Quantification of Mycobacterium avium subsp. paratuberculosis (MAP) survival in monocyte-derived macrophages.
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.
Mitchell
Rebecca M
RM
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. rmm37@cornell.edu
Gollnick
Nicole S
NS
Sreevatsan
Srinand
S
Russell
David G
DG
Schukken
Ynte H
YH
D000818:Animals; D002417:Cattle / Q000276:immunology / Q000382:microbiology; D002460:Cell Line; D008264:Macrophages / Q000382:microbiology* / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000662:veterinary; D016927:Mycobacterium avium subsp. paratuberculosis / Q000502:physiology*; D010587:Phagocytosis / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000662:veterinary