Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/8/2005
Publication Date: 10/1/2005
Citation: Shigetoshi, E., Elliot, B., Scott, M.C., Waters, W.R., Bannantine, J.P., Speer, C.A. 2005. New Method of Serological Testing for Mycobacterium Avium subsp. Paratuberculosis (Johne's Disease) by Flow Cytometry. Foodborne Pathogens and Disease. 2(3):250-62. Interpretive Summary: The entire intact bacterium of Mycobacterium avium subsp. paratuberculosis (MAP) has never been used in a flow cytometric assay. Other diagnostic assays have used only the broken bacterium, but never intact. The present study uses this novelty to show that by keeping the bacterium intact, surface markers on the bacterium can be used to create a diagnostic test that is both sensitive AND specific. While the test could have been an ELISA based assay, a flow cytometric assay can be more readily automated and amendable for high throughput. This new assay was tested using sera from cattle in several herds with known histories of Johne’s disease. The results show that this test can reliably detect infected animals earlier and with more accuracy than other serological tests currently in use for Johne’s disease.
Technical Abstract: Johne’s disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. MAP has also been suspected as an etiologic agent of Crohn’s disease in humans. Attempts to control JD have proven inordinately difficult due to low levels of sensitivity by currently available tests, which are also incapable of detecting preclinical MAP infections. In the present work, we describe the use of a flow cytometry method (FCM) for the serological diagnosis of preclinical and clinical JD in cattle. The FCM is highly sensitive and subspecies-specific, easily distinguishing MAP-infected from MAP-non-infected cattle as well as MAP from M. scrofulaceum and M. avium subsp. avium. Sensitivity of the FCM was compared to a commercially available ELISA in two experiments involving 88 and 94 serum samples from MAP-positive and MAP-negative dairy and beef cattle farms that were separated into the following groups: 1) sera from a JD-free farm; 2) sera from a JD-negative farm with one recently detected JD-positive cow; 3) sera from JD-positive farms that had tested negative by ELISA; 4) sera from JD-positive farms that were considered “suspect” by ELISA; and 5) sera from JD-positive farms that tested JD-positive by ELISA. Testing by FCM showed that groups 1-5 were 0, 62.5, 90, 100 and 96.7% positive for MAP, respectively. The FCM was significantly more sensitive than the ELISA and capable of detecting MAP infections in serum samples as early as at 170 days after intratonsilar inoculation of MAP. IgG from cattle inoculated 240 days earlier with MAP bound specifically to MAP, whereas negative binding occurred with serum from control cattle or cattle experimentally infected with M. avium subsp. avium or M. bovis. The FCM assay is rapid and is completed in less than 4 h. Moreover, the FCM is objective, technically easy and can be automated for handling large numbers of samples. This novel assay may form the basis of a highly sensitive and subspecies-specific test for the diagnosis of JD.