A direct qPCR screening approach to improve the efficiency of Mycoplasma bovis isolation in the frame of a broad surveillance study

Culturing Mycoplasma bovis is laborious and unpredictable with most laboratories relying on molecular methods for its detection and identification. However, bacterial culture is still necessary to relate phenotypic characteristics to genotypic traits within and between individual strains. Thus, the main objective of this study was to develop a procedure that saved time and consumables during the culturing of M. bovis within the scope of a broad antimicrobial resistance surveillance project. Deep nasopharyngeal swabs (DNPS) collected from feedlot cattle upon arrival at 10 Southern Alberta feedlots were enriched in broth and an aliquot of the culture was directly used in a M. bovis-specific quantitative PCR (qPCR) assay. Only qPCR-positive cultures were plated onto agar media for the isolation of M. bovis. The detection of M. bovis from broth culture by direct-culture-qPCR proved to be more sensitive (1.61 x 10(2) CFU/mL) than using a commercial kit (1.61 x 10(3) CFU/mL) to extract DNA from pure cultures of M. bovis. When isolation of M. bovis from broth-enriched DNPS (n = 208 samples) was used as the gold standard for diagnostics, the qPCR screening approach showed 100% sensitivity, 87.27% specificity, and a kappa index = 0.87 (strong agreement). In contrast, qPCR of DNPS samples (n = 58) exhibited 100% sensitivity, 42.86% specificity, and a kappa index = 0.49 (weak agreement). The qPCR protocol described here together with a high throughput direct-culture-qPCR approach for sample testing made it possible to reduce the labor and cost of M. bovis isolation by eliminating the need to process 97.3% of M. bovis-negative samples. This was possible through the use of qPCR Ct values as a predictive tool of the likelihood of M. bovis isolation. This new procedure could be evaluated for its use in antimicrobial resistance surveillance programs that focus on Mycoplasma species.