A Highly Sensitive and Subspecies-Specific Surface Antigen Enzyme- Linked Immunosorbent Assay for Diagnosis of Johne's Disease

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 samples by ethanol vortex enzyme-linked immunosorbent assay (EVELISA) were obtained with surface antigens dislodged from 50 g/well of bacilli treated with 80% ethanol followed by a 30-second interval of agitation by vortex. The diagnostic specificity and sensitivity of the EVELISA were 97.4% and 100%, respectively. EVELISA plates that had been vacuum-sealed and then tested 7 weeks later (the longest interval tested) had diagnostic specificity and sensitivity rates of 96.9 and 100%, respectively. In a comparative study involving serum samples from 64 fecal culture-positive cattle, the EVELISA identified 96.6% of the low-level fecal shedders and 100% of the midlevel and high-level shedders, whereas the Biocor ELISA detected 13.7% of the low-level shedders, 25% of the mid-level shedders, and 96.2% of the high-level shedders. Thus, the EVELISA was substantially superior to the Biocor ELISA, especially in detecting low-level and midlevel shedders. The EVELISA may form the basis for a highly sensitive and subspecies-specific test for the diagnosis of JD.

Evaluation of Diagnostic Tests for Johne's Disease (Mycobacterium Avium Subspecies Paratuberculosis) in New Zealand Dairy Cows

“…36 A substantial research effort has been dedicated to the improvement and validation of diagnostic tests for JD. 2,7,9,15,23,24,26,27,31,32 Research has focused on the tests most commonly used: the enzyme-linked immunosorbent assay (ELISA) for serum antibody and culture of MAP from the feces. However, 2 challenges to evaluating diagnostic tests for JD exist that cannot easily be overcome by sampling methods or laboratory technology.…”

Induction of B Cell Responses upon Experimental Infection of Neonatal Calves with Mycobacterium avium subsp. paratuberculosis

“…Surface antigens of M. avium subsp. paratuberculosis (strain Linda) were extracted using 80% ethanol as previously described (8). Strain Linda, an isolate from a human patient with Crohn's disease (ATCC, Manassas, VA), resulted in more sensitive detection of antibody in JD-positive sera than in strain K-10 extracts (unpublished results).…”

Trends and advances in the diagnosis and control of paratuberculosis in domestic livestock

“…Removal of cross-reacting antibodies by absorbing sera with Mycobacterium phlei has improved the specificity of ELISA (Yokomizo et al 1983). Some studies have reported 98.8% (Cox et al 1991), 99.7% (Reichel et al 1999) and 97.4% (Eda et al 2006) specificity, but it compromises with assay sensitivity. In general, highest sensitivity of serological tests have been recorded in animals with clinical signs of the disease since presence of antibodies is higher in the later stages of infection (Sweeney et al 1995;Nielsen & Toft 2008).…”