Johne's disease is a chronic wasting condition of ruminants caused by Mycobacterium avium subsp. paratuberculosis (M. ptb.). A proportion of infected adult animals develop clinical disease more than 12 months after infection. More knowledge regarding the pathogenesis of this disease, including apoptotic responses during the course of infection, could allow advances towards earlier detection and control of the disease and identification of resistant sheep. Merino sheep aged 7 months were orally dosed with three doses of 0, 4x10⁴, 4x10⁵ or 4x10⁷ M. ptb./dose (2 animals per group) and samples were taken from ileal (ILN), jejunal (JLN) and prescapular (PLN) lymph nodes 15.5 weeks later. Isolated lymph node cells (LNC) were incubated with medium alone, M. ptb. antigen (10 µg/ml) or Con A (10 µg/ml) for up to 6 days. Caspase activity, a marker of apoptosis, was determined by flow cytometry on days 0, 2, 4 and 6. Regardless of disease status similar levels of caspase activity were observed in LNC immediately after isolation. At this very early stage in the development of Johne's disease, in vitro incubation with M. ptb. antigen did not induce apoptosis. There were no significant differences in the percentage of caspase positive cells when LNC were incubated in the presence of medium alone or M. ptb. antigen. These results suggest that M. ptb. antigen responses in vitro in ILN and JLN cells may not prove useful in identifying very early exposure to M. ptb. in sheep. Further studies are being undertaken to assess apoptotic contributions in ovine peripheral blood mononuclear cells during the course of M. ptb. infection.