The tracking of strains around farms has been a major and successful strategy for targeting interventions against organisms like Salmonella in poultry flocks. However, for campylobacters, such a strategy is limited by the ubiquitous nature of the organism in this environment and its poor recoverability from such sources. An alternative and novel approach has therefore been developed. This approach exploits the genetic diversity of these organisms and aims to use real-time PCR technology to identify DNA specific for the flock-colonizing strain in environmental samples.
The approach adopted was to develop a LightCycler assay based on oligonucleotide probes targeting the SVR of the flaA
gene of the Campylobacter
strain colonizing the target poultry flock. Two striking advantages of real-time PCR are its rapidity and the low cost of analysis. The specificity of such assays is dependent on the similarity between the melting curves of the probe and homologous DNA compared with various test samples. Real-time PCR assays for the detection of Campylobacter
) or C
) in poultry fecal samples and carcass rinses (7
) have recently been reported. However, the real-time PCR described in this report aims to detect DNA specific to a target campylobacter strain.
The SVR of the flaA
gene was selected as the target for identifying the target strain. The genetic variability of this DNA region is well recognized, has previously been adopted for strain-typing purposes (25
), and has recently been used to improve the discriminatory power of multilocus sequence typing (9
). One possible issue with such a method is the propensity for genetic instability in this region. However, given the geographical and temporal limitations involved, genetic instability should be minimized.
The specificity of the assay was investigated both in silico against known sequences from 10 strains and then in the laboratory with those strains. The probes could detect one to five nucleotide differences on the basis of the melting curves observed. This variation was also determined by the locations, as well as the number, of the polymorphisms. The sensitivity of the method was determined with samples spiked with homologous strains to be 1 × 102
to 4 × 102
CFU/ml with two different probes (or 1 to 12 genome equivalents per reaction) for bacterial cultures (40
). Estimates of 5 × 102
to 1 × 105
campylobacters/ml (g) in food and environmental samples (20
) have been previously reported with similar assays, so this level of test sensitivity appears appropriate.
The purpose of this assay development was to identify potential reservoirs and sources of strains colonizing the poultry flock in the farm environment. The major advantage of a real-time PCR approach is that samples or isolates can be tested in large numbers rapidly and cheaply compared with other typing-based methods. The approach was therefore tested on a single farm to provide proof of principle. The strategy adopted was to sample a target flock, its farm environs, and any associated flocks throughout the flock's life. The poultry farm selected (farm A) was complex, with a directly adjacent poultry farm (farm B) having shared access and an associated poultry farm (farm C). In addition, there were other domestic animal species in adjacent fields. Ceca from all three flocks were sampled at slaughter, as well as feces from flocks on farms A and B during the growing period. The environmental sampling included six visits to the farm and the collection of 507 samples, which were then enriched and cultured, and any isolates and the enrichment broth samples were stored frozen in the presence of a cryopreservative. Of these environmental samples, only 39 yielded campylobacter isolates and three of these isolates were unrecoverable from storage, supporting previous experience indicating that environmental campylobacter isolates are highly stressed and poorly culturable.
Sixteen isolates were recovered from the target flock at slaughter. All were of the same PFGE type, supporting previous evidence that in intensively reared broiler flocks in the United Kingdom colonization is generally restricted to one or two strains (3
). The SVR of a representative flock strain was sequenced, and an sSVR probe (probe P1) was designed. This probe identified identical strains colonizing flocks on farm B at least 6 days earlier and on farm C at or about the time of slaughter but not in the previous flock on farm A, indicating that colonization may have been initiated on farm B and then transmitted to the adjacent poultry houses on farm A and that the previous flock on farm A was not a source. The specificity of the probe in these cases was confirmed by PFGE on the various isolates.
Subsequently, all of the culture-positive environmental enrichment broth samples were tested with probe P1 by the real-time PCR method. Surprisingly, particularly in the earlier samples collected, no Campylobacter DNA was detected in a number of these broth samples, despite evidence of a positive culture. However, investigation suggested that the handling and storage of the broth samples for future DNA testing were not optimal, which could have explained this anomaly. In particular, it is considered vital that aliquots of enrichment broth samples be made and frozen as soon as possible following completion of incubation. For later visits to the farm, the recovery of probe-positive samples was substantially improved and at least three probe-positive but culture-negative samples were identified, suggesting that, in some instances, the sensitivity of the probe approach was greater than that of the culture method.
A variety of melting curves were obtained, indicating the presence of at least four strains (Fig. ) detectable by probe P1 and that the melting curve characteristic of the target flock strain (P1.T1) could be detected in many of the environmental samples. PFGE of the available isolates confirmed this observation but indicated that the same melting curve was detectable in isolates with a clearly different PFGE pattern (A5), suggesting incomplete specificity of this probe. This was confirmed to be a consequence of similarity in the SVR sequences of these two strains. Subsequently, a second probe, P4, with significantly improved discriminatory power was designed on the basis of another part of the SVR of the A5 strain.
With the combined discriminatory power of the two probes, the strain, which eventually colonized the target flock, could be tracked around the farm environment. Clearly, this strain was persistently carried by cattle in an adjacent field. It has previously been established that cattle are frequently colonized by C
) and that the species and serotypes observed in cattle strains are similar to those that can affect humans. A link between cattle colonization and flock colonization has also been identified (6
), although the direction of transfer could not be established and the presence of other animals on the farm is considered a high-risk factor for campylobacter-positive flocks (1
This investigation has also provided evidence that campylobacter contamination of the farmer's boots provided the route of transmission from the cattle in the adjacent field to the poultry farm environment, probably causing colonization in houses on adjacent farm B first and then leading to colonization of the flock on farm A. Campylobacters recovered from farmers’ boots were identified in a recent study as a likely source of flock infection (22
), and a report from the Food Standards Agency entitled Evidence for the Effectiveness of Biosecurity to Exclude Campylobacter from Poultry Flocks
(available at http://www.food.gov.uk/safereating/microbiology/flocks/
), as well as a systematic review of the literature (1
), clearly indicates that farmers are the major vehicle for tracking campylobacters into poultry houses. There is also a possible role for the feed truck in transmission as a P1.T1-positive signal was recovered from a wheel well, but there were many other strains also present and a clear relationship to the poultry-colonizing strain could not be confirmed.
In conclusion, a real-time PCR assay using the LightCycler platform for the identification of specific C. jejuni SVR types in DNA-containing lysates from enrichment broth samples has been developed. This method has been used to investigate the on-farm sources of campylobacters colonizing a target poultry flock. By retrospectively testing environmental samples which had been collected, enriched, and stored frozen throughout the flock-growing period, the approach could overcome the problems associated with poor recoverability of environmental campylobacter strains and the major resource costs associated with strain typing. In the farm investigated, cattle in a field adjacent to the target poultry houses were carrying a strain identical to that which later colonized the flock. The evidence from this preliminary study was supported by the characterization of isolates from the flock and the farm environment. A larger trial of multiple farms has recently been completed, and preliminary data confirm the proof of principle of the probe approach. These results may allow the environmental sources of campylobacters in poultry flocks to be prioritized and to enable targeted interventions to be developed.