Dissertation
Development of attenuated mutants of Mycobacterium avium subsp. paratuberculosis: Evaluation of mutants as potential live vaccines for bovine paratuberculosis
Washington State University
Doctor of Philosophy (PhD), Washington State University
05/2010
DOI:
https://doi.org/10.7273/000006111
Abstract
Mycobacterium avium subsp. paratuberculosis (Map) is the causative pathogen of Johne's disease, a chronic inflammatory wasting disease in ruminants. The disease is prevalent in the world and causes a substantial economic loss. Johne's disease has been difficult to control because of the lack of an effective vaccine. To address this problem we examined the potential of targeted gene disruption as a method to develop candidate vaccines with impaired capacity to survive in the host for use as a vaccine. We selected relA and pknG, known to be important virulence genes in Mycobacterium tuberculosis and Mycobacterium bovis, to explore this possibility. Deletion mutants were made in both of a clinical isolate of Map (K10) and its recombinant strain containing the green fluorescent protein gene (K10-GFP). Comparison of survival in an in vitro assay revealed deletion of either gene reduced survival in monocyte derived macrophages (M[phi]) compared to survival of wild-type K10. In contrast, survival in vivo was mainly affected by deletion of relA. The bacteria were detected in all three wild-type infected animals and three of five pknG mutant infected calves by bacterial culture or PCR at three months post infection (PI). In contrast, no bacteria were detected in any tissue sample from five calves infected with the relA mutant (P < 0.05). The percentage of Map culture positive tissues (nine different sites from each animal) at the time of necropsy were 48.1 % (28.7 - 68.1, 95 % CI) for wild type, 24.4 % (12.9 - 39.6) for the pknG mutant, and 0 % (0 - 7.87) for the relA mutant infected groups, respectively. Flow cytometric analysis of the immune response to the wild-type K10-GFP strain of Map and the mutant strains showed deletion of either gene did not affect their capacity to elicit a strong proliferative response to Map antigens or live Map. Quantitative RT-PCR revealed that genes encoding IFN-[gamma], IL-17, IL-22, T-bet, RORC, and granulysin were up-regulated in stimulated PBMCs with Map at three months PI compared to those at preinfection. The findings show deletion of relA alone may be sufficient for development of a vaccine.
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Details
- Title
- Development of attenuated mutants of Mycobacterium avium subsp. paratuberculosis
- Creators
- Kun Taek Park
- Contributors
- William C. Davis (Chair)Douglas Ruben Call (Committee Member) - Washington State University, Paul G. Allen School for Global Animal HealthThomas Eugene Besser (Committee Member) - Washington State University, Department of Veterinary Microbiology and PathologyNancy S Magnuson (Committee Member) - Washington State University, School of Molecular Biosciences
- Awarding Institution
- Washington State University
- Academic Unit
- College of Veterinary Medicine
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 87
- Identifiers
- 99901055118901842
- Language
- English
- Resource Type
- Dissertation