Genomics Attributes Associated with Host Adaptation in Campylobacter Jejuni and Campylobacter Coli from Poultry and Swine

Non-Technical Summary: Campylobacter is currently a leading bacterial cause for human diarrheal illness in the United States, and is, in addition, associated with severe autoimmune sequelae such as Guillain-Barre Syndrome. Poultry are a leading vehicle for human infections, and other food production animals are frequently colonized as well. However, the ecology of Campylobacter in the farm environment and in its natural niche (the gastrointestinal tract of animals) is remarkably complex, and still poorly understood. A troublesome outcome of this limited understanding is the unabated high pre-harvest prevalence of the pathogen in poultry and other food production animals. In spite of the well-known zoonotic nature of the pathogen, there is currently only limited and fragmented information on possible host adaptations that underlie the repeatedly observed predominance of certain Campylobacter species (e.g. C. jejuni vs. C. coli) and certain strain types in different meat animals. These gaps in knowledge compromise our ability to fully understand mechanisms mediating colonization of the animals pre-harvest, and to thus design effective, science based mitigation strategies, including effective vaccine design and validation programs. Genome sequence data are available from only meat animal-derived strains: two from chicken carcasses and one of likely bovine origin. Only fragmented genetic information is available for campylobacters from turkeys, increasingly recognized as an important reservoir for the pathogen in the United States, or for swine, which are primarily (and extensively) colonized with C. coli. In this project, we will obtain genome sequence information for a C. jejuni strain prevalent in turkeys and two strains of C. coli, one predominant in turkeys and one in swine. Genomic features prevalent in strains from specific meat animals (chickens, turkeys, or swine) will be identified, and host associations will be confirmed following screening of a panel of strains prevalent in different meat animals. The role of selected host-associated genes in colonization will be evaluated using oral infections of chickens and turkeys. The findings of the project will provide critically needed information on genetic attributes associated with prevalence of certain Campylobacter species and strains in specific meat animals. Such information will be crucial for science-based intervention strategies for eventual reduction of Campylobacter load in meat animals, especially poultry and swine. <P> Approach: We will sequence the genome of two strains from conventionally grown turkeys, one C. jejuni NC116 and one C. coli, and one strain from conventionally grown swine. Prior to genome sequencing, the colonizing potential of these strains will be confirmed in infections of one-day old chicks and turkeys. The genomes of these three Campylobacter strains described above will be sequenced using the latest pyrosequencing techniques. The automated annotation of completed genomes will be manually verified to provide the highest confidence level. Genomic comparisons will provide insights in strain and group (host) specific genes and alleles, leading to identification of host-association signatures. Special attention will be paid to the genome content of hypervariable regions, including variable genes involved in LOS and capsule biosynthesis, and in restriction - modification systems. The genome sequences of the three strains will be compared to those from other animal-derived strains (C. jejuni RM1221 and C. coli RM2228, both from chicken and C. jejuni 81-176, of likely bovine origin). Sequences found to be unique to the turkey-derived strains, or harbored in common with chicken-derived strains will be identified for further determination of their host-specific nature (specific to turkeys, or possibly specific to poultry (chicken and turkeys). Sequences found unique to the swine-derived C. coli will also be examined to determine their possible specificity to swine. DNA probes specific to unique sequences will be hybridized with genomic DNA from a diversity panel of strains of C. jejuni and C. coli from food production animals, constructed so as to represent maximal diversity per host. The host-specific genes will be tested for ability to confer host specificity and impact in colonization. Genes found to be host-associated will be deleted and replaced with a chloramphenicol resistance cassette and the possible role of unique genes will be thus investigated by testing isogenic wildtype and deletion mutants. Candidate host-associated (variable) alleles will be cloned and used to replace the corresponding allele in the strain from the heterologous host (swine or turkey), thus creating hybrid strains. These deletion constructs and hybrid strains will then be tested in two separate chick and turkey colonization challenges. Initial tests for colonization will use the direct crop inoculation. Deletion constructs or hybrid strains that still fully colonize the birds will be also assayed via the horizontal transmission assay, which monitors the ability of various strains to be passed from infected to uninfected birds. For this assay, one group of day-old poults will be inoculated by oral gavage and a week later will be distributed into batteries containing uninoculated poults.