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Previous Article | Next Article 
Applied and Environmental Microbiology, June 2008, p. 3626-3633, Vol. 74, No. 12
0099-2240/08/$08.00+0 doi:10.1128/AEM.01669-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Department of Medical Microbiology, School of Medicine, University of Manchester, Clinical Sciences Building, Manchester Royal Infirmary, Manchester M13 9WL, United Kingdom,1 Health Protection Agency North West Laboratory, Clinical Sciences Building, Manchester Royal Infirmary, Manchester M13 9WZ, United Kingdom,2 Epidemiology Group, Faculty of Veterinary Science, University of Liverpool, Neston CH64 7TE, United Kingdom,3 FEMS North West, Royal Preston Hospital, Sharoe Green Lane, Preston PR2 9HG, United Kingdom,4 Institute of Veterinary, Animal and Biomedical Sciences, College of Sciences, Massey University, Palmerston North, New Zealand5
Received 20 July 2007/ Accepted 3 March 2008
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ABSTRACT |
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INTRODUCTION |
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C. jejuni is a common commensal in the gastrointestinal tracts of wild and farm animals and is ubiquitous in the natural environment. The routes of zoonotic transmission to humans are complex but are believed to be primarily food borne, with various food-producing animals including poultry (6) and cattle and sheep (31) implicated as important reservoirs. Disease outbreaks are rare and cases are often sporadic with the source of infection unidentified (24). However, when outbreaks occur, unpasteurized or contaminated cow's milk is a well-documented source (10, 14, 18, 25, 26). In a survey of infectious intestinal diseases in England and Wales, 26% of milk-borne outbreaks were attributable to campylobacters during an 8-year period (13). Fecal contamination (27), direct udder excretion (15, 23), and wild birds pecking milk bottle tops (30) are among the reported mechanisms by which milk becomes contaminated with campylobacters.
The epidemiology of C. jejuni in animal populations or their products which enter the food chain remains poorly understood, due in part to problems such as the difficulties of interlaboratory comparisons, inadequate discrimination, and poor reproducibility associated with strain characterization methods. Multilocus sequence typing (MLST), a robust and accurate molecular characterization method, has been shown to be a valuable tool for investigating the epidemiology and the population structure of C. jejuni (7), and several studies have delineated the C. jejuni populations of isolates from various food-producing animals (5, 19).
Hence, the purpose of the present study was to investigate the role and the contribution of cattle on dairy farms as a source for zoonotic C. jejuni infections in humans. A longitudinal study of five farms in the northwest of England over an 11-month period was undertaken to explore several aspects of C. jejuni epidemiology within dairy farm populations. We first estimated the prevalence of Campylobacter spp. in the cattle population and their variations between farms and/or over time. With MLST, the diversity and prevalence of C. jejuni genotypes from the cattle were investigated. Further, the C. jejuni clonal complexes identified were analyzed for possible between-group differences in their distributions among the farms, seasons, and cattle management groups, to aid the understanding of potential underlying relationships for the elucidation of C. jejuni epidemiology in cattle on dairy farms.
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MATERIALS AND METHODS |
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Information on cattle management groups was recorded for each sample. The definitions for the different management groups were as follows: unweaned, any calf having access to milk; weaned, any calf not having access to milk but of prebreeding age; heifer, any cow that was of breeding age but not yet calved; lactating, any cow producing milk; and dry, any previously calved cow that was not producing milk. Each farm was situated less than 3 km away from the University of Liverpool field station in Leahurst. Farms 1 and 4 were immediately adjacent to each other, with farm 2 approximately 1 km away. Farms 3 and 5 were 1 km apart from each other and 5 km away from farms 1, 2, and 4. In terms of cattle herd sizes, farms 1 and 3 were the largest, with roughly 100 and 200 cattle, respectively. Farms 2, 4, and 5 had smaller herd sizes, with roughly 50 cattle each. Subsequently, approximately 50 and 25 cattle fecal samples were collected in each sampling month from farms 1 and 3 and from farms 2, 4, and 5, respectively. The months of sampling were January through July, September, and November in 2003, except for farm 4 (on which sampling ceased in April due to the sale of the farm) and farm 5 (on which sampling was not performed for September and November).
Bacterial growth conditions.
Fecal samples (1 g) were inoculated into campylobacter enrichment broth (LAB135; Lab M, Bury, United Kingdom) containing selective supplement (X131; Lab M) and incubated at 37°C for 24 h followed by a further 24 h at 42°C. A loopful of enrichment culture was then inoculated onto campylobacter blood-free selective agar (CM0739, SR0155; Oxoid, Basingstoke, United Kingdom) and incubated for 24 to 48 h at 42°C under microaerobic conditions using an atmosphere generation system (CN0025; Oxoid). Growth on the selective agar was subcultured onto Columbia blood agar (CBA) (CM0331; Oxoid) and incubated for 24 h under the same conditions for the selective agar. On the basis of growth and colony morphology, oxidase reaction, and Gram stain, Campylobacter spp. were presumptively identified and inoculated into 20% (vol/vol) glycerol brain heart infusion broths (BHIB) for storage at –80°C until required for DNA extraction.
Preparation of chromosomal DNA.
Isolates were revived by culturing on CBA. A loopful of the glycerol brain heart infusion broths was inoculated onto CBA plates for discrete colonies and incubated under the conditions described above. Two to three colonies of growth were removed from the pure culture and emulsified by vortexing in 250 µl of molecular biology-grade water in a 1.5-ml microcentrifuge tube. Bacterial cells were killed and lysed by heating the suspension at 100°C for 25 min in a heat block followed by a freezing treatment at –20°C for 5 min. The centrifugation of the suspension at 13,000 x g for 10 min results in bacterial cell debris forming a pellet and DNA being suspended in the supernatant.
Differentiation of Campylobacter species.
A real-time PCR assay for the differentiation of C. jejuni and Campylobacter coli using TaqMan PCR (PE Applied Biosystems, Foster City, CA) was used to confirm the presumptively identified Campylobacter spp. isolates as described by Best and colleagues (4). Subsequently, only C. jejuni isolates were included in the study.
MLST.
Internal fragments of seven gene targets were amplified by PCR and their nucleotide sequences determined with primers and reaction conditions in accordance with the published MLST scheme for C. jejuni (9). Briefly, the internal fragments of the seven target gene loci (aspA [aspartase A], glnA [glutamine synthase], gltA [citrate synthase], glyA [serine hydroxymethyltransferase], pgm [phosphoglucomutase], tkt [transketolase], and uncA [ATP synthase alpha subunit]) were amplified by PCR and purified with a MultiScreen PCR filter plate and a MultiScreen vacuum manifold (Millipore Corporation, Billerica, MA) according to the manufacturer's instructions. For each of the seven target gene loci, the dideoxy-termination sequencing reaction was performed at least once on each of the amplified forward and reverse DNA strands with BigDye reaction mix version 3.1 and 5x sequencing buffer (PE Applied Biosystems). Reaction products were purified and unincorporated dye terminators were removed by precipitation with 95% and 70% ethanol and sequenced using an ABI Prism 377 automated DNA sequencer. Chromatograms were imported into the Sequencher software version 4.0.5 (Gene Codes Corporation) for sequence editing and assembly. Allele identification and sequence type (ST) and clonal complex assignments for each isolate were done by interrogating the Campylobacter MLST database (http://pubmlst.org/campylobacter).
Statistical analysis.
The associations between C. jejuni genotypes and the respective farms, seasons, and cattle management groups were analyzed with statistical tests using SPSS software version 14.0. A multivariate model was built to adjust for the possible confounding effects between variables using logistic regression, and where appropriate, a risk statistic (adjusted odds ratio) was included to indicate the strength of the relationship. P values of 
0.05 were the criteria chosen for statistical significance and, where appropriate, more-stringent criteria for statistical significance were used (Bonferroni correction) to adjust for multiple comparisons. Paired data (isolates which were sampled from the same cattle from different months) as well as data from farms 4 and 5 were excluded from these analyses due to the absence of data in certain months, in order to avoid the introduction of bias to the model.
The genotypic diversity for each farm, season, and management group, as evaluated by the number of clonal complexes present in each group, along with the number of isolates representing each genotype, was normalized and quantified by calculating the Simpson's index of diversity (D) (28), where D is given by the following formula: D = 1 – 
, where n is the number of isolates belonging to a particular clonal complex and N is the total number of isolates in a given group. The value of the index ranges between 0 and 1, where 0 represents no diversity and 1 represents infinite diversity. The index also represents the probability that two individual isolates taken randomly from a given group will have a different clonal complex.
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RESULTS |
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C. jejuni genotypes.
From a total of 297 C. jejuni isolates, 41 STs were identified, of which 22 appeared only once in the data set (Table 1). The most-common STs in the study were ST-61 (23.2%), ST-42 (18.2%), ST-21 (8.8%), ST-48 (8.4%), ST-22 (6.4%), and ST-257 (4.4%), which collectively represented 69% of the data set.
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Genotypic diversity and distribution of genotypes among farms, seasons, and management groups.
The number of distinct C. jejuni STs or clonal complexes present on each farm ranged from 2 on farm 5 to 19 on farm 3. The genotypic diversity of each farm, as quantified by the Simpson's index of diversity (28), based on the number of clonal complexes present, highlighted the diversity of farms 1 to 4, while farm 5 was the least diverse (Table 2).
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The distribution of different C. jejuni clonal complexes in each of the four seasons demonstrated slight variations (Table 3). Clonal complexes ST-61, ST-21, ST-42, ST-48, ST-22, ST-257, and ST-508 were the genotypes present in all four seasons. The ST-61 complex was more prevalent in spring (28.9%), whereas the ST-21 complex appeared to be more prevalent in winter (27.0%) and autumn (30.6%) and the ST-42 complex demonstrated a relatively consistent prevalence throughout. Clonal complexes ST-48, ST-22, and ST-257 were most prevalent in summer, spring, and winter, respectively.
Similarly, the number of STs presented in the cattle management or age groups ranged from 11 (8 clonal complexes) in the dry group to 22 (11 clonal complexes) in heifers. The Simpson's index of diversity, with a range of 0.803 to 0.887, also indicated a consistently high diversity among the management groups, although C. jejuni isolates originating from older cattle appeared to be the most diverse (Table 2).
Clonal complexes ST-61, ST-21, ST-42, ST-22, and ST-257 were present in C. jejuni isolates from cattle from all five management groups, and a progressive change in the distribution of C. jejuni clonal complexes in each of the five cattle groups was observed (Table 3). Collectively, the ST-61, ST-42, and ST-21 complexes appeared to be more prevalent in younger cattle. Conversely, older cattle appeared to harbor uncommon genotypes more frequently.
Associations between ST-61, ST-21, and ST-42 clonal complexes and the respective farms, seasons, and cattle management groups.
The apparent nonrandom associations in the distributions of three predominant clonal complexes were explored with a multivariate logistic regression model with isolates from farms 1 to 3 (Table 4). Data from farms 4 and 5 were omitted from these analyses due to the absence of data in certain months, in order to avoid the introduction of bias to the model.
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The adjusted odds ratio for temporal effects indicated that there was a significantly increased probability of isolating the ST-61 complex in feces collected in spring compared to summer (P = 0.03). A nonsignificant positive statistical association was seen for the ST-61 complex in feces collected in winter compared to summer (P = 0.079). No other significant temporal effects were seen for isolating the ST-21 and ST-42 complexes under this model.
There were also no statistically significant differences in the distributions of individual clonal complexes among the five cattle management groups under the multivariate model (data not shown).
Changes in genotype carriage.
Thirty animals were sampled on two or more occasions at different months during the study and were characterized by MLST (Table 5). Of the 30 cows, 25 were sampled twice and 5 were sampled three times. Sixteen cows carried a different genotype on successive sampling occasions (animals 1 to 16), including one which was sampled three times (animal 13). Of these, 13 animals (animals 1 to 13) carried a different clonal complex on each occasion, while three animals carried a different ST of the same clonal complex (animals 14 to 16). Twelve cows possessed the same ST on successive sampling occasions (animals 19 to 30), including two which were sampled on three occasions (animals 19 and 20). The two remaining cows (animals 17 and 18) were sampled three times and carried an identical ST in the first two samples with a change in clonal complex for the third sample. ST-61, ST-22, ST-48, ST-42, ST-19, and ST-49 were the only STs which were carried on successive occasions.
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DISCUSSION |
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Depending on a range of factors, including sampling type and size, recovery methods, herd type, season, and geography, wide discrepancies in the overall prevalence of C. jejuni in cattle have been reported in past studies. Nonetheless, the prevalence rates of 39.5% (34), 38.6% (3), and 23.0% (22) for thermophilic campylobacters described in several studies involving fecal samples of dairy cattle were generally in concordance with our findings (35.9%). Temporal fluctuations in prevalence were observed with a distinct peak in June (50.8%; Fig. 1a), coinciding with the seasonal peak traditionally seen in human infections in England and Wales (http://www.hpa.org.uk/infections) and consistent with the pronounced late spring peak observed in the shedding numbers of thermophilic campylobacters in cattle in a previous study (32). Although it is a widely held view that poultry is the predominant source of campylobacter infections, the findings from this study suggest that cattle may play a role in the seasonal peak in human infections or that the exposure to a common source of contamination may exist. Furthermore, a number of recent studies, including a 6-year survey, have shown evidence against a direct causal link between the seasonality of campylobacter prevalence in chickens and human infections (20, 36).
Several studies have delineated the C. jejuni populations of isolates from various food-producing animals, which provided increasing evidence that members of the ST-61 complex are more restricted to cattle/bovine sources (8, 12, 19). Our study further confirms the hypothesis by clearly demonstrating the dominance of the ST-61 complex in a large cattle data set. Interestingly, clonal complexes ST-21 and ST-42 were also frequently isolated, and clonal complexes ST-48, ST-22, and ST-257 were moderately common. Whereas the ST-42 complex was found to be strongly associated with ovine sources in previous studies (5, 8, 19), this study provided the first description of a high prevalence in cattle. This finding raises the possibility that there may be similarities in the physiological features of the gastrointestinal tracts of cattle and sheep that cause ruminants to be more likely to carry similar C. jejuni genotypes. Additionally, the previous report of cattle and sheep isolates sharing similar pulsed-field gel electrophoresis and flagellin genotypes fits well with this speculation (11). Further investigation involving more ruminant isolates may well provide the key to explain certain aspects of the host specificity for C. jejuni ST-61 and ST-42 clonal complexes. Indeed, there is evidence that the guts of cattle may provide a stable environment for bacterial strains, as there were instances where organisms were found to be genetically stable, dominant cattle strains. For example, certain pathogenic Salmonella STs have been found to be associated exclusively with bovine sources (1). In addition, C. coli strains isolated from cattle were found to be highly clonal in a previous study, where more than 80% of the strains belonged to a single ST (21). Incidentally, this group of C. coli strains possessed the uncA allele 17, which is found in the C. jejuni genotypes belonging to the ST-61 clonal complex found in high numbers in this study.
To put into a public health context the contribution of the C. jejuni genotypes identified in this study, we interrogated the Campylobacter MLST database, which revealed that all of the C. jejuni clonal complexes and at least 66% (27/41) of the STs identified in this study have been isolated from human disease in the past. In addition, all of the six most-common clonal complexes (ST-21, ST-45, ST-206, ST-61, ST-48, and ST-257) identified in the largest human data set to date (8) have all been isolated from cattle in this study. Further, 13 of the clonal complexes reported here were present in an investigation of human campylobacter isolates conducted in the northwest of England in 2003, where the ST-21 complex was clearly the dominant genotype (29). However, the genotypes identified in the present study are distinct from those recovered from environmental sources, where isolates belonging to the ST-45 complex and various uncommon genotypes were more frequently isolated (12). In addition, it has been found that new and unassigned genotypes in humans were predominantly associated with swimming in natural bodies of water (17). This suggests that the transmission pathway of campylobacters from cattle to humans may not necessarily include environmental sources or routes.
This is the first study to outline and compare the genotypic diversity of C. jejuni in cattle herds with a set of spatially and temporally related isolates using MLST. We have demonstrated that C. jejuni genotypes were highly diverse in cattle, with 90% of this data set represented by only 6 clonal complexes while the remaining 10% were represented by 11 complexes. These observations are in agreement with the notion that C. jejuni has a weakly clonal population structure (9, 33). The similarities in genotypic diversity observed between different seasons according the Simpson's index of diversity (Table 2) were somewhat surprising, as these similarities were in contrast to the hypothesis that the characteristic seasonal variation, which is the hallmark of C. jejuni epidemiology, would be reflected to a certain extent by variations in the diversity of the genotypes. On the other hand, the apparent differences in the genotypic diversity of C. jejuni between farms were consistent with the fact that there were geographical differences recorded for C. jejuni clonal complexes, as discussed below. The evaluation of the differences in the genotypic diversity of C. jejuni within and between various sources, regions, and time frames may provide additional insights into the ecological aspects of C. jejuni populations and may, for example, assist in the identification of specific animals or environments that are potential drivers for generating novel genotypes.
To better define the C. jejuni epidemiology in cattle on dairy farms, C. jejuni clonal complexes were analyzed for differences in their distributions among farms, seasons, and cattle management groups.
We have demonstrated that the distribution of C. jejuni genotypes was not random among farms (Tables 3 and 4), suggesting a strong geographical association among genotypes. Consequently, these findings led to the observation that two groups of farms which were 5 km apart (farms 3 and 5 and farms 1, 2, and 4) appeared to have maintained different genotypes, while farms within the same group (no more than 1 km apart) appeared to possess similar C. jejuni genotypes. This observation suggests that campylobacters may be readily transmitted between distances of approximately 1 km, but transmission may be limited on a larger geographical scale. This is consistent with the previous report that C. jejuni isolates from distances of less than 1 km were genetically more similar than isolates separated by greater distances, independent of sample or host types (12).
Certain dynamics were also noted among clonal complexes between farms, in particular, the presence of the ST-61 complex and ST-42 complex genotypes were almost mutually exclusive; these strains were not present on any of the farms with comparable prevalence. This may be explained by a potential ecological competition between strains within the bovine gut, although further studies are required to corroborate this observation. In addition, although the results of this study and past studies have suggested that the ST-61 complex may be a cattle-adapted C. jejuni genotype, the apparent geographical associations shown in this study have revealed that there are indeed cattle farms with only a few ST-61 complex isolates. More importantly, this raises the question of how a strain that is well adapted to cattle can also exist in very low numbers in nearby groups of cattle within a defined geographic area. It is possible that the variations of genotypes observed were due to farm management factors, which may possibly be explained only by scrutinizing the particular differences in farming practices or the diets of animals on individual farms.
Surprisingly, a temporal pattern was detected only for one of the clonal complexes in the logistic regression model (Table 4), where the probability of recovering ST-61 complex isolates was marginally increased in winter and significantly increased in spring, compared to summer. Consequently, this may suggest that no particular genotypes are exclusively responsible for the seasonal peak seen in summer. Nonetheless, the elucidation of the temporal characteristics of genotypes from different sources may help clarify the role of particular genotypes and their attribution to the rise of human infections during specific time frames.
Although there were no significant differences in the distributions of individual genotypes among cattle management groups, it appeared that the dominant genotypes (ST-61, ST-21, and ST-42 complexes) were more prevalent in younger cattle and gradually became relatively less prevalent in older cattle with higher proportions of uncommon genotypes. This observation agrees with a previous report which showed that adult animals carry a broader range of C. jejuni serotypes than calves (22).
We examined strain carriage over time in a small number of animals and observed no striking patterns, except for ST-61, which appeared to be more persistent in cattle and was carried on two sampling occasions in four animals, as well as on all three occasions from two animals. However, the number of animals involved was too small for this observation to be considered significant. Further investigations into this aspect of C. jejuni epidemiology could provide insights into the ecological dynamics of different genotypes within animal hosts.
This is the first report of a longitudinal study of C. jejuni in dairy cattle herds using MLST, and the results have provided important advances in knowledge in several key aspects of C. jejuni epidemiology, indicating that dairy cattle and their products may have a significant role as sources or transmission routes for human campylobacter infections.
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ACKNOWLEDGMENTS |
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We thank the following people for their assistance: Chris Roberts for his valuable advice on statistical analyses; Steve Keeney and Pam Watson for allowing access to their sequencing facilities; and all the farmers who participated in this study.
This study made use of the Campylobacter jejuni Multi Locus Sequence Typing website (http://pubmlst.org/campylobacter/) developed by Keith Jolley and Man-Suen Chan and hosted by the University of Oxford (16). The development of this site has been funded by the Wellcome Trust.
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FOOTNOTES |
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Published ahead of print on 18 April 2008.
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