A. J. H. Leatherbarrow

Tracing the Source of Campylobacteriosis

Campylobacter jejuni is the leading cause of bacterial gastro-enteritis in the developed world. It is thought to infect 2–3 million people a year in the US alone, at a cost to the economy in excess of US

Rapid Evolution and the Importance of Recombination to the Gastroenteric Pathogen Campylobacter jejuni

4 billion. C. jejuni is a widespread zoonotic pathogen that is carried by animals farmed for meat and poultry. A connection with contaminated food is recognized, but C. jejuni is also commonly found in wild animals and water sources. Phylogenetic studies have suggested that genotypes pathogenic to humans bear greatest resemblance to non-livestock isolates. Moreover, seasonal variation in campylobacteriosis bears the hallmarks of water-borne disease, and certain outbreaks have been attributed to contamination of drinking water. As a result, the relative importance of these reservoirs to human disease is controversial. We use multilocus sequence typing to genotype 1,231 cases of C. jejuni isolated from patients in Lancashire, England. By modeling the DNA sequence evolution and zoonotic transmission of C. jejuni between host species and the environment, we assign human cases probabilistically to source populations. Our novel population genetics approach reveals that the vast majority (97%) of sporadic disease can be attributed to animals farmed for meat and poultry. Chicken and cattle are the principal sources of C. jejuni pathogenic to humans, whereas wild animal and environmental sources are responsible for just 3% of disease. Our results imply that the primary transmission route is through the food chain, and suggest that incidence could be dramatically reduced by enhanced on-farm biosecurity or preventing food-borne transmission.

Prevalence and genetic diversity of Campylobacter spp. in environmental water samples from a 100-square-kilometer predominantly dairy farming area.

Responsible for the majority of bacterial gastroenteritis in the developed world, Campylobacter jejuni is a pervasive pathogen of humans and animals, but its evolution is obscure. In this paper, we exploit contemporary genetic diversity and empirical evidence to piece together the evolutionary history of C. jejuni and quantify its evolutionary potential. Our combined population genetics–phylogenetics approach reveals a surprising picture. Campylobacter jejuni is a rapidly evolving species, subject to intense purifying selection that purges 60% of novel variation, but possessing a massive evolutionary potential. The low mutation rate is offset by a large effective population size so that a mutation at any site can occur somewhere in the population within the space of a week. Recombination has a fundamental role, generating diversity at twice the rate of de novo mutation, and facilitating gene flow between C. jejuni and its sister species Campylobacter coli. We attempt to calibrate the rate of molecular evolution in C. jejuni based solely on within-species variation. The rates we obtain are up to 1,000 times faster than conventional estimates, placing the C. jejuni–C. coli split at the time of the Neolithic revolution. We weigh the plausibility of such recent bacterial evolution against alternative explanations and discuss the evidence required to settle the issue.

Molecular Epidemiology and Characterization of Campylobacter spp. Isolated from Wild Bird Populations in Northern England

ABSTRACT Water samples were taken systematically from a 100-km2 area of mainly dairy farmland in northwestern England and examined for Campylobacter spp. Pulsed-field gel electrophoresis-restriction fragment length polymorphism (PFGE-RFLP) and flaA strain typing of Campylobacter jejuni and Campylobacter coli isolates were done. Data on the water source and the adjacent environment were recorded and examined as explanatory variables. Campylobacter spp. were isolated from 40.5% (n = 119) of the water samples tested. C. jejuni was isolated from 14.3%, C. coli was isolated from 18.5%, and Campylobacter lari was isolated from 4.2% of the samples. Campylobacter hyointestinalis was not isolated from any water source. The difference in prevalence between water types (trough, running, and standing) was significant (P = 0.001). C. jejuni was the species most commonly isolated from trough-water and running-water sources, while C. coli was the most frequently isolated from standing water (P < 0.001). No association was found between the presence of Escherichia coli and that of Campylobacter spp. The final multivariable logistic regression model for Campylobacter spp. included the following variables: water source, soil type, aspect, and amount of cattle fecal material in the environment (fecal pat count). Strain typing demonstrated a diverse population of C. jejuni and the presence of a common C. coli flaA type that was widely distributed throughout the area. Most of the isolates within the common flaA type were discriminated by PFGE-RFLP. These findings suggest a possible role for environmental water in the epidemiology of Campylobacter spp. in a farming environment.

Genotypic and Antibiotic Susceptibility Characteristics of a Campylobacter coli Population Isolated from Dairy Farmland in the United Kingdom

ABSTRACT Campylobacter infections have been reported at prevalences ranging from 2 to 50% in a range of wild bird species, although there have been few studies that have investigated the molecular epidemiology of Campylobacter spp. Consequently, whether wild birds are a source of infection in humans or domestic livestock or are mainly recipients of domestic animal strains and whether separate cycles of infection occur remain unknown. To address these questions, serial cross-sectional surveys of wild bird populations in northern England were carried out over a 2-year period. Fecal samples were collected from 2,084 wild bird individuals and screened for the presence of Campylobacter spp. A total of 56 isolates were recovered from 29 birds sampled at 15 of 167 diverse locales. Campylobacter jejuni, Campylobacter lari, and Campylobacter coli were detected by PCR, and the prevalences of different Campylobacter spp. in different avian families ranged from 0% to 33%. Characterization of 36 C. jejuni isolates by multilocus sequence typing revealed that wild birds carry both livestock-associated and unique strains of C. jejuni. However, the apparent absence of unique wild bird strains of C. jejuni in livestock suggests that the direction of infection is predominantly from livestock to wild birds. C. lari was detected mainly in wild birds sampled in an estuarine or coastal habitat. Fifteen C. lari isolates were analyzed by macrorestriction pulsed-field gel electrophoresis, which revealed genetically diverse populations of C. lari in Eurasian oystercatchers (Haematopus ostralegus) and clonal populations in magpies (Pica pica).

Temporal and farm-management-associated variation in the faecal-pat prevalence of Campylobacter jejuni in ruminants.

ABSTRACT Campylobacter infections are the most common cause of bacterial enteritis in humans, and nearly 8% of such infections are caused by Campylobacter coli. Most studies have concentrated on Campylobacter jejuni, frequently isolated from intensively farmed poultry and livestock production units, and few studies have examined the spread and relatedness of Campylobacter across a range of geographical and host boundaries. Systematic sampling of a 100-km2 area of mixed farmland in northwest England yielded 88 isolates of C. coli from a range of sample types and locations, and water was heavily represented. Screening for antibiotic resistance revealed a very low prevalence of resistance, while genotyping performed by using three methods (flaA PCR restriction fragment length polymorphism [RFLP], pulsed-field gel electrophoresis [PFGE], and fluorescent amplified fragment length polymorphism [fAFLP]) provided insights into the genomic relatedness of isolates from different locations and hosts. Isolates were classified into 23 flaA groups, 34 PFGE groups, and five major fAFLP clusters. PFGE banding analysis revealed a high level of variability and no clustering by sample type. fAFLP and flaA analyses successfully grouped the isolates by sample type. We report preliminary findings suggesting that there is a strain of C. coli which may have become adapted to survival or persistence in water and that there is a group of mainly water-derived isolates from which unusual flaA PCR fragments were recovered.

Typing of Campylobacter jejuni Isolates from Dogs by Use of Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis

In a 2-year longitudinal study of adult animals on 15 dairy farms and four sheep farms in Lancashire, UK. C. jejuni was isolated from all farms, although not on every occasion. Faecal samples were collected and cultured using standard techniques for isolation of Campylobacter. Assignment to species was via PCR assays. Peak prevalence of C. jejuni in both cattle and sheep was observed during the summer and in cattle this apparent seasonality was associated with grazing pasture [odds ratio (OR) 2.14], while in sheep it was independent of grazing. Increased prevalence was associated with increased milk yield (OR 1.05) and herd size (OR 1.01) in dairy cattle, and with increased stocking density (OR 1.29) and pasture quality (OR 2.16) in sheep. There was considerable variation in prevalence between farms but no evidence of large-scale spatial variation. The association between C. jejuni prevalence and diet in dairy cattle deserves further investigation.

Exploring the diversity of Arcobacter butzleri from cattle in the UK using MLST and whole genome sequencing.

ABSTRACT Campylobacter is a major cause of human gastroenteritis worldwide. Risk of Campylobacter infection in humans has been associated with many sources, including dogs. This study aimed to investigate whether C. jejuni carried by dogs could potentially be a zoonotic risk for humans and if there were common sources of C. jejuni infection for both humans and dogs. Multilocus sequence typing (MLST) together with macrorestriction analysis of genomic DNA using SmaI and pulsed-field gel electrophoresis (PFGE) were both used to analyze 33 C. jejuni isolates obtained from various dog populations, including those visiting veterinary practices and from different types of kennels. MLST data suggested that there was a large amount of genetic diversity between dog isolates and that the majority of sequence types found in isolates from these dogs were the same as those found in isolates from humans. The main exception was ST-2772, which was isolated from four samples and could not be assigned to a clonal complex. The most commonly identified clonal complex was ST-45 (11 isolates), followed by ST-21 (4 isolates), ST-508 (4 isolates), and ST-403 (3 isolates). The profiles obtained by macrorestriction PFGE were largely in concordance with the MLST results, with a similar amount of genetic diversity found. The diversity of sequence types found within dogs suggests they are exposed to various sources of C. jejuni infection. The similarity of these sequence types to C. jejuni isolates from humans suggests there may be common sources of infection for both dogs and humans. Although only a small number of household dogs may carry C. jejuni, infected dogs should still be considered a potential zoonotic risk to humans, particularly if the dogs originate from kennelled or hunt kennel dog populations, where the prevalence may be higher.

Spatio-temporal epidemiology of Campylobacter jejuni enteritis, in an area of Northwest England, 2000-2002.

Arcobacter butzleri is considered to be an emerging human foodborne pathogen. The completion of an A. butzleri genome sequence along with microarray analysis of 13 isolates in 2007 revealed a surprising amount of diversity amongst A. butzleri isolates from humans, animals and food. In order to further investigate Arcobacter diversity, 792 faecal samples were collected from cattle on beef and dairy farms in the North West of England. Arcobacter was isolated from 42.5% of the samples and the diversity of the isolates was investigated using multilocus sequence typing. An A. butzleri whole genome sequence, obtained by 454 shotgun sequencing of an isolate from a clinically-healthy dairy cow, showed a number of differences when compared to the genome of a human-derived A. butzleri isolate. PCR-based prevalence assays for variable genes suggested some tentative evidence for source-related distributions. We also found evidence for phenotypic differences relating to growth capabilities between our representative human and cattle isolates. Our genotypic and phenotypic observations suggest that some level of niche adaptation may have occurred in A. butzleri.

Molecular epidemiology and genetic diversity of Campylobacter jejuni in ruminants

A total of 969 isolates of Campylobacter jejuni originating in the Preston, Lancashire postcode district over a 3-year period were characterized using multi-locus sequence typing. Recently developed statistical methods and a genetic model were used to investigate temporal, spatial, spatio-temporal and genetic variation in human C. jejuni infections. The analysis of the data showed statistically significant seasonal variation, spatial clustering, small-scale spatio-temporal clustering and spatio-temporal interaction in the overall pattern of incidence, and spatial segregation in cases classified according to their most likely species-of-origin.