title: Comparison of polyclonal antibodies to three different preparations of Mycobacterium paratuberculosis in immunohistochemical diagnosis of Johne's disease in cattle.
abstract: Polyclonal antisera were raised in rabbits against preparations of live and heat-killed Mycobacterium 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 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 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.
authors.affiliation: National Animal Disease Center, USDA-ARS, Ames, IA 50010, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Stabel
authors.forename: M R
authors.initials: MR
authors.lastname: Ackermann
authors.forename: J P
authors.initials: JP
authors.lastname: Goff
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D002417:Cattle; D002418:Cattle Diseases; D006099:Granuloma / Q000473:pathology; D007082:Ileum; D007150:Immunohistochemistry; D008198:Lymph Nodes / Q000382:microbiology / Q000473:pathology; D008264:Macrophages / Q000382:microbiology / Q000473:pathology; D016927:Mycobacterium avium subsp. paratuberculosis / Q000276:immunology / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000276:immunology / Q000473:pathology*; D011817:Rabbits; D012189:Retrospective Studies
chemical_list: D000907:Antibodies, Bacterial