A better understanding of the epidemiology of C. jejuni
is clearly necessary as it continues to be the most common bacterial cause of human gastroenteritis. Indeed, the routes of disease transmission and the relative significance of infection sources to the burden on human disease have largely remained elusive. Nonetheless, several previous studies have greatly improved the understanding of C. jejuni
epidemiology by investigating C. jejuni
isolate populations from a range of animal hosts using geographically and temporally diverse isolate collections which were characterized by MLST (5
). However, the dynamics and interactions of C. jejuni
isolate populations between animal reservoirs and their immediate environment have not been established. Given the important role of cattle in C. jejuni
), this study addresses the molecular epidemiology of C. jejuni
isolate populations in a dairy farmland environment, with the aim of elucidating the dynamics of horizontal transmission of C. jejuni
genotypes among different sources within a defined area, specifically whether cattle would acquire C. jejuni
isolates from the environment and vice versa. Insight into such interactions would advance the knowledge of C. jejuni
epidemiology to inform on the relative importance of potential reservoirs for human infection.
This study has demonstrated that clonal complexes ST-21, ST-45, and ST-61 were the most common C. jejuni
genotypes in the dairy farming environment under study, comprising 60% of the entire data set (Table ; Fig. ). This finding may have significant implications for disease control and prevention, as not only do these strains have the capacity to cause disease, they are also the most frequently isolated genotypes in humans (9
). Moreover, seven further clonal complexes identified have also been associated with human infections, albeit on a smaller scale (9
). These observations clearly highlight the need to recognize that cattle and their associated environment could act as important reservoirs for human disease.
The genotypic composition of the C. jejuni
population in cattle was found to be consistent with that delineated from a sizeable number of isolates in a previous longitudinal study (22
), where nine clonal complexes overlapped between studies. Clonal complexes ST-21 and ST-61 were highly prevalent, and the ST-48 complex was moderately common in both cases, although a relatively lower number of ST-42 complex isolates and higher number of ST-45 complex isolates were observed in this study, whereas strains which did not overlap accounted for less than 4% of the isolates in each data set. These findings therefore confirmed the major C. jejuni
genotypes found in cattle from the longitudinal study; they further reinforce the hypothesis that ST-61 complex isolates may be from a cattle-adapted C. jejuni
lineage, especially in the United Kingdom, as suggested in previous studies (5
). Further, since all but one of the clonal complexes (ST-1332) identified in cattle in this study have been associated with human infections in the past, there is evidence to suggest that cattle may serve as a major reservoir for C. jejuni
infections. A longitudinal aspect was also included in the cattle isolates in this study, where isolates obtained on two occasions separated by 12 months had similar genotype distributions (Fig. ). This indicates that C. jejuni
genotypes in dairy cattle were largely stable over time, which would concur with the finding that there were minimal seasonal patterns in C. jejuni
genotypes in cattle from the previous longitudinal study (22
The ST-45 complex was found to be the most prevalent (31.3%) and the only dominant genotype among wild birds in this study, which was apparent by comparing the ST-45 complex prevalence to that of the next most common lineages, the ST-21 complex (6.3%) and the ST-677 complex (6.3%) (Table ). Despite this, however, the ST-45 lineage was found to be highly diverse and comprised of various sequence types with similar prevalence. Likewise, the distribution of isolates among sequence types of other lineages was fairly even throughout the data set, while the overall population structure of C. jejuni
isolates in wild birds appeared to be highly diverse (Table ). This feature was somewhat unique to the wild bird data set, since it has been observed that C. jejuni
isolates from other sources often belong to dominant sequence types within clonal lineages, including the ST-45 complex. However, this observation may support the suggestion that the relatively high body temperature of birds could provide an optimal growth environment for campylobacters (19
), and therefore, isolates may be genetically more diverse (25
). Adding to this observation was the presence of a large group of genetically distinct isolates with novel sequence types that were unassigned to clonal complexes (35.9%) (Fig. ), which contributed to 52% of all such isolates of the entire data set. This observation may also be a reflection of the high diversity of C. jejuni
isolates in wild birds but warrants further investigation. The ST-45 complex (5
) and unassigned sequence types (25
) were found to be highly prevalent in poultry sources in previous studies, and the results of this study suggest that this may also be true in wild birds. The coincidence of such findings in poultry and wild birds could suggest that similar mechanisms may exist in the gastrointestinal tract of avian animals to facilitate the niche adaptation of similar C. jejuni
Given the association of the ST-45 complex between poultry and human infections (9
), the high prevalence of the ST-45 complex observed in this data set strongly suggests that wild birds may play a part in the role of disease transmission and should also be regarded as a potential reservoir for C. jejuni
infections. Additionally, seven other complex strains with lower prevalence in the wild bird data set have also caused human gastroenteritis in the past. However, these observations are in contrast to previous studies that demonstrated a significantly limited degree of overlap between C. jejuni
genotypes found in certain wild bird species and those found in human clinical disease using pulsed-field gel electrophoresis (2
). Possible explanations for inconsistencies may include geographical differences, typing methods used, and the difference in bird taxa investigated between studies, where it has been demonstrated that C. jejuni
strains could differ according to species and/or specific feeding habits and ecology (3
The C. jejuni
genotypes found in environmental water closely resembled those found in the wild bird data set, where the ST-45 complex and unassigned sequence types were predominant. Unlike in birds, however, these C. jejuni
isolates appeared to be less diverse, as the ST-45 complex only included two sequence types and presented fewer unassigned sequence types. This may indicate that, while environmental water is contaminated with C. jejuni
isolates from wild animals, only a proportion of genotypes were more adapted to survive and persist in the environment. Further, the low diversity of sequence types observed in water may also be, in part, a reflection of the fact that Campylobacter
spp. do not replicate outside hosts (21
), hence the lack of means by which genetic variation could be generated following contamination.
There are implications for human infection from the finding that six of the eight clonal complexes isolated from environmental water in this area matched those that have caused disease in humans and that geographical areas such as the location studied are used for recreational activities such as water sports, camping, and picnicking. This suggestion, however, also conflicts with two previous studies that were conducted in New Zealand, which have concluded that genotypes found in environmental water did not overlap with those that cause human disease, although different characterization methods were used in these studies (7
The wild mammal isolates included in this study consisted mainly of rabbit and a number of badger samples, where the ST-45 complex was predominant, although the ST-21 clonal complex and unassigned sequence types were also highly prevalent in rabbits. Interestingly, rabbit was the only source where there was comparable prevalence between a genotype that was found to be dominant in cattle (the ST-21 complex) and genotypes that were found to be dominant in wild birds and water (ST-45 and unassigned sequence types). In addition, similar to that found in water samples, the ST-45 complex from these sources was also less diverse and had fewer unassigned sequence types than that found in wild birds. The most prevalent genotypes in the wild mammal data set were also the most relevant to human infections, in particular, clonal complexes ST-45, ST-21, ST-61, ST-42 (9
), and ST-677 (20
), which collectively represented 73.7% of rabbit isolates.
The associations of clonal complexes between isolation sources were compared, and distinct as well as overlapping genotypes were identified among different sources within the study area. For example, the ST-61 complex was significantly associated with cattle, while the ST-21 complex was also found in significantly higher numbers compared to that found in other sources except rabbits. A set of common genotypic characteristics among wildlife and environmental isolates that was distinct from cattle isolates was also apparent, which was the high prevalence of clonal complexes ST-45, ST-677, and ST-952 and unassigned sequence types.
The finding that ST-45 complex isolates were significantly associated with sources from wildlife and the environment is in contrast with the suggestion from previous studies that it is predominantly a poultry-adapted strain. This may indicate that the ST-45 complex was also widespread in the natural environment, or it may be a reflection of contamination of the environment from animal sources. The latter, however, may be a more plausible explanation; wild birds could be a likely source of environmental contamination considering the hypothesis that, as previously discussed, the ST-45 complex may be an avian-adapted C. jejuni
), although additional studies would be necessary to form robust conclusions. Nonetheless, the high prevalence of the ST-45 complex observed in both wild birds and in environmental water suggests that this strain is capable of withstanding marked differences between the high temperatures of birds and the ambient temperatures of environmental waters.
In addition, despite the relatively lower prevalence, the ST-677 and ST-952 complex strains were predominantly isolated from wildlife and environmental water sources, and only one isolate belonging to the ST-677 complex was found in cattle. While the ST-952 complex was a newly identified lineage in this study, in a previous study, infection with the ST-677 complex was found to be associated with drinking unchlorinated water or water from natural sources (20
). An increase in the sample size may reveal a higher number of these strains from water and wildlife sources.
A considerable number of uncommon and genetically distinct sequence types, many of which were newly identified in this study, have been observed from wildlife and environmental sources. This observation is in-line with a previous study, where infections with novel and unassigned sequence types were associated with swimming in natural bodies of water (20
). Therefore, it is suspected that these isolates may in fact represent a group of closely related isolates for which the genetic links are yet to be identified due to the small numbers recovered to date. Further studies on a larger scale may be able to identify the relationship between these strains and genotypes that are commonly found in the environment, such as the ST-952 complex, or as new emerging C. jejuni
clonal groups associated with the environment.
The finding of low numbers of the ST-21 complex from wildlife and environmental sources was somewhat surprising, since it has been considered to be a stable cluster of C. jejuni
isolates that is ubiquitous and has adapted to a wide range of hosts and environments (5
). However, due to the limited number of environmental isolates that has been investigated using MLST to date, the question of whether the ST-21 complex is indeed less widespread in the natural environment deserves further research.
The associations between clonal complexes and sources were confirmed by performing a gene flow analysis based on 3,309-bp-long concatenated nucleotide sequences from all seven housekeeping loci (Table ). Considering that an FST
value of 0.932 was found between the two Campylobacter
species (C. jejuni
and C. coli
) in a previous study (8
), the FST
values of 0.199 and 0.175 found within C. jejuni
isolates are noteworthy and are indicative of a limited gene flow between C. jejuni
isolate populations in cattle with those in wild birds and the environment, respectively. This is in agreement with the segregated distribution of clonal complexes observed between these sources, suggesting that transmission from cattle to humans is perhaps through nonenvironmental routes, that is, through the food chain.
Our primary objective was to investigate the potential role of wildlife and the environment as a source of C. jejuni infection in cattle by comparing the distribution of genotypes from diverse sources. These observations have demonstrated that C. jejuni isolate populations in wildlife and environmental samples were notably distinct from those in cattle within this geographical area. The finding that certain clonal complexes were predominantly shared among isolates from wildlife and environmental sources is indicative of common or interconnecting horizontal transmission pathways of C. jejuni isolates within these sources. However, the dissemination of genotypes between cattle and the environment and wildlife appeared to be considerably restricted, and we suggest that C. jejuni isolates from the environment have limited significance as reservoirs for infection of the cattle population currently under study.
From the public health viewpoint, the finding that the majority of the C. jejuni isolate genotypes identified in this study have been associated with human infections emphasizes the need to recognize cattle and their associated environment as important potential reservoirs for human disease, in particular for clonal complex strains ST-21, ST-45, and ST-61. Furthermore, the distinct distribution of these genotypes among samples from different sources observed in this study would imply that their transmission to humans is perhaps via independent routes. This situation would have implications in the design of control strategies.
Investigations that have addressed the importance of the potential transmission of C. jejuni isolates between cattle and environmental sources have been scarce. Based on the systematic sampling of isolates in this study, a population snapshot that mirrored the dynamics of C. jejuni isolates within a dairy farmland microcosm was explored with MLST, a characterization tool proven to be valuable for discerning the molecular epidemiology of C. jejuni. This has offered further insight into C. jejuni epidemiology, where both widely distributed genotypes and potentially host-associated genotypes have been identified. The added knowledge and understanding of such relationships would further our ability to recognize the sources of campylobacteriosis. To corroborate these findings, and given the high degree of significance of the C. jejuni genotypes identified in this study with respect to human disease, further studies involving a larger number of isolates and focusing on wildlife and environmental samples should be an essential research priority.