Quantification of Mycobacterium avium subsp. paratuberculosis (MAP) survival in monocyte-derived macrophages

Abstract

Real-time PCR assays were developed to quantitate Mycobacterium avium subsp. paratuberculosis (MAP) in bovine monocyte-derived macrophages. We measured the absolute number of both host cells and bacteria in in vitro challenge assays. Results obtained from real-time quantitative PCR (qPCR) DNA copy counts were compared to visual quantitation of fluorescent-stained MAP in macrophages. Conclusions from our original visual analysis were supported by the second (qPCR) methodology; however, the qPCR assay proved to be more consistent between samples and was easier to perform. There was a strain-to-strain difference in growth curves between fluorescent quantitation (FQ) and qPCR that we believe to be a consequence of bacterial growth characteristics in FQ. In summary, real-time PCR assays provided a more accurate and precise method for evaluating intracellular growth dynamics when comparing strains of MAP.