2007 Annual Report 1a.Objectives (from AD-416) A: 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. B: Determine, using DNA hybridization analyses, the distribution of DNA sequences identified as variable in subtraction hybridization experiments. C: Inactivate identified genes using insertional mutagenesis/homologous recombination techniques. D: Perform cell invasion assays for mutant and wild type isolates to determine the contribution of different genes for virulence in C. jejuni. 1b.Approach (from AD-416) 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. B: In an effort to determine the distribution of differentially identified gene sequences, SlotBlot hybridization will be performed. C: As “patterns” of host specific genes presence/absence emerge, those genes will be evaluated for inactivation by gene disruption/homologous recombination mutagenesis. 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. 3.Progress Report This report documents research conducted under a trust agreement between ARS and the U.S. Poultry and Egg Association. Additional details of reserach can be found in the report for the in-house associated project 6612-32000-055-00D, Molecular Characterization and Gastrointestinal Tract Ecology of Commensal Human Food-borne Bacterial Pathogens in the Chicken. A competitive grant was funded by, the U.S Poultry and Egg Association on June 01, 2007. To date, a temporary technical support person was hired and subtractive hybridization investigations were initiated. Campylobacter spp. is the leading bacterial etiology of acute gastroenteritis in humans. Epidemiological evidence implicates poultry as source of the organism that causes human illness. However, recent comparative genomic investigations at our laboratory identified specific biomarkers present in Campylobacter spp. isolates recovered from human illness that were not necessarily present in isolates recovered from poultry. Additionally, investigations demonstrated that non-invasive Campylobacter spp. strains were isolated from patients with non-inflammatory disease, while invasive strains were isolated from patients with inflammatory diarrhea. An extensive epidemiologic investigation conducted in Iceland by our laboratory provided a library of epidemiologically well defined Campylobacter spp. isolates recovered from both poultry and human infections. DNA sequence analyses of the isolates revealed that certain flaA SVR subtypes were recovered from chickens as well as from humans with campylobacteriosis. However, there were also subtypes that were predominate in poultry, but were never recovered from humans. The reason for this phenomenon is not well understood. Consequently, subtractive hybridization will be performed using Campylobacter spp. isolates with flaA SVR subtypes from chickens only, as well as using DNA from Campylobacter spp. isolates with subtypes recovered from both chickens and humans. Nucleotide sequences determined to differ will be used as probes in DNA hybridization analyses to determine the distribution of differentially identified gene sequences among other isolates from our Iceland investigation. Furthermore, genes that to vary between isolates will be inactivated by insertional mutagenesis and compared to the wild type Campylobacter spp. in eukaryotic cell invasion assays to ascertain the contribution to virulence. The identification of biomarkers predictive of human infection, as well as severity of infection, will allow for development of intervention strategies such that more virulent Campylobacter spp. isolates are targeted for reduction.