Quantifying Mycobacterium avium subspecies paratuberculosis infection of bovine monocyte derived macrophages by confocal microscopy

Quantification of Mycobacterium avium subspecies paratuberculosis (MAP) during in vitro infection experiments is challenging due to limitations of currently utilised methods, such as colony counting. Here we describe quantifying MAP infection of bovine macrophages (M phi) using confocal microscopy. Bovine monocyte derived macrophages were infected with MAP at a high or low dose and the number of intracellular bacteria calculated at 2 h post infection using confocal microscopy. Bacteria within simultaneously infected M phi were quantified by colony counting in order to compare confocal microscopy results with results obtained by an established method. Confocal microscopy provided a robust alternative quantification method that allowed for assessment of the infection at the individual M phi level. This demonstrated that MAP infection was not homogeneous, and that there were higher numbers of both infected M phi and intracellular bacteria and bacterial aggregates at the high dose compared to the low dose, potentially impacting the M phi response to infection. Confocal microscopy can therefore provide a level of detail regarding the infection unobtainable by other quantification methods.