Distribution and Genetic Profiles of Campylobacter in Commercial Broiler Production from Breeder to Slaughter in Thailand

Poultry and poultry products are commonly considered as the major vehicle of Campylobacter infection in humans worldwide. To reduce the number of human cases, the epidemiology of Campylobacter in poultry must be better understood. Therefore, the objective of the present study was to determine the distribution and genetic relatedness of Campylobacter in the Thai chicken production industry. During June to October 2012, entire broiler production processes (i.e., breeder flock, hatchery, broiler farm and slaughterhouse) of five broiler production chains were investigated chronologically. Representative isolates of C. jejuni from each production stage were characterized by flaA SVR sequencing and multilocus sequence typing (MLST). Amongst 311 selected isolates, 29 flaA SVR alleles and 17 sequence types (STs) were identified. The common clonal complexes (CCs) found in this study were CC-45, CC-353, CC-354 and CC-574. C. jejuni isolated from breeders were distantly related to those isolated from broilers and chicken carcasses, while C. jejuni isolates from the slaughterhouse environment and meat products were similar to those isolated from broiler flocks. Genotypic identification of C. jejuni in slaughterhouses indicated that broilers were the main source of Campylobacter contamination of chicken meat during processing. To effectively reduce Campylobacter in poultry meat products, control and prevention strategies should be aimed at both farm and slaughterhouse levels.     

Molecular Epidemiology of Campylobacter jejuni Isolates from Wild-Bird Fecal Material in Children's Playgrounds

In many countries relatively high notification rates of campylobacteriosis are observed in children under 5 years of age. Few studies have considered the role that environmental exposure plays in the epidemiology of these cases. Wild birds inhabit parks and playgrounds and are recognized carriers of Campylobacter, and young children are at greater risk of ingesting infective material due to their frequent hand-mouth contact. We investigated wild-bird fecal contamination in playgrounds in parks in a New Zealand city. A total of 192 samples of fresh and dried fecal material were cultured to determine the presence of Campylobacter spp. Campylobacter jejuni isolates were also characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), and the profiles obtained were compared with those of human isolates. C. jejuni was isolated from 12.5% of the samples. MLST identified members of clonal complexes ST-45, ST-682, and ST-177; all of these complexes have been recovered from wild birds in Europe. PFGE of ST-45 isolates resulted in profiles indistinguishable from those of isolated obtained from human cases in New Zealand. Members of the ST-177 and ST-682 complexes have been found in starlings (Sturnus vulgaris) in the United Kingdom, and these birds were common in playgrounds investigated in New Zealand in this study. We suggest that feces from wild birds in playgrounds could contribute to the occurrence of campylobacteriosis in preschool children. Further, the C. jejuni isolates obtained in this study belonged to clonal complexes associated with wild-bird populations in the northern hemisphere and could have been introduced into New Zealand in imported wild garden birds in the 19th century.     

Campylobacteriosis in Urban versus Rural Areas: A Case-Case Study Integrated with Molecular Typing to Validate Risk Factors and to Attribute Sources of Infection

Campylobacter infection is a leading cause of bacterial gastroenteritis worldwide, and most clinical cases appear as isolated, sporadic infections for which the source is rarely apparent. From July 2005 to December 2007 we conducted a prospective case-case study of sporadic, domestically-acquired Campylobacter enteritis in rural versus urban areas and a prevalence study of Campylobacter in animal and environmental sources in the Eastern Townships, Quebec. Isolates were typed using Multilocus Sequence Typing (MLST) to reinforce the case-case findings and to assign a source probability estimate for each human isolate. The risk of human campylobacteriosis was 1.89-fold higher in rural than urban areas. Unconditional multivariate logistic regression analysis identified two independent risk factors associated with human Campylobacter infections acquired in rural area: occupational exposure to animals (OR = 10.6, 95% CI: 1.2–91, p = 0.032), and household water coming from a private well (OR = 8.3, 95% CI: 3.4–20.4, p<0.0001). A total of 851 C. jejuni isolates (178 human, 257 chicken, 87 bovine, 266 water, 63 wild bird) were typed using MLST. Among human isolates, the incidence rates of clonal complexes (CC) CC-21, CC-45, and CC-61 were higher in rural than urban areas. MLST-based source attribution analysis indicated that 64.5% of human C. jejuni isolates were attributable to chicken, followed by cattle (25.8%), water (7.4%), and wild birds (2.3%). Chicken was the attributable source for the majority of cases, independent of residential area, sex and age. The increased incidence in rural compared to urban areas was associated with occupational exposure to animals, particularly cattle among those aged 15–34 years, and with consumption of private well water. Both bovine and water exposure appeared to contribute to the seasonal variation in campylobacteriosis. These results provide a basis for developing public education and preventive programs targeting the risk factors identified.     

Frequency and Spatial Distribution of Environmental Campylobacter spp.

Humans are exposed to Campylobacter spp. in a range of sources via both food and environmental pathways. For this study, we explored the frequency and distribution of thermophilic Campylobacter spp. in a 10- by 10-km square rural area of Cheshire, United Kingdom. The area contains approximately 70, mainly dairy, farms and is used extensively for outdoor recreational activities. Campylobacter spp. were isolated from a range of environmental samples by use of a systematic sampling grid. Livestock (mainly cattle) and wildlife feces and environmental water and soil samples were cultured, and isolates were presumptively identified by standard techniques. These isolates were further characterized by PCR. Campylobacter jejuni was the most prevalent species in all animal samples, ranging from 11% in samples from nonavian wildlife to 36% in cattle feces, and was isolated from 15% of water samples. Campylobacter coli was commonly found in water (17%) and sheep (21%) samples, but rarely in other samples. Campylobacter lari was recovered from all sample types, with the exception of sheep feces, and was found in moderate numbers in birds (7%) and water (5%). Campylobacter hyointestinalis was only recovered from cattle (7%) and birds (1%). The spatial distribution and determinants of C. jejuni in cattle feces were examined by the use of model-based spatial statistics. The distribution was consistent with very localized within-farm or within-field transmission and showed little evidence of any larger-scale spatial dependence. We concluded that there is a potentially high risk of human exposure to Campylobacter spp., particularly C. jejuni, in the environment of our study area. The prevalence and likely risk posed by C. jejuni-positive cattle feces in the environment diminished as the fecal material aged. After we took into account the age of the fecal material, the absence or presence of rain, and the presence of bird feces, there was evidence of significant variation in the prevalence of C. jejuni-positive cattle feces between grazing fields but no evidence of spatial clustering beyond this resolution. The spatial pattern of C. jejuni is therefore consistent with that for an organism that is ubiquitous in areas contaminated with cattle feces, with a short-scale variation in infection intensity that cannot be explained solely by variations in the age of the fecal material. The observed pattern is not consistent with large-scale transmission attributable to watercourses, wildlife territories, or other geographical features that transcend field and farm boundaries.     

Typing of Campylobacter jejuni and Campylobacter coli isolated from live broilers and retail broiler meat by flaA-RFLP, MLST, PFGE and REP-PCR

We analyzed 100 Campylobacter spp. isolates (C. jejuni and C. coli) from Grenada, Puerto Rico and Alabama, which were collected from live broilers or retail broiler meat. We analyzed these isolates with four molecular typing methods: restriction fragment length polymorphism of the flaA gene (flaA-RFLP), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and automated repetitive extragenic palindromic polymerase chain reaction (REP-PCR) using the DiversiLab system. All methods performed similarly for the typing of C. jejuni and C. coli. The DNA extraction method appears to influence the results obtained with REP-PCR. This method was better for the typing of C. jejuni than C. coli, however both REP-PCR and flaA-RFLP generated types that were indistinguishable between C. jejuni and C. coli and appeared to be random, without any relationship to species, location, or source of isolates. PFGE and MLST generated typing results that had a better correlation with the geographic location of the isolates and showed higher concordance with the Wallace coefficient. The adjusted Rand coefficient did not show higher concordance among the methods, although the PFGE/MLST combination exhibited the highest concordance. PFGE and MLST revealed a better discriminatory power for C. coli isolates than REP-PCR or flaA-RFLP. The use of readily available online tools to calculate the confidence interval of the Simpson's index of diversity and the adjusted Rand and Wallace coefficients helped estimate the discriminatory power of typing methods. Further studies using different C. jejuni and C. coli strains may expand our understanding of the benefits and limitations of each of these typing methods for epidemiological studies of Campylobacter spp.     

Prevalence and Pathogenic Potential of Campylobacter Isolates from Free-Living,Human-Commensal American Crows

Recent studies have suggested a potential role for wild birds in zoonotic transmission of Campylobacter jejuni, the leading cause of gastroenteritis in humans worldwide. In this study, we detected Campylobacter spp. in 66.9% (85/127) of free-ranging American crows (Corvus brachyrhyncos) sampled in the Sacramento Valley of California in 2012 and 2013. Biochemical testing and sequence analysis of 16S rRNA revealed that 93% of isolates (n = 70) were C. jejuni, with cytolethal distending toxin (CDT) and flagellin A genes detected by PCR in 20% and 46% of the C. jejuni isolates (n = 59), respectively. The high prevalence of C. jejuni, coupled with the occurrence of known virulence markers CDT and flagellin A, demonstrates that crows shed Campylobacter spp. in their feces that are potentially pathogenic to humans. Crows are abundant in urban, suburban, and agricultural settings, and thus further study to determine their role in zoonotic transmission of Campylobacter will inform public health.     

Clonal Population Structure and Antimicrobial Resistance of Campylobacter jejuni in Chicken Meat from Belgium

Campylobacter jejuni is one of the most important causes of human diarrhea worldwide. In the present work, multilocus sequence typing was used to study the genotypic diversity of 145 C. jejuni isolates from 135 chicken meat preparations sampled across Belgium. Isolates were further typed by pulsed-field gel electrophoresis, and their susceptibilities to six antimicrobials were determined. Fifty-seven sequence types (STs) were identified; 26.8% of the total typed isolates were ST-50, ST-45, or ST-257, belonging to clonal complex CC-21, CC-45, or CC-257, respectively. One clonal group comprised 22% (32/145) of all isolates, originating from five different companies and isolated over seven sampling months. Additionally, 53.1% of C. jejuni isolates were resistant to ciprofloxacin, and 48.2% were resistant to tetracycline; 28.9% (42/145) of all isolates were resistant to both ciprofloxacin and tetracycline. The correlation between certain C. jejuni clonal groups and resistance to ciprofloxacin and tetracycline was notable. C. jejuni isolates assigned to CC-21 (n = 35) were frequently resistant to ciprofloxacin (65.7%) and tetracycline (40%); however, 90% (18/20) of the isolates assigned to CC-45 were pansusceptible. The present study demonstrates that certain C. jejuni genotypes recur frequently in the chicken meat supply. The results of molecular typing, combined with data on sample sources, indicate a possible dissemination of C. jejuni clones with high resistance to ciprofloxacin and/or tetracycline. Whether certain clonal groups are common in the environment and repeatedly infect Belgian broiler flocks or whether they have the potential to persist on farms or in slaughterhouses needs further investigation.Campylobacter jejuni is among the most common bacterial causes of human gastroenteritis worldwide (4, 23). Infected humans exhibit a range of clinical symptoms from mild, watery diarrhea to severe inflammatory diarrhea (14). In addition, C. jejuni has been identified as an important infectious trigger for Guillain-Barré syndrome, the most common cause of acute flaccid paralysis in polio-free regions (16). Another issue of concern regarding Campylobacter is the increase in antimicrobial resistance appearing in various regions around the world (1). Infection with an antimicrobial-resistant Campylobacter strain may lead to a suboptimal outcome of antimicrobial treatment or even to treatment failure (11).Consumption of contaminated water and raw milk has been implicated in campylobacteriosis outbreaks (23). However, the majority of human cases are sporadic, and consumption or mishandling of contaminated raw or undercooked poultry meat is believed to be an important source of infection. Risk assessment studies, outbreak investigations, and case-control reports all incriminate chicken meat as a major source, perhaps the major source, of food-borne transmission (14, 17, 32, 48). In Belgium in 1999, a controlled withdrawal of poultry products from sale due to alleged dioxin contamination resulted in a 40% reduction in the frequency of human campylobacteriosis (44). Thereafter and since the year 2000, the Campylobacter contamination of Belgian poultry carcasses and meat has been monitored by the Federal Agency for the Safety of the Food Chain, and the rate of positive samples is regarded as high. In 2006, 55.5% of cecal samples (n = 6,443) from Belgian broilers at slaughter tested positive for Campylobacter (3). In 2007, an industry-focused survey reported that 48% of Belgian chicken meat preparations (n = 656) were contaminated with Campylobacter (19).Molecular typing is an important tool in elucidating the diversity and transmission routes of Campylobacter isolates contaminating the food chain. In the United States, molecular analysis of Campylobacter spp. from poultry production and processing environments showed that many of the clones found within a flock are present in the final products, although the diversity of Campylobacter isolates in the final product was lower than that observed in the flock (22). Furthermore, numerous molecular epidemiological studies indicate that the genotypes of C. jejuni isolated from human cases overlap those of poultry origin (17, 47). Various molecular typing methods for the study of the population structure of Campylobacter are currently available (46). Among these, the multilocus sequence typing (MLST) approach is an emerging tool for research on the population structure and molecular epidemiology of Campylobacter. The technique is highly reproducible, portable, and easy to interpret, and results can be shared through a publicly accessible online database (31, 34). As such, MLST is becoming an important tool for studying the molecular epidemiology of Campylobacter in a global context. The accumulation of sequence typing data generated from different countries and settings could allow the creation of more-sophisticated models of the epidemiology and evolution of bacterial pathogens and the development of improved approaches for combating their spread (41).In Belgium, there is a paucity of information regarding the population structure of Campylobacter in the chicken meat supply. No population-based surveys have been conducted to investigate the molecular epidemiology of C. jejuni in chicken meat at points close to human consumption. In this study, MLST and pulsed-field gel electrophoresis (PFGE) were used to characterize the diversity of, and clonal relationships among, 145 C. jejuni isolates from Belgian chicken meat preparations. In addition, we characterized the antimicrobial resistance in this collection and correlated it with C. jejuni genotypes.     

Use of PCR for Direct Detection of Campylobacter Species in Bovine Feces

This study reports on the use of PCR to directly detect and distinguish Campylobacter species in bovine feces without enrichment. Inhibitors present in feces are a major obstacle to using PCR to detect microorganisms. The QIAamp DNA stool minikit was found to be an efficacious extraction method, as determined by the positive amplification of internal control DNA added to bovine feces before extraction. With nested or seminested multiplex PCR, Campylobacter coli, C. fetus, C. hyointestinalis, and C. jejuni were detected in all fecal samples inoculated at ≈10⁴ CFU g⁻¹, and 50 to 83% of the samples inoculated at ≈10³ CFU g⁻¹ were positive. At ≈10² CFU g⁻¹, C. fetus, C. hyointestinalis, and C. jejuni (17 to 50% of the samples) but not C. coli were detected by PCR. From uninoculated bovine feces, a total of 198 arbitrarily selected isolates of Campylobacter were recovered on four commonly used isolation media incubated at three temperatures. The most frequently isolated taxa were C. jejuni (152 isolates) and C. lanienae (42 isolates), but isolates of C. fetus subsp. fetus, Arcobacter butzleri, and A. skirrowii also were recovered (≤2 isolates per taxon). Considerable variability was observed in the frequency of isolation of campylobacters among the four media and three incubation temperatures tested. With genus-specific primers, Campylobacter DNA was detected in 75% of the fecal samples, representing an 8% increase in sensitivity relative to that obtained with microbiological isolation across the four media and three incubation temperatures tested. With nested primers, C. jejuni and C. lanienae were detected in 25 and 67% of the samples, respectively. In no instance was DNA from either C. coli, C. fetus, or C. hyointestinalis detected in uninoculated bovine feces. PCR was more sensitive than isolation on microbiological media for detecting C. lanienae (17%) but not C. jejuni. Campylobacters are a diverse and fastidious group of bacteria, and the development of direct PCR not only will increase the understanding of Campylobacter species diversity and their frequency of occurrence in feces but also will enhance the knowledge of their role in the gastrointestinal tract of livestock and of the factors that influence shedding.     

Predominant Campylobacter jejuni Sequence Types Persist in Finnish Chicken Production

Consumption and handling of chicken meat are well-known risk factors for acquiring campylobacteriosis. This study aimed to describe the Campylobacter jejuni population in Finnish chickens and to investigate the distribution of C. jejuni genotypes on Finnish chicken farms over a period of several years. We included 89.8% of the total C. jejuni population recovered in Finnish poultry during 2004, 2006, 2007, 2008, and 2012 and used multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) to characterize the 380 isolates. The typing data was combined with isolate information on collection-time and farm of origin. The C. jejuni prevalence in chicken slaughter batches was low (mean 3.0%, CI_95% [1.8%, 4.2%]), and approximately a quarter of Finnish chicken farms delivered at least one positive chicken batch yearly. In general, the C. jejuni population was diverse as represented by a total of 63 sequence types (ST), but certain predominant MLST lineages were identified. ST-45 clonal complex (CC) accounted for 53% of the isolates while ST-21 CC and ST-677 CC covered 11% and 9% of the isolates, respectively. Less than half of the Campylobacter positive farms (40.3%) delivered C. jejuni-contaminated batches in multiple years, but the genotypes (ST and PFGE types) generally varied from year to year. Therefore, no evidence for a persistent C. jejuni source for the colonization of Finnish chickens emerged. Finnish chicken farms are infrequently contaminated with C. jejuni compared to other European Union (EU) countries, making Finland a valuable model for further epidemiological studies of the C. jejuni in poultry flocks.     

Antimicrobial Susceptibility Profiles and Molecular Typing of Campylobacter jejuni and Campylobacter coli Isolates from Ducks in South Korea

Campylobacter is a food-borne zoonotic pathogen that causes human gastroenteritis worldwide. Campylobacter bacteria are commensal in the intestines of many food production animals, including ducks and chickens. The objective of the study was to determine the prevalence of Campylobacter species in domestic ducks, and the agar dilution method was used to determine resistance of the isolates to eight antibiotics. In addition, multilocus sequence typing (MLST) was performed to determine the sequence types (STs) of selected Campylobacter isolates. Between May and September 2012, 58 duck farms were analyzed, and 56 (96.6%) were positive for Campylobacter. Among the isolates, 82.1% were Campylobacter jejuni, 16.1% were C. coli, and one was unidentified by PCR. Of the 46 C. jejuni isolates, 87.0%, 10.9%, and 21.7% were resistant to ciprofloxacin, erythromycin, and azithromycin, respectively. Among the C. coli isolates, all 9 strains were resistant to ampicillin, and 77.8% and 33.3% were resistant to ciprofloxacin and azithromycin, respectively. The majority of the Campylobacter isolates were classified as multidrug resistant. Twenty-eight STs were identified, including 20 STs for C. jejuni and 8 STs for C. coli. The most common clonal complexes in C. jejuni were the ST-21 complex and the ST-45 complex, while the ST-828 complex predominated in C. coli. The majority of isolates were of STs noted in ducks and humans from earlier studies, along with seven STs previously associated only with human disease. These STs overlapped between duck and human isolates, indicating that Campylobacter isolates from ducks should be considered potential sources of human infection.     

Detection of Cytolethal Distending Toxin Activity and cdt Genes in Campylobacter spp. Isolated from Chicken Carcasses

This study was designed to determine whether isolates from chicken carcasses, the primary source of Campylobacter jejuni and Campylobacter coli in human infections, commonly carry the cdt genes and also whether active cytolethal distending toxin (CDT) is produced by these isolates. Campylobacter spp. were isolated from all 91 fresh chicken carcasses purchased from local supermarkets. Campylobacter spp. were identified on the basis of both biochemical and PCR tests. Of the 105 isolates, 70 (67%) were identified as C. jejuni, and 35 (33%) were identified as C. coli. PCR tests amplified portions of the cdt genes from all 105 isolates. Restriction analysis of PCR products indicated that there appeared to be species-specific differences between the C. jejuni and C. coli cdt genes, but that the restriction patterns of the cdt genes within strains of the same species were almost invariant. Quantitation of active CDT levels produced by the isolates indicated that all C. jejuni strains except four (94%) had mean CDT titers greater than 100. Only one C. jejuni strain appeared to produce no active CDT. C. coli isolates produced little or no toxin. These results confirm the high rate of Campylobacter sp. contamination of fresh chicken carcasses and indicate that cdt genes may be universally present in C. jejuni and C. coli isolates from chicken carcasses.     

Production of a Monoclonal Antibody Specific for the Major Outer Membrane Protein of Campylobacter jejuni and Characterization of the Epitope

Campylobacter species are important enteric pathogens causing disease in humans and animals. There is a lack of a good immunological test that can be used routinely to separate Campylobacter jejuni from other Campylobacter species. We produced monoclonal antibodies (MAbs) directed against the major outer membrane protein (MOMP) of C. jejuni using recombinant MOMP as the antigen. One MAb, designated MAb5C4 and of the immunoglobulin G1 isotype, was found to be potentially specific for C. jejuni. Dot blots demonstrated that MAb5C4 reacted with all 29 isolates of C. jejuni tested but did not react with 2 C. jejuni isolates, 26 other Campylobacter spp. isolates, and 19 non-Campylobacter isolates. Western blotting showed that MAb5C4 bound to a single protein band approximately 43 kDa in size, corresponding to the expected size of C. jejuni MOMP. The detection limit of MAb5C4 in a dot blot assay was determined to be about 5 × 10³ bacteria. The epitope on the MOMP was mapped to a region six amino acids in length with the sequence ²¹⁶GGQFNP²²¹, which is 97% conserved among C. jejuni strains but divergent in other Campylobacter spp.; a GenBank search indicated that 95% of C. jejuni isolates will be able to be detected from non-Campylobacter spp. based on the highly specific and conserved region of the GGQFNP polypeptide. The epitope is predicted to be located in a region that is exposed to the periplasm. MAb5C4 is a potentially specific and sensitive MAb that can be used for the specific detection and identification of C. jejuni.     

Campylobacter insulaenigrae Isolates from Northern Elephant Seals (Mirounga angustirostris) in California

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42°C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.     

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

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).Infection with Campylobacter spp. continues to be the leading cause of human infectious intestinal disease in the United Kingdom and has a significant economic impact (39). Consequently, there is a continuing effort to identify effective control methods. The majority of human infections (∼90%) are caused by Campylobacter jejuni subsp. jejuni (46). Other Campylobacter species, including Campylobacter coli and Campylobacter lari, can also cause enteritis in humans, but their prevalence is lower. Most C. jejuni infections are believed to result from consumption of contaminated food, including poultry meat (27, 40), red meat (52), and milk (13), which is thought to be contaminated primarily by feces. It is well established that most livestock species, including poultry, ruminants, and pigs, carry C. jejuni asymptomatically (27), making control at the farm level difficult. However, the epidemiology of C. jejuni cannot be explained solely by food-borne exposure; C. jejuni has also been isolated from a range of environmental samples, including samples of soil, water, sand, and the feces of a number of wildlife species, including wild birds (1-3). However, the role that non-food-borne exposure plays in the epidemiology of C. jejuni is currently not well defined.High prevalences of Campylobacter species infections have been found in a wide range of wild bird species, although there is great variation between taxa (2, 4, 7, 16, 35, 47, 48). Given their ability to fly long distances and their ubiquity, wild birds have the potential to play an important role in the epidemiology and evolution of Campylobacter spp. However, whether wild birds are a source of infection for humans or domestic livestock or are mainly recipients of domestic animal strains or, indeed, whether separate cycles of infection occur remain unknown. These questions remain unanswered in part because investigations of the epidemiology of Campylobacter spp. have been complicated by their high inter- and intraspecies genetic diversity (6).The methods that have been routinely used to characterize Campylobacter isolates are restricted due to genomic instability in Campylobacter populations (10, 38, 45). Multilocus sequence typing (MLST) is a method that has the advantage of being objective since it is sequence based, which allows comparison of isolates from different laboratories and accurate determination of relationships between isolates from diverse sources (11). MLST studies of C. jejuni in farm animals and the environment, including wildlife, suggest that some strains may be associated with particular host groups (6, 10, 15, 30). However, in the same studies other strains were found to occur in several host species or habitats. Few studies have investigated the molecular epidemiology of Campylobacter infection in wild bird populations using MLST, and because only a relatively small number of isolates from wild birds have been characterized by MLST, conclusions have not been drawn yet about how wild bird isolates fit into the overall phylogenetic scheme or whether wild birds act as reservoirs, amplifiers, or merely indicators of infection of domestic animals with zoonotic genotypes.In the current study a large cross-sectional survey of wild bird populations in northern England was undertaken to investigate the epidemiology of Campylobacter infection. Previous studies that have focused on the epidemiology of Campylobacter spp. solely in wild birds have investigated either a narrow range of taxonomic groups (2, 5, 17, 23, 29, 33, 43, 50) or wild birds from a limited range of habitats (18, 25, 48). Studies that have investigated a broad range of wild bird species have used Campylobacter characterization techniques that do not allow conclusions about possible host associations to be drawn or comparison of the genetic diversity of isolates between studies (21, 25, 34, 47, 53). Therefore, the aims of this study were (i) to determine the host range and prevalence of Campylobacter spp. in a wild bird population and (ii) through molecular characterization of isolates to determine whether wild birds were a likely source of infection in humans or domestic livestock and whether separate cycles of infection with host-adapted strains of Campylobacter spp. were maintained in the wild bird population.     

Prevalence and Genetic Diversity of Campylobacter spp. in Environmental Water Samples from a 100-Square-Kilometer Predominantly Dairy Farming Area

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.     

Comparison of PCR Binary Typing (P-BIT), a New Approach to Epidemiological Subtyping of Campylobacter jejuni,with Serotyping,Pulsed-Field Gel Electrophoresis,and Multilocus Sequence Typing Methods

To overcome some of the deficiencies with current molecular typing schema for Campylobacter spp., we developed a prototype PCR binary typing (P-BIT) approach. We investigated the distribution of 68 gene targets in 58 Campylobacter jejuni strains, one Campylobacter lari strain, and two Campylobacter coli strains for this purpose. Gene targets were selected on the basis of distribution in multiple genomes or plasmids, and known or putative status as an epidemicity factor. Strains were examined with Penner serotyping, pulsed-field gel electrophoresis (PFGE; using SmaI and KpnI enzymes), and multilocus sequence typing (MLST) approaches for comparison. P-BIT provided 100% typeability for strains and gave a diversity index of 98.5%, compared with 97.0% for SmaI PFGE, 99.4% for KpnI PFGE, 96.1% for MLST, and 92.8% for serotyping. Numerical analysis of the P-BIT data clearly distinguished strains of the three Campylobacter species examined and correlated somewhat with MLST clonal complex assignations and with previous classifications of “high” and “low” risk. We identified 18 gene targets that conferred the same level of discrimination as the 68 initially examined. We conclude that P-BIT is a useful approach for subtyping, offering advantages of speed, cost, and potential for strain risk ranking unavailable from current molecular typing schema for Campylobacter spp.Campylobacter species, particularly C. jejuni subsp. jejuni (hereafter C. jejuni), represent the most commonly reported bacterial cause of gastroenteritis in humans in the developed world (47), with New Zealand having one of the highest rates of infection (55). The sheer scale of infection makes concerted epidemiological studies difficult, as does the extremely wide distribution of the organism, found in all major avian and mammalian food animals, their products, and indeed environments. Moreover, many Campylobacter spp. are susceptible to spontaneous genetic change through a variety of mechanisms that can result in conflicting data for genetic typing methods aiming to establish a molecular epidemiological link between strains (reviewed by On and colleagues [47]).The poor discrimination of phenotypic typing methods led to intense developments in molecular epidemiological tools for more accurate data. Although a wide range of genotypic methods have been described (47), two methods are now more commonly used by laboratories worldwide. The availability of standardized protocols for macrorestriction profiling with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) have facilitated major contributions to our understanding of the epidemiology of these bacteria. Nonetheless, issues remain, notably relating to the speed, cost, and ease of data analysis from these methods. Furthermore, although MLST has proven useful in evaluating the original host of a given strain, no current methods provide information on the relative risk to human health from individual strains. Various studies, including those identifying stable clones found in humans and various animals as well as strain types only in a particular animal host (5, 13, 38, 48, 61), and whole-genome microarray-based comparisons revealing a correlation between genome content and stress survival (46) indicate that not all strains are of equal risk to humans.In this study, we designed a range of specific PCR assays and investigated the distribution of 68 genes associated with epidemicity factors in C. jejuni, to establish the basis of a novel PCR binary typing (P-BIT) system that is inexpensive, rapid, and highly portable. We compared our data with MLST and PFGE (using restriction enzymes SmaI and KpnI) results for the same isolates of C. jejuni (n = 58), C. coli (n = 2), and C. lari (n = 1).     

Differentiation of Campylobacter Populations as Demonstrated by Flagellin Short Variable Region Sequences

The DNA sequence of the flaA short variable region (SVR) was used to analyze a random population of Campylobacter isolates to investigate the weakly clonal population structure of members of the genus. The SVR sequence from 197 strains of C. jejuni and C. coli isolated from humans, bovine, swine, and chickens identified a group of 43 strains containing disparate short variable region sequences compared to the rest of the population. This group contains both C. jejuni and C. coli strains but disproportionately consisted of bovine isolates. Relative synonymous codon usage analysis of the sequences identified two groups: one group typified C. jejuni, and the second group was characteristic for C. coli and the disparate alleles were not clustered. The data show that there is significant differentiation of Campylobacter populations according to the source of the isolate even without considering the disparate isolates. Even though there is significant differentiation of chicken and bovine isolates, the bovine isolates did not show any difference in ability to colonize chickens. It is possible that disparate sequences were obtained through the lateral transfer of DNA from Campylobacter species other than C. jejuni and C. coli. It is evident that recombination within the flaA SVR occurs rapidly. However, the rate of migration between populations appears to limit the distribution of sequences and results in a weakly clonal population structure.     

Evaluation of novel agars for the enumeration of Campylobacter spp. in poultry retail samples

The purpose of this study was to examine the performance of novel agars for the identification and enumeration of Campylobacter species. The analytical sensitivity and specificity of Campylobacter Selective agar (CASA), Brilliance CampyCount agar (BCCA) and CampyFoodIDagar (CFA) for 84 Campylobacter spp. isolates and 50 non-Campylobacter spp. isolates from 37 distinct genera were of 100% sensitivity, with a 98% specificity for BCCA and CFA, and a 100% specificity for CASA. The application of these selective agars for Campylobacter spp. enumeration in comparison to the conventional agars, modified charcoal cefoperazonedeoxycholate agar (mCCDA) and Campy-Cefex (CCA) was examined using Campylobacter jejuni and Campylobacter coli inoculated samples. From C. jejuni inoculated samples, recovery on BCCA was significantly greater than other media (p < 0.05). Recovery on CASA was not significantly different from mCCDA and CCA (p > 0.05). With C. coli inoculated samples, recovery was significantly greater on BCCA and CASA than with other media (p < 0.05). The recovery of both C. jejuni and C. coli from inoculated samples with CFA was significantly less than with other media (P < 0.05). CASA was able to effectively inhibit and differentiate Campylobacter spp. from background microflora while false positive organisms occurred with BCCA and CFA. An examination of 483 randomly selected suspect Campylobacter colonies from naturally contaminated samples demonstrated a colony confirmation rate for CCA, CFA, BCCA, mCCDA, and CASA, of 84%, 87%, 88%, 90%, and 100%, respectively. The media evaluated present an alternative to conventional selective agars for the identification and enumeration of thermotolerant Campylobacter spp. from samples of poultry origin through the farm to fork continuum.     

Lack of Evidence for Vertical Transmission of Campylobacter spp. in Chickens

Campylobacter jejuni is a major cause of bacterial food-borne infection in the industrial world. There is evidence that C. jejuni is present in eggs and hatchery fluff, opening the possibility for vertical transmission from hens to progeny. Poultry operations in Iceland provide an excellent opportunity to study this possibility, since breeding flocks are established solely from eggs imported from grandparent flocks in Sweden. This leaves limited opportunity for grandparents and their progeny to share isolates through horizontal transmission. While Campylobacter was not detected in all grandparent flocks, 13 of the 16 egg import lots consisted of eggs gathered from one or more Campylobacter-positive grandparent flocks. No evidence of Campylobacter was found by PCR in any of the 10 relevant quarantine hatchery fluff samples examined, and no Campylobacter was isolated from the parent birds through 8 weeks, while they were still in quarantine rearing facilities. After the birds were moved to less biosecure rearing facilities, Campylobacter was isolated, and 29 alleles were observed among the 224 isolates studied. While three alleles were found in both Sweden and Iceland, in no case was the same allele found both in a particular grandparent flock and in its progeny. We could find no evidence for vertical transmission of Campylobacter to the approximately 60,000 progeny parent breeders that were hatched from eggs coming from Campylobacter-positive grandparent flocks. If vertical transmission is occurring, it is not a significant source for the contamination of chicken flocks with Campylobacter spp.     

Effect of Incubation Temperature on the Detection of Thermophilic Campylobacter Species from Freshwater Beaches,Nearby Wastewater Effluents,and Bird Fecal Droppings

This large-scale study compared incubation temperatures (37°C versus 42°C) to study the detection of thermophilic Campylobacter species, including Campylobacter jejuni, C. coli, and C. lari, in various surface water samples and bird fecal droppings around Hamilton Harbor, Lake Ontario. The putative culture isolates obtained from incubation temperatures of 37 and 42°C were confirmed by Campylobacter genus- and species-specific triplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer (ITS) region. A total of 759 water, wastewater, and bird fecal dropping samples were tested. Positive amplification reactions for the genus Campylobacter were found for 454 (60%) samples incubated at 37°C, compared to 258 (34%) samples incubated at 42°C. C. jejuni (16%) and C. lari (12%) were detected significantly more frequently at the 42°C incubation temperature than at 37°C (8% and 5%, respectively). In contrast, significantly higher rates of C. coli (14%) and other Campylobacter spp. (36%) were detected at the 37°C incubation temperature than at 42°C (8% and 7%, respectively). These results were consistent across surface water, wastewater, and bird fecal dropping samples. At times, Campylobacter spp. were recovered and detected at 37°C (3% for C. jejuni, 10% for C. coli, and 3% for C. lari) when the same samples incubated at 42°C were negative. A significantly higher rate of other Campylobacter spp. was detected only at 37°C (32%) than only at 42°C (3%). These results indicate that incubation temperature can significantly influence the culturability and detection of thermophilic and other fastidious Campylobacter spp. and that a comprehensive characterization of the Campylobacter spp. in surface water, wastewaters, or bird fecal droppings will require incubation at both 37 and 42°C.