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.
Publication Date: 2004-03-20 PubMed ID: 15031644DOI: 10.1023/B:ANTO.0000020383.66622.4dGoogle Scholar: Lookup
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- Journal Article
Summary
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The research discusses a new method using Polymerase Chain Reaction to quickly identify genotypes of the Rhodococcus equi bacteria, responsible for causing fatal respiratory diseases in animals like foals. The study investigated the distribution of the vapA and vapB genes within 35 randomly chosen Rhodococcus equi strains from various sources.
Understanding Rhodococcus equi
- Rhodococcus equi is a facultative pathogen that can cause severe pneumonia, often leading to death in foals. The bacterium also infects pigs, cattle, humans, and is present in the environment.
- The strains that are especially virulent to horses possess the vapA gene, which codes for a 17.4 kD polypeptide. Its significance beyond association with virulence is unknown.
- Alternatively, a similar gene called vapB, coding for an 18.2 kDa polypeptide, has been identified in pig and human strains of the bacteria. Both vapA and vapB genes have more than 80% sequence identity, but they seem to result in different pathogenicity profiles.
Development of Polymerase Chain Reaction (PCR) Technique
- A new PCR technique was developed to allow for the selective amplification of the vapA and vapB genes.
- This method was employed to determine the distribution of the two genes across 35 randomly selected Rhodococcus equi isolates from various animal sources and the environment.
- The genotypes identified using this innovative PCR technique were vapA+, vapB+, or vap-, with the latter indicating that neither of the two genes was present. None of the isolates demonstrated positivity for both genes.
Observations and Result Analysis
- It was observed that all equine isolates were vapA+, reinforcing the fact that vapA is an accurate marker for equine virulence.
- Human isolates exhibited all three genotypes, while porcine isolates were either vapB+ or vap-; there were no vapA+ isolates in pigs.
- The high occurrence of vapA+ among equine isolates, as confirmed by Fisher Exact test, was statistically significant. However, due to a small sample size, no statistically significant conclusions were drawn about the distribution of genotypes in other animal groups.
Cite This Article
APA
Oldfield C, Bonella H, Renwick L, Dodson HI, Alderson G, Goodfellow M.
(2004).
Rapid determination of vapA/vapB genotype in Rhodococcus equi using a differential polymerase chain reaction method.
Antonie Van Leeuwenhoek, 85(4), 317-326.
https://doi.org/10.1023/B:ANTO.0000020383.66622.4d Publication
Researcher Affiliations
- Molecular Biology Research Group, MicroScience Technologies Ltd, Pentlands Science Park, Edinburgh EH26 0PZ, UK.
MeSH Terms
- Actinomycetales Infections / veterinary
- Animals
- Antigens, Bacterial / genetics
- Bacterial Proteins / genetics
- Base Sequence
- DNA-Binding Proteins / genetics
- Environmental Microbiology
- Genotype
- Horse Diseases / microbiology
- Horses
- Humans
- Membrane Glycoproteins / genetics
- Molecular Sequence Data
- Polymerase Chain Reaction / methods
- Rhodococcus equi / genetics
- Sequence Alignment
- Virulence Factors / genetics
Citations
This article has been cited 6 times.- Żychska M, Witkowski L, Klementowska A, Rzewuska M, Kwiecień E, Stefańska I, Czopowicz M, Szaluś-Jordanow O, Mickiewicz M, Moroz A, Bonecka J, Kaba J. Rhodococcus equi-Occurrence in Goats and Clinical Case Report. Pathogens 2021 Sep 4;10(9).
- Valero-Rello A, Hapeshi A, Anastasi E, Alvarez S, Scortti M, Meijer WG, MacArthur I, Vázquez-Boland JA. An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi. Infect Immun 2015 Jul;83(7):2725-37.
- Monego F, Maboni F, Krewer C, Vargas A, Costa M, Loreto E. Molecular characterization of Rhodococcus equi from horse-breeding farms by means of multiplex PCR for the vap gene family. Curr Microbiol 2009 Apr;58(4):399-403.
- Letek M, Ocampo-Sosa AA, Sanders M, Fogarty U, Buckley T, Leadon DP, González P, Scortti M, Meijer WG, Parkhill J, Bentley S, Vázquez-Boland JA. Evolution of the Rhodococcus equi vap pathogenicity island seen through comparison of host-associated vapA and vapB virulence plasmids. J Bacteriol 2008 Sep;190(17):5797-805.
- Rodríguez-Lázaro D, Lewis DA, Ocampo-Sosa AA, Fogarty U, Makrai L, Navas J, Scortti M, Hernández M, Vázquez-Boland JA. Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi. Appl Environ Microbiol 2006 Jun;72(6):4256-63.
- Ghielmetti G, Stevens MJA, Schmitt S, Kittl S, Cernela N, Biggel M, Schulthess B, Keller PM, Schrenzel J, Stephan R. Multi-host distribution of Rhodococcus equi (Prescottella equi) strains and their phylogenomic clustering. BMC Microbiol 2025 Jul 21;25(1):447.
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