Campylobacter jejuni, Campylobacter coli, and cytolethal distending toxin genes in laying hens

As no data are available on the prevalence of cytolethal distending toxin (cdt) genes carried by Campylobacter spp. in laying hens, this study was conducted with the aim to evaluate the prevalence of both Campylobacter spp. and cdt genes in 1680 laying hens from four different farms. The samples were analyzed by culture methods and by polymerase chain reaction. Campylobacter spp. were isolated from 1097/1680 cloacal swabs. Among the isolates, 913 were identified as Campylobacter jejuni whereas 345 were identified as Campylobacter coli. All isolates carried cdt genes. The results presented here confirm the very common occurrence of C. jejuni and C. coli in laying hens and underline that the cdt genes may also be frequently present in both C. jejuni and C. coli isolates from laying hens.     

Effect of cytolethal distending toxin of Campylobacter jejuni on adhesion and internalization in cultured cells and in colonization of the chicken gut

Campylobacter jejuni produces cytolethal distending toxin (CDT) that causes host cells to arrest during their cell cycle and that is involved in the pathogenesis of inflammatory diarrhea in humans. To assess the role of CDT in adherence and invasion of different cultured host cells (HeLa and HD-11) and in colonization of the chicken intestine, the genes of C jejuni NCTC11168 encoding the toxin subunits (cdtA, cdtB, and cdtC) were inactivated by insertional mutagenesis. No significant difference was found in adhesion of the wild-type C. jejuni and the isogenic mutants to HeLa and HD-11 cells. All of the mutants exhibited a decrease (>10-fold) in the ability to invade HeLa cells, but no significant difference was noticed for HD-11 cells. The ability of mutants to colonize birds either directly or by horizontal transfer was unchanged. These data indicated that although the production of cytotoxin does not play a role in the adherence to either human or avian cells, it may play a role in the invasion, survival, or both of C. jejuni in human cells, which are more susceptible to C. jejuni internalization. The CDT also does not seem to play a role in the colonization of poultry.     

A cytolethal distending toxin gene-based multiplex PCR assay for detection of Campylobacter spp. in stool specimens and comparison with culture method

In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacter-positive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248).     

The cytolethal distending toxin among Helicobacter pullorum strains from human and poultry origin

Helicobacter pullorum has been associated with diarrhoea, gastroenteritis and liver disease in humans and with hepatitis and enteritis in poultry. The purpose of the present study was to examine whether cytolethal distending toxin was present among 10 poultry and three human H. pullorum isolates and whether a different level of cytolethal distending toxin activity was noted. A PCR assay was performed to detect the cdtB gene. In addition, epithelial Hep-2 cells inoculated with sonicate from all strains were observed microscopically and DNA analysis of these cells was done by flow cytometry. All H. pullorum isolates harboured the cdtB gene, but functional cytolethal distending toxin activity was only demonstrated in the human H. pullorum strain CCUG 33839. A significant number of cells treated with sonicate from this strain were enlarged. The nuclei were distended proportionally. Giant cells and multinucleated cells were observed as well. In addition, stress fibers accumulated. DNA analysis by flow cytometry revealed 31.0% of these cells at the S/G2 stage of the cell cycle. The tested poultry and human H. pullorum isolates all possess the cdtB gene, but under the circumstances adopted in this study only the human strain CCUG 33839 seems to show biological activity typically for CDT in vitro.     

副猪嗜血杆菌细胞致死膨胀毒素CdtC亚基的克隆表达及细胞毒性分析

细胞致死膨胀毒素(CDT)由CdtA、CdtB和CdtC三个亚基构成,是细菌的一种重要毒力因子.为研究副猪嗜血杆菌(H.parasuis) CDT的CdtC亚基的功能,本研究根据GenBank中cdtC基因序列设计引物,扩增cdtC基因,并克隆到原核表达载体pET-28a(+)中进行诱导表达.SDS-PAGE和western blot检测目的蛋白主要以包涵体形式存在,分子量约为25 ku.将纯化后的重组蛋白复性后进行体外细胞毒性试验.结果表明,CdtC蛋白对Vero细胞和PK-15细胞有较强的细胞毒性作用.本研究为研究细胞致死膨胀毒素CDT的致病机制奠定了基础.     

Occurrence of virulence genes among Campylobacter jejuni and Campylobacter coli isolates from domestic animals and children

The presence of the flaA, cadF, cdtB and iam genes of Campylobacter spp. was determined with the PCR method. The materials to investigate were 56 C. jejuni and 23 C. coli strains isolated from clinical samples (children and domestic animals). It was found that all of the Campylobacter spp. isolates from children with diarrhoea and domestic animals had cadF gene, responsible for adherence. The flaA gene was present in all Campylobacter spp. isolates derived from children and cats. Occurrence of flaA gene was confirmed in 100% of C. jejuni strains obtained from dogs. The high prevalence of the cdtB gene associated with toxin production was observed in this study (100%-Campylobacter spp. isolates obtained from dogs and cats, 97.9%-Campylobacter spp. isolates from children). The isolates showed a wide variation for the presence of iam gene. The lowest prevalence (23.5%) was detected in Campylobacter spp. obtained from dogs. The highest rates of iam detection (91.6%) were revealed in C. coli isolates from children.     

Campylobacter jejuni and Campylobacter coli inoculation of neonatal calves

Three groups of neonatal calves were inoculated orally with pathogenic strains of Campylobacter jejuni or C coli. The calves developed a mild, self-limiting enteritis characterized by thick mucoid feces. Bacteremia and fecal shedding of Campylobacter were sporadic in all inoculated calves. Two groups of calves were killed 1 to 3 weeks after inoculation to study the pathogenesis of infection. Postmortem culture of tissues revealed C jejuni or C coli most frequently in the ileum, cecum, colon, and blood. Clinical or pathologic differences between C jejuni-inoculated and C coli-inoculated calves were minimal.     

Fecal shedding of Campylobacter jejuni and Campylobacter coli among feral pigs in Texas

The population and range of feral pigs in the United States are rapidly expanding, yet key knowledge gaps exist regarding their role in the ecology and transmission of foodborne pathogens. Our objectives were to estimate the prevalence of Campylobacter jejuni and Campylobacter coli shedding among feral pigs throughout Texas and to identify risk factors for positive status. Faecal samples were collected from feral pigs in Texas from February 2014 through May 2015, and target organisms were detected using PCR assays. The prevalence of C. jejuni shedding was 1.6% (6/370), and the prevalence of C. coli shedding was 3.5% (13/370). C. coli shedding was significantly more common (p = .008) among female pigs than among male pigs. Feral pigs may represent a source of human campylobacteriosis.     

Pathogenicity of Campylobacter jejuni and Campylobacter coli strains in the pregnant guinea pig model

Pathogenicity of 17 Campylobacter isolates for pregnant guinea pigs was investigated. Of 14 isolates, 12 (86%) produced rates of abortion ranging from 13% to 87%. Two isolates did not produce abortion. Reference strains of C fetus subsp venerealis produced abortion in 60% to 87% and C fetus subsp fetus produced abortion in 60% of the guinea pigs. Inoculated organisms were recovered from uterus, blood, liver, kidney, spleen, and gallbladder of the guinea pigs at rates as high as 83% for 2 ovine isolates and as low as 13% for 2 bovine and 1 human isolates. Most isolations were from the uterus. Two avian isolates were not recovered. Within the C jejuni and C coli group, the ovine and the human isolates appear to be more pathogenic. Swine, bovine, and avian isolates were less pathogenic. Seemingly, the pregnant guinea pig was a suitable and practical model for evaluating the pathogenicity of Campylobacter organisms, regardless of their host of origin.     

Combined Campylobacter jejuni and Campylobacter coli Rapid Testing and Molecular Epidemiology in Conventional Broiler Flocks

Campylobacter spp. are important causes of bacterial zoonosis, most often transmitted by contaminated poultry meat. From an epidemiological and risk assessment perspective, further knowledge should be obtained on Campylobacter prevalence and genotype distribution in primary production. Consequently, 15 Austrian broiler flocks were surveyed in summer for their thermophilic Campylobacter spp. contamination status. Chicken droppings, dust and drinking water samples were collected from each flock at three separate sampling periods. Isolates were confirmed by PCR and subtyped. We also compared three alternative methods (culture‐based enrichment in Bolton broth, culture‐independent real‐time PCR and a lateral‐flow test) for their applicability in chicken droppings. Twelve flocks were found to be positive for thermophilic Campylobacter spp. during the entire sampling period. Seven flocks (46.6%) were contaminated with both, C. jejuni and C. coli, five flocks harboured solely one species. We observed to a majority flock‐specific C. jejuni and C. coli genotypes, which dominated the respective flock. Flocks within a distance <2 km shared the same C. jejuni genotypes indicating a cross‐contamination event via the environment or personnel vectors. Multilocus sequence typing (MLST) of C. jejuni revealed that the majority of isolates were assigned to globally distributed clonal complexes or had a strong link to the human interface (CC ST‐446 and ST4373). The combination of techniques poses an advantage over risk assessment studies based on cultures alone, as, in the case of Campylobacter, occurrence of a high variety of genotypes might be present among a broiler flock. We suggest applying the lateral‐flow test under field conditions to identify ‘high‐shedding’ broiler flocks at the farm level. Consequently, poultry farmers and veterinarians could improve hygiene measurements and direct sanitation activities, especially during the thinning period. Ultimately, real‐time PCR could be applied to quantify Campylobacter spp. directly from chicken droppings and avoid non‐interpretable results achieved by culture‐dependent methods.     

Molecular detection of Campylobacter species and Cytolethal distending toxin isolated from chicken livers in Tabriz

Introduction and purpose: Campylobacter jejuni and coli are zoonotic bacteria commonly associated With human diarrhea and usually transmit through consumption of meat and poultry contaminated products such as heart and liver. Cytolethal distending toxin (cdt) in Campylobacter spp. is among the significant virulence factors of these bacteria in the intestine. The purpose of this study was to determine the prevalence of Campylobacter spp. and presence of cdt genes among isolated bacteria. Materials and methods: In this cross sectional study, 100 chicken livers were examined. Detection, bacterial enumeration and isolation of Campylobacter spp. was done using Campylobacter selective agar media containing Campylobacter growth supplement, gram staining, catalase and oxidase production, hippurate hydrolysis and PCR molecular technique. Also the presence of cdt genes were detected using PCR assay. Results: From 100 studied liver samples, 43 were contaminated with Campylobacter spp. Among them 31(72 %) samples had Campylobacter jejuni and 12 (28 %) had Campylobacter coli. All Campylobacter jejuni isolates contained cdtA ،cdtB and cdtC genes. However, all of these genes detected in 9 (75 %) of isolated coli. Conclusion: The results of this study showed that great percentages of chicken livers in Tabriz were contaminated with Campylobacter.     

Fla-DGGE analysis of Campylobacter jejuni and Campylobacter coli in cecal samples of broilers without cultivation

In a commercial broiler flock during rearing multiple genotypes of Campylobacter jejuni may be present as well as in gastrointestinal tracts of individual birds. The aim of this study was to optimize and apply a denaturing gradient gel electrophoresis assay of the flagellin gene (fla-DGGE) for analysis of C. jejuni and Campylobacter coli in cecal samples of broilers without prior cultivation. One C. coli and 21 C. jejuni strains isolated from broiler flocks, of which 14 typed as unique by restriction fragment length polymorphism of flaA and two undefined strains, were clustered into 9 groups when applying fla-DGGE. Spiking of cecal samples revealed that fla-DGGE is able to detect at least 4.55-5.96logCFUCampylobacter/mlcecal material. The presence of 3 strains spiked in cecal material was demonstrated by fla-DGGE as the corresponding bands were visible on the DGGE gel. Naturally contaminated cecal samples were shown to contain different types of C. jejuni and C. coli. Fla-DGGE has some potential as a cultivation-independent fast primary subtyping method for C. jejuni and C. coli in cecal samples of broilers.     

Risk factors for Campylobacter jejuni and Campylobacter coli in young cattle on English and Welsh farms

Campylobacter jejuni and C. coli are the most prevalent causes of bacterial diarrhoea in most of the Western World. In Great Britain, the source remains unknown for the majority of cases, though poultry is considered the main source of infection. Molecular typing methods identify cattle as a potential source of a proportion of the non-source-attributed cases, mainly through direct contact, environmental contamination or milk, but little is known about the epidemiology of Campylobacter in cattle. A cross-sectional study was undertaken on young cattle 3-17 months of age on 56 cattle farms in England and Wales to identify association between the presence of C. jejuni and C. coli and farm characteristics and management practices. Campylobacter was detected on 62.5% of the farms and the presence of dairy cows (OR: 3.7, CI(95%): 1.2; 11.7), indoor housing (OR: 4.6, CI(95%): 1.8; 12.0), private water supply (OR: 2.5, CI(95%): 1.2; 5.4), presence of horses (OR: 3.2, CI(95%):1.5; 6.9) and feeding hay (OR: 2.9, CI(95%):1.6; 5.5) were associated with detection. The model's goodness-of-fit was improved when herd size was forced in the model without being statistically significant (p=0.34).     

An outbreak of infectious hepatitis in commercially reared ostriches associated with Campylobacter coli and Campylobacter jejuni

A disease causing high morbidity and mortality was observed in young ostriches from six properties in southeast Queensland, Australia. The disease affected birds from 2–8 weeks of age and was characterised clinically by bright-green urates and pathologically by severe necrotic hepatitis. The liver lesions resembled those of vibrionic hepatitis in other avian species. Campylobacter coli was isolated from the livers of affected ostriches from five of the six properties. Campylobacter jejuni subsp. jejuni was isolated from birds from the remaining property. Pulsed-field gel electrophoresis-based (PFGE) typing of representative isolates indicated that trade of infected birds between farms was an important factor in the spread of C. coli. Phenotypic and genotypic data suggest a clonal variant of the principal outbreak type may account for the remaining cases from which C. coli was found. Conventional biochemical test results and PFGE clearly distinguished the C. jejuni strain isolated from the geographically remote farm from the outbreak of C. coli type. We believe this to be the first definitive report of avian hepatitis associated with C. coli.     

Survival of Escherichia coli,Enterococci and Campylobacter jejuni in Canada Goose Faeces on Pasture

Freshly excreted Canada goose faeces pose a public health risk as they contain pathogenic microorganisms. Accordingly, a study was carried out on the growth and survival of resident indicator bacteria (enterococci and Escherichia coli) and inoculated Campylobacter jejuni in freshly excreted faeces over summer and winter. Canada goose faeces were collected, mixed thoroughly and inoculated with 10⁸ g^?1C. jejuni. The faeces were mixed again before making the Canada goose dropping. The simulated goose droppings (N = 70) were placed on pasture, and the concentrations of E. coli, enterococci and the pathogen, C. jejuni, were monitored. In summer only, the molecular marker of E. coli LacZ and the avian‐associated bacteria E2 was also monitored. Results of the survival study indicated that significant growth of enterococci and E. coli occurred in summer, before concentrations decreased to less than 15% of the original concentration (day 77) for enterococci and 0.01% for E. coli. LacZ followed a similar pattern to E. coli, while the E2 marker dropped to below 0.1% of the original concentration within 4 days. In winter, enterococci grew slightly, while no growth of E. coli occurred. In both summer and winter, C. jejuni was rapidly inactivated. This research highlights the ability of bacterial indicators to replicate and survive in the environment when harboured by avian faeces, and the limited risk aged Canada goose faeces pose as an environmental source of Campylobacter spp.