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Development and Characterization of Salmonella Mutants for Use as Live Vaccine


The objectives of this research proposal are to develop and characterized an attenuated strain of the food-borne pathogen Salmonella typhimurium. We will examine the ability of Salmonella yqhC mutant to cause infections in vitro cell culture system as well as in animal model of infection. Most importantly, this study will investigate the potential use of such mutant as live attenuated vaccine for protection against Salmonella infections in animals. Reduction of Salmonella colonization and outbreaks in animals will significantly reduce incidences of salmonellosis in humans. Further, this proposal will define the immunological basis of host protection afforded by Salmonella yqhC mutant, which will provide valuable information necessary for effective vaccine designing. Besides providing training to a postdoctoral scientist, this study will help summer students develop research skills and generate interest in the field of food-safety. This study will significantly contribute to the development of food-safety research group at Tuskegee University and certainly to the overall national food-safety which is the ultimate goal of USDA/CSREES.

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NON-TECHNICAL SUMMARY: A: Salmonella infection, most reported food borne diseases in the united states, is due to consumption of contaminated food specifically meat products. B: Prevent or reduce Salmonella infections in animals using live attenuated vaccine against Salmonella developed by creating attenuated strains of Salmonella.<P>
APPROACH: In objective 1, we will test the ability of yqhC mutant to bind and invade T84 intestinal epithelial cells. Cell death and cytotoxic effect of the mutant on macrophages and intestinal epithelial cells will be determined using microscopic examination and LDH release assay. Levels of inflammatory cytokines/chemokines in RAW264.7 macrophages and T84 cells infected with the wild type Salmonella and its mutant will be measured using BioPlex assay. This objective is expected to be completed in 10-12 month in the first year of funding period. Objective 2 will investigate the effect of YqhC deletions in an in vivo mouse model of salmonellosis. We will determine LD50, and induction of inflammatory cytokines, systemic bacterial load and clearance, and histopathology will also be evaluated in Swiss Webster mice. The ability of each mutant to invade, survive and replicate in vivo will be determined. In addition, samples from liver and spleen will be saved in 10% buffered formalin for H&E staining and histopathological examination. To evaluate the level of cytokines in mice following inoculation with the mutant and the WT, blood samples will be taken at day 0, 1, 3, and 7 post-infection to determine TNF-alpha, IFN-gamma, IL-1, IL-6, and IL-8 levels in serum using Bioplex method. Total RNA will be isolated from liver and spleen and used to quantitate cytokine transcripts using quantitative real-time RT-PCR. This objective will be initiated at the beginning of the second year and will continue to the end of the year. Finally, objective 3 will start in the 8th month of the second year and continue to the end of the 3rd year. In this aim we will determine if mice immunized with such mutant would protect animals against lethal dose challenge of the wild-type (WT) Salmonella. We believe that the YqhC mutant will be avirulent/less virulent in mice. We will examine whether mice infected with such mutant will be protected when challenged with 5-100 times the lethal challenge dose of WT Salmonella. The immunized mice will be challenged with the WT counterpart 8 weeks after the mutated bacteria are cleared from the animals so that the innate immunity is back to normal. It is important to examine the immunological basis of protection in animals that have been immunized with the yqhC mutant. Such studies are crucial in determining the effectiveness of any attenuated bacterial strain to be used as a vaccine candidate. To determine the role of cell-mediated immunity in providing protection, lymphocyte proliferation assay will be performed by measuring [3H]-thymidine incorporation during the last 18 hr of culture. Culture supernatants also will be assessed for levels of IFN-gamma versus IL-4 using ELISA to determine the contribution of Th1 and Th2 related immune responses in providing protection against lethal infection. Additionally, we will determine the distribution of leukocyte subpopulations CD4/CD8 in the draining lymph nodes and spleens at selected points in time after immunization with mutant strain before challenge with wild type Salmonella.

Fadl, Amin
Tuskegee University
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