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<OL> <LI> Complete the molecular characterization of selected isolates of the pathogens Campylobacter spp., Salmonella spp. and Clostridium perfringens from poultry, utilizing repetitive-sequence PCR. Make a comparison regarding cost and efficiency of identification with other methods of differentiating bacteria, such as, multi-locus sequence analysis or pulsed-field gel. <LI> Identify host and pathogen genes important to colonization and/or toxin formation by Campylobacter jejuni and Clostridium perfringens in poultry, and monitor host and pathogen gene expression by RNA microarray analysis. Complete comparative genomic analyses between robust and poor colonizers to identify gene targets that could be disrupted to decrease pathogen presence in the gut environment. <LI> Qualitatively and quantitatively identify and compare selected microbial populations in the chicken gastrointestinal and reproductive tracts and in the internal organs of healthy and of diseased birds. Use biophotonics models where appropriate, and develop approaches for processing biofilms to provide for quantitative measurement of bacterial populations in situ.
APPROACH: In an effort to further characterize food-borne bacteria, we will (1) perform rep-PCR analysis of Campylobacter spp., Salmonella spp, and Clostridium perfringens isolates, formally recovered from various stages of well-defined poultry production and processing operations; (2) determine if rep-PCR technology can speciate Campylobacter spp.; (3) perform comparisons regarding cost, technical difficulty and efficiency of rep-PCR technology relative to previously established subtyping methods (DNA sequence analysis including multi-locus sequence typing [MLST] and pulse field gel electrophoresis [PFGE]) for each pathogen; and (4) develop an internet accessible database for each poultry associated pathogen assayed by rep-PCR. In an effort to identify factors responsible for colonization of poultry by Campylobacter jejuni, (1) pathogen gene content and (2) differential gene expression will be monitored by suppressive subtractive hybridization and RNA microarray analysis, respectively. Genes and gene products identified during these experiments will be further characterized and investigated for the development of possible intervention strategies. In an effort to further our understanding of how environmental changes affect microbial populations associated with poultry production and processing environments we will (1) quantitatively characterize chicken intestinal tract populations in the presence or absence of antimicrobial growth promoters (AGP?s) provided in poultry feed and (2) develop biophotonic methods and techniques to investigate microbial populations in the chicken intestinal tract. Environmental factors (such as AGP in feed) determined to produce changes in microbial populations will be identified and further investigated for the development of possible alternative intervention strategies such as bacteriocins in chicken feed (Stern et al., 2005; 2006). In an effort to further characterize the role of the fertilized egg in the transmission of Campylobacter spp. in chickens, we will introduce a genetically characterized strain of Campylobacter jejuni (Hiett, et al., 2002), to day-of hatch chicks and test for this particular isolate in the inoculated chickens for a 70 week period. Detection of bacterial pathogens in novel locations in the chicken will allow for the development of more targeted intervention strategies. In an effort to further our understanding of biofilm formation and persistence in poultry operations, we will (1) develop techniques to label pathogens present in biofilms, especially Listeria monocytogenes, and (2) develop methods for the production of biofilms. Environmental factors contributing to biofilm formation will be identified and further investigated for the development of possible intervention strategies.