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

Rebecca M. Mitchell, Nicole S. Gollnick, Srinand Sreevatsan, David G. Russell, Ynte H. Schukken

Research output: Contribution to journalArticle

3 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)73-78
Number of pages6
JournalVeterinary immunology and immunopathology
Volume139
Issue number1
DOIs
StatePublished - Jan 1 2011

Keywords

  • Fluorescence
  • Mycobacterium avium subsp. paratuberculosis
  • PBMC
  • Quantitative PCR

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