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.
Bibliographical noteFunding Information:
We acknowledge the excellent technical support of Suzanne Klaessig. We also wish to thank Kenneth W. Simpson, Nancy A. Lorr, Thomas L. Olson, and Wilhelm H. Elmore for provision of laboratory facilities and technical support. Further we would like to thank all the members of the Russell lab for their advice and training along the development of the quantitative PCR assay. Both Laura Brunengraber and Kaori Sakamoto were instrumental in training on real-time PCR techniques. We also gratefully acknowledge the owner of the animals included in this study for his collaboration. Financial support for this work was provided in part by the USDA Agricultural Research Service (Agreement No. 58-1265-3-156) for the Regional Dairy Quality Management Alliance. Funding was also provided by the Johne's Disease Integrated Program (USDA contract 45105). Johne's disease research in the QMPS laboratory is supported by JDIP and USDA-NRI grants.
- Mycobacterium avium subsp. paratuberculosis
- Quantitative PCR