized that the enhanced inflammatory response documented in vitro in cells costimulated with STXs and LPS could be blunted with anti-LPS antibodies in vivo. Mice immunized with E. coli O111:B4 were challenged with LPS and STXs following the confirmation of circulating anti-LPS IgG antibodies. There was no difference in mortality in immunized mice following STX + LPS challenge; however, the immunized mice failed to suppress circulating TNFα following intravenous LPS challenge compared to naïve mice, suggesting that the immunization did not prevent pattern recognition receptor activation by circulating LPS [ 125 ]. Mice pretreated with immunosuppressive doses of dexamethasone exhibited greater survival following STX2 challenge, reduced numbers of Gb3-positive CNS cells, reduced damage to the blood brain barrier, and a reduction in the number of activated astrocytes compared to controls [ 126 ]. The vasoactive drug anisodamine, an inhibitor of TNFα secretion, improved the survival of mice injected with lethal doses of STX. The improved survival was reversed via a concurrent injection of recombinant TNFα with STX, suggesting that the suppression of TNFα by anisodamine provided significant protection from STX-induced disease [ 127 ]. The pretreatment of mice with MCP-1-, MIP-1α-, or RANTES-neutralizing antibodies protected against renal fibrin deposition following STX + LPS injections [ 128 ]. Isogai et al. found that germ-free mice pretreated with anti-TNF antibodies and infected with STEC were protected from clinical signs of morbidity, reduced renal pathology on histology, and reduced renal IL-1β, TNFα, and IL-6 protein concentrations despite similar levels of intestinal colonization and fecal STX content compared to controls [ 129 ]. A broad range of diseases are either driven through inflammatory responses or are complicated by inflammatory dysfunction. Anti-cytokine and chemokine therapeutics are being developed for use in diseases ranging from rheumatoid arthritis to inflammatory bowel disease, with multiple commercially approved drugs already available in the United States [ 130 , 131 , 132 , 133 ]. General anti-inflammatory drugs, such as glucocorticoids and NSAIDs, have been available for decades to treat acute inflammatory diseases. Further preclinical studies are necessary to characterize the role of inflammation in the pathogenesis of STX-induced HUS, as the role of the host response to STEC infection in the pathogenesis of HUS remains unclear [ 10 , 11 , 12 ]. Further work is necessary to determine if specific host cytokine responses are necessary for the development of HUS to determine the suitability of RSR elements as therapeutic targets for clinical study ( Figure 3 ). 10. Future Directions Research focusing on traditional toxin neutralizers has yielded several candidate molecules successful in animal models, but none have achieved FDA approval for use in patients. The epidemiologic profile of STEC outbreaks complicates pharmaceutical development due to its acute, sporadic, and geographically dispersed distribution of cases. Focusing on the downstream molecular impacts of STX, such as the UPR and RSR, have the advantage of utilizing therapeutics developed for use in common chronic diseases that have a greater financial incentive for pharmaceutical development. Further preclinical study is required to determine the roles of the UPR and RSR during development of HUS in order to identify and validate potential novel therapeutic targets to improve STEC and HUS patient outcomes.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198012/

Silencing of Kir2 channels by caveolin-1: cross-talk with cholesterol

A growing number of studies show that different types of ion channels localize in caveolae and are regulated by the level of membrane cholesterol. Furthermore, it has been proposed that cholesterol-induced regulation of ion channels might be attributed to partitioning into caveolae and association with caveolin-1 (Cav-1). We tested, therefore, whether Cav-1 regulates the function of inwardly rectifying potassium channels Kir2.1 that play major roles in the regulation of membrane potentials of numerous mammalian cells. Our earlier studies demonstrated that Kir2.1 channels are cholesterol sensitive. In this study, we show that Kir2.1 channels co-immunoprecipitate with Cav-1 and that co-expression of Kir2.1 channels with Cav-1 in HEK293 cells results in suppression of Kir2 current indicating that Cav-1 is a negative regulator of Kir2 function. These observations are confirmed by comparing Kir currents in bone marrow-derived macrophages isolated from Cav-1 −/− and wild-type animals. We also show, however, that Kir2 channels maintain their sensitivity to cholesterol in HEK293 cells that have very low levels of endogenous Cav-1 and in bone marrow-derived macrophages isolated from Cav-1 −/− knockout mice. Thus, these studies indicate that Cav-1 and/or intact caveolae are not required for cholesterol sensitivity of Kir channels. Moreover, a single point mutation of Kir2.1, L222I that abrogates the sensitivity of the channels to cholesterol also abolishes their sensitivity to Cav-1 suggesting that the two modulators regulate Kir2 channels via a common mechanism.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375772/

Progress towards the Development of a NEAT Vaccine for Anthrax II: Immunogen Specificity and Alum Effectiveness in an Inhalational Model

Bacillus anthracis is the causative agent of anthrax disease, presents with high mortality, and has been at the center of bioweapon efforts. The only currently U.S. FDA-approved vaccine to prevent anthrax in humans is anthrax vaccine adsorbed (AVA), which is protective in several animal models and induces neutralizing antibodies against protective antigen (PA), the cell-binding component of anthrax toxin. However, AVA requires a five-course regimen to induce immunity, along with an annual booster, and is composed of undefined culture supernatants from a PA-secreting strain.

PMC