The occurrence and characterization of Campylobacter spp. in silver gulls (Larus argentatus), three-toed gulls (Rissa tridactyla) and house sparrows (Passer domesticus)

Altogether 16 Campylobacter (C.) isolates could be recovered from 65 Herring gulls: 5 x C. laridis, 2 x C. jejuni biovar 1, 4 x C. jejuni biovar 2 and 5 x C. coli. Campylobacter spp. were isolated from 15 out of 51 samples from Kittiwakes: 2 x C. jejuni biovar 1 and 13 x C. laridis. All C. coli isolates grew on agar containing 1.5% NaCl. Two Campylobacter isolates from 50 House sparrows differed from all other isolates by a distinct beta-hemolysis and other phenotypic characteristics and could not be associated with a certain Campylobacter species. Epidemiological aspects and the possible role of the examined birds as a source of infection for man and domestic animals are discussed.     

Prevalence of gastrointestinal bacterial pathogens in a population of zoo animals

Faecal prevalence of gastrointestinal bacterial pathogens, including Campylobacter, Escherichia coli O157:H7, Salmonella, Shigella, Yersinia, as well as Arcobacter, were examined in 317 faecal specimens from 44 animal species in Belfast Zoological Gardens, during July-September 2006. Thermophilic campylobacters including Campylobacter jejuni, Campylobacter coli and Campylobacter lari, were the most frequently isolated pathogens, where members of this genus were isolated from 11 animal species (11 of 44; 25%). Yersinia spp. were isolated from seven animal species (seven of 44; 15.9%) and included, Yersinia enterocolitica (five of seven isolates; 71.4%) and one isolate each of Yersinia frederiksenii and Yersinia kristensenii. Only one isolate of Salmonella was obtained throughout the entire study, which was an isolate of Salmonella dublin (O 1,9,12: H g, p), originating from tiger faeces after enrichment. None of the animal species found in public contact areas of the zoo were positive for any gastrointestinal bacterial pathogens. Also, water from the lake in the centre of the grounds, was examined for the same bacterial pathogens and was found to contain C. jejuni. This study is the first report on the isolation of a number of important bacterial pathogens from a variety of novel host species, C. jejuni from the red kangaroo (Macropus rufus), C. lari from a maned wolf (Chrysocyon brachyurus), Y. kristensenii from a vicugna (Vicugna vicugna) and Y. enterocolitica from a maned wolf and red panda (Ailurus fulgens). In conclusion, this study demonstrated that the faeces of animals in public contact areas of the zoo were not positive for the bacterial gastrointestinal pathogens examined. This is reassuring for the public health of visitors, particularly children, who enjoy this educational and recreational resource.     

Heterogeneity of Campylobacter jejuni and Campylobacter coli strains from healthy sheep

The objectives of this study were to identify, at species level, thermophilic campylobacters isolated from clinically healthy sheep by a multiplex polymerase chain reaction (mPCR). The heterogeneity among Campylobacter jejuni and C. coli isolates was also investigated using a restriction fragment length polymorphism (RFLP) analysis of the flagellin (flaA) gene. Samples of intestinal contents, gall bladders and faeces were collected from 610 healthy sheep. While gall bladder samples were plated directly onto Preston agar, an enrichment stage was applied for intestinal and faecal samples. Of the 610 samples, 302 (49.5%) were positive for Campylobacter spp. Using a mPCR assay for species identification, 103 (34.1%) were positive with C. jejuni-specific primers, while 100 (33.1%) were positive with C. coli-specific primers. Additionally, 16 (11.9%) of the intestinal content samples were positive for both species by mPCR. All the isolates identified as C. jejuni and C. coli were successfully subtyped by flaA typing. Of 203 isolates tested, 48 different flaA types were found. Twenty-six flaA types were identified among C. jejuni isolates and the remaining 22 from C. coli isolates.     

Incidence of zoonoses in petting zoos and evaluation of hygiene measures to prevent the transmission to humans

In summer 2003, a study was performed in thirty Swiss petting zoos with the objective to determine the prevalence of zoonotic agents, and to describe hygiene measures implemented to reduce the risk of human infection. Fecal samples from different animal species were collected from the floor of pens to determine the prevalence of Salmonella spp., Campylobacter spp., verocytotoxin producing E. coli/ VTEC and Francisella tularensis. A questionnaire on hygiene measures, number of animals per species, housing system, care procedures and feeding was administered to every petting zoo to estimate exposure of visitors to zoonotic microorganisms. In total, 423 fecal samples were examined. Of these samples, 41 were positive for Campylobacter spp., which were mainly isolates from pigs and poultry (35% positive samples from each species). In pigs, 50% of the positive samples (6 samples) were typed as C. jejuni. The others were typed as C. coli (3) and C lan' (3), respectively. Five poultry isolates were typed as C. jejuni, and two as C. coli. Two samples were positive for Salmonella spp. Salmonella typhimurium was isolated from a goat, the other isolate could not be identified by serotyping. Neither Francisella tularensis nor verocytotoxin producing E. coli/ VTEC were found. The low prevalence of zoonotic microorganisms in Swiss petting zoos could be attributed to the cleanness of enclosures and animals, low stocking rates and good animal care. However, there is room for improvement concerning visitors' information on hygiene and hand washing. Furthermore, a strict separation between picnic - areas and animals should be enforced.     

Bulk growth procedures and a button agglutination test for Campylobacter

Bulk-cell yields were obtained from 4 Campylobacter spp incubated aerobically without the use of a special atmosphere. A button agglutination test was developed for quantitation of blood serum antibodies against C fetus subsp venerealis, C fetus subsp fetus, C jejuni, and "C hyointestinalis." The test was sensitive, and different individuals reading it usually attained the same titers. Cells of C fetus subsp venerealis, C fetus subsp fetus, and "C hyointestinalis" grown aerobically in broth made satisfactory antigens for the button test, but some cell pools of C jejuni had a tendency to autoagglutinate. Cells of C jejuni grown on blood agar had less tendency to autoagglutinate.     

Antimicrobial resistance of thermophilic Campylobacter spp. isolated from cattle in Poland

Campylobacter species are among the most frequently identified bacterial causes of human gastroenteritis. Because Campylobacter spp. harbored by cattle can be transmitted to humans, in this study we investigated antimicrobial resistance of thermophilic Campylobacter isolated from cows. Our study included 150 strains of Campylobacter (143 strains of C. jejuni and 7 strains of C. coli) isolated from cows in South-Western Poland. The minimal inhibitory concentration (MIC) to ciprofloxacin, erythromycin, gentamicin and tetracycline were determined using the agar dilution methodology. All strains of C. coli were susceptible to all four drugs studied. The most frequently detected resistance of C. jejuni was to ciprofloxacin (26 strains 18.2%). Resistance to tetracycline was observed in 5 strains (3.5%). All strains of C. jejuni were susceptible to erythromycin and gentamicin.     

Detection of Campylobacter upsaliensis in diarrheic dogs and cats, using a selective medium with cefoperazone.

Using a newly formulated selective medium containing cefoperazone, we isolated 72 Campylobacter strains in fecal samples from 397 diarrheic dogs and cats. Of these, 39 were thermophilic catalase-negative Campylobacter species. We identified these Campylobacter strains by DNA:DNA hybridization, using digoxigenin-labeled total genomic DNA of 4 Campylobacter reference strains (C jejuni, C coli, C lari, and C upsaliensis) as a probe. The labeling was done with a commercially available kit. We could identify 66 of the 72 Campylobacter isolates to the species level with this method; identification with probes always agreed with conventional test results. Of the 66 identified strains, 33 were C upsaliensis and 33 were C jejuni. Six isolates could not be assigned to a known species with probes or conventional tests. On the basis of our findings, C upsaliensis is more resistant to cefoperazone than to cephalothin, thereby explaining the unexpected recovery of these campylobacters on cephalosporin-containing media.     

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).     

Antimicrobial susceptibility patterns of thermotolerant Campylobacter strains isolated from food animals in Ethiopia

Thermotolerant Campylobacter spp. are frequent causes of diarrhoea in humans worldwide mostly originating from poultry. It has been suggested that extensive veterinary use of antibiotics is largely responsible for resistance in human isolates. During a 4-month period from January to April 2004, 192 Campylobacter spp. were isolated from fecal samples of 485 healthy food animals. The in vitro susceptibility to 12 antibiotics was determined by the agar disk diffusion method. Among the 192 Campylobacter spp. isolated, 135 (70.3%) were identified to be C. jejuni, 51 (26.6%) were C. coli and 6 (3.1%) were C. lari. C. jejuni was the most prevalent species in chickens (80.8%) versus 16.2% C. coli and 3.0% C. lari. All isolates found in pigs were C. coli. All strains were sensitive to chloramphenicol and ciprofloxacin and all were resistant to cephalothin. More than 90% of the strains were sensitive to clindamycin, erythromycin, gentamicin, nalidixic acid, norfloxacin, streptomycin and tetracycline. Resistance was found against ampicillin in 20% and trimethoprim-sulphamethoxazole in 37.5%. Resistance was not statistically different among C. jejuni, C. coli and C. lari (p>0.05). Multidrug resistance to two or more drugs was detected in 14.5% of strains. In conclusion, the study showed that antimicrobial resistance is found only at relatively low frequencies for most antimicrobial agents tested except for ampicillin and trimethoprim-sulphamethoxazole. The low percentages of resistance to most antimicrobial agents tested in this study may be the result of low/no usage of these agents as a growth promoters or treatment in the Ethiopian animal farm setting. The detection of multidrug resistant isolates may pose a threat to humans and further limits therapeutic options.     

Immuno-histochemical and -cytochemical evidence suggesting the presence of Campylobacter jejuni and Campylobacter coli in cases of porcine intestinal adenomatosis

Antisera against a number of Campylobacter species were used in immuno-histochemical and -cytochemical studies on cases of porcine intestinal adenomatosis. Avidin-biotin-complex (ABC) and streptavidin immunoperoxidase methods were used on formalin-fixed, paraffin-embedded and frozen sections. Protein A gold method was used on formaldehyde fixed and frozen sections for immuno-cytochemistry. The antisera used were raised in rabbits by subcutaneous or intravenous injection of living or formalin treated organisms. Anti-sera against different serotypes of the thermotolerant, catalase positive campylobacters, Campylobacter jejuni and Campylobacter coli, gave positive reactions in the immuno-histochemical studies. The staining was found in intestinal epithelial cells both in the ileum and in the colon and was restricted to the apical cytoplasm of adenomatous epithelial cells. The staining had a granular pattern, the positive structures sometimes having the shape of Campylobacter. Epithelial cells in areas with normal differentiation of goblet cells did not stain. In contrast, no staining resulted with antisera against Campylobacter sputorum subsp. mucosalis and Campylobacter hyointestinalis. Immuno-cytochemistry, using antisera against Campylobacter jejuni, showed that the positive staining in altered epithelial cells were restricted to intracellular organisms having a structure resembling Campylobacter spp.     

Detection of Campylobacter jejuni strains in the water lines of a commercial broiler house and their relationship to the strains that colonized the chickens

Campylobacter jejuni is frequently present in the intestinal tract of commercial broiler chickens, and their drinking water has been proposed to be an initial source of bacteria for newly hatched chicks. We studied three sequential commercial broiler flocks raised in a house from which we had cultured C. jejuni from the nipple waters prior to placement of the first flock. Campylobacter cells were detected by immunofluorescence in the biofilm of the drinking nipples during the weeks when the flock was colonized with C. jejuni but not during weeks when the birds were negative. Campylobacter jejuni was isolated from the drinking water during the growth of the first flock and was present in the birds from all three flocks. Randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) typing with primer OPA11 indicated that seven distinct strains were present within the broiler house. One strain found in drinking water was similar to a strain found in birds in the second flock; however, RAPD-PCR with primer HLW85 showed that the strains were not identical. These results suggest that although the watering system is a potential source of C. jejuni in broiler flocks, the waterborne strain in this study was not detected in the birds.     

Serum agglutinin titers against somatic and flagellar antigens of Campylobacter jejuni and isolation of Campylobacter spp in chickens

From June 1983 to June 1984, two hundred twenty-five 3- to 30-month-old chickens (hens) on 10 different farms were examined for Campylobacter spp. Watering trays and fly vectors also were examined for Campylobacter spp on 6 of the 10 farms. Campylobacter jejuni was isolated from fecal specimens from 64 hens (28.4%), C coli was isolated from 6 hens (2.7%), and C laridis was isolated from 9 hens (4%). The isolation rate of C jejuni was 6.7% to 46.7% for 9 of the 10 farms. On 2 farms, agglutinin titers greater than or equal to 1:40 against somatic and flagellar antigen of C jejuni were detected in hens from which the bacteria were isolated. Hens having titers greater than or equal to 1:40 against C jejuni or hens from which C jejuni had been isolated often occupied adjacent pens. Campylobacter jejuni was isolated from a watering tray on 1 farm and from fly vectors on 2 farms.     

Campylobacter succession in broiler chickens

The competitive ability of Campylobacter coli OR12 over C. jejuni OR1 has been examined in experimental broiler chickens following the observation that C. coli replaced an established C. jejuni intestinal colonisation within commercial chicken flocks reared outdoors [El-Shibiny, A., Connerton, P.L., Connerton, I.F., 2005. Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens. Appl. Environ. Microbiol. 71, 1259-1266]. Co-cultures of C. coli OR12 with C. jejuni OR1, revealed that the two species were able to grow together at similar growth rates in exponential growth phase but if the disparity of the inoculum ratios were >log(10)4 in favour of C. coli OR12, C. jejuni OR1 was observed to prematurely enter decline phase. Chickens were pre-colonised with C. jejuni OR1 at 21-days-old to examine succession in vivo. The birds were inoculated between 2 and 12 days later with C. coli OR12, to determine if the second isolate could efficiently colonise and compete with an established C. jejuni strain. C. coli OR12 were able to co-colonise before replacing C. jejuni OR1 as the dominant species when the birds were more than 27 days of age at the time of administration over a 4-day period. If these criteria were met C. coli OR12 became the dominant isolate otherwise co-colonisation occurred until they were met. C. coli OR12 was also found to displace three alternative C. jejuni strains from pre-colonised chickens challenged with C. coli OR12 at 30 days of age and tested at 40 days. These data raise the possibility of manipulating populations of Campylobacter colonising chickens through competition.     

Detection of thermophilic Campylobacter from sparrows by multiplex PCR: the role of sparrows as a source of contamination of broilers with Campylobacter

The best combination of primers and the annealing temperature of multiplex PCR for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari were examined. The multiplex PCR was able to detect type strains of the three species. All results of identification of wild strains (30 strains of C. jejuni, 20 strains of C. coli, and 4 strains of C. lari) by the multiplex PCR coincided with those of the conventional biochemical identification tests, suggesting that the multiplex PCR can simultaneously differentiate C. jejuni, C. coli, and C. lari from wild strains of campylobacters easily and rapidly. Campylobacters were detected from sparrow feces by the multiplex PCR and antimicrobial sensitivities of the strains were determined to discuss the role of sparrows in contamination of broilers with C. jejuni. Three out of 13 strains of C. jejuni isolated from sparrow feces showed quinolone resistance. From the frequent use of quinolones for treatment of industrial animals like chickens, pigs, and cows, the three strains of quinolone-resistant C. jejuni in sparrows must have been originated from those industrial animals. Sparrows that have quinolone-resistant C. jejuni were considered to have contacted with industrial animals or thier feed. It may be presumed, on the contrary, that C. jejuni in sparrows could be a potential source of contamination of broilers.     

Serotype and genotype diversity and hatchery transmission of Campylobacter jejuni in commercial poultry flocks.

We investigated the genotype and serotype diversity of Campylobacter coli and C. jejuni in two parent flocks of adult hens and their offspring over two rotations in order to evaluate the role of hatchery mediated transmission and/or vertical transmission of campylobacters in broiler flocks. In total, 314 C. jejuni and 32 C. coli isolates from parent and broiler flocks and from the surroundings of broiler houses were typed by flagellin gene PCR/RFLP (fla-typing), and selected isolates were also typed by serotyping and macrorestriction profiling using PFGE (MRP/PFGE). The combined typing results showed that the broiler flocks could be colonised by 1-3 different Campylobacter clones and parent flocks could be colonised by 2-6 different clones. C. coli was isolated from up to 36% of birds in one parent flock, whereas only C. jejuni was isolated from broiler flocks. C. jejuni clones from different flocks were clearly discriminated by fla-typing as well as by MRP/PFGE, except for a few cases where individual isolates belonging to two different clones were found to have altered fla-types. Similarly, one C. coli clone showed pronounced fla-type variation. The present results lead to the conclusion that vertical transmission or horizontal transmission via the hatchery are not significant transmission routes of C. jejuni to broiler chickens under Danish conditions. In the cases where more than one Campylobacter clone simultaneously colonised flocks, we found that the different clones coexisted in flocks rather than excluding each other.     

The occurrence of Campylobacter species in Hungarian broiler chickens from farm to slaughter

The reported number of human enteric diseases caused by thermotolerant campylobacters increased in the last few decades worldwide. The microorganism gets into the food chain mostly with poultry meat or meat products. We are not aware of the way the campylobacters infect the broiler flocks, and there is little information about the real prevalence, about the reaction of thermotolerant campylobacters to the environmental factors and about the possibilities of elimination of the bacteria from the food chain. As a part of the long study, samples were collected from a broiler flock from the first day of life to the slaughter of the animals, in summer and in winter. In the summer period, at the first two sampling days (days 0 and 12) all of the samples were negative. At day 26, one cloaca sample, one sample from the surface of the wall near the ventilation aperture and an insect-sample were positive. At day 42, we found Campylobacter spp. on every sampling point at the slaughterhouse. In the winter period, we could not find Campylobacter spp. either from 0 day old, or from 10- and 31-day-old chickens, but we found them at 42 days of age on the slaughter plant. At the slaughtering place, 93.3% of the live birds were infected with Campylobacter spp., and at the end of the processing line, the infection rate was 100%. We could isolate campylobacters from the hands of the workers and from the processing environment as well. Out of the positive samples, 95.5% was contaminated with Campylobacter jejuni.     

Distribution and genetic variability among Campylobacter spp. isolates from different animal species and humans in Switzerland

In Switzerland, a national database with 1028 Campylobacter isolates from poultry, pigs, cats, dogs, cattle, humans, zoo animals and water has been created. The database contains the genetic fingerprint and background information of each Campylobacter isolate. Dominant species could be identified in the different sources with a majority of Campylobacter jejuni in poultry (73%), humans (79%), cattle (95%), zoo animals (40%) and water (100%), of Campylobacter coli in pigs (72%), and of Campylobacter upsaliensis/helveticus in cats and dogs (55%). The comparison of three genotyping methods, amplified fragment length polymorphism (AFLP), pulsed field gel electrophoresis and restriction fragment length polymorphism, revealed that AFLP allows discrimination between the different Campylobacter species and is the most appropriate method to distinguish specific strains within the same species. Genotyping analysis demonstrated that the Campylobacter population is heterogeneous among the different sources and that no dominant clone is spread in the country. Genotyping and the resulting database are useful tools to trace back future Campylobacter infections.     

Prevalence and clonal diversity of Campylobacter jejuni from dairy farms and urban sources

AIM: To investigate the role of free-living animals such as sparrows, rodents and flies as potential reservoirs of Campylobacter spp on a dairy farm, and to assess the genetic diversity among Campylobacter isolates from the farm and an urban source. METHODS: A total of 290 samples (bovine, passerine and rodent faeces, and whole flies) were collected from a large commercial dairy farm in the Manawatu district in New Zealand, and from faeces from urban sparrows in a nearby city. Other samples collected from the dairy farm included five from silage, two from aprons worn by workers during milking, two from workers' boots and two from water in troughs in a paddock. Isolates of thermophilic Campylobacter spp were identified morphologically and phenotypically and further characterised molecularly using pulsed-field gel electrophoresis (PFGE) and the restriction enzyme SmaI. RESULTS: Campylobacter jejuni was the only Campylobacter species isolated from all samples. The highest prevalence was found in faeces from dairy cows (54%), followed by faeces from sparrows from the urban area (40%) and the farm (38%), and from rodents (11%) and whole flies (9%). Other samples from the farm environment such as silage, trough water, and workers' aprons and boots were also positive for C. jejuni. Of the 22 restriction patterns obtained, seven were common to more than one source. CONCLUSIONS: Cattle, sparrows, rodents and flies are potential reservoirs of C. jejuni on dairy farms. Identical clones of C. jejuni carried by cattle, sparrows, flies and rodents probably indicate a common source of infection. The high level of asymptomatic carriage of C. jejuni by healthy dairy cows could be sufficient to maintain infections within the dairy farm surroundings via environmental contamination.     

Comparison of Campylobacter carriage rates in diarrheic and healthy pet animals.

To explore the clinical significance of campylobacter infections for dogs and cats we compared intestinal carriage rates for Campylobacter sp. between animals with gastroenteritis and healthy controls. We cultured fecal specimens of 405 diarrheic dogs and 203 cats as well as 71 asymptomatic dogs and 35 cats using a selective medium in addition to filtration on a non-selective blood agar plate. We identified 224 campylobacter isolates using conventional phenotypic tests and DNA hybridization. There were 112 isolates, of C. upsaliensis, 43 C. jejuni and 69 other Campylobacter sp. For cats, there was no association between campylobacter carriage and disease, irrespective of the animals age. For dogs older than 12 months there was also no difference in campylobacter carriage rates between diarrheic and healthy animals. However, in younger dogs 44% of animals with diarrhea shed campylobacters in their feces, more than twice the rate in asymptomatic controls (21%), a significant difference. C. jejuni and C. upsaliensis contributed equally to this association between diarrhea and campylobacter prevalence. The parallel use of two culture methods enabled us to show that the recovery of Campylobacter sp. by filtration may be less than optimal and that filtering is probably unsuitable as a reference method for culturing C. upsaliensis. Finally we found that almost half of the campylobacter isolates from cats belong to a phenotypically homogeneous group of strains closely resembling C. upsaliensis but hybridizing only weakly with C. upsaliensis DNA.     

Characterization of Campylobacter spp. from wild birds

Bacteria of the genus Campylobacter were isolated from 28 Rooks (Corvus frugilegus), 1 Red Kite (Milvus milvus), 1 Lapwing (Vanellus vanellus), 1 Coot (Fulica atra), 1 Common Moorhen (Gallinula chloropus) and 1 Northern Mallard (Anas platyrhynchos). Altogether, C. jejuni biovar 1, was isolated 19x, C. jejuni biovar 2 8x and C. coli 5x. Among C. jejuni biovar 1 and 2 there were 5 isolates tolerating a content of 1.5% NaCl in the medium. H2S proof of 3 C. jejuni biovar 2 and 1 C. coli isolates resulted positive or negative dependent on incubation time of the used bacterial inoculum. Concerning Rooks the findings indicate that nestlings are more often infected with campylobacters than older birds. Only 1 campylobacter isolate could be recovered from altogether 54 birds of prey although 16 Buzzards (Buteo buteo) were investigated as nestlings.