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.
Methods: In 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.
Results: A 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)].
Conclusions: The ability of the quadruplex assay to discriminate between C. pseudotuberculosis biovar Ovis and
Equi strains enhances its usefulness in the clinical microbiology laboratory.
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