Role of surface antigens of Mycobacterium spp. in Diagnosis

Mycobacterial species are ubiquitous in nature and a worldwide concern for human and animal health. The major mycobacterial infections in animals are Johne’s disease (JD) and bovine tuberculosis (bTB). Controlling these infections is difficult due to the lack of highly sensitive and sensitive diagnostic test. Currently available diagnostic tests have to be carried out in laboratory settings with well experienced and trained examiners. My goal is to develop a sensitive on-site (in-field) device for diagnosis of Johne’s disease and bovine tuberculosis. The specific aims of this thesis were (1) to review currently-used or recently developed diagnostic tests for mycobacterial infections, (2) to optimize a milk-based enzyme linked immunosorbant assay (ELISA) for diagnosis of JD, (3) to evaluate a serum-based ELISA for detection of bTB in red deer and (4) to develop a bead-based microfluidic assay as a prototype of on-site diagnostic device for JD.

Previous reports on currently-used or recently developed diagnostic tests for mycobacterial infections were reviewed to summarize challenges and opportunities in development of sensitive on-site diagnostic devices. Most of the current serological tests for mycobacterial infections utilize crude extract of the pathogen or single molecule causing low diagnostic specificity and specificity. Use of multiple antigens was shown to be effective in improving test accuracy.

Ethanol extract of Mycobacterium avium subspecies paratuberculosis (MAP) was used to optimize a serum ELISA to test milk samples for JD. Using the optimized conditions, the average of ELISA values in the JD-positive milk samples was found to be significantly higher than that in the JD-negative milk samples.

Using ethanol extract from Mycobacterium bovis (MB), an ELISA was developed to detect anti-MB antibodies in serum of farmed red deer. The tentative diagnostic sensitivity and specificity was estimated to be 90% and 93.3%, respectively.

Magnetic beads coated with ethanol extract of MAP were used to develop a microfluidic immunoassay for diagnosis of JD. The antigen-coated magnetic beads were tested in the microfluidic system using bovine serum samples and a high level of antibody binding in JD-positive serum was observed.