Genetic diversity of Campylobacter in a major milkshed and development of novel molecular epidemiologic approaches for source tracking

Campylobacter infections are the leading cause of foodborne bacterial gastroenteritis worldwide. Dairy products, especially raw milk, and untreated water are the most common causes of campylobacteriosis outbreaks while the majority of sporadic cases are linked to consumption of contaminated poultry products. However, the source of Campylobacter infections is often unknown. There is an increasing need to understand sources and mechanisms of contamination for Campylobacter and other foodborne pathogens across the food system continuum, and implement prevention strategies. C. jejuni and C. coli are amendable to source tracking through the use of robust genetic approaches; however, multiple typing approaches are often necessary due to the weakly clonal population structure and genetic instability of some strains. To advance our knowledge of the molecular epidemiology of Campylobacter in farm environments, and provide new information on high resolution subtyping approaches, we characterized ∼400 Campylobacter isolates by using an 8-locus MLST/MOMP typing scheme; 3 primer sets that amplify different fragments of the porA allele were compared. A subset of strains was also subtyped by PFGE analysis using the SmaI enzyme. The isolates originated from outbreak investigation and prevalence surveys conducted in major agriculture production regions of California from 2006-2007. The sources included bovine feces, dairy wastewater, human stools, raw milk, and feral swine feces and tissues. As a proof of concept, we first applied an 8-locus MLST/MOMP typing approach during farm investigations following two milkborne outbreaks traced to dairies in the Central Valley of California, a major U.S. milkshed. Over 100 C. jejuni isolates from dairy farm samples were screened by MOMP typing and the outbreak strains were found in flush alley water and bovine feces, respectively. Subsequent to the outbreak investigations, we characterized C. jejuni and C. coli strains isolated from bovine waste and raw milk at 10 other dairy farms and raw milk sourced from 2 creameries in the Central Valley representing over 600 dairies. We found high genetic diversity among C. jejuni populations in the dairy environments compared with a remarkably clonal C. coli population isolated from the same environments. C. jejuni strains isolated from tanker truck raw milk shipped to creameries showed the most allelic diversity followed by strains from bovine feces and dairy flush alley water collected on individual farms. Overall, the combination of MLST, PFGE (SmaI enzyme) and the full-length porA sequence provided the most discriminatory power for subtyping C. jejuni isolates. In a separate survey conducted near spinach and other leafy green fields, multiple Campylobacter species were isolated from a feral swine population. C. jejuni strains from feral swine were closely related to ST-21, a common MLST subtype associated with human campylobacteriosis. In summary, multiple subtyping approaches can be used to source track Campylobacter through the farm environment and elucidate important sources and potential transport mechanisms to the food supply. The findings from this work provide data that support efforts to protect the milk and produce supply from contamination with Campylobacter originating from zoonotic sources in California agriculture landscapes.