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Barletta, R.; Stabel, Ju, R..; Bannantine, Jo, .
University of Alaska
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Johne's Disease (JD) is one of the most significant problems in terms of animal health and food security. The potential linkage with Crohn's disease makes MAP a concern as a zoonotic and/or food-borne pathogen, causing problems for both national and international trade, as well as public health and national agrosecurity. These issues underscore the initiatives to develop improved control strategies including diagnostic tests and vaccines. Herein, we propose a unique and innovative approach to develop second-generation MAP live-attenuated vaccine(s) coupling strain attenuation with the development of DIVA diagnostic capabilities based on the same gene deletion (e.g., either MAP 1152 or MAP 1156). The ultimate aim of this research is to produce a live-attenuated vaccine(s) with DIVA capabilities to be used in cattle to control JD.Our hypothesis is that protective immunity can be elicited by a live-attenuated MAP strain with DIVA capabilities that activates T-cell responses. To test this hypothesis we will:1. Generate attenuated unmarked in-frame deletion mutants with DIVA capabilities: New unmarked deletion mutants in MAP 1152 and MAP 1156 from MAP strain wild type K-10 will be generated. We have demonstrated that the original marked DMAP52 and DMAP56, already available in our laboratories, are attenuated in bovine macrophages. These mutants will be complemented and tested for invasion and intracellular survival in monocyte-derived macrophages (MDMs).2. Test novel antigens for DIVA capabilities: We will develop and implement new generations of DIVA capable diagnostic tests for JD to work in conjunction with the candidate vaccine mutants. Recombinant capture antigens MAP 1152 and MAP 1156 synthesized in E. coli have been shown to differentiate between seropositive and seronegative samples as determined by parallel testing using a commercially available JD ELISA. Herein, we will expand the humoral immunity test to a greater number of samples from JD natural and experimentally infected calves, and develop tests of cellular immunity.3. Assess the immunogenicity and pathogenicity of unmarked MAP mutants and their complemented strains in calves: We will assess the immunogenicity and pathogenicity of the unmarked mutants in calves in relation to the wild type K-10 and complemented strains. We will determine histopathological lesion scores, intestinal colonization, fecal shedding and cell-mediated immune responses.We expect this project will result in the development of a second-generation live-attenuated vaccine against JD with commercial and agrosecurity potential. The overall impact is the embodiment of a JD vaccine with DIVA capabilities. Achievement of this goal would be of significant importance in ameliorating the negative impact of JD on national/international trade and the security of agricultural assets.
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Nat'l. Inst. of Food and Agriculture
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Food Defense and Integrity
Natural Toxins
Bacterial Pathogens