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Comparison of Polyclonal Antibodies to Three Different Preparations of Mycobacterium Paratuberculosis in Immunohistochemical Diagnosis of Johne's Disease in Cattle

Polyclonal antisera were raised in rabbits against preparations of live and heat-killed Mycobac-terium paratuberculosis and cell-wall proteins of M. paratuberculosis and were evaluated as diagnostic tools in immunohistochemical staining of bovine tissue. Live preparations of M. paratuberculosis (LMp) were inoculated intraperitoneally or intravenously at 109/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 hemocyanin to improve antigenicity (KLH-CWPMp). The HKMp and KLH-CWPMp preparations were emulsified in incomplete Freund's adjuvant before subcutaneous inoculation of rabbits. Antibody titers in the terminal blood 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. bovis-infected tissues. Sensitivity and specificity of immunohistochemical detection of Johne's disease (paratuberculosis) from bovine tissues was 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 both intestine and lymph node.