Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi

Abstract

Background 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. 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.Electronic supplementary materialThe online version of this article (10.1186/s12917-017-1210-5) contains supplementary material, which is available to authorized users.

Skin disease in donkeys (Equus asinus): a retrospective study from four veterinary schools

“…Sterile panniculitis, diagnosed with histological investigation and negative bacterial culture, was diagnosed in three donkeys in our study but only reported once previously . Corynebacterium pseudotuberculosis, a pathogen well investigated in horses, was isolated from three cases in our study, one of which was reported in a review article . Another rarely reported pathogen in donkeys, Sporothrix schenckii, was seen in one of our cases .…”

Molecular Characterization of Corynebacterium pseudotuberculosis Isolated over a 15-Year Period in Switzerland

“…C. pseudotuberculosis can be distinguished into two biovars based on its ability to reduce nitrate. Nitrate-negative strains are classified as biovar Ovis and nitrate-positive ones as biovar Equi [ 20 , 21 , 22 ]. In sheep and goats, CLA is predominantly caused by biovar Ovis, whereas the nitrate-positive biovar is the causative agent of abscesses and ulcerative lymphangitis in horses and oedematous skin disease (OSD) in buffaloes [ 2 , 4 , 7 , 23 ].…”

Atypical Multibacterial Granulomatous Myositis in a Horse: First Report in Italy

“…Quadruplex PCR reactions were performed with a final volume of 50 µL, containing 20 ng of genomic DNA, 0.25 mM dNTPs, 0.1 unit of Taq DNA polymerase (Life Technologies, Carlsbad, CA, USA), 2 mM MgCl 2, enzyme buffer (Life Technologies, Carlsbad, CA, USA), 0.5 µM of each primer oligonucleotide, and water to a final volume of 50 µL. The primers used in this study anneal to the 16s rRNA, rpoB, narG, and pld gene sequences of Corynebacterium pseudotuberculosis, in accordance with Almeida et al [24]. The amplified products were resolved through electrophoresis on 1.0% (w/v) agarose gels and stained using ethidium bromide.…”