Previous studies have reported that peripheral blood mononuclear cells (PBMCs) from cows with a different Johne's disease status challenged with Mycobacterium avium subsp. paratuberculosis (MAP) show a difference in gene expression profiles. These studies have not evaluated if PBMCs show a difference in MAP killing ability as well. Other in vitro studies looked at gene expression patterns of macrophages from Johne's disease-negative animals when exposed to MAP and Mycobacterium avium subsp. avium. Neither of the studies looked at differences in bacterial survival and propagation after infecting PBMCs or macrophages with strains of MAP from different host species. In this study we investigated the ability of bovine monocyte-derived macrophages from naturally infected cows in different stages of Johne's disease to kill MAP isolates from different host species. We tested the hypotheses that infection history of cows does not affect macrophage killing ability and that killing capacity of bovine macrophages is not dependent on the MAP strain. PBMCs were obtained from low-shedding and high-shedding Johne's disease-positive (n = 3 per group) and Johne's disease-negative (n = 3) multiparious cows. Following differentiation, macrophages where challenged in vitro with MAP strains of different host specificity (bovine, sheep, bison and human). Macrophages were harvested at 2 hours, 4 days, 7 days and 10 days following infection. For each time point ingestion and intracellular survival of MAP strains were determined by fluorescent staining of the bacteria with carboxyfluorescein diacetate. Furthermore, the approximate number of surviving bacteria was estimated by quantification of the MAP hsp65 gene by real-time PCR. Results of this study will advance the understanding of the bovine immune system as it pertains to infections with MAP, the possibility of inherent susceptibility of cattle to Johne's disease and the bovine species as a reservoir for MAP strains found in other species, including humans.