Rhodococcus equi causes severe pyogranulomatous pneumonia in foals. This facultative intracellular pathogen produces similar lesions in immunocompromised humans, particularly in AIDS patients. Virulent strains of R. equi bear a large plasmid that is required for intracellular survival within macrophages and for virulence in foals and mice. Only two plasmid-encoded proteins have been described previously; a 15-to 17-kDa surface protein designated virulence-associated protein A (VapA) and an antigenically related 20-kDa protein (herein designated VapB). These two proteins are not expressed by the same R. equi isolate. We describe here the substantial similarity between VapA and VapB. Moreover, we identify three additional genes carried on the virulence plasmid, The nocardioform actinomycete, Rhodococcus equi, is an important pulmonary pathogen in foals and in human patients with AIDS. This gram-positive bacterium is a facultative intracellular pathogen that persists and multiplies within macrophages. Intracellular survival is considered to be necessary for the development of disease, which is characterized by severe and sometimes fatal pneumonia in both humans and foals (5,17).Clinical isolates of R. equi contain a large plasmid ranging in size from 80 to 90 kb in equine or 30 to 100 kb in human AIDS isolates (25,30). The large plasmid is essential for virulence in mice and foals and for intracellular survival in murine and equine macrophages (9). Plasmid curing by repeated passage during in vitro culture at 37°C eliminates the virulent phenotype (9, 27). These findings indicate that the plasmid encodes proteins that are necessary for virulence.Only two proteins encoded by R. equi virulence plasmids have been described to date. Equine isolates express a 15-to 17-kDa protein (VapA) that appears as a characteristic broad band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (20,26,29). Human R. equi isolates from AIDS patients generally express either VapA or an antigenically related 20-kDa protein (25). The 20-kDa protein is expressed by isolates from pigs as well as humans. No R. equi isolate has been shown to express both VapA and the 20-kDa protein. However, antigenic cross-reactivity between VapA and the 20-kDa protein has been demonstrated using immunoblots and serum from infected foals (25). The similarity of VapA and the 20-kDa plasmid-encoded proteins raises the possibility that the two proteins have analogous functions in different strains of R. equi.VapA and the 20-kDa protein are located on the bacterial surface, and expression is reported to be thermally and pH regulated (22,24). Specifically, VapA and the 20-kDa protein can be detected when R. equi is cultured at 38°C but not when cultured at 30°C, and expression at 38°C is observed only if the pH of the medium is decreased below 8 (22). These characteristics suggest that expression is upregulated in the mammalian host and intracellularly where VapA or the 20-kDa protein would play a role in the pathogenesis of rhodococcal p...

Findings suggest that administration of human albumin solution in healthy dogs with normal serum albumin concentrations may result in signs of a type III hypersensitivity reaction.

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Results suggested that dogs developed a pronounced IgG response following exposure to HA and that some dogs with no history of HA administration were positive for anti-HA IgG.

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Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines requires testing of additional antigens, optimization of the vaccine formulation and a better understanding of the protective immune response.

Improving the ability of DNA-based vaccines to induce potent Type1/Th1 responses against intracellular pathogens in large outbred species is essential. Rhodoccocus equi and equine infectious anemia virus (EIAV) are two naturally occurring equine pathogens that also serve as important large animal models of neonatal immunity and lentiviral immune control. Neonates present a unique challenge for immunization due to their diminished immunologic capabilities and apparent Th2 bias. In an effort to augment R. equi- and EIAV-specific Th1 responses induced by DNA vaccination, we hypothesized that a dual promoter plasmid encoding recombinant equine IL-12 (rEqIL-12) would function as a molecular adjuvant. In adult horses, DNA vaccines induced R. equi- and EIAV-specific antibody and lymphoproliferative responses, and EIAV-specific CTL and tetramer-positive CD8+ T lymphocytes. These responses were not enhanced by the rEqIL-12 plasmid. In neonatal foals, DNA immunization induced EIAV-specific antibody and lymphoproliferative responses, but not CTL. The R. equi vapA vaccine was poorly immunogenic in foals even when co-administered with the IL-12 plasmid. It was concluded that DNA immunization was capable of inducing Th1 responses in horses; dose and route were significant variables, but rEqIL-12 was not an effective molecular adjuvant. Additional work is needed to optimize DNA vaccine-induced Th1 responses in horses, especially in neonates.

Immune adult horses have CD8 + cytotoxic T lymphocytes (CTLs) that recognize and lyse Rhodococcus equi-infected cells in an equine lymphocyte alloantigen (ELA)-A [classical major histocompatibility complex (MHC) class I]-unrestricted fashion. As protein antigens are MHC class I-restricted, the lack of restriction suggests that the bacterial antigens being recognized by the host are not proteins. The goals of this study were to test the hypothesis that these CTLs recognize unique R. equi cell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of soluble R. equi antigen with broadly reactive proteases did not significantly diminish the ability of the antigen to stimulate R. equi-specific CTLs. R. equi-specific CTLs were also shown to lyse target cells (equine macrophages) pulsed with an R. equi lipid extract. Analysis of the R. equi lipid by TLC and MS (MALDI-TOF and ES) indicated that the extracted antigen consisted of three primary fractions: trehalose monomycolate (TMM), trehalose dimycolate (TDM) and cardiolipin (CL). ELA-A-mismatched cells pulsed with purified TMM and CL, but not the TDM fraction, were recognized and lysed by R. equi-specific CTLs. Because of their role in immune clearance and pathogenesis, transcription of the cytokines gamma interferon (IFN-c) and interleukin-4 (IL-4) was also measured in response to R. equi lipids by using real-time PCR; elevated IFN-c, but not IL-4, was associated with host clearance of the bacteria. The whole-cell R. equi lipid and all three R. equi lipid fractions resulted in marked increases in IFN-c transcription, but no increase in IL-4 transcription. Together, these data support the hypothesis that immune recognition of unique lipids in the bacterial cell wall is an important component of the protective immune response to R. equi. The results also identify potential lipid antigens not previously shown to be recognized by CTLs in an important, naturally occurring actinomycete bacterial pathogen.