Identification of Diagnostic Molecular Markers for Detection of Bovine TuberculosisByAilam LimBovine tuberculosis (bTB) is distributed worldwide, creating a public health risk and causing substantial economic loss to cattle producers. Hence, control of bTB is important for both animal and human health. The tuberculin skin tests (TST) and whole blood interferon gamma ELISA (IFN-g assay) are the principal antemortem diagnostic assays used worldwide for bTB surveillance and in the "test and slaughter" based bTB control and eradication programs. The currently used TST and IFN-g; assays are less than perfect in test sensitivity and specificity, consequently false negative and false positive test results are common. Presently, in the state of Michigan, only 1-2% of the antemortem test-positive cattle culled for postmortem examination have bTB. To reduce the number of healthy cattle slaughtered for confirmation of bTB, diagnostic procedures capable of differentiating test-false positive cattle from true bTB infected cattle are needed. The objectives for the studies presented in this dissertation were: 1) identify differences in gene expression profiles between cattle that have bTB and those that are antemortem test-false positive, 2) analyze the differences in gene expression profiles to select potential molecular markers capable of differentiating bTB infected cattle from antemortem test-false positive cattle, 3) begin a validation process to confirm that the selected molecular markers are able to differentiate bTB infected cattle from antemortem test-false positive cattle.To accomplish those objectives, microarray hybridization experiments were used to examine gene expression profiles from small groups of bTB infected and antemortem test-false positive cattle. Quantitative real-time PCR (qPCR) assays were used to validate data derived from the microarray hybridizations and to test selected molecular markers for disease on larger groups of bTB infected and antemortem test-false positive cattle.In the initial study, cattle were grouped as single antemortem test-false positive (SFP), double antemortem test-false positive (DFP), and postmortem confirmed bTB infected (bTB). Whole blood from the cattle was stimulated for 4 hours with purified protein derivative made from cultures of Mycobacterium bovis (bPPD). Unique gene expression profiles were identified from the microarray hybridizations and qPCR assays confirmed that the gene expression profiles differentiated cattle in the bTB group from cattle in the DFP group, but not the SFP group.In subsequent studies, the period of antigen stimulation of whole blood was extended to overnight; the microarray hybridization library was changed to provide thousands of additional genes for testing. Also, a new group of cattle was created that consisted of animals that were from bTB free herds and were single antemortem test-false positive (SR). The overall results showed that differential gene expression profiles exist between test-false positive and true bTB infected cattle. Furthermore, a predictor model was constructed using seven genes (IL-1R2, ATR, BOLA-DRB4, CCNG1, CXCL2, IL-10 and TARS). This model was tested in a preliminary study and showed high sensitivity (95%), specificity (92.9%), positive, and negative predictive values (90.5% and 96.3% respectively) for diagnosis of bTB. In conclusion, there is great potential for using several genes identified in this group of studies as the basis for a differential diagnostic test for detection of bTB, which may limit the unnecessary culling of test-false positive cattle as bTB suspects.
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