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The long term goal of our study is to eliminate intestinal carriage of Salmonella in chickens. In this initial project we propose to use a novel strategy to identify the genes involved in persistence that can then be targeted for vaccines or drug discovery. <P>
We have designed a novel high-throughput method to identify candidate genes involved in persistence in the chicken cecum. We expect Aim 1, screening of our mutant banks, to be completed in the first six months of this project, and screening on contents and tissue ex vivo (Aim 2) will be completed within the first year of this project. As we can house large numbers of chicks (several hundred) concurrently, we will verify multiple mutants simultaneously (as many as 12 simultaneously). In this way Aim 3, verification and complementation, will be completed in years 2 and 3. <P>
At the end of our study, we expect to have confirmed 15 - 20 genes as having individual persistence phenotypes in chicken cecum but not defective in cranial sections of the chick GI tract. We will have determined which of these mutants has altered binding to the chick cecal epithelium. <P>Future work will include experiments to determine where the genes identified are expressed during intestinal infection in the chick, and whether the mutants we identify here are able to form biofilms on chick intestinal tissue. <P>Identification of Typhimurium genes involved in these important biological processes is an essential stepping stone to our long term goal of developing novel strategies to eliminate cecal carriage of this organism in chickens.
NON-TECHNICAL SUMMARY: Salmonella is responsible for 1.4 million annual cases of salmonellosis in humans in the United States, and poultry is a major source. We hypothesize that Salmonella possesses genes that allows it to colonize/persist in the intestinal tract of poultry. The corresponding gene products are potential targets for improved vaccines and novel drugs, to reduce cecal carriage of Salmonellae on the farm. Using the most prevalent pathogenic serovar, Typhimurium (STm), we have generated a library of defined non-polar deletions in groups of non-essential genes as well as in non-essential individual genes. AIM 1: We will screen our library for genes involved in intestinal persistence in chicks. This library has the lowest complexity combined with the highest gene coverage of any mutant library tested to date for any bacterium in any host, a critical feature for genetic screening because Salmonella numbers in the intestine of chicks may transiently fall to very low numbers. Next, individual genes responsible within each deleted region will be identified using a custom pool of individual gene mutants. AIM 2: Genes required for initial colonization and for resistance to crop and gizzard contents will be determined in vitro, and excluded from further consideration. AIM 3: We will verify and complement `persistence' genes in the chick.<P>
APPROACH: The goal of our study is to eliminate intestinal carriage of Salmonella in chickens. We will use a novel strategy for genetic screening to identify the genes involved in persistence that can then be targeted for vaccines or drug discovery. Libraries of mutants in multi-gene regions, as well as targeted mutations in individual non-essential genes will be pooled and used together to identify the bacterial factors that are important for intestinal persistence of STm in chicks (AIMS 1 and 3). We will also use these cutting edge tools to identify all genes responsible for resistance to gizzard and crop contents, and for tissue association/ invasion in ex vivo chick tissue (AIM 2). Colonization and persistence genes identified in this study are logical targets for the development of improved vaccines, novel drugs, pro-biotic, or nutritional changes that will reduce Salmonella prevalence on the farm.