Clostridium perfringens Produces an Adhesive Pilus Required for the Pathogenesis of Necrotic Enteritis in Poultry.

Clostridium perfringens type G strains cause necrotic enteritis (NE) in poultry, an economically important disease that is a major target of in-feed antibiotics. NE is a multifactorial disease, involving not only the critically important NetB toxin but also additional virulence and virulence-associated factors. We previously identified a C. perfringens chromosomal locus (VR-10B) associated with disease-causing strains that is predicted to encode a sortase-dependent pilus. In the current study, we sought to provide direct evidence for the production of a pilus by C. perfringens and establish its role in NE pathogenesis. Pilus structures in virulent C. perfringens strain CP1 were visualized by transmission electron microscopy (TEM) of immunogold-labeled cells. Filamentous structures were observed extending from the cell surface in wild-type CP1 but not from isogenic pilin-null mutant strains. In addition, immunoblotting of cell surface proteins demonstrated that CP1, but not the null mutant strains, produced a high molecular weight ladder-like pattern characteristic of a pilus polymer. Binding to collagen types I, II, and IV was significantly reduced (Tukey's test, P < 0.01) in all three pilin mutants compared to CP1 and could be specifically blocked by CnaA and FimA antisera, indicating that these pilins participate in adherence. Furthermore, fimA and fimB null mutants were both severely attenuated in their ability to cause disease in an in vivo chicken NE challenge model. Together, these results provide the first direct evidence for the production of a sortase-dependent pilus by C. perfringens and confirm its critical role in NE pathogenesis and collagen binding.IMPORTANCE In necrotic enteritis (NE), an intestinal disease of chickens, Clostridium perfringens cells adhere tightly to damaged intestinal tissue, but the factors involved are not known. We previously discovered a cluster of C. perfringens genes predicted to encode a pilus, a hair-like bacterial surface structure commonly involved in adherence. In the current study, we have directly imaged this pilus using transmission electron microscopy (TEM). We also show that inactivation of the pilus genes stops pilus production, significantly reducing the bacterium's ability to bind collagen and cause disease. Importantly, this is the first direct evidence for the production of a sortase-dependent pilus by C. perfringens, revealing a promising new target for developing therapeutics to combat this economically important disease.

Nonculture molecular techniques for diagnosis of bacterial disease in animals: a diagnostic laboratory perspective.

The past decade has seen remarkable technical advances in infectious disease diagnosis, and the pace of innovation is likely to continue. Many of these techniques are well suited to pathogen identification directly from pathologic or clinical samples, which is the focus of this review. Polymerase chain reaction (PCR) and gene sequencing are now routinely performed on frozen or fixed tissues for diagnosis of bacterial infections of animals. These assays are most useful for pathogens that are difficult to culture or identify phenotypically, when propagation poses a biosafety hazard, or when suitable fresh tissue is not available. Multiplex PCR assays, DNA microarrays, in situ hybridization, massive parallel DNA sequencing, microbiome profiling, molecular typing of pathogens, identification of antimicrobial resistance genes, and mass spectrometry are additional emerging technologies for the diagnosis of bacterial infections from pathologic and clinical samples in animals. These technical advances come, however, with 2 caveats. First, in the age of molecular diagnosis, quality control has become more important than ever to identify and control for the presence of inhibitors, cross-contamination, inadequate templates from diagnostic specimens, and other causes of erroneous microbial identifications. Second, the attraction of these technologic advances can obscure the reality that medical diagnoses cannot be made on the basis of molecular testing alone but instead through integrated consideration of clinical, pathologic, and laboratory findings. Proper validation of the method is required. It is critical that veterinary diagnosticians understand not only the value but also the limitations of these technical advances for routine diagnosis of infectious disease.

Identification of accessory genome regions in poultry Clostridium perfringens isolates carrying the netB plasmid.

Necrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. NE pathogenesis involves the NetB toxin, which is encoded on a large conjugative plasmid within a 42-kb pathogenicity locus. Recent multilocus sequence type (MLST) studies have identified two predominant NE-associated clonal groups, suggesting that host genes are also involved in NE pathogenesis. We used microarray comparative genomic hybridization (CGH) to assess the gene content of 54 poultry isolates from birds that were healthy or that suffered from NE. A total of 400 genes were variably present among the poultry isolates and nine nonpoultry strains, many of which had putative functions related to nutrient uptake and metabolism and cell wall and capsule biosynthesis. The variable genes were organized into 142 genomic regions, 49 of which contained genes significantly associated with netB-positive isolates. These regions included three previously identified NE-associated loci as well as several apparent fitness-related loci, such as a carbohydrate ABC transporter, a ferric-iron siderophore uptake system, and an adhesion locus. Additional loci were related to plasmid maintenance. Cluster analysis of the CGH data grouped all of the netB-positive poultry isolates into two major groups, separated according to two prevalent clonal groups based on MLST analysis. This study identifies chromosomal loci associated with netB-positive poultry strains, suggesting that the chromosomal background can confer a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism, and plasmid maintenance.

Veterinary antimicrobial stewardship in North America.

Major changes are occurring in veterinary antimicrobial stewardship (AMS) in food animals in Canada and the USA. Advances have been ending the use of medically important antimicrobials (MIAs) as growth promoters and bringing all MIAs for food animals under veterinary prescription in Canada (2018) or MIAs in feed or water under veterinary prescription (2017) in the USA. The USA proposes bringing all MIAs for food and companion animals under veterinary oversight, to reduce the duration of preventive use for food animals and to develop a strategy for companion animals. Both countries are taking a 'One Health' approach as part of their national strategies on addressing AMS. Federal state or province jurisdictional issues have impeded development and implementation of regulation-based stewardship approaches. Veterinary regulatory bodies in some of the larger states and provinces are active in AMS. Both the American and Canadian veterinary medical associations are independently heavily engaged in promoting AMS, as are, variably, the different veterinary 'specialty' groups. Regulatory changes and market demand are markedly reducing the use of antimicrobials in food animals. The promotion of veterinary AMS is happening at an increasing pace.

Immunization with subunits of a novel pilus produced by virulent Clostridium perfringens strains confers partial protection against necrotic enteritis in chickens.

Necrotic enteritis (NE) is an economically important disease of broiler chickens that is caused primarily by Clostridium perfringens strains that produce the NetB toxin. It is controlled in North America principally through the application of in-feed antimicrobials, but alternative control methods, such as vaccination, are urgently needed. We previously identified a cluster of C. perfringens genes prevalent in disease-causing strains, denominated VR-10B, that is predicted to encode a pilus. The current study evaluated the ability of three predicted pilin structural subunits (CnaA, FimA, FimB) to protect against NE in two immunization studies. In the first study, young broiler chickens were immunized twice intramuscularly (i.m.) with CnaA or FimA, which resulted in only a weak serum antibody response, and no reduction in the severity of intestinal lesions following experimental challenge with C. perfringens strain CP1. In the second study, chickens were injected subcutaneously (s.c.) with CnaA, FimB, or a combination of all three proteins, on days 7, 14 and 19, which resulted in a marked antibody response specific to each antigen. Chickens immunized with either CnaA or FimB had significantly reduced NE lesion severity, whereas immunization with all three proteins in combination did not provide protection. Western blot experiments using serum from immunized birds were also performed, providing the first experimental evidence to suggest that this locus may in fact encode a functional pilus structure.

Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations.

Clostridium perfringens is an important pathogen of animals and humans and is the causative agent of necrotic enteritis (NE) in poultry. This study focuses on the typing of intestinal C. perfringens isolates (n = 61) from outbreaks of NE collected from several areas of Southern Ontario, using a recently developed multilocus sequence typing (MLST) technique. For comparison, C. perfringens isolates from healthy birds were also obtained and typed. An additional locus, the pfoS locus, was included in our analysis, in an attempt to increase the discriminatory ability of the method previously published. Birds were collected from two major poultry processors in Canada, and isolates from processor 2 formed a distinct MLST cluster. Isolates from healthy birds also collected from the outbreak flocks clustered together with isolates from the birds with NE. Although isolates from eight outbreaks clustered together, MLST types were also occasionally different between outbreaks. Strong linkage disequilibrium was observed between loci, suggesting a clonal C. perfringens population structure. Detection assays for toxin genes cpb2 (beta-2 toxin), tpeL, and the newly described netB (NetB toxin) were also performed. netB was almost always found in outbreak isolates, whereas cpb2 was found exclusively in healthy bird isolates. The toxin gene tpeL, which has not been previously identified in C. perfringens type A strains, was also found, but only in the presence of netB. Resistance to bacitracin was found in 34% of isolates from antimicrobial agent-free birds and in 100% of isolates from conventionally raised birds.

Performance of a whole blood interferon-gamma assay for detection and eradication of caseous lymphadenitis in sheep.

Caseous lymphadenitis (CLA), a chronic bacterial disease of sheep and goats caused by Corynebacterium pseudotuberculosis, could be controlled by eradication of infected carriers. This study aimed at validation of a whole blood interferon-gamma (IFN-gamma) enzyme immunoassay (EIA) (Bovigam, Pfizer) in naturally infected sheep for use in eradication of infection from a flock. This assay used formalin-inactivated whole bacterial cells as antigen. The sensitivity of the whole cell assay was improved by increasing both the volume of blood and the number of bacterial cells. The assay was validated in experimentally infected sheep and in a flock of known-negative sheep, as well as in a naturally infected flock, a proportion of which was vaccinated with a commercial CLA vaccine. An optical density (540nm) (OD) cut-off of 0.09 was effective in classifying animals as test positive or negative in the naturally infected flock, although there was variation in OD between visits, notably with weakly reacting animals. The test had a sensitivity of 91% and a specificity of 98%. Postmortem data supported the results in test-negative animals. Visit-to-visit variation in IFN-gamma EIA OD in the naturally infected flock as well as CLA disease status was used to develop an algorithm for the eradication of CLA from a known infected flock. The whole blood IFN-gamma assay shows promise for eradication of caseous lymphadenitis from sheep flocks.

Typing of Clostridium perfringens by multiple-locus variable number of tandem repeats analysis.

Clostridium perfringens is a well-characterized bacterial species which can be both commensal and pathogenic in humans and many animals. Genetic typing of the bacterium is often used for molecular epidemiological purposes, and can be useful for observing population structures as well. Analysis of the variable number of tandem repeats (VNTRs) within the genome, called multiple-locus VNTR analysis (MLVA) provides genetic information useful for molecular typing. A MLVA typing method has been developed recently by Sawires and Songer [Sawires, Y.S., Songer, J.G., 2005. Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens. Anaerobe 11, 262-272] for C. perfringens. A novel MLVA protocol is described here, with the aim of investigating the discriminatory potential of the method, and to obtain preliminary data on the population structure of C. perfringens from a wide variety of C. perfringens sources. This protocol uses new loci in noncoding regions of the chromosome, and also makes use of capillary electrophoresis for more precise results and for high-throughput typing. DNA sequencing of amplicons was performed to ensure inclusion of conserved tandem repeats within each locus. Fifty-four epidemiologically unrelated isolates from a local collection obtained from 11 different animal species were typed at 6 loci. Thirty-five unique MLVA types were obtained, resulting in a Simpson's index of diversity of 0.975. Epidemiologically related isolates (n=27) previously typed by pulsed-field gel electrophoresis (PFGE) were also examined with MLVA and the congruency of the two methods was found to be very high. All 81 isolates were successfully typed with MLVA, and polymerase chain reactions (PCR) were automated using robotics and 96-well plates, with PCR product sizes determined using capillary electrophoresis. Reproducibility was also shown to be very high.

Genetic diversity of Clostridium perfringens isolated from healthy broiler chickens at a commercial farm.

Clostridium perfringens is an important commensal and bacterial pathogen of many animal species. It has particular significance in poultry, where it may cause necrotic enteritis. Our objective was to characterize the population diversity of C. perfringens colonizing healthy birds, and to observe how diversity changed over time. Isolates were obtained from broiler chicken cecal samples in two barns on a single farm, on days 7, 14, 22, 27, 30 and 34 of a single 42-day rearing cycle. Bacitracin was used as a feed additive in one of the barns and withdrawn from the second barn for the duration of the experiment. Each isolate was typed using pulsed-field gel electrophoresis (PFGE) using SmaI restriction endonuclease. A total of 205 cecal isolates from 49 birds were typed, as well as 93 isolates from the barn environment (bedding, drinking water and feces). Eight major PFGE types and 17 subtypes were found in the 298 total isolates. The results show that an optimal sampling strategy would involve a large number of birds, with only a few isolates sampled per bird. The diversity of C. perfringens in this study appears to be low within a single bird, and increases as the bird matures. There was no significant difference in genetic diversity between the two barns. In addition, isolates from fresh fecal samples appear to represent the cecal C. perfringens population accurately, although this was not proven statistically. Antimicrobial susceptibility testing was performed on selected isolates (n=41) representing a cross-section of PFGE types. Based on minimum inhibitory concentration distributions, 95% of the isolates tested were deemed resistant to bacitracin, with a 16 microg/mL breakpoint. Three new cpb2 (beta2 toxin gene) variants were found in the study.

Live attenuated vaccine-based control of necrotic enteritis of broiler chickens.

A vaccine for necrotic enteritis (NE) of chickens would reduce the current need to prevent or treat the disease in broiler chickens with antimicrobial drugs. The objective of this study was to understand aspects of immunity to the disease. The first experiment examined the virulence of six strains of Clostridium perfringens isolated from cases of NE in broiler chickens. Using a 5-day experimental oral infection of 2-week-old broiler chickens, four of the six strains were found to be virulent. Pulsed-field gel electrophoresis and PCR showed that virulence was not associated with a plasmid encoding the beta2 toxin gene, cpb2, since this was present in virulent and one of the two avirulent strains. In the second experiment, two virulent and one avirulent strains were tested for their ability to immunize ("infection-immunization") chickens through the oral route. The procedure used experimental infection for 5 days followed by bacitracin treatment for 9 days, and then re-challenge 2 days later with a virulent strain, CP4. Infection-immunization with the virulent isolates protected chickens from subsequent virulent challenge, whereas the infection-immunization with the avirulent isolate did not. In a third experiment, two of four alpha-toxin-negative mutants of CP4 protected birds from experimental NE after oral immunization. These two mutants were also attenuated for virulence. We conclude that it is possible to immunize chickens successfully against NE and that immunogen(s) other than alpha-toxin are important in protective immunity against oral infection.

Assessment in mice of vapA-DNA vaccination against Rhodococcus equi infection.

There is a need to produce a vaccine against Rhodococcus equi pneumonia in foals in which immunity against infection is largely based on a type 1, cell-mediated, immune response. The VapA protein of the virulence plasmid of R. equi is highly immunogenic. To assess the potential of vapA-DNA to produce immunity, C57BL/6 and BALB/c mice were immunized with a DNA vaccine constructed from vapA incorporated into pcDNA3.1. The plasmid construct expressed VapA in a COS-7 cell line. Intramuscular immunization of mice resulted in enhanced clearance of R. equi from the liver of intravenously challenged mice compared to non-immunized controls. This effect was more marked when pORF-IL-12, a plasmid expressing murine IL12, was included with the vaccine. Antibody developed to VapA, with an IgG2a response being more marked in mice immunized with pcDNA-vapA than in non-immunized or in mice immunized with the mixed vapA and IL-12 plasmid constructs. In conclusion, this study has shown for the first time that DNA immunization with vapA enhances the immune responses of mice against R. equi infection, that the IgG subisotype response is consistent with a type 1-based immune response, and that this can be enhanced by injection of the IL-12 gene.

Epidemiology of Rhodococcus equi infection in horses.

Current understanding of the epidemiology of Rhodococcus equi infection on horse farms is reviewed. Infection is widespread in herbivores and their environment, because herbivore manure supplies the simple organic acid substrates on which the organism thrives. There is a progressive development of infection in the soil on horse farms with prolonged use, because: (1) there is a continual supply of nutrients; (2) the organism multiplies progressively as temperatures rise; (3) the bacterium has a robust nature. While this aerobic organism fails to multiply in the largely anaerobic intestine of the adult horse, multiplication to very large numbers may occur in the intestine of a foal in its first 8-12 weeks of life. Farms used for foal breeding over many years may thus become particularly dangerous for foals. Areas for future study include the effectiveness of decontamination, manure-removal programs and dust reduction in reducing challenge to susceptible foals.

Outer membrane proteins of three pathogenic Leptospira species.

The outer membrane proteins of seven reference strains of pathogenic Leptospira (L. alstoni serovar grippotyphosa, L. borgpetersenii serovar hardjo, and L. interrogans serovars autumnalis, bratislava, canicola, icterohaemorrhagiae, and pomona) were investigated to identify common surface-exposed outer membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sodium-N-lauroylsarcosinate-insoluble outer membrane enriched fractions of the reference serovars and two field isolates of serovars hardjo and pomona revealed six common protein bands with approximate molecular masses of 77, 66, 42, 35.5, 24, and 18 kDa. At times the 35.5 kDa endoflagellar band resolved into two distinct bands, 35.5 kDa and 34 kDa. Immunoblotting of the same fractions using rabbit leptospiral antibodies showed six bands to be common (66, 59.5, 44, 42, 35.5, and 18 kDa). The 44 kDa band stained poorly with Coomassie blue but prominently by immunoblotting. Four reference strains (serovars bratislava, canicola, icterohaemorrhagiae, pomona), and two field isolates of serovar pomona and one of serovar bratislava were grown in low iron media to which the iron chelators 2,2'-dipyridyl or ethylenediaminehydroxyphenylacetic acid were added. No iron-dependent expression of outer membrane proteins was observed. The only difference observed between the outer membrane proteins when reference serovars of canicola or pomona were grown in dialysis bags in the peritoneum of swine or in vitro was the loss of the 77 kDa band from in vivo grown organisms. Treatment of whole leptospires with proteinase K did not remove the 77, 66, 59.5, or 42 kDa protein; these proteins may not be surface expressed or are inaccessible to the proteinase K. The 44 kDa band could not be evaluated by this method and the 18 kDa band was proteinase K resistant.

Serodiagnosis of leptospirosis in pigs using an axial filament enzyme-linked immunosorbent assay.

The axial filament (AF) from Leptospira interrogans serovar canicola was isolated by cesium chloride density gradient centrifugation of 2% sarcosyl treated whole cells. Isolation of AF was confirmed by electron microscopic examination, by protein-A immunogold labelling, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting. Analysis by SDS-PAGE of the purified preparation showed relatively weak bands of molecular size 41 kDa and 21 kDa, and strong bands of 35 kDa and 34.5 kDa. Immunoblot analysis using antiserum to the AF against sonicated leptospires of a variety of serovars showed prominent reaction against the 41, 35, and 34.5 kDa protein bands, as well as against minor bands of molecular weight 43, 39, and 37 kDa. Antisera prepared against leptospiral serovars also identified minor bands at 33 and 32 kDa. Immunoblots with antiserum to whole cells of serovar bratislava detected the 35 and 34.5 kDa AF bands of Borrelia burgdorferi moderately and of Treponema hyodysenteriae only slightly in comparison to leptospiral AF. Antibody to B. burgdorferi did not detect the leptospiral AF antigen. Immunoblots with antiserum to T. hyodysenteriae showed a marked reaction with a 41 kDa band of B. burgdorferi but only a very minor reaction with leptospiral AF. The AF was tested in an AF-ELISA against sera from 260 pigs, many of which reacted in the microscopic agglutination test (MAT) against one or more leptospiral serovars. A sensitivity of 97.1% and a specificity of 93.1% was determined in comparison to the MAT. Only moderate correlation was observed between titres detected in the AF-ELISA and the MAT (r = 0.4). When sonicated whole cells (WC) of serovar canicola were used in an ELISA (WC-ELISA), high correlation was observed between AF-ELISA and WC-ELISA (r = 0.97). These findings show that the AF-ELISA can be used effectively as a species-specific antigen for the serological diagnosis of leptospirosis in swine and that sonicated whole cells can substitute excellently for purified AF as the antigen source. These findings may be extrapolated to the use of AF in immunodiagnosis of leptospirosis in other species.

Clinical manifestations, diagnosis, treatment, and prevention of Rhodococcus equi infections in foals.

Since the 1986 Rhodococcus equi workshop, there have been major breakthroughs in understanding the epidemiology of, the virulence of, and the immune response to, this intriguing pathogen. However, with the exception of the use of hyperimmune plasma for the prevention of the disease (Martens et al., 1989; Madigan et al., 1991) the clinical aspects of R. equi infections have essentially remained unchanged. This article reviews the various clinical manifestations and summarizes recent advances in diagnosis, treatment and prevention of R. equi infections in foals.

Role of antibody to extracellular proteins of Rhodococcus equi in protection against R. equi pneumonia in foals.

Rhodococcus equi produces two exoenzymes (REE), a cholesterol oxidase in large amounts and a phospholipase C, which cause lysis of sheep red blood cells (SRBC) sensitized with Staphylococcus aureus beta toxin. Two immunization studies were done in foals to determine the role of antibody to REE in protection against R. equi pneumonia. In the first study, three foals (mean age 10 days) were vaccinated four times at 2-week intervals with over 1 million units of partially purified exoenzymes (PREE). In the second study, three foals (mean age 19 days) were administered plasma from an adult horse vaccinated with PREE. Relatively low titres (16-32) of neutralizing antibody were detected in the foals of the former group, and passive transfer of neutralizing antibody (titres 32-64) occurred in the latter. Following immunization, principal foals and an equal number of similarly aged nonimmunized foals were challenged by aerosol with 1 x 10(10) live R. equi per day for 5 consecutive days. No severe clinical pneumonia developed in either group and, with one exception, only minor and resolving lung abscesses developed in these foals. These studies showed that antibody response of foals to immunization with PREE was poor, antibody to PREE did not prevent foals from developing lung abscesses following experimental infection, and that foals even as young as 3 weeks of age may be largely refractory to aerosol challenge with virulent R. equi.

Association with HeLa cells by Rhodococcus equi with and without the virulence plasmid.

HeLa cell association was examined in eight Rhodococcus equi isolates containing 80-85 kb plasmids and their plasmid negative derivatives, and in two other plasmid negative R. equi strains. Seven of the eight plasmid positive strains possessed an 85 kb plasmid and produced the virulence-associated protein (VapA) detected by monoclonal antibody staining in immunoblots; one of the eight had an 80 kb plasmid but did not produce VapA. Curing of the plasmids by repetitive subcultures at 42 degrees C in broth was confirmed by colony and DNA dot blot hybridization with a 7.5 kb BamHI-HindIII plasmid fragment probe, by attempted isolation of plasmid DNA, and by demonstration of lack of VapA. Most strains associated well with HeLa cells and no relationship was found with plasmid status and possession of VapA. Association with HeLa cells was significantly greater for strains with a dry colony type than for those with a mucoid colony, a result which correlated with hydrophobicity of the colonies. HeLa cell association does not correlate with the presence of the virulence plasmid in R. equi.

Comparison of an interferon-gamma to a phospholipase D enzyme-linked immunosorbent assay for diagnosis of Corynebacterium pseudotuberculosis infection in experimentally infected goats.

The optimal method of control of caseous lymphadenitis of goats caused by Corynebacterium pseudotuberculosis is eradication of infection by identification and removal of infected carrier animals. The objective of this study was to compare detection of C. pseudotuberculosis experimentally infected goats using a commercially available bovine interferon-gamma (IFN-gamma) whole blood enzyme-linked immunosorbent assay (ELISA) to serological response to a recombinant phospholipase D (PLD) ELISA. The tests were assessed repeatedly over 1 year in three infected and three non-infected goats. Using a IFN-gamma optical density cut-off at 0.10 as positive under the conditions used, the test accurately detected C. pseudotuberculosis experimentally infected goats over a 363 day period with a reliability of 89.2% and non-infected goats with a reliability of 97.1%. Using a cut-off value of the mean for negative samples plus two standard deviations, the PLD ELISA detected C. pseudotuberculosis experimentally infected goats over this period with a reliability of 81.0% and non-infected goats with a reliability of 97.0%. The PLD ELISA was however more predictive than the IFN-gamma ELISA of the presence of lesions observed at postmortem examination of infected goats.

Protective effect against Rhodococcus equi infection in mice of IgG purified from horses vaccinated with virulence associated protein (VapA)-enriched antigens.

IgG was purified from horses immunized with repeated doses of virulence associated (VapA) enriched antigens extracted with Triton X-114 from the surface of a virulent strain of R. equi. This IgG were administered to mice immunosuppressed by prior treatment with indomethacin. Mice administered the higher dose were completely protected against intraperitoneal infection with R. equi; mice given the lower dose were partially protected. By contrast, mice administered concentrated nonimmune equine IgG were not protected. This study demonstrates that VapA may be an important antigen involved in humoral protective immunity in R. equi infections caused by foal virulent strains.