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.     

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.     

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.     

Prevalence in Bulk Tank Milk and Epidemiology of Campylobacter jejuni in Dairy Herds in Northern Italy

Thermotolerant Campylobacter spp. are frequently the cause of human gastroenteritis and have assumed more importance in Italy following the increased consumption of raw milk. Our objectives were to determine the prevalence and genotypes of Campylobacter spp. in dairy herds and to investigate the possible sources of bulk milk contamination. Bulk milk from dairy herds (n = 282) was cultured for Campylobacter spp. and Enterobacteriaceae. At three Campylobacter jejuni-positive farms, bovine feces, pigeon intestines, milk, and water points were also investigated. Isolates were identified by PCR and genotyped using multilocus sequence typing (MLST). C. jejuni was detected in 34 (12%) bulk milk samples. The strains belonged to 14 sequence types, and the most common clonal complexes were CC-21, CC-48, and CC-403. No association was demonstrated between the presence of C. jejuni and high levels of Enterobacteriaceae in bulk milk. At the three farms examined, C. jejuni was isolated from bovine feces (25/82 [30.5%]), pigeon intestines (13/60 [21.7%]), bulk milk (10/24 [41.7%]), and water points (4/16 [25%]). MLST revealed lineages that were common between milk and bovine feces but distinct between cattle and pigeons. In one herd, C. jejuni with the same genotype was isolated repeatedly from bulk milk and a cow with an udder infection. Our results showed a high prevalence of C. jejuni in bulk milk and suggested that udder excretion, in addition to fecal matter, may be a route of bulk milk contamination. MLST analysis indicated that pigeons are probably not relevant for the transmission of C. jejuni to cattle and for milk contamination.     

Operationalising Factors That Explain the Emergence of Infectious Diseases: A Case Study of the Human Campylobacteriosis Epidemic

A framework of general factors for infectious disease emergence was made operational for Campylobacter utilising explanatory variables including time series and risk factor data. These variables were generated using a combination of empirical epidemiology, case-case and case-control studies, time series analysis, and microbial sub-typing (source attribution, diversity, genetic distance) to unravel the changing/emerging aetiology of human campylobacteriosis. The study focused on Scotland between 1990–2012 where there was a 75% increase in reported cases that included >300% increase in the elderly and 50% decrease in young children. During this period there were three phases 1990–2000 a 75% rise and a 20% fall to 2006, followed by a 19% resurgence. The rise coincided with expansions in the poultry industry, consumption of chicken, and a shift from rural to urban cases. The post-2000 fall occurred across all groups apart from the elderly and coincided with a drop of the prevalence of Campylobacter in chicken and a higher proportion of rural cases. The increase in the elderly was associated with uptake of proton pump inhibitors. During the resurgence the increase was predominantly in adults and the elderly, again there was increasing use of PPIs and high prevalences in chicken and ruminants. Cases associated with foreign travel during the study also increased from 9% to a peak of 16% in 2006 before falling to an estimated 10% in 2011, predominantly in adults and older children. During all three periods source attribution, genetic distance, and diversity measurements placed human isolates most similar to those in chickens. A combination of emergence factors generic for infectious diseases were responsible for the Campylobacter epidemic. It was possible to use these to obtain a putative explanation for the changes in human disease and the potential to make an informed view of how incidence rates may change in the future.     

Evidence of land‐sea transfer of the zoonotic pathogen Campylobacter to a wildlife marine sentinel species

Environmental pollution often accompanies the expansion and urbanization of human populations where sewage and wastewaters commonly have an impact on the marine environments. Here, we explored the potential for faecal bacterial pathogens, of anthropic origin, to spread to marine wildlife in coastal areas. The common zoonotic bacterium Campylobacter was isolated from grey seals (Halichoerus grypus), an important sentinel species for environmental pollution, and compared to isolates from wild birds, agricultural sources and clinical samples to characterize possible transmission routes. Campylobacter jejuni was present in half of all grey seal pups sampled (24/50 dead and 46/90 live pups) in the breeding colony on the Isle of May (Scotland), where it was frequently associated with histological evidence of disease. Returning yearling animals (19/19) were negative for C. jejuni suggesting clearance of infection while away from the localized colony infection source. The genomes of 90 isolates from seals were sequenced and characterized using a whole‐genome multilocus sequence typing (MLST) approach and compared to 192 published genomes from multiple sources using population genetic approaches and a probabilistic genetic attribution model to infer the source of infection from MLST data. The strong genotype‐host association has enabled the application of source attribution models in epidemiological studies of human campylobacteriosis, and here assignment analyses consistently grouped seal isolates with those from human clinical samples. These findings are consistent with either a common infection source or direct transmission of human campylobacter to grey seals, raising concerns about the spread of human pathogens to wildlife marine sentinel species in coastal areas.     

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.     

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.     

Influencing Factors and Applicability of the Viability EMA-qPCR for a Detection and Quantification of Campylobacter Cells from Water Samples

In recent years, increasing numbers of human campylobacteriosis cases caused by contaminated water have been reported. As the culture-based detection of Campylobacter is time consuming and can yield false-negative results, the suitability of a quantitative real-time PCR method in combination with an ethidium monoazide pretreatment of samples (EMA-qPCR) for the rapid, quantitative detection of viable Campylobacter cells from water samples was investigated. EMA-qPCR has been shown to be a promising rapid method for the detection of viable Campylobacter spp. from food samples. Application of membrane filtration and centrifugation, two methods frequently used for the isolation of bacteria from water, revealed a mean loss of up to 1.08 log₁₀ cells/ml from spiked samples. Both methods used alone lead to a loss of dead bacteria and accumulation of viable bacteria in the sample as shown by fluorescence microscopy. After filtration of samples, no significant differences could be detected in subsequent qPCR experiments with and without EMA pretreatment compared to culture-based enumeration. High correlations (R² = 0.942 without EMA, R² = 0.893 with EMA) were obtained. After centrifugation of samples, qPCR results overestimated Campylobacter counts, whereas results from both EMA-qPCR and the reference method were comparable. As up to 81.59% of nonviable cells were detected in pond water, EMA-qPCR failed to detect correct quantities of viable cells. However, analyses of spiked tap water samples revealed a high correlation (R² = 0.863) between results from EMA-qPCR and the reference method. After membrane filtration, EMA-qPCR was successfully applied to Campylobacter field isolates, and results indicated an advantage over qPCR by analysing defined mixtures of viable and nonviable cells. In conclusion, EMA-qPCR is a suitable method to detect viable Campylobacter from water samples, but the isolation technique and the type/quality of the water sample impact the results.     

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.     

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.     

A Systematic Review Characterizing On-Farm Sources of Campylobacter spp. for Broiler Chickens

Campylobacter and antimicrobial-resistant Campylobacter are frequently isolated from broiler chickens worldwide. In Canada, campylobacteriosis is the third leading cause of enteric disease and the regional emergence of ciprofloxacin-resistant Campylobacter in broiler chickens has raised a public health concern. This study aimed to identify, critically appraise, and synthesize literature on sources of Campylobacter in broilers at the farm level using systematic review methodology. Literature searches were conducted in January 2012 and included electronic searches in four bibliographic databases. Relevant studies in French or English (n = 95) conducted worldwide in any year and all study designs were included. Risk of Bias and GRADE criteria endorsed by the Cochrane collaboration was used to assess the internal validity of the study and overall confidence in the meta-analysis. The categories for on-farm sources were: broiler breeders/vertical transfer (number of studies = 32), animals (n = 57), humans (n = 26), environment (n = 54), and water (n = 63). Only three studies examined the antimicrobial resistance profiles of Campylobacter from these on-farm sources. Subgroups of data by source and outcome were analyzed using random effect meta-analysis. The highest risk for contaminating a new flock appears to be a contaminated barn environment due to insufficient cleaning and disinfection, insufficient downtime, and the presence of an adjacent broiler flock. Effective biosecurity enhancements from physical barriers to restricting human movement on the farm are recommended for consideration to enhance local on-farm food safety programs. Improved sampling procedures and standardized laboratory testing are needed for comparability across studies. Knowledge gaps that should be addressed include farm-level drug use and antimicrobial resistance information, further evaluation of the potential for vertical transfer, and improved genotyping methods to strengthen our understanding of Campylobacter epidemiology in broilers at the farm-level. This systematic review emphasizes the importance of improved industry-level and on-farm risk management strategies to reduce pre-harvest Campylobacter in broilers.     

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.     

Correlation between Genotypic Diversity,Lipooligosaccharide Gene Locus Class Variation,and Caco-2 Cell Invasion Potential of Campylobacter jejuni Isolates from Chicken Meat and Humans: Contribution to Virulotyping

Significant interest in studying the lipooligosaccharide (LOS) of Campylobacter jejuni has stemmed from its potential role in postinfection paralytic disorders. In this study we present the results of PCR screening of five LOS locus classes (A, B, C, D, and E) for a collection of 116 C. jejuni isolates from chicken meat (n = 76) and sporadic human cases of diarrhea (n = 40). We correlated LOS classes with clonal complexes (CC) assigned by multilocus sequence typing (MLST). Finally, we evaluated the invasion potential of a panel of 52 of these C. jejuni isolates for Caco-2 cells. PCR screening showed that 87.1% (101/116) of isolates could be assigned to LOS class A, B, C, D, or E. Concordance between LOS classes and certain MLST CC was revealed. The majority (85.7% [24/28]) of C. jejuni isolates grouped in CC-21 were shown to express LOS locus class C. The invasion potential of C. jejuni isolates possessing sialylated LOS (n = 29; classes A, B, and C) for Caco-2 cells was significantly higher (P < 0.0001) than that of C. jejuni isolates with nonsialylated LOS (n = 23; classes D and E). There was no significant difference in invasiveness between chicken meat and human isolates. However, C. jejuni isolates assigned to CC-206 (correlated with LOS class B) or CC-21 (correlated with LOS class C) showed statistically significantly higher levels of invasion than isolates from other CC. Correlation between LOS classes and CC was further confirmed by pulsed-field gel electrophoresis. The present study reveals a correlation between genotypic diversity and LOS locus classes of C. jejuni. We showed that simple PCR screening for C. jejuni LOS classes could reliably predict certain MLST CC and add to the interpretation of molecular-typing results. Our study corroborates that sialylation of LOS is advantageous for C. jejuni fitness and virulence in different hosts. The modulation of cell surface carbohydrate structure could enhance the ability of C. jejuni to adapt to or survive in a host.Campylobacter jejuni is an important human enteric pathogen worldwide (3, 7, 26). Infected humans exhibit a range of clinical spectra, from mild, watery diarrhea to severe inflammatory diarrhea (28). Factors influencing the virulence of C. jejuni include motility, chemotaxis, the ability to adhere to and invade intestinal cells, intracellular survival, and toxin production (28, 30, 52). Besides its role in human enteric illnesses, C. jejuni is a predominant infectious trigger of acute postinfectious neuropathies, such as Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) (1). Significant interest in studying the structure and biosynthesis of the core lipooligosaccharide (LOS) of C. jejuni has resulted from its potential role in these paralytic disorders. Many studies have now provided convincing evidence that molecular mimicry between C. jejuni LOS and gangliosides in human peripheral nerve tissue plays an important causal role in the pathogenesis of GBS/MFS (16, 17, 19, 21).Initial comparative studies of C. jejuni LOS structure and the corresponding DNA sequences of the LOS biosynthesis loci identified eight different LOS locus classes. Three of these classes, A, B, and C, harbor sialyltransferase genes involved in incorporating sialic acid into the LOS (42). Sialylation of the LOS core was found to be associated with ganglioside mimicry and also to affect immunogenicity and serum resistance (21). Recently, Parker et al. (43) identified 11 additional LOS classes on the basis of the sequence at the LOS biosynthesis locus. Their investigation also suggested that the LOS loci of C. jejuni strains are hot spots for genetic exchange, which can lead to mosaicism.Despite evidence on locus variation within C. jejuni LOS classes, PCR-based screening of a collection of 123 clinical and environmental strains showed that almost 60% of C. jejuni strains belong to class A, B, or C (42). Additionally, Godschalk et al. (16) found that 53% (9/17) of GBS-associated C. jejuni strains possessed LOS of class A, while 64% (35/55) of the non-GBS-associated isolates possessed LOS of class A, B, or C, and 62% (13/21) of enteritis-associated Campylobacter strains expressed LOS of class A, B, or C, as well. This relative representation of sialylated LOS classes A, B, and C was hypothesized to be advantageous for C. jejuni in the colonization and infection of various hosts (42, 49). Recently, Louwen et al. (34) demonstrated that C. jejuni strains possessing sialylated LOS (class A, B, or C) invade Caco-2 cells significantly better than nonsialylated strains (with class D or E). Knockout mutagenesis of the LOS sialyltransferase Cst-II in three C. jejuni strains revealed a significant reduction in the invasion potentials of the mutant strains (34). The possible role of LOS in adhesion and invasion was previously highlighted in the work of Perera et al. (44) and Kanipes et al. (29), where a C. jejuni waaF mutant strain showed significant reductions in levels of adherence to and invasion of INT-407 cells.LOS class diversity in C. jejuni strains isolated from chicken meat, an important source of human campylobacteriosis (6, 7, 26), has hardly been studied at all. In addition, the role of LOS class variation in the invasion potential of C. jejuni strains from chicken meat still needs to be explored. The epidemiological relevance of C. jejuni LOS gene screening can be further elaborated by correlating its results with results from other molecular-typing tools (e.g., multilocus sequence typing [MLST] and pulsed-field gel electrophoresis [PFGE]). In the present study, we screened a diverse collection of C. jejuni isolates, from consumer-packaged chicken meats and from sporadic human cases of diarrhea, by PCR for five LOS classes (A, B, C, D, and E). Then we correlated the LOS classes assigned by PCR screening with the genotypes assigned by PFGE and MLST. Finally, we tested the invasion potentials of a representative subset of C. jejuni isolates in relation to their LOS classes and genotypic diversity.     

Wild Griffon Vultures (Gyps fulvus) as a Source of Salmonella and Campylobacter in Eastern Spain

The existence of Campylobacter and Salmonella reservoirs in wildlife is a potential hazard to animal and human health; however, the prevalence of these species is largely unknown. Until now, only a few studies have evaluated the presence of Campylobacter and Salmonella in wild griffon vultures and based on a small number of birds. The aim of this study was to evaluate the presence of Campylobacter and Salmonella in wild griffon vultures (n = 97) during the normal ringing programme at the Cinctorres Observatory in Eastern Spain. In addition, the effect of ages of individuals (juveniles, subadult and adult) on the presence were compared. Campylobacter was isolated from 1 of 97 (1.0%) griffon vultures and identified as C. jejuni. Salmonella was isolated from 51 of 97 (52.6%) griffon vultures. No significant differences were found between the ages of individuals for the presence of Salmonella. Serotyping revealed 6 different serovars among two Salmonella enterica subspecies; S. enterica subsp. enterica (n = 49, 96.1%) and S. enterica subsp. salamae (n = 2, 3.9%). No more than one serovar was isolated per individual. The serovars isolated were S. Typhimurium (n = 42, 82.3%), S. Rissen (n = 4, 7.8%), S. Senftenberg (n = 3, 5.9%) and S. 4,12:b[-] (n = 2, 3.9%). Our results imply that wild griffon vultures are a risk factor for Salmonella transmission, but do not seem to be a reservoir for Campylobacter. We therefore rule out vultures as a risk factor for human campylobacteriosis. Nevertheless, further studies should be undertaken in other countries to confirm these results.     

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.     

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.     

Tracing the source of campylobacteriosis

Campylobacter jejuni is the leading cause of bacterial gastro-enteritis in the developed world. It is thought to infect 2–3 million people a year in the US alone, at a cost to the economy in excess of US $4 billion. C. jejuni is a widespread zoonotic pathogen that is carried by animals farmed for meat and poultry. A connection with contaminated food is recognized, but C. jejuni is also commonly found in wild animals and water sources. Phylogenetic studies have suggested that genotypes pathogenic to humans bear greatest resemblance to non-livestock isolates. Moreover, seasonal variation in campylobacteriosis bears the hallmarks of water-borne disease, and certain outbreaks have been attributed to contamination of drinking water. As a result, the relative importance of these reservoirs to human disease is controversial. We use multilocus sequence typing to genotype 1,231 cases of C. jejuni isolated from patients in Lancashire, England. By modeling the DNA sequence evolution and zoonotic transmission of C. jejuni between host species and the environment, we assign human cases probabilistically to source populations. Our novel population genetics approach reveals that the vast majority (97%) of sporadic disease can be attributed to animals farmed for meat and poultry. Chicken and cattle are the principal sources of C. jejuni pathogenic to humans, whereas wild animal and environmental sources are responsible for just 3% of disease. Our results imply that the primary transmission route is through the food chain, and suggest that incidence could be dramatically reduced by enhanced on-farm biosecurity or preventing food-borne transmission.     

Campylobacter Infection as a Trigger for Guillain-Barré Syndrome in Egypt

Background

Most studies of Campylobacter infection triggering Guillain-Barré Syndrome (GBS) are conducted in western nations were Campylobacter infection and immunity is relatively rare. In this study, we explored Campylobacter infections, Campylobacter serotypes, autoantibodies to gangliosides, and GBS in Egypt, a country where Campylobacter exposure is common.

Methods

GBS cases (n = 133) were compared to age- and hospital-matched patient controls (n = 374). A nerve conduction study was performed on cases and a clinical history, serum sample, and stool specimen obtained for all subjects.

Results

Most (63.3%) cases were demyelinating type; median age four years. Cases were more likely than controls to have diarrhea (29.5% vs. 22.5%, Adjusted Odds Ratio (ORa) = 1.69, P = 0.03), to have higher geometric mean IgM anti-Campylobacter antibody titers (8.18 vs. 7.25 P<0.001), and to produce antiganglioside antibodies (e.g., anti-Gd1a, 35.3 vs. 11.5, ORa = 4.39, P<0.0001). Of 26 Penner:Lior Campylobacter serotypes isolated, only one (41:27, C. jejuni, P = 0.02) was associated with GBS.

Conclusions

Unlike results from western nations, data suggested that GBS cases were primarily in the young and cases and many controls had a history of infection to a variety of Campylobacter serotypes. Still, the higher rates of diarrhea and greater antibody production against Campylobacter and gangliosides in GBS patients were consistent with findings from western countries.