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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC AND IMMUNOLOGICAL CHARACTERISTICS OF JOHNE'S DISEASE Title: Absorbed EVELISA: a diagnostic test with improved specificity for Johne's Disease in cattle

Authors
item Scott, M -
item BANNANTINE, JOHN
item Kaneko, Yumiko -
item Branscum, Adam -
item Whitlock, Robert -
item Mori, Yasuyuki -
item Speer, Clarence -
item Eda, Shigetoshi -

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 1, 2010
Publication Date: November 11, 2010
Repository URL: http://hdl.handle.net/10113/45952
Citation: Scott, M.C., Bannantine, J.P., Kaneko, Y., Branscum, A.J., Whitlock, R.H., Mori, Y., Speer, C.A., Eda, S. 2010. Absorbed EVELISA: a diagnostic test with improved specificity for Johne's Disease in cattle. Foodborne Pathogens and Disease. 7(11):1291-1296.

Interpretive Summary: This manuscript represents a continuing collaboration with Dr. Eda at the University of Tennessee on the ethanol vortex ELISA (EVELISA) test, which has shown promise as an improved serological test for Johne’s disease, which is caused by the bacterium Mycobacterium avium subsp paratuberculosis. In this communication, we examined the potential for false positive reactions using the novel EVELISA test on a known Johne’s disease negative dairy herd from Japan. A 70% positive test rate was discovered in this known negative herd! Not only did we see this with our EVELISA test, but also with the current commercially available ELISA test. The cause of the false positive results were due to environmental mycobacteria such as M. scrofulaceum, M. szulgai, M. gordonae, M. kansasii and M. phlei. By mixing cattle serum samples with a protein extract of M. phlei cells (a process called absorption), we demonstrate a remarkable lowering of the false positive reaction with the cattle sera negative for Johne’s disease. We conclude that this additional step will greatly enhance the specificity of the test, but will not lower the sensitivity of the test. This information is very useful to animal producers and veterinarians who want to test cattle for Johne’s disease.

Technical Abstract: Johne's disease (JD) is one of the most economically important infectious diseases in the dairy industry. Since there is no practical vaccine or chemotherapeutics for JD, the use of enzyme-linked immunosorbent assays (ELISA) to identify cattle for follow-up fecal culture testing and/or culling is recommended for Johne's disease control in dairy herds. However, several reports have estimated the diagnostic sensitivities of currently available ELISAs as being less than 30%. Also, recent reports indicated that infection of cattle with environmental mycobacteria caused false positive reactions in current ELISA tests. In 2006, we developed a novel ELISA test for Johne's disease using antigens extracted from Mycobacterium avium subsp. paratuberculosis. This new ELISA, named EVELISA, had much higher diagnostic sensitivity than that of commercial ELISA tests. To further investigate EVELISA performance, we obtained 38 serum samples from cattle on a Johne's disease-free Japanese herd with suspected cases of serological false positive reactions. When these samples were tested using commercial ELISA and EVELISA tests, more than 70% of the samples were falsely identified as JD positive by both methods. Antibodies in the serum samples reacted strongly with antigens of various environmental mycobacteria, suggesting that the false positive reactions in EVELISA were due to the presence of antibodies cross-reacting with M. paratuberculosis antigens. The propensity for false positive reactions from the EVELISA was inhibited markedly by the use of M. phlei antigens for pre-absorption of cross-reactive antibodies, without reducing the sensitivity (true positive reaction) of the test. This study documents false positive reactions in two serological diagnostic methods for Johne's disease, and indicates that the modified EVELISA with M. phlei absorption may form a basis for the development of a sensitive diagnostic test with a lower level of false positive reactions than that of a current commercial ELISA test.

Last Modified: 9/29/2014