Abstract
BackgroundCorynebacterium pseudotuberculosis is classified into two biovars, nitrate-negative biovar Ovis which is the etiologic agent of caseous lymphadenitis in small ruminants and nitrate-positive biovar Equi, which causes abscesses and ulcerative lymphangitis in equines. The aim of this study was to develop a quadruplex PCR assay that would allow simultaneous detection and biovar-typing of C. pseudotuberculosis.MethodsIn the present study, genomes of C. pseudotuberculosis strains were used to identify the genes involved in the nitrate reduction pathway to improve a species identification three-primer multiplex PCR assay. The nitrate reductase gene (narG) was included in the PCR assay along with the 16S, rpoB and pld genes to enhance the diagnosis of the multiplex PCR at biovar level.ResultsA novel quadruplex PCR assay for C. pseudotuberculosis species and biovar identification was developed. The results of the quadruplex PCR of 348 strains, 346 previously well-characterized clinical isolates of C. pseudotuberculosis from different hosts (goats, sheep, horse, cattle, buffalo, llamas and humans), the vaccine strain 1002 and the type strain ATCC 19410T, were compared to the results of nitrate reductase identification by biochemical test. The McNemar’s Chi-squared test used to compare the two methods used for C. pseudotuberculosis biovar identification showed no significant difference (P = 0.75) [95% CI for odds ratio (0.16–6.14)] between the quadruplex PCR and the nitrate biochemical test. Concordant results were observed for 97.13% (338 / 348) of the tested strains and the kappa value was 0.94 [95% CI (0.90–0.98)].ConclusionsThe ability of the quadruplex assay to discriminate between C. pseudotuberculosis biovar Ovis and Equi strains enhances its usefulness in the clinical microbiology laboratory.
Highlights
Corynebacterium pseudotuberculosis is classified into two biovars, nitrate-negative biovar Ovis which is the etiologic agent of caseous lymphadenitis in small ruminants and nitrate-positive biovar Equi, which causes abscesses and ulcerative lymphangitis in equines
Corynebacterium pseudotuberculosis is a facultative intracellular bacterium that is the causative agent of caseous lymphadenitis (CLA) in goats and sheep, abscesses and ulcerative lymphangitis in horses and oedematous skin disease (OSD) in Buffalo
Since the strains 1/06-A, 316, 162, and CIP52.97 were nitrate reductase positive in biochemical test and the genes were not identified in their genomes, primers to target narKGHJI cluster and narT gene were designed (Table 2)
Summary
Corynebacterium pseudotuberculosis is classified into two biovars, nitrate-negative biovar Ovis which is the etiologic agent of caseous lymphadenitis in small ruminants and nitrate-positive biovar Equi, which causes abscesses and ulcerative lymphangitis in equines. Direct and indirect tests to detect C. pseudotuberculosis have already been proposed, such as complement fixation test [10], synergistic hemolysis inhibition test [11], microagglutination assay [12], phospholipase D (PLD) antigen-based ELISA [13] and a multiplex PCR developed by our research group [14]. While these tests are useful for clinical diagnosis in diseased animals, none can differentiate the two biovars of C. pseudotuberculosis, which is currently only accomplished by biochemical tests. Since cattle can be infected by strains of both biovars, which may have different tissue preferences: biovar Ovis infects the mammary gland [17] and skin [18] and biovar Equi causes ulcerative lymphangitis and coronet lesions [9, 19, 20]
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