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B-Cell epitope mapping of the VapA protein of Rhodococcus equi: implications for early detection of R. equi disease in foals.

ABSTRACT: Linear B-cell epitopes of the Rhodococcus equi virulence-associated protein (VapA) were mapped using a synthetic peptide bank in this study. The peptides were screened in an enzyme-linked immunosorbent assay (ELISA) with a total of 70 sera from foals with current R. equi disease (51 sera), as well as from foals that had either recovered from R. equi infection 10 months previously (3 sera) or that had no known history of R. equi disease (16 sera). An epitope with the sequence NLQKDEPNGRA was identified and was universally recognized by all 51 sera from foals with R. equi disease and was not recognized by any of the other sera. There was poor reactivity between all sera and peptides relating to other areas of the VapA protein. It is proposed that an ELISA based upon a defined peptide epitope may be used in an improved serological diagnostic test for R. equi infection in foals.

SUBMITTER: Vanniasinkam T

PROVIDER: S-EPMC87987 | biostudies-literature | 2001 Apr

REPOSITORIES: biostudies-literature

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B-Cell epitope mapping of the VapA protein of Rhodococcus equi: implications for early detection of R. equi disease in foals.

Vanniasinkam T T Barton M D MD Heuzenroeder M W MW

Journal of clinical microbiology 20010401 4

Linear B-cell epitopes of the Rhodococcus equi virulence-associated protein (VapA) were mapped using a synthetic peptide bank in this study. The peptides were screened in an enzyme-linked immunosorbent assay (ELISA) with a total of 70 sera from foals with current R. equi disease (51 sera), as well as from foals that had either recovered from R. equi infection 10 months previously (3 sera) or that had no known history of R. equi disease (16 sera). An epitope with the sequence NLQKDEPNGRA was iden ...[more]

PMID: 11283104

Similar Datasets

VapA of Rhodococcus equi binds phosphatidic acid.

Project description:Rhodococcus equi is a multihost, facultative intracellular bacterial pathogen that primarily causes pneumonia in foals less than six months in age and immunocompromised people. Previous studies determined that the major virulence determinant of R. equi is the surface bound virulence associated protein A (VapA). The presence of VapA inhibits the maturation of R. equi-containing phagosomes and promotes intracellular bacterial survival, as determined by the inability of vapA deletion mutants to replicate in host macrophages. While the mechanism of action of VapA remains elusive, we show that soluble recombinant VapA32-189 both rescues the intramacrophage replication defect of a wild type R. equi strain lacking the vapA gene and enhances the persistence of nonpathogenic Escherichia coli in macrophages. During macrophage infection, VapA was observed at both the bacterial surface and at the membrane of the host-derived R. equi containing vacuole, thus providing an opportunity for VapA to interact with host constituents and promote alterations in phagolysosomal function. In support of the observed host membrane binding activity of VapA, we also found that rVapA32-189 interacted specifically with liposomes containing phosphatidic acid in vitro. Collectively, these data demonstrate a lipid binding property of VapA, which may be required for its function during intracellular infection.

| S-EPMC5777868 | biostudies-literature

The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis.

Project description:Rhodococcus equi (R. equi) is an important pulmonary pathogen in foals that often leads to the death of the horse. The bacterium harbors a virulence plasmid that encodes numerous virulence-associated proteins (Vaps) including VapA that is essential for intracellular survival inside macrophages. However, little is known about the precise function of VapA. Here, we demonstrate that VapA causes perturbation to late endocytic organelles with swollen endolysosome organelles having reduced Cathepsin B activity and an accumulation of LBPA, LC3 and Rab7. The data are indicative of a loss of endolysosomal function, which leads cells to upregulate lysosome biogenesis to compensate for the loss of functional endolysosomes. Although there is a high degree of homology of the core region of VapA to other Vap proteins, only the highly conserved core region of VapA, and not VapD of VapG, gives the observed effects on endolysosomes. This is the first demonstration of how VapA works and implies that VapA aids R. equi survival by reducing the impact of lysosomes on phagocytosed bacteria.

| S-EPMC5387311 | biostudies-literature

Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi.

Project description:We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA+ and vapA-negative (including vapB+) plasmid genotypes. The vapA+ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to approximately 10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA+) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA+ "horse-pathogenic" genotype determination.

| S-EPMC1489618 | biostudies-literature

Identification of genomic loci associated with Rhodococcus equi susceptibility in foals

Project description:aCGH performed to identify copy number variants in Quarter Horses and perform casec-control GWAS

2014-05-10 | GSE57510 | GEO

Identification of genomic loci associated with Rhodococcus equi susceptibility in foals.

Project description:Pneumonia caused by Rhodococcus equi is a common cause of disease and death in foals. Although agent and environmental factors contribute to the incidence of this disease, the genetic factors influencing the clinical outcomes of R. equi pneumonia are ill-defined. Here, we performed independent single nucleotide polymorphism (SNP)- and copy number variant (CNV)-based genome-wide association studies to identify genomic loci associated with R. equi pneumonia in foals. Foals at a large Quarter Horse breeding farm were categorized into 3 groups: 1) foals with R. equi pneumonia (clinical group [N = 43]); 2) foals with ultrasonographic evidence of pulmonary lesions that never developed clinical signs of pneumonia (subclinical group [N = 156]); and, 3) foals without clinical signs or ultrasonographic evidence of pneumonia (unaffected group [N = 49]). From each group, 24 foals were randomly selected and used for independent SNP- and CNV-based genome-wide association studies (GWAS). The SNP-based GWAS identified a region on chromosome 26 that had moderate evidence of association with R. equi pneumonia when comparing clinical and subclinical foals. A joint analysis including all study foals revealed a 3- to 4-fold increase in odds of disease for a homozygous SNP within the associated region when comparing the clinical group with either of the other 2 groups of foals or their combination. The region contains the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) gene, which is involved in neutrophil function. No associations were identified in the CNV-based GWAS. Collectively, these data identify a region on chromosome 26 associated with R. equi pneumonia in foals, providing evidence that genetic factors may indeed contribute to this important disease of foals.

| S-EPMC4043894 | biostudies-literature

Immunogenicity of an electron beam inactivated Rhodococcus equi vaccine in neonatal foals.

Project description:Rhodococcus equi is an important pathogen of foals that causes severe pneumonia. To date, there is no licensed vaccine effective against R. equi pneumonia of foals. The objectives of our study were to develop an electron beam (eBeam) inactivated vaccine against R. equi and evaluate its immunogenicity. A dose of eBeam irradiation that inactivated replication of R. equi while maintaining outer cell wall integrity was identified. Enteral administration of eBeam inactivated R. equi increased interferon-γ production by peripheral blood mononuclear cells in response to stimulation with virulent R. equi and generated naso-pharyngeal R. equi-specific IgA in newborn foals. Our results indicate that eBeam irradiated R. equi administered enterally produce cell-mediated and upper respiratory mucosal immune responses, in the face of passively transferred maternal antibodies, similar to those produced in response to enteral administration of live organisms (a strategy which previously has been documented to protect foals against intrabronchial infection with virulent R. equi). No evidence of adverse effects was noted among vaccinated foals.

| S-EPMC4143214 | biostudies-literature

Identification of genomic loci associated with Rhodococcus equi susceptibility in foals

Project description:aCGH performed to identify copy number variants in Quarter Horses and perform casec-control GWAS Two-condition experiment, All samples were compared to a single Quarter Horse reference to identify copy number variants to be used in the CNV GWAS

2014-05-10 | E-GEOD-57510 | biostudies-arrayexpress

Development of septic polysynovitis and uveitis in foals experimentally infected with Rhodococcus equi.

Project description:Rhodococcus equi is one of the most important causes of disease in foals. Infection is typically characterized by pyogranulomatous pneumonia although extrapulmonary infections occur occasionally. Uveitis and polysynovitis have been reported in foals naturally infected with R. equi and are thought to be the result of an immune-mediated process. However, the pathogenesis of these conditions is poorly understood. The objectives of this study were to document the occurrence of uveitis and polysynovitis after experimental infection with R. equi and to determine if these disorders are the direct result of infection at these sites. Foals between 3 and 4 weeks of age were infected intratracheally with virulent R. equi using inocula of 1×108 CFU (high inoculum; n = 16) or 1×107 CFU (low inoculum; n = 12). Foals were monitored twice daily and necropsy was performed 14 days post-infection. Aqueous humor and synovial fluid were collected aseptically and the percentage of affected lung was calculated. The mean (± SD) percentage of affected lung was significantly higher with the high inoculum (31.8 ± 14.6%) than with the low inoculum (14.4 ± 11.4%). Fourteen of 25 foals developed uveitis and 20 of 28 foals developed polysynovitis. R. equi was cultured from the aqueous humor of 11 foals and from the synovial fluid of 14 foals. The risk of development of polysynovitis and protein concentration in the aqueous humor were significantly higher in foals that received the high inoculum. These results indicate that polysynovitis and uveitis are septic complications associated with the severity of lung disease.

| S-EPMC5802921 | biostudies-literature

Identification of a VapA virulence factor functional homolog in Rhodococcus equi isolates housing the pVAPB plasmid.

Project description:Rhodococcus equi is a facultative intracellular bacterium of macrophages and is an important pathogen of animals and immunocompromised people wherein disease results in abcessation of the lungs and other sites. Prior work has shown that the presence of the major virulence determinant, VapA, encoded on the pVAPA-type plasmid, disrupts normal phagosome development and is essential for bacterial replication within macrophages. pVAPA- type plasmids are typical of R. equi strains derived from foals while strains from pigs carry plasmids of the pVAPB-type, lacking vapA, and those from humans harbor various types of plasmids including pVAPA and pVAPB. Through the creation and analysis of a series of gene deletion mutants, we found that vapK1 or vapK2 is required for optimal intracellular replication of an R. equi isolate carrying a pVAPB plasmid type. Complementation analysis of a ?vapA R. equi strain with vapK1 or vapK2 showed the VapK proteins of the pVAPB-type plasmid could restore replication capacity to the macrophage growth-attenuated ?vapA strain. Additionally, in contrast to the intracellular growth capabilities displayed by an equine R. equi transconjugant strain carrying a pVAPB-type plasmid, a transconjugant strain carrying a pVAPB-type plasmid deleted of vapK1 and vapK2 proved incapable of replication in equine macrophages. Cumulatively, these data indicate that VapK1 and K2 are functionally equivalent to VapA.

| S-EPMC6171844 | biostudies-literature

Randomized, controlled trial comparing Rhodococcus equi and poly-N-acetyl glucosamine hyperimmune plasma to prevent R equi pneumonia in foals.

Project description:BackgroundHyperimmune plasma raised against β-1→6-poly-N-acetyl glucosamine (PNAG HIP) mediates more opsonophagocytic killing of Rhodococcus equi (R equi) than does R equi hyperimmune plasma (RE HIP) in vitro. The relative efficacy of PNAG HIP and RE HIP to protect foals against R equi pneumonia, however, has not been evaluated.HypothesisTransfusion with PNAG HIP will be superior to RE HIP in foals for protection against R equi pneumonia in a randomized, controlled, blinded clinical trial.AnimalsFour hundred sixty Quarter Horse and Thoroughbred foals at 5 large breeding farms in the United States.MethodsA randomized, controlled, blinded clinical trial was conducted in which foals were transfused within 24 hours after birth with 2 L of either RE HIP or PNAG HIP. Study foals were monitored through weaning for clinical signs of pneumonia by farm veterinarians. The primary outcome was the proportion of foals that developed pneumonia after receiving each type of plasma.ResultsThe proportion of foals that developed pneumonia was the same between foals transfused with RE HIP (14%; 32/228) and PNAG HIP (14%; 30/215).Conclusions and clinical importanceResults indicate that PNAG HIP was not superior to a commercially available, United States Department of Agriculture-licensed RE HIP product for protecting foals against R equi pneumonia under field conditions.

| S-EPMC8692225 | biostudies-literature

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