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.     

Multilocus Sequence Typing Confirms Wild Birds as the Source of a Campylobacter Outbreak Associated with the Consumption of Raw Peas

From August to September 2008, the Centers for Disease Control and Prevention (CDC) assisted the Alaska Division of Public Health with an outbreak investigation of campylobacteriosis occurring among the residents of Southcentral Alaska. During the investigation, pulsed-field gel electrophoresis (PFGE) of Campylobacter jejuni isolates from human, raw pea, and wild bird fecal samples confirmed the epidemiologic link between illness and the consumption of raw peas contaminated by sandhill cranes for 15 of 43 epidemiologically linked human isolates. However, an association between the remaining epidemiologically linked human infections and the pea and wild bird isolates was not established. To better understand the molecular epidemiology of the outbreak, C. jejuni isolates (n = 130; 59 from humans, 40 from peas, and 31 from wild birds) were further characterized by multilocus sequence typing (MLST). Here we present the molecular evidence to demonstrate the association of many more human C. jejuni infections associated with the outbreak with raw peas and wild bird feces. Among all sequence types (STs) identified, 26 of 39 (67%) were novel and exclusive to the outbreak. Five clusters of overlapping STs (n = 32 isolates; 17 from humans, 2 from peas, and 13 from wild birds) were identified. In particular, cluster E (n = 7 isolates; ST-5049) consisted of isolates from humans, peas, and wild birds. Novel STs clustered closely with isolates typically associated with wild birds and the environment but distinct from lineages commonly seen in human infections. Novel STs and alleles recovered from human outbreak isolates allowed additional infections caused by these rare genotypes to be attributed to the contaminated raw peas.     

Identification and characterization of insect-specific proteins by genome data analysis

Background

Previous studies have sought to identify a link between the distribution of variable genes amongst isolates of Campylobacter jejuni and particular host preferences. The genomic sequence data available currently was obtained using only isolates from human or chicken hosts. In order to identify variable genes present in isolates from alternative host species, five subtractions between C. jejuni isolates from different sources (rabbit, cattle, wild bird) were carried out, designed to assess genomic variability within and between common multilocus sequence type (MLST) clonal complexes (ST-21, ST-42, ST-45 and ST-61).

Results

The vast majority (97%) of the 195 subtracted sequences identified had a best BLASTX match with a Campylobacter protein. However, there was considerable variation within and between the four clonal complexes included in the subtractions. The distributions of eight variable sequences, including four with putative roles in the use of alternative terminal electron acceptors, amongst a panel of C. jejuni isolates representing diverse sources and STs, were determined.

Conclusion

There was a clear correlation between clonal complex and the distribution of the metabolic genes. In contrast, there was no evidence to support the hypothesis that the distribution of such genes may be related to host preference. The other variable genes studied were also generally distributed according to MLST type. Thus, we found little evidence for widespread horizontal gene transfer between clonal complexes involving these genes.     

Epidemiological relationships of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> strains isolated from humans and chickens in South Korea

Thirty-nine human isolates of Campylobacter jejuni obtained from a national university hospital during 2007–2010 and 38 chicken isolates of C. jejuni were collected from poultry farms during 2009–2010 in South Korea were used in this study. Campylobacter genomic species and virulence-associated genes were identified by PCR. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed to compare their genetic relationships. All isolates were highly resistant to ciprofloxacin, nalidixic acid, and tetracycline. Of all isolates tested, over 94% contained seven virulence associated genes (flaA, cadF, racR, dnaJ, cdtA, cdtB, and cdtC). All isolates were classified into 39 types by PFGE clustering with 90% similarity. Some chicken isolates were incorporated into some PFGE types of human isolates. MLST analysis for the 39 human isolates and 38 chicken isolates resulted in 14 and 23 sequence types (STs), respectively, of which 10 STs were new. STs overlapped in both chicken and human isolates included ST-21, ST-48, ST-50, ST-51, and ST-354, of which ST-21 was the predominant ST in both human and chicken isolates. Through combined analysis of PFGE types and STs, three chicken isolates were clonally related to the three human isolates associated with food poisoning (VII-ST-48, XXII-ST-354, and XXVIII-ST-51). They were derived from geographically same or distinct districts. Remarkably, clonal spread of food poisoning pathogens between animals and humans was confirmed by population genetic analysis. Consequently, contamination of campylobacters with quinolone resistance and potential virulence genes in poultry production and consumption may increase the risk of infections in humans.     

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.     

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.     

Antimicrobial Susceptibility Profiles and Strain Type Diversity of Campylobacter jejuni Isolates from Turkeys in Eastern North Carolina

Campylobacter jejuni is one of the most common bacterial causes of human gastroenteritis, and recent findings suggest that turkeys are an important reservoir for this organism. In this study, 80 C. jejuni isolates from eastern North Carolina were characterized for resistance to nine antimicrobials, and strain types were determined by fla typing, pulsed-field gel electrophoresis (PFGE) with SmaI and KpnI, and (for 41 isolates) multilocus sequence typing (MLST). PFGE analysis suggested that many of the isolates (37/40 [ca. 93%]) in a major genomic cluster had DNA that was partially methylated at SmaI sites. Furthermore, 12/40 (30%) of the isolates in this cluster were completely resistant to digestion by KpnI, suggesting methylation at KpnI sites. MLST of 41 isolates identified 10 sequence types (STs), of which 4 were new. Three STs (ST-1839, ST-2132 and the new ST-2934) were predominant and were detected among isolates from different farms. The majority of the isolates (74%) were resistant to three or more antimicrobials, and resistance to ciprofloxacin was common (64%), whereas resistance to the other drug of choice for treatment of human campylobacteriosis, erythromycin, was never encountered. Most (33/34) of the kanamycin-resistant isolates were also resistant to tetracycline; however, only ca. 50% of the tetracycline-resistant isolates were also kanamycin resistant. Isolates with certain antimicrobial resistance profiles had identical or closely related strain types. Overall, the findings suggest dissemination of certain clonal groups of C. jejuni isolates in the turkey production industry of this region.     

Genetic Diversity of Campylobacter jejuni Isolates from Farm Animals and the Farm Environment

The genetic diversity of Campylobacter jejuni isolates from farm animals and their environment was investigated by multilocus sequence typing (MLST). A total of 30 genotypes, defined by allelic profiles (assigned to sequence types [STs]), were found in 112 C. jejuni isolates originating in poultry, cattle, sheep, starlings, and slurry. All but two of these genotypes belonged to one of nine C. jejuni clonal complexes previously identified in isolates from human disease and retail food samples and one clonal complex previously associated with an environmental source. There was some evidence for the association of certain clonal complexes with particular farm animals: isolates belonging to the ST-45 complex predominated among poultry isolates but were absent among sheep isolates, while isolates belonging to the ST-61 and ST-42 complexes were predominant among sheep isolates but were absent from the poultry isolates. In contrast, ST-21 complex isolates were distributed among the different isolation sources. Comparison with MLST data from 91 human disease isolates showed small but significant genetic differentiation between the farm and human isolates; however, representatives of six clonal complexes were found in both samples. These data demonstrate that MLST and the clonal complex model can be used to identify and compare the genotypes of C. jejuni isolates from farm animals and the environment with those from retail food and human disease.     

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.     

Source Attribution of Human Campylobacter Isolates by MLST and Fla-Typing and Association of Genotypes with Quinolone Resistance

Campylobacteriosis is the most frequent zoonosis in developed countries and various domestic animals can function as reservoir for the main pathogens Campylobacter jejuni and Campylobacter coli. In the present study we compared population structures of 730 C. jejuni and C. coli from human cases, 610 chicken, 159 dog, 360 pig and 23 cattle isolates collected between 2001 and 2012 in Switzerland. All isolates had been typed with multi locus sequence typing (MLST) and flaB-typing and their genotypic resistance to quinolones was determined. We used complementary approaches by testing for differences between isolates from different hosts with the proportion similarity as well as the fixation index and by attributing the source of the human isolates with Bayesian assignment using the software STRUCTURE. Analyses were done with MLST and flaB data in parallel and both typing methods were tested for associations of genotypes with quinolone resistance. Results obtained with MLST and flaB data corresponded remarkably well, both indicating chickens as the main source for human infection for both Campylobacter species. Based on MLST, 70.9% of the human cases were attributed to chickens, 19.3% to cattle, 8.6% to dogs and 1.2% to pigs. Furthermore we found a host independent association between sequence type (ST) and quinolone resistance. The most notable were ST-45, all isolates of which were susceptible, while for ST-464 all were resistant.     

Genetic Diversity and Quinolone Resistance in Campylobacter jejuni Isolates from Poultry in Senegal

We used the multilocus sequence typing (MLST) method to evaluate the genetic diversity of 46 Campylobacter jejuni isolates from chickens and to determine the link between quinolone resistance and sequence type (ST). There were a total of 16 ST genotypes, and the majority of them belonged to seven clonal complexes previously identified by using isolates from human disease. The ST-353 complex was the most common complex, whereas the ST-21, ST-42, ST-52, and ST-257 complexes were less well represented. The resistance phenotype varied for each ST, and the Thr-86-Ile substitution in the GyrA protein was the predominant mechanism of resistance to quinolone. Nine of the 14 isolates having the Thr-86-Ile substitution belonged to the ST-353 complex. MLST showed that the emergence of quinolone resistance is not related to the diffusion of a unique clone and that there is no link between ST genotype and quinolone resistance. Based on silent mutations, different variants of the gyrA gene were shown to exist for the same ST. These data provide useful information for understanding the epidemiology of C. jejuni in Senegal.     

Comparative Genotyping of Campylobacter jejuni Strains from Patients with Guillain-Barré Syndrome in Bangladesh

Background

Campylobacter jejuni is a common cause of acute gastroenteritis and is associated with post-infectious neuropathies such as the Guillain-Barré syndrome (GBS) and the Miller Fisher syndrome (MFS). We here present comparative genotyping of 49 C. jejuni strains from Bangladesh that were recovered from patients with enteritis or GBS. All strains were serotyped and analyzed by lipo-oligosaccharide (LOS) genotyping, amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE).

Methodology/Principal Findings

C. jejuni HS:23 was a predominant serotype among GBS patients (50%), and no specific serotype was significantly associated with GBS compared to enteritis. PCR screening showed that 38/49 (78%) of strains could be assigned to LOS classes A, B, C, or E. The class A locus (4/7 vs 3/39; p<0.01) was significantly associated in the GBS-related strains as compared to enteritis strains. All GBS/oculomotor related strains contained the class B locus; which was also detected in 46% of control strains. Overlapping clonal groups were defined by MLST, AFLP and PFGE for strains from patients with gastroenteritis and GBS. MLST defined 22 sequence types (STs) and 7 clonal complexes including 7 STs not previously identified (ST-3742, ST-3741, ST-3743, ST-3748, ST-3968, ST-3969 and ST-3970). C. jejuni HS:23 strains from patients with GBS or enteritis were clonal and all strains belonged to ST-403 complex. Concordance between LOS class B and ST-403 complex was revealed. AFLP defined 25 different types at 90% similarity. The predominant AFLP type AF-20 coincided with the C. jejuni HS:23 and ST-403 complex.

Conclusion/Significance

LOS genotyping, MLST, AFLP and PFGE helped to identify the HS:23 strains from GBS or enteritis patients as clonal. Overall, genotypes exclusive for enteritis or for GBS-related strains were not obtained although LOS class A was significantly associated with GBS strains. Particularly, the presence of a clonal and putative neuropathogenic C. jejuni HS:23 serotype may contribute to the high prevalence of C. jejuni related GBS in Bangladesh.     

Campylobacter spp. Recovered from the Upper Oconee River Watershed, Georgia in a 4-Year Study

Waterways should be considered in the migration routes of Campylobacter, and the genus has been isolated from several water sources. Inferences on migration routes can be made from tracking genetic types in populations found in specific habitats and testing how they are linked to other types. Water samples were taken over a 4-year period from waterways in the Upper Oconee River Watershed, Georgia, to recover isolates of thermophilic Campylobacter. The isolates were typed by multilocus sequence typing (MLST) and analyzed to determine the overall diversity of Campylobacter in that environment. Forty-seven independent isolates were recovered from 560 samples (8.4 %). Two (~4 %) isolates were Campylobacter coli, three (~6 %) isolates were putatively identified as Campylobacter lari, and the remaining 42 (~90 %) were Campylobacter jejuni. The C. jejuni and C. coli isolates were typed by the Oxford MLST scheme. Thirty sequence types (STs) were identified including 13 STs that were not found before in the MLST database, including 24 novel alleles. Of the 17 previously described STs, 10 have been isolated from humans, 6 from environmental water, and 6 from wild birds (five types from multiple sources). Seven sites had multiple positive samples, and on two occasions, the same ST was isolated at the same site. The most common type was STST61 with four isolates, and the most common clonal complex was CC179 with nine isolates. CC179 has been commonly associated with environmental water. Although some Campylobacter STs that were found in the Oconee River engage in widespread migration, most are tightly associated with or unique to environmental water sources.     

Novel Clonal Complexes with an Unknown Animal Reservoir Dominate Campylobacter jejuni Isolates from River Water in New Zealand

Campylobacter jejuni is widely distributed in the environment, and river water has been shown to carry high levels of the organism. In this study, 244 C. jejuni isolates from three river catchment areas in New Zealand were characterized using multilocus sequence typing. Forty-nine of the 88 sequence types identified were new. The most common sequence types identified were ST-2381 (30 isolates), ST-45 (25 isolates), and ST-1225 (23 isolates). The majority of the sequence types identified in the river water could be attributed to wild bird fecal contamination. Two novel clonal complexes (CC) were identified, namely, CC ST-2381 (11 sequence types, 46 isolates) and CC ST-3640 (6 sequence types, 12 isolates), in which all of the sequence types were new. CC ST-2381 was the largest complex identified among the isolates and was present in two of the three rivers. None of the sequence types associated with the novel complexes has been identified among human isolates. The ST-2381 complex is not related to complexes associated with cattle, sheep, or poultry. The source of the novel complexes has yet to be identified.Contamination of the environment by bacterial pathogens is a significant health concern, as it provides a continuous source of organisms for the infection and reinfection of humans and animals. Enteric pathogens gain entry into the environment through the discharge of sewage into water and via contamination from animal feces (22). Fecal contamination is responsible for the continued presence and spread of a range of pathogenic organisms, including Campylobacter, norovirus, and Escherichia coli O157. Determining the roles of various environmental sources in human enteric disease requires an understanding of the distribution, survival, population structure, and pathogenic potential of the pathogens in the environment.Campylobacter is the most common cause of gastrointestinal illness in the industrialized world (17), imposing significant economic costs on health systems, and is associated with a number of neurological sequelae (32, 33). The majority of human campylobacter infections are caused by Campylobacter jejuni (90%), with Campylobacter coli mostly responsible for the remainder. Although Campylobacter has been isolated from a wide range of animals (41) and birds (47, 48), contaminated poultry and poultry products remain the most significant sources of human infections (10, 38, 50, 51). Campylobacter is a spiral gram-negative organism that grows best under low-oxygen conditions at 42°C. The organism is unable to grow outside an animal host, and survival in the environment is dependent on ambient temperature, oxygen levels, and sunlight.Studies worldwide examining rivers and waterways show that there is significant contamination by Campylobacter, with the sources being sewage outflow, direct fecal deposition, and pasture runoff (12, 22, 34, 37, 39). Similarly, coastal waters and estuaries can be contaminated by either sewage or bird fecal deposition (23, 35). The inability of Campylobacter to grow in the environment and its sensitivity to sunlight are thought to ensure that the organism is eventually purged from the system. However, the high levels of the organism identified in water systems have been highlighted as a risk for human infection.The characterization of campylobacter populations by multilocus sequence typing (MLST) has shown that the organism is weakly clonal and that certain clonal complexes are associated with particular animals (5, 9, 26). Isolates from human cases of infection show a wide variety of sequence types and many clonal complexes. Source attribution studies using MLST have identified poultry as causing approximately 60% of human infections (14, 38, 50). Cattle have been identified as a potential source of infection due to the high level of similarity between bovine and human strains (18, 19). There remains, however, a significant number of infections for which the source is not certain.New Zealand has one of the highest rates of campylobacteriosis in the developed world. This is due to the significant quantity of fresh chicken consumed coupled with high levels of contamination found in poultry products (1, 10, 51, 52). Campylobacter has been isolated from a range of environmental sources within New Zealand, including its rivers and streams (12, 37). Isolation rates for rivers in New Zealand range from 55 to 90%, comparable to results of studies overseas, and show the same seasonal variation as that seen elsewhere in the world (20). Pulsed-field gel electrophoresis (PFGE) analysis identified indistinguishable macrorestriction profiles for cattle, human, and river isolates, suggesting river water as a potential source of infection (8). In this study, C. jejuni isolates from three rivers in New Zealand, two on the South Island and one on the North Island, were characterized using MLST.     

Application of Streptococcus uberis Multilocus Sequence Typing: Analysis of the Population Structure Detected among Environmental and Bovine Isolates from New Zealand and the United Kingdom

We recently developed a multilocus sequence typing (MLST) scheme to differentiate S. uberis isolates and facilitate an understanding of the population biology of this pathogen. The scheme was initially used to study a collection of 160 bovine milk isolates from the United Kingdom and showed that the majority of isolates were from one clonal complex (designated the ST-5 complex). Here we describe the MLST analysis of a collection of New Zealand isolates. These were obtained from diverse sources, including bovine milk, other bovine anatomical sites, and environmental sources. The complete allelic profiles of 253 isolates were determined. The collection was highly diverse and included 131 different sequence types (STs). The New Zealand and United Kingdom populations were distinct, since none of the 131 STs were represented within the previously studied collection of 160 United Kingdom S. uberis isolates. However, seven of the STs were members of the ST-5 clonal complex, the major complex within the United Kingdom collection. Two new clonal complexes were identified: ST-143 and ST-86. All three major complexes were isolated from milk, other bovine sites, and the environment. Carriage of the hasA gene, which is necessary for capsule formation, correlated with clonal complex and isolation from clinical cases of mastitis.     

Molecular Epidemiology of Campylobacter jejuni in a Geographically Isolated Country with a Uniquely Structured Poultry Industry

In New Zealand the number of campylobacteriosis notifications increased markedly between 2000 and 2007. Notably, this country''s poultry supply is different than that of many developed countries as the fresh and frozen poultry available at retail are exclusively of domestic origin. To examine the possible link between human cases and poultry, a sentinel surveillance site was established to study the molecular epidemiology of Campylobacter jejuni over a 3-year period from 2005 to 2008 using multilocus sequence typing. Studies showed that 60.1 to 81.4% of retail poultry carcasses from the major suppliers were contaminated with C. jejuni. Differences were detected in the probability and level of contamination and the relative frequency of genotypes for individual poultry suppliers and humans. Some carcasses were contaminated with isolates belonging to more than one sequence type (ST), and there was evidence of both ubiquitous and supplier-associated strains, an epidemiological pattern not recognized yet in other countries. The common poultry STs were also common in human clinical cases, providing evidence that poultry is a major contributor to human infection. Both internationally rare genotypes, such as ST-3069 and ST-474, and common genotypes, such as ST-45 and ST-48, were identified in this study. The dominant human sequence type in New Zealand, ST-474, was found almost exclusively in isolates from one poultry supplier, which provided evidence that C. jejuni has a distinctive molecular epidemiology in this country. These results may be due in part to New Zealand''s geographical isolation and its uniquely structured poultry industry.Campylobacteriosis is a leading enteric zoonosis in the developed world, and the majority of cases are caused by Campylobacter jejuni (22). Poultry sources are suspected to be the major source of human infection with C. jejuni (22, 43, 49), and this conclusion is supported by high levels of contamination of poultry and the detection of identical C. jejuni genotypes in human cases and poultry samples (25). However, C. jejuni can be isolated from a variety of sources, including ruminants (17) and environmental water (41). Due to the complex epidemiology of this pathogen, there is still uncertainty about the relative contributions of individual pathways to the human disease burden (8). In New Zealand the number of notified campylobacteriosis cases increased markedly in the last decade, peaking in 2006 at a total of 15,873 notified cases (422.4 cases per 100,000 population) (1) and costing the economy an estimated US$32 million annually (42). Although there are a number of factors that may have contributed to this increase, it has been noted that the rise in campylobacteriosis cases coincided with a marked increase in the sale and consumption of fresh poultry between 1992 and 2005, while the sale of frozen poultry remained relatively static (1).New Zealand has one of the highest enteric infectious disease rates in industrialized countries (26), and the high ratio of domestic production animals to humans and the frequent use of rural water supplies in New Zealand have been postulated to be underlying causes (9). In addition, this country''s poultry industry is uniquely structured; it is almost entirely focused on the domestic market, and no raw, fresh or frozen, poultry products are imported because of biosecurity threats. Due to its geographical isolation and tight border controls, New Zealand has remained free of poultry diseases endemic in other countries, such as diseases caused by Salmonella enterica serovar Enteritidis PT4 and S. enterica serovar Typhimurium DT 104, Newcastle disease, and infectious bursal disease.The production of poultry meat in New Zealand is highly integrated; only three companies supply 90% of the chicken meat, which represents 95% of the poultry meat consumed. The remaining 5% of poultry meat consumed includes species such as turkey and duck. The chicken processors own or control most stages of production, processing, and distribution. One of the three dominant companies has one processing plant that distributes nationwide, and one company has multiple plants that tend to be more localized in their distribution, except when they make specialty products, which are distributed nationally. The other companies distribute primarily locally. The broilers are commonly barn raised, not free range, and animal welfare standards require a maximum stocking density of 38 kg (live weight) of broiler chickens per m². There are approximately 160 broiler farms in a number of specific areas of New Zealand. These farms are usually located near the slaughterhouses that they supply.To enable regulators to implement food safety programs to reduce human campylobacteriosis, there has been great interest in understanding the importance and epidemiology of C. jejuni in the New Zealand poultry production system. A sentinel surveillance site was therefore established in the Manawatu region to quantify the contributions of different sources, including poultry suppliers, to the human disease burden and to study the molecular epidemiology of C. jejuni (33, 34). Isolates were typed using multilocus sequence typing (MLST), a method that has major advantages over other methods of genotyping when the long-term epidemiology of a disease is studied. However, other methods may be more appropriate in other settings, such as outbreak investigations (21), where a higher degree of discrimination may be required. MLST offers a large web-based archive of isolates from many sources and countries: the Campylobacter PubMLST database (10). Sequence typing by MLST is now internationally recognized as a valuable approach for national and international epidemiological characterization and source tracking of major pathogenic microorganisms, such as C. jejuni (46, 48).The use of integrated surveillance across human, domestic animal, and wildlife populations has been identified as a key component of strategies aimed at prevention and control of emerging pathogens, particularly when the population dynamics of multihost pathogens are poorly understood (52). At the Manawatu surveillance site, samples from human clinical cases, animal-derived food products, and the environment were gathered in a defined geographical area of New Zealand over a 3-year period (19, 34) and genotyped using MLST (11). The resulting data set contained a total of 969 typed samples, 502 of which were from human cases. The temporal and spatial scale of the data allowed us to obtain a more in-depth understanding of local transmission dynamics compared with the results of previous research (43, 50). The application of novel risk attribution approaches to these data previously identified poultry as the major contributor to the human disease burden, with widely varying contributions from different suppliers (33).In this study we extended the findings of risk attribution and epidemiological studies of human cases (33, 34). Data from the sentinel surveillance site were used to study the epidemiology of C. jejuni for the individual producers that comprise the poultry sector in New Zealand and to better understand the contributions of the producers to the human campylobacteriosis burden. Our study included an investigation of both the probability of contamination and the level of contamination of poultry carcasses and a study of human and poultry MLST sequence types (STs). The resulting data were compared to make inferences about the epidemiology of C. jejuni in the New Zealand poultry industry and to identify determinants for the high number of human cases attributed to this food source.     

Multilocus Sequence Typing for Comparison of Veterinary and Human Isolates of Campylobacter jejuni

Multilocus sequence typing (MLST) has been applied to 266 Campylobacter jejuni isolates, mainly from veterinary sources, including cattle, sheep, poultry, pigs, pets, and the environment, as well as isolates from human cases of campylobacteriosis. The populations of veterinary and human isolates overlap, suggesting that most veterinary sources should be considered reservoirs of pathogenic campylobacters. There were some associations between source and sequence type complex, indicating that host or source adaptation may exist. The pig isolates formed a distinct group by MLST and may well represent a potential pig-adapted clone of C. jejuni. A subset (n = 82) of isolates was reanalyzed with a second MLST scheme which provided a unique set of isolates that had been analyzed at a total of 12 loci. The distribution of isolates among the complexes in each of the two schemes was similar but not identical. In addition to isolates from human outbreaks, one group of isolates that were not epidemiologically linked was also identical at all 12 loci. This group of isolates is believed to represent another stable strain of C. jejuni.     

Risk factors for campylobacteriosis of chicken, ruminant, and environmental origin: a combined case-control and source attribution analysis

Background

Campylobacteriosis contributes strongly to the disease burden of food-borne pathogens. Case-control studies are limited in attributing human infections to the different reservoirs because they can only trace back to the points of exposure, which may not point to the original reservoirs because of cross-contamination. Human Campylobacter infections can be attributed to specific reservoirs by estimating the extent of subtype sharing between strains from humans and reservoirs using multilocus sequence typing (MLST).

Methodology/Principal Findings

We investigated risk factors for human campylobacteriosis caused by Campylobacter strains attributed to different reservoirs. Sequence types (STs) were determined for 696 C. jejuni and 41 C. coli strains from endemic human cases included in a case-control study. The asymmetric island model, a population genetics approach for modeling Campylobacter evolution and transmission, attributed these cases to four putative animal reservoirs (chicken, cattle, sheep, pig) and to the environment (water, sand, wild birds) considered as a proxy for other unidentified reservoirs. Most cases were attributed to chicken (66%) and cattle (21%), identified as the main reservoirs in The Netherlands. Consuming chicken was a risk factor for campylobacteriosis caused by chicken-associated STs, whereas consuming beef and pork were protective. Risk factors for campylobacteriosis caused by ruminant-associated STs were contact with animals, barbecuing in non-urban areas, consumption of tripe, and never/seldom chicken consumption. Consuming game and swimming in a domestic swimming pool during springtime were risk factors for campylobacteriosis caused by environment-associated STs. Infections with chicken- and ruminant-associated STs were only partially explained by food-borne transmission; direct contact and environmental pathways were also important.

Conclusion/Significance

This is the first case-control study in which risk factors for campylobacteriosis are investigated in relation to the attributed reservoirs based on MLST profiles. Combining epidemiological and source attribution data improved campylobacteriosis risk factor identification and characterization, generated hypotheses, and showed that genotype-based source attribution is epidemiologically sensible.     

Longitudinal Study of Campylobacter jejuni Bacteriophages and Their Hosts from Broiler Chickens

A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages. Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn. Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3. The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content. All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE). PFGE and multilocus sequence typing indicated that this C. jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C. jejuni types insensitive to the resident bacteriophages. All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct. These results are significant because this is the first study of an environmental population of C. jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens. The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens. In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance.     

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.