Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/20/2006
Publication Date: 5/20/2006
Citation: Eda, S., Scott, M.C., Bannantine, J.P., Waters, W.R., Yoshiyuki, M., Whitlock, R.H., Speer, C.A. 2006. A Novel ELISA for the Diagnosis of Mycobacterium avium subsp. paratuberculosis infections (Johne's disease) in Cattle [abstract]. American Society for Microbiology 106th Annual Meeting. Interpretive Summary:
Technical Abstract: Background: Johne’s disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), has a significant impact on the US dairy and beef cattle industries. Currently, the fecal culture test and ELISAs are the most widely used tests for JD, however, ELISAs have relatively low sensitivity for MAP, detecting approximately 50% of MAP infections identified by fecal culture, which in itself is capable of detecting only 40% of JD-positive animals. The fecal culture and PCR tests are dependent on the presence of MAP in the feces and cannot differentiate between “pass through” MAP and MAP arising from active infections. Methods: ELISAs for the diagnosis of JD, caused by MAP, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treating MAP bacilli with formaldehyde and then sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD–negative cattle or from calves experimentally inoculated with MAP, M. avium subsp.avium or M. bovis. Because the results obtained from the WELISA and SELISA were similar, most of the experiments were performed with the SELISA. Results: To optimize the SELISA test, various concentrations of formaldehyde (3.7 to 37%) and intervals of sonication (2-300 seconds) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in non-specific binding by the SELISA. SELISAs prepared by treating MAP with 37% formaldehyde and then a two seconds of sonication produced the greatest difference (7X) between MAP-negative and MAP-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of MAP infections in calves experimentally inoculated with MAP or other mycobacteria. Conclusion: Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD.