We used MOMP typing to screen a substantial number of farm isolates collected after two milk-borne C. jejuni outbreaks and successfully identified the clinical outbreak strains. This study demonstrates that sequencing the C. jejuni porA allele is a promising target for rapid environmental screening to identify specific clones prior to conducting labor-intensive molecular typing methods following outbreaks.
By implementing a single-allele screening approach, public health laboratories could decrease time and labor costs associated with C. jejuni
molecular epidemiologic studies in the food supply continuum. Specifically, sequencing of the porA
gene and construction of a phylogenetic tree can be accomplished in under 24 h compared with MLST and 2-enzyme PFGE confirmation, which may take at least a week to complete in public health laboratories with limited staff and resources. The screen is also amendable to retrospective high-throughput sequencing of strains by boiling single MicroBank freezer stocks to extract DNA directly from the bead, thus eliminating the need to regrow isolates by culture (24
). We used the bead extraction technique to rapidly resequence porA
alleles using the Oxford primer set published subsequently to our original analysis as described below (29
). Laboratories could further streamline the screen by developing an allelic discrimination real-time PCR assay to detect single nucleotide polymorphisms in the porA
sequence specific to the clone (clinical outbreak strain) of interest (30
). This approach provides a method for screening a large number of clinical and, possibly, environmental isolates in investigations for rapid bacterial source tracking and selection of strains for next-generation whole-genome sequencing for high-resolution differentiation (31
In 2008, an extended 8-locus MLST-porA
typing scheme was published by researchers in Oxford, England (29
). The Oxford porA
product (570 bp, minimum; 738 bp, maximum) overlaps with the fragment we analyzed in this study. Cody et al. (2009) used the Oxford primer set to type a C. jejuni
strain collection and demonstrated that the porA
allele has sufficient genetic diversity and stability for use as a molecular epidemiologic tool (32
). Results from our study are comparable and show that the Oxford typing scheme discriminated an additional three porA
This study has some limitations that indicate the need for further investigation. For example, and unfortunately for effective trace-back investigations, many clinical laboratories often discard C. jejuni
isolates after confirmation of species, leaving few human isolates available for analysis. Even in this very large outbreak, isolates were not saved, thus limiting our ability to evaluate the robustness of MOMP typing in discriminating clinical MLST and PFGE subtypes. Our analysis also emphasizes the importance of selecting multiple pure colonies from individual samples during C. jejuni
outbreak investigations for successful molecular subtyping, as described previously in the human and veterinary literature (33
). Indeed, our results confirmed that multiple C. jejuni
subtypes in individual bovine fecal and wastewater samples are not uncommon (). However, the regional prevalence of the C. jejuni
outbreak strains at dairy A (ST-21) and dairy B (ST-1244) was unknown at the time of the investigations. Follow-up studies of multiple dairies in the central valley indicate that ST-1244 is relatively common from bovine sources, including bulk tank milk (M. T. Jay-Russell, unpublished data). Noteworthy is a report by Kwan et al. (2008) of ST-1244 as predictive potentially of strains that are bovine adapted, although further studies will be required to determine whether this is a marker for host-adapted C. jejuni
Public health implications.
An important goal of this study was to show the utility of MOMP typing to source-track genetically related strains in the context of an outbreak and identify potential on-farm risk factors and prevention strategies. Although C. jejuni
was not isolated from milk samples during either outbreak investigation, our molecular typing results support strongly the epidemiologic findings implicating milk products produced by the dairies as the sources of both outbreaks (16
is notoriously difficult to isolate from milk following outbreaks, possibly due to die-off during the lag time from consumption to recognition of the outbreak, uneven distribution of the bacteria in bulk tank milk, or inadequate culture and isolation methods for milk.
The exact mechanism of contamination at dairy A remains unclear, but it might have been the result of cross-contamination after pasteurization or heavy loads of Campylobacter before pasteurization. Based on the ease of recovery of C. jejuni from flush alley water, we speculate it is possible that cross-contamination with environmental dairy wastewater during milk processing occurred. Notably, the dairy A pasteurization room was located adjacent to the milking parlor and ~100 m from the dairy lagoon. Moreover, the recovery of C. jejuni from flush alley water and lagoon samples at dairy A, approximately 3 months after the outbreak, suggests that the outbreak strain was predominant at this dairy and the lagoon could be an important environmental reservoir. The high numbers of indicator bacteria (coliforms and E. coli) identified in lagoon and flush alley water are consistent with the pathogen prevalence. Additional research is needed to define the spread of Campylobacter in recycled wastewater systems and strategies to minimize contamination at dairies that use these systems for manure management.
Similar to the results at dairy A, the clinical outbreak strain was cultured from bovine feces during the environmental investigation of the dairy B outbreak linked to organic raw milk and raw chocolate colostrum. Dairy B also had an on-site creamery adjacent to cattle areas. Raw dairy products are not subjected to a “kill step,” so it is plausible to suggest that viable C. jejuni from bovine fecal material contaminated the milk/colostrum sometime during production or processing. Cross-contamination with environmental waters represents another possible mechanism of contamination. Indeed, a different pathogen, C. coli, was isolated from milking parlor discharge water, suggesting the possibility of cross-contamination.
The results obtained by MOMP typing the environmental isolates ultimately facilitated more intensive investigation into on-farm food safety practices at the implicated dairies and were used by state agriculture and public health officials to reinforce the importance of sanitation and education of dairy workers about best practices in dairy production (16
). Additionally, the California state legislature passed a bill that became effective on 1 January 2008 requiring no more than 10 coliform bacteria per milliliter in grade A raw milk for human consumption, the same coliform limit required for pasteurized milk (36
In summary, our study highlights the potential for ubiquitous dissemination of C. jejuni in the dairy farm environment and the utility of using MOMP typing as an epidemiologic tool for screening C. jejuni isolates to identify specific strains during dairy-related outbreak investigations to support implementation of on-farm prevention strategies. The approach could be applied during investigation of other potential food and water sources where there is a need to prioritize a large number of C. jejuni environmental isolates before confirmation by other molecular methods such as MLST, PFGE, or whole-genome sequencing analyses.