Polyclonal antisera were raised in rabbits against preparations of live and heat-killed Mycobacterium paratuberculosis as well as cell-wall proteins of M. paratuberculosis and evaluated as diagnostic tools in immunohistochemical staining of bovine tissue. Live preparations of M. paratuberculosis (LMp) were inoculated at 109/ml either intraperitoneal or intravenous. Heat-killed M. paratuberculosis (HKMp) was prepared by treatment of bacteria at 85C for 10 minutes. Cell-wall proteins were isolated from M. paratuberculosis and conjugated to keyhole limpet hemocyanin to improve antigenicity (KLH-CWPMp). Heat-killed and KLH-CWPMp preparations were emulsified in incomplete Freund's adjuvant before subcutaneous inoculation of rabbits. At the terminal bleed, antibody titers were higher for HKMp and KLH-CWPMp rabbits compared to the LMp inoculum (1:1024 vs 1:64). The KLH-CWPMp antibody did not cross-react with M. bovis-infected tissues. Sensitivity and specificity of immunohistochemical detection of paratuberculosis in bovine tissues was much higher for the KLH-CWPMp polyclonal antibody.