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<OL type="A"> <LI> Determine variability in gene content, using suppressive subtractive hybridization analyses, between DNA from Campylobacter jejuni recovered from chicken flocks and humans, versus those recovered from chickens only. <LI> Determine, using DNA hybridization analyses, the distribution of DNA sequences identified as variable in subtraction hybridization experiments. <LI> Inactivate identified genes using insertional mutagenesis/homologous recombination techniques. <LI> Perform cell invasion assays for mutant and wild type isolates to determine the contribution of different genes for virulence in C. jejuni.
APPROACH: <BR>A: Suppressive subtractive hybridization reactions will be performed for the identification of differential gene content. Clones obtained from subtractive hybridization analyses will be sequenced to identify genes unique to each flaA SVR subtype. <BR><BR>B: In an effort to determine the distribution of differentially identified gene sequences, SlotBlot hybridization will be performed. <BR><BR>C: As 'patterns' of host specific genes presence/absence emerge, those genes will be evaluated for inactivation by gene disruption/homologous recombination mutagenesis. <BR><BR>D: Mutant and wild type Campylobacter spp. isolates will be examined for motility using phase contrast microscopy as well for the ability to invade eukaryotic cells during in vitro culture. Adherent bacteria will be expressed as a percent of the total, while the invasion index will be expressed as a percent of the total adherent bacteria.