Veterinary and Biomedical Sciences, Department of


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A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Veterinary Science, Under the Supervision of Professor Raul G. Barletta. Lincoln, Nebraska: December, 2010
Copyright 2010 Avery L. Paulson


Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease (JD) in ruminants. Development of genetic tools and completion of the MAP genome sequencing project expanded opportunities for antigen discovery. In this thesis, I review the current trends in diagnosis and disease control of JD and present the results of the studies on the seroreactivity of two proteins encoded for by the MAP1152-MAP1156 gene cluster. MAP1152 encodes for a PPE protein and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Maltose-binding protein (MBP) tagged recombinant MAP proteins were purified from Escherichia coli. Western immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of immunized mice and rabbits, and naturally infected cattle. MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme linked immunosorbent assay (ELISA) for the recombinant proteins was developed and used to test pre-classified positive and negative serum samples from naturally infected and non-infected cattle. Samples, with one exception, displayed no seroreactivity against MBP-LacZ (P > 0.05), the negative control antigen. MAP1152 displayed seroreactivity against all positive sera, but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between non- infected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, MAP infected cattle mount a humoral response to both MAP1152 and MAP1156. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immuno-pathogenesis of JD.

Adviser: Raul G. Barletta