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Identification of virulent Rhodococcus equi by amplification of gene coding for 15- to 17-kilodalton antigens
1Department of Animal Hygiene, School of Veterinary Medicine and Animal Sciences, Kitasato University, Aomori, Japan.
Abstract:
During a survey of the prevalence of virulent Rhodococcus equi at horse-breeding farms by plasmid and protein profiles, cryptic plasmids of various sizes were found in 66 (3.8%) of 1,725 isolates from feces of horses and 129 (5.9%) of 2,200 isolates from soil. Twenty-two isolates, which contained cryptic plasmids of different sizes, were found by plasmid profiles, and their protein profiles and mouse pathogenicities were examined. Of the 22 isolates, 7 were virulent R. equi, contained both virulence and cryptic plasmids, and expressed 15- to 17-kDa antigens. The remaining 15 isolates were avirulent and did not express the antigens: 6 strains contained cryptic plasmids of two different sizes and 9 strains contained cryptic plasmids of various sizes. A PCR assay was developed for the rapid identification of virulence plasmids of R. equi. Oligonucleotide primers, derived from the sequence of a gene coding for the 15- to 17-kDa virulence-associated antigens of R. equi, amplified a 564-bp product from all the tested isolates harboring a virulence plasmid. This PCR product hybridized with virulence plasmid DNA in the Southern hybridization assay. Virulence plasmid-cured derivatives and all of the tested isolates harboring cryptic plasmids only were negative. The PCR is a rapid, sensitive, and specific test for the identification of virulent R. equi from environmental isolates compared with standard techniques, such as plasmid and protein profiles and the mouse pathogenicity test, and is considered to be a useful tool for epidemiological studies.
Insights
A new PCR assay rapidly identifies virulent Rhodococcus equi, a bacterium affecting horses. This method is more efficient than traditional techniques for detecting R. equi in environmental samples.
Area of Science:
- Veterinary Microbiology
- Bacterial Pathogenesis
- Molecular Diagnostics
Background:
- Rhodococcus equi is a significant pathogen in foals, causing severe pneumonia.
- Identifying virulent strains is crucial for disease control and epidemiological studies.
- Traditional methods for R. equi identification are time-consuming and labor-intensive.
Purpose of the Study:
- To develop and validate a rapid Polymerase Chain Reaction (PCR) assay for identifying virulent R. equi.
- To compare the efficacy of the PCR assay with conventional methods like plasmid profiling, protein analysis, and mouse pathogenicity testing.
Main Methods:
- Plasmid and protein profiling were used to characterize R. equi isolates from horse feces and soil.
- A PCR assay was designed using primers specific to the virulence-associated antigen gene (15-17 kDa).
- Southern hybridization was employed to confirm PCR results with virulence plasmid DNA.
Main Results:
- Cryptic plasmids were found in 3.8% of fecal and 5.9% of soil isolates.
- Seven virulent R. equi isolates expressed 15-17 kDa antigens and harbored both virulence and cryptic plasmids.
- The developed PCR assay successfully amplified a 564-bp product from all virulent R. equi isolates harboring virulence plasmids, demonstrating high specificity and sensitivity.
Conclusions:
- The PCR assay provides a rapid, sensitive, and specific method for identifying virulent R. equi.
- This molecular tool is advantageous over traditional methods for epidemiological surveillance of R. equi in environmental samples.
- The findings support the use of PCR for efficient detection and control of R. equi outbreaks.