Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/2/2006
Publication Date: 8/10/2006
Citation: Eda, S., Bannantine, J.P., Waters, W.R., Mori, Y., Whitlock, R.H., Scott, M.C., Speer, C.A. 2006. A Highly Sensitive and Subspecies-Specific Surface Antigen Enzyme-Linked Immunosorbent Assay for the Diagnosis of Johne's Disease. Clinical and Vaccine Immunology.13(8)837-844. Interpretive Summary: In this study, we attempted to shake loose surface proteins of Mycobacterium avium subsp. paratuberculosis (MAP) using a gentle agitation. After testing a variety of methods and solutions, we found that simple vortex mixing in ethanol was the most effective means to shake loose surface proteins from the bacteria. This “extract” of surface proteins was found to be highly immunogenic and useful for development of a highly sensitive ELISA to diagnose Johne’s disease in cattle. 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, is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Control of JD could be accomplished by diagnosis and good animal husbandry, but this is currently not feasible because commercially available diagnostic tests have low sensitivity levels and are incapable of diagnosing prepatent infections. In this study, a highly sensitive and subspecies-specific enzyme-linked immunosorbent assay was developed for the diagnosis of JD by using antigens extracted from the surface of M. avium subsp. paratuberculosis. Nine different chemicals and various intervals of agitation by vortex were evaluated for their ability to extract the surface antigens. Various quantities of surface antigens per well in a 96 well microtiter plate were also tested. The greatest differences in distinguishing between JD-positive and JD-negative serum by ethanol vortex enzyme-linked immunosorbent assay (EVELISA) were obtained with surface antigens dislodged from 50 ug/well of bacilli treated with 80% ethanol followed by a 30 second interval of agitation by vortex. Diagnostic specificity and sensitivity of the EVELISA were 97.4% and 100%, respectively. EVELISA plates that had been vacuum-sealed and then tested seven weeks later (the longest interval tested) had diagnostic specificity and sensitivity rates of 96.9 and 100%, respectively. In a comparative study involving serum from 64 fecal culture-positive cattle, the EVELISA identified 96.6% of the low fecal-shedders and 100% of the mid- and high-shedders, whereas the Biocor ELISA detected 13.7% of the low-shedders, 25% of the mid-shedders and 96.2% of the high-shedders. Thus, the EVELISA was substantially superior to the Biocor ELISA, especially in detecting low- and mid-shedders. The EVELISA may form the basis for a highly sensitive and subspecies-specific test for the diagnosis of JD.