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<OL> <LI> Complete molecular characterization of the pathogens Campylobacter spp., Salmonella spp., and Clostridium perfringens from poultry utilizing repetitive extragenic palindromic (rep)-PCR and related methods. <LI>Identify host and pathogen genes important to colonization by Campylobacter jejuni and Clostridium perfringens in poultry, and monitor host and pathogen gene expression by RNA and DNA microanalyses. <LI>Quantitatively and qualitatively identify microbial populations associated with the chicken gastrointestinal tract, reproductive tract, and internal organs. Develop biophotonics models to use for real time in situ colonization investigations. Develop approaches for processing biofilm samples to allow quantitative measurement in situ.
APPROACH: The molecular subtype methodology of repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) will be employed to characterize genomes of Campylobacter spp., Salmonella spp., and Clostridium perfringens in well characterized poultry production and processing environments. Additionally, DNA sequence, MLST and PFGE will be utilized for comparative characterization of Campylobacter spp. The comparative genomics techniques of suppressive-subtractive hybridization accompanied by RNA and DNA microarray analyses will be used, in combination, to identify genes differentially expressed between Campylobacter jejuni isolates previously characterized as robust or poor colonizers of poultry. Quantitative real-time PCR, using group-specific 16S rDNA primer sets, will be used to characterize microbial populations in the chicken gastrointestinal tract upon exposure to different feed conditions. Biophotonic models will be developed using existing strains of genetically modified Salmonella typhimurium that express, either constitutively or under specific controls. These marked bacteria will be utilized to colonize young or day-of-hatch chicks and a model will be developed for the colonization process based on whole, live-bird, low-level photon imaging of luminescent bacteria used for the initial challenge. Techniques for labeling pathogens, especially Campylobacter jejuni, and sampling biofilms will be tested to accurately represent the biofilm community within the poultry environment. A lab model of biofilm formation that uses a batch reactor to produce biofilms that include target pathogens will be established. Methods will be developed for producing biofilms and observing target pathogens, labeled with antibodies or fluorophores, within biofilm models. The lab-produced or natural biofilms will be analyzed by scanning electron, confocal scanning laser epifluorescence, and atomic force microscopy. Companion software for image analysis will be used to measure the surface coverage of the biofilm and differences in area and depth parameters with and without pathogens.