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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC AND IMMUNOLOGICAL CHARACTERISTICS OF JOHNE'S DISEASE Title: Development and Characterization of attenuated Mutant Candidate Vaccines for Control of Paratuberculosis

Authors
item Park, Kun - WASHINGTON STATE UNIV.
item Dahl, John - WASHINGTON STATE UNIV.
item Bannantine, John
item Barletta, Raul - UNIV. OF NEBRASKA
item Allen, Andrew - WASHINGTON STATE UNIV.
item Hamilton, Mary - WASHINGTON STATE UNIV.
item Park, Min - WASHINGTON STATE UNIV.
item Davis, William - WASHINGTON STATE UNIV.

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: October 28, 2007
Publication Date: October 28, 2007
Citation: Park, K.T., Dahl, J.L., Bannantine, J.P., Barletta, R.G., Allen, A.J., Hamilton, M.J., Park, M.K., Davis, W.C. 2007. Development and Characterization of attenuated Mutant Candidate Vaccines for Control of Paratuberculosis [abstract]. 9th International Colloquium on Paratuberculosis. Abstract No. 20A, p. 106.

Technical Abstract: Mycobacterium avium subsp. paratuberculosis (Map) is the causative pathogen of Johne’s disease, a chronic inflammatory wasting disease in ruminants. The disease has been difficult to control because of the lack of an effective vaccine. To develop a live attenuated vaccine for Map, as well as for the study of specific gene function, an efficient method for generating targeted gene mutation is urgently needed. Here we report an efficient allelic exchange mutagenesis system in Map using an in-vitro generated specialized transducing bacteriophage (phAE87). Three genes were selected for mutagenesis: pknG and relA, genes known to be important virulence factors in mycobacteria and lsr2, a gene regulating lipid biosynthesis. Mutants were successfully generated in the sequencing project virulent strain K10 and in a recombinant strain expressing the green fluorescent protein gene, gfp. With the optimized conditions we developed, we obtained allelic exchange frequencies of 78-100% with a transduction frequency of 9.5 x 10**-8 – 1.6 x 10**-7. The improved efficiency of disrupting selected genes in Map should accelerate development of additional mutants for vaccine testing and functional studies.

Last Modified: 11/25/2014
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