Simultaneous detection of Waddlia chondrophila and Listeria monocytogenes in aborted ruminant samples by real-time quantitative PCR.

Abstract Waddlia chondrophila and Listeria monocytogenes are well known emerging pathogens that cause ruminants' abortion around the world. Zoonotic infections caused by these bacteria are mostly underestimated due to difficulties of diagnosis resulting from their intracellular growth. The purpose of this study was to develop and validate a dual real-time quantitative PCR (qPCR) assay for the simultaneous quantification of W . chondrophila and L . monocytogenes in biological samples from aborted ruminants. Technical performance was examined using linear control plasmids. A total of 211 veterinary samples (15 placental tissues, 50 blood, 53 milk and 93 vaginal swab samples) were used to compare the qPCR with standard culture methods. The limit of detection was 2 plasmid template copies per reaction with approximately 5000-fold differences in concentrations of the two competing templates. Coefficients of variation for positive control plasmids were less than 1.8%. Both intra- (1.01–2.13% and 1.13–1.71%) and inter- (1.02–2.24% and 1.2–1.66%) assay variations of qPCR for W . chondrophila and L . monocytogenes plasmids were within the acceptable limits, implying high reproducibility and repeatability of the assay. The use of the qPCR assay resulted in 53 positive identifications among 211 veterinary samples against only 19 when standard cultural methods were used. On the basis of these results, we determined 100% sensitivity and 100% specificity for our new qPCR. Dual abortigenic agents were identified in 8.6% and 26.6% of vaginal swab samples and placental tissues, respectively. To conclude, this new qPCR will be of great value in the simultaneous and rapid diagnosis of W . chondrophila and L . monocytogenes in large-scale screening programs and also during outbreaks.