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United States Department of Agriculture

Agricultural Research Service

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

item Stabel, Judith
item Ackermann, Mark
item Goff, Jesse

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 1, 1996
Publication Date: N/A

Interpretive Summary: Paratuberculosis, a chronic inflammation of the lower intestine in cattle, is caused by the bacterium, Mycobacterium paratuberculosis. The incidence of paratuberculosis may be as high as 20% in some states in the United States with considerable economic loss associated with the presence of this disease. An effective vaccine to prevent paratuberculosis is not available at this time and bacterium is fairly resistant to antibiotics and other therapeutics, making treatment of the disease difficult. In addition, accurate diagnosis of paratuberculosis in cattle is often laborious and by culture may require up to 12 weeks for detection. Immunohistochemistry is a diagnostic tool which can be used to detect the presence of the bacterium in tissues of infected animals. This technique is performed by using an antibody to detect organisms within tissue sections and can be performed within 6 hours. In this study, we evaluated three different antibody preparations on their ability to detect M. paratuberculosis in tissues. This method provides a more sensitive, efficient method of detecting paratuberculosis in cattle allowing for more rapid assessment of infection status in a herd.

Technical Abstract: 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 10*9/ml either IP or IV. Heat-ki i85C for 10 min. Cell-wall proteins were isolated from M. paratuberculosis and conjugated to keyhole limpet hemocyanin to improve antigenicity (KLH-CW 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. 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.

Last Modified: 4/20/2015
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