Cell death by apoptosis is a part of normal development. Apoptosis also controls the immune response and is involved in the cytotoxic killing of infected cells. Cell-mediated immune responses play an important role in mycobacterial disease. Increasing our current knowledge of the immunological mechanisms involved in disease progression, including apoptotic responses, may allow advancements in the area of early diagnosis, identification of resistant animals and disease control.

For experimental infection, Merino lambs (randomly drafted into groups of 20) were challenged with either 3.36x10⁸ CFU of a clonal inoculum (Telford) or 3.76x10⁸ CFU of a gut homogenate from an animal with clinical ovine Johne's disease (OJD). One group received no treatment. Sequential blood samples were taken over a period of 12 months. Faecal shedding and presence of Mycobacterium avium subsp. paratuberculosis (Mptb) in tissue samples were determined by culture in a radiometric system (Bactec). Diseased sheep were categorised based on histological lesion type. In addition, samples were taken from sheep sourced from OJD-infected and disease-free farms.

Mononuclear cells were isolated from peripheral blood using density gradient centrifugation. Isolated cells were incubated with medium alone, Mptb 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 day 6 of culture. Phenotype markers were also used to identify specific apoptotic lymphocyte subpopulations. Apoptosis in intestinal tissue was studied by TUNEL assay and by the expression of apoptosis-related genes by Q-PCR.

Mptb antigen-driven apoptosis of mononuclear cells in experimentally challenged animals varies as disease progresses. There were also significant differences in the presence of apoptotic cells, as well as expression of apoptosis-related genes, in intestinal tissues from disease-free sheep and those with histological lesions.