Rapid determination of vapA/vapB genotype in Rhodococcus equi using a differential polymerase chain reaction method

Abstract

Rhodococcus equi is a facultative pathogen of foals. Infection causes an often fatal pulmonary pneumonia. The organism has also been isolated from pigs, cattle, humans and the environment. Equine virulence has a high positive correlation with the expression of a 17.4 kD polypeptide of unknown function, VapA, the product of the plasmid-encoded vapA gene. More recently an isogene of vapA, referred to as vapB and encoding an 18.2 kDa polypeptide, has been identified among pig and human isolates. The two genes share > 80% sequence identity, yet their host strains apparently exhibit different pathogenicity profiles (for example by reference to virulence in mouse model system and host specificity). In this study, a polymerase chain reaction (PCR) technique was developed that permits the selective amplification of vapA and vapB. Using this technique the distribution of the two genes among 35 randomly selected isolates of Rhodococcus equi from various animal and environmental sources was determined. Using this technique the genotype of each isolate could be unambiguously assigned as vapA+, vapB+ or vap- (i.e., scoring negative for both vapA and vapB). No isolate scored positive for both vapA and vapB. 100% of equine isolates scored vapA+, confirming the status of vapA as a reliable marker of equine virulence. All three genotypes were found among human isolates; porcine isolates scored either vapB+ or vap- and no vapA+ isolates were present in this sample. Rigorous statistical analysis using the Fisher Exact test confirmed that the high frequency of vapA+ among equine isolates is significant; however the sample size was too small to draw statistically significant conclusions regarding the distribution of genotypes among within other animal groups.

Evolution of theRhodococcus equi vapPathogenicity Island Seen through Comparison of Host-AssociatedvapAandvapBVirulence Plasmids

“…Thus, vapA and vapB seem to be divergent allelic variants of the same ancestral vap gene, consistent with the observed mutual exclusivity of their corresponding PCR gene markers among R. equi isolates (38,39). Similarly, vapG and vapL, both of which are transcribed in the opposite direction relative to the other vap genes, appear to be derived from a common ancestral gene despite mapping to very different sites in their corresponding PAIs (Fig.…”

“…Six vap family genes were identified in the pVAPB1593 PAI: the previously described vapB (39,49), plus five new vap multigene family members, which we named vapJ, vapK1, vapK2, vapM, and vapL ( Fig. 1; also see Tables S1 and S2 in the supplemental material).…”

“…Nonequine R. equi isolates often produce a larger (20-kDa) variant surface lipoprotein antigen, VapB (10,49), which is very similar in primary structure to VapA (78% amino acid sequence identity) (39). VapA and VapB (and corresponding genes) are mutually exclusive, i.e., they do not occur in the same isolate, suggesting that they are allelic variants of one locus that has divergently evolved in two different plasmid subpopulations (38).…”

An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi

“…Early studies showed that VapA-encoding virulence plasmids were typical of equine strains (28,29), while a second plasmid type, encoding VapB, a VapA variant (24), was common among nonequine (pig and human) isolates (10,(30)(31)(32)(33). Recently, the existence of a third type of R. equi virulence plasmid was identified in bovine and human isolates that were initially deemed to be "plasmidless" (because vapA and vapB negative) but that tested positive for a traA plasmid conjugal transfer gene marker (34).…”

Biotechnological tools for diagnosis of equine infectious diseases

“…It can also be used for detection of many equine diseases (Oldfield et al, 2004;Ocampo-Sosa et al, 2007;Pusterla et al, 2007;Letek et al, 2008;Monego et al, 2009). Polymerase chain reaction (PCR)-based diagnostic tests can allow rapid and sensitive detection of equine infectious pathogen (Paxson, 2008).…”