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

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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.

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Authors and Affiliations

  1. Molecular Biology Research Group, MicroScience Technologies Ltd, Pentlands Science Park, Edinburgh EH26 0PZ, UK

    Christopher Oldfield, Hal Bonella & Lynne Renwick

  2. Department of Biomedical Sciences, Bradford University, Bradford, BD7 1DP, UK

    Hilary I. Dodson & Grace Alderson

  3. School of Biology, University of Newcastle, Newcastle-upon-Tyne, NE1 7RU, UK

    Michael Goodfellow

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  1. Christopher Oldfield
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  2. Hal Bonella
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  3. Lynne Renwick
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  4. Hilary I. Dodson
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  5. Grace Alderson
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  6. Michael Goodfellow
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Oldfield, C., Bonella, H., Renwick, L. et al. Rapid determination of vapA/vapB genotype in Rhodococcus equi using a differential polymerase chain reaction method. Antonie Van Leeuwenhoek 85, 317–326 (2004). https://doi.org/10.1023/B:ANTO.0000020383.66622.4d

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