COMPARISON OF POLYCLONAL ANTIBODIES TO 3 DIFFERENT PREPARATIONS OF MYCOBACTERIUM-PARATUBERCULOSIS IN IMMUNOHISTOCHEMICAL DIAGNOSIS OF JOHNES-DISEASE IN CATTLE

Polyclonal antisera were raised in rabbits against :preparations of li ve and heat-killed Mycobacterium paratuberculosis and cell-wall protei ns of M. paratuberculosis and were evaluated as diagnostic tools in im munohistochemical staining of bovine tissue. Live preparations of M. p aratuberculosis (LMp) were inoculated intraperitoneally or intravenous ly at 10(9)/ml. Heat-killed M. paratuberculosis (HKMp) was prepared by treatment of bacteria at 85 C for 10 minutes. Cell-wall proteins were isolated from M. paratuberculosis and conjugated to keyhole limpet he mocyanin to improve antigenicity (KLH-CWPMp). The HKMp and KLH-CWPMp p reparations were emulsified in incomplete Freund's adjuvant before sub cutaneous inoculation of rabbits. Antibody titers in the terminal bloo d sample were higher for HKMp and KLH-CWPMp than for LMp rabbits (1:1, 024 vs. 1:64). The KLH-CWPMp antibody did not cross-react with M. bovi s-infected tissues. Sensitivity and specificity of immunohistochemical detection of Johne's disease (paratuberculosis) from bovine tissues w as much higher for the KLH-CWPMp polyclonal antibody. Immunoreactivity of the antibody resulted in staining of bacteria in the cytoplasm of macrophages, mononuclear giant cells, and extracellular bacteria in bo th intestine and lymph node.