Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/3/2004
Publication Date: 1/20/2005
Citation: lHuntley, J., Whitlock, R., Bannantine, J.P., Stabel, J.R. 2005. Comparison of diagnostic detection methods for mycobacterium avium subsp. paratuberculosis from North American bison. Journal of Veterinary Diagnostic Investigation. 42(1):42-51. Interpretive Summary: Johne's disease is a chronic, debilitating intestinal disorder in cattle,sheep and wild ruminants, characterized by diarrhea, reduced feed intake, weight loss and death. Animals usually become infected when they are young by ingesting feces containing the causative bacteria. However, symptoms of disease do not usually present themselves until the animals reach 3 to 5 years of age or even older. During this time the animal is infected and may be shedding the organism in its feces without showing any clinical signs of disease. In addition to reduced production by these animals through reduced milk production, they also present a potential infective threat to the rest of the herd. Johne's disease is difficult to diagnose and therefore to control. Development of accurate and sensitive diagnostic tests is essential for controlling the spread of this disease. In this paper, we present results from different types of tissue staining protocols to identify infected bison. Further, we compare these staining methods with a gene detection method. Results of this study suggest that the gene detection method is much more sensitive for identification of infected bison. Being able to accurately detect infected animals will help allay the spread of this disease in wildlife and domesticated animals.
Technical Abstract: Tissues were collected from 14 North American bison (Bison bison) that were suspected of having Johne's disease and analyzed for the presence of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis). Sections of ileum, ileal-cecal lymph node, and three sequential sections of jejunum with their associated mesenteric lymph nodes were taken from each animal. The tissues were processed for Ziehl-Neelsen acid-fast staining, auramine O/acridine orange fluorescent staining, immunohistochemical staining, and polymerase chain reaction (PCR) detection of M. paratuberculosis-specific DNA. The non-specific Ziehl-Neelsen and auramine O techniques identified infection in a relatively low percentage of animals. Ziehl-Neelsen staining identified 50% (7/14) of the animals and 21.4% (24/112) of the tissues as positive, whereas auramine O staining identified 35.7% (5/14) of the animals and 25% (28/112) of the tissues as positive. Immunohistochemical analyses of bison tissues, using anti-sera collected from rabbits immunized with four different preparations of M. paratuberculosis, identified a greater percentage of infected animals (ranging from 57 to 93%) and tissues (ranging from 28 to 46%). Tissue samples from the middle ileum were most frequently positive by all staining techniques. As a final diagnostic test, PCR amplification of the IS900 insertion sequence identified 100% (14/14) of the animals and 81.2% (91/112) of the tissues as positive. Collectively, these data indicate that DNA-based detection of M. paratuberculosis was more efficient than staining, identified all of the bison as positive, and detected the greatest number of positive tissues within each animal. Additionally, the sample preparation for PCR detection takes less time than the staining methods and PCR positive samples are easily identified.