In this study in vitro survival assays of multiple strains of Mycobacterium avium subsp. paratuberculosis (MAP) were used to examine whether species-specific MAP strains have different survival rates and cytotoxicity to macrophages. Six strains of MAP were used to infect mouse Balb/C bone marrow derived macrophages at a multiplicity of infection of 10:1. The strains of MAP included several bovine strains, bison, sheep and one ovine strain. A no-infection negative control and a M. avium complex (Mac) positive control were utilized. Macrophages were harvested at 4 timepoints following infection - 2 hours, 4 days, 7 days and 10 days. MAP cytotoxicity to macrophages was evaluated by counting multiple visual fields and comparing density of attached macrophages to the non-infected control population. Live/dead staining of MAP with carboxyfluorescein diacetate (CFDA) and counterstaining with Evan's Blue stained viable bacteria while quenching background macrophage fluorescence. Presence of MAP hsp65 DNA relative to macrophage GAPDH DNA isolated from infected macrophages allowed a more quantitative determination of bacterial copies per macrophage.Our null hypothesis would indicate that all strains of MAP are equally viable in the mouse macrophage system, with no difference in number of bacteria successfully invading macrophages or pattern of propagation of bacteria once invasion occurs. Our working alternative hypothesis was that we would find multiple survival patterns among strains of MAP, with differences in both levels of successful invasion (percent macrophages staining positive for MAP) and propagation once within the cells. Infections with the two bovine strains of MAP were expected to have results more similar to each other than to infections with other strains tested.