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.     

The occurrence of Campylobacter subtypes in environmental reservoirs and potential transmission routes

AIM: To identify potential reservoirs and transmission routes of human pathogenic Campylobacter spp. METHODS AND RESULTS: An enrichment PCR method for the detection and identification of Campylobacter jejuni and/or Campylobacter coli in faecal, food and river water samples was applied to 1450 samples of 12 matrix types obtained from a defined geographical area. PCR-positive samples were cultured to yield isolates for typing, and the data for 616 C. jejuni isolates obtained. Serotyping and SmaI macrorestriction profiling using pulsed field gel electrophoresis revealed a high level of diversity within the isolates from each matrix. Campylobacter jejuni and C. coli subtypes indistinguishable from those obtained from human cases were detected in most of the matrices examined. No Campylobacter isolates were isolated from possum faeces. CONCLUSIONS: Ten of the 12 matrices examined may be involved in the transmission of human campylobacteriosis as they contained Campylobacter subtypes also isolated from clinical cases. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate that, for this rural population, a range of potential transmission routes that could lead to campylobacteriosis exist. Their relative importance needs to be assessed from an exposure assessment standpoint.     

Enteropathogenic bacteria in frozen chicken.

Eighty-two samples of frozen chicken from retail stores were examined for the presence of Campylobacter, Yersinia enterocolitica, and salmonellae. Aerobic plate counts and numbers of coliform bacteria at 37 degrees C were determined. Campylobacter fetus subsp. jejuni was found in 22% of the samples, Y. enterocolitica was found in 24.5% and Salmonella typhimurium was found in one sample (1.2%). The isolated strains of Y. enterocolitica belonged to serotypes 4, 5b, 6, and 8. Aerobic plate counts and numbers of coliform bacteria at 37 degrees C were not found to be noticeably higher in samples containing pathogens than in pathogen-free samples. This investigation showed that chicken does contain other pathogenic bacteria than salmonellae. Campylobacter and Y. enterocolitica were isolated in much higher frequencies than Salmonella.     

The heterogeneity of Campylobacter jejuni and Campylobacter coli strains isolated from healthy cattle

AIMS: To identify campylobacters isolated from clinically healthy cattle at species level by a multiplex polymerase chain reaction (m-PCR). The heterogeneity among Campylobacter jejuni and Campylobacter coli isolates was also investigated by using a restriction fragment length polymorphism (RFLP) analysis of flagellin (flaA) gene. METHODS AND RESULTS: Samples of intestinal contents, gall bladders, liver and faeces were collected from a total number of 1154 healthy cattle. The samples were inoculated onto Preston enrichment broth and agar. Of 1154 samples, 301 (26.1%) were positive for Campylobacter spp. Using an m-PCR assay for species identification, 179 (59.5%) were positive with C. jejuni specific primers while 30 (10%) were positive with C. coli specific primers. None of the liver samples examined was positive for C. jejuni or C. coli by mPCR. All the isolates identified as C. jejuni and C. coli were successfully subtyped by flaA typing. Of the 209 isolates tested, 28 different flaA types were found. Twenty-three flaA types were identified among 179 C. jejuni isolates and the remaining five from C. coli isolates. CONCLUSIONS: Although the overall results suggest that the degree of heterogeneity among the flaA genes of thermophilic Campylobacter strains isolated from healthy cattle is relatively high, they should be treated cautiously as the number of band types for C. coli was low and band type 8 in C. jejuni was represented by a high percentage (%58). SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study suggest that healthy cattle can play role in the contamination of environment and human food chain by Campylobacter spp.     

MAMA-PCR assay for the detection of point mutations associated with high-level erythromycin resistance in Campylobacter jejuni and Campylobacter coli strains

SYNOPSIS: Twenty Campylobacter jejuni and 16 Campylobacter coli strains isolated from humans and food/animals, including 17 isolates resistant to erythromycin, were analyzed. A combined mismatch amplification mutation assay-PCR technique was developed to detect the mutations A 2074 C and A 2075 G in the 23S rRNA gene associated with erythromycin resistance. All high-level erythromycin-resistant strains examined by DNA sequencing carried the transition mutation A 2075 G, whereas no isolate carried the A 2074 C mutation. No mutations were found among the susceptible and low-level erythromycin-resistant strains.     

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

Water samples were taken systematically from a 100-km² area of mainly dairy farmland in northwestern England and examined for Campylobacter spp. Pulsed-field gel electrophoresis-restriction fragment length polymorphism (PFGE-RFLP) and flaA strain typing of Campylobacter jejuni and Campylobacter coli isolates were done. Data on the water source and the adjacent environment were recorded and examined as explanatory variables. Campylobacter spp. were isolated from 40.5% (n = 119) of the water samples tested. C. jejuni was isolated from 14.3%, C. coli was isolated from 18.5%, and Campylobacter lari was isolated from 4.2% of the samples. Campylobacter hyointestinalis was not isolated from any water source. The difference in prevalence between water types (trough, running, and standing) was significant (P = 0.001). C. jejuni was the species most commonly isolated from trough-water and running-water sources, while C. coli was the most frequently isolated from standing water (P < 0.001). No association was found between the presence of Escherichia coli and that of Campylobacter spp. The final multivariable logistic regression model for Campylobacter spp. included the following variables: water source, soil type, aspect, and amount of cattle fecal material in the environment (fecal pat count). Strain typing demonstrated a diverse population of C. jejuni and the presence of a common C. coli flaA type that was widely distributed throughout the area. Most of the isolates within the common flaA type were discriminated by PFGE-RFLP. These findings suggest a possible role for environmental water in the epidemiology of Campylobacter spp. in a farming environment.     

Comparison of the survival of Campylobacter jejuni and Campylobacter coli in culturable form in surface water

Six Campylobacter jejuni and six Campylobacter coli strains were isolated from cows and pigs, and their survival in lake water was compared by viable counts. Campylobacter jejuni survived longer in culturable form than C. coli in untreated and membrane-filtered water both at 4 and 20 degrees C. This difference in survival time may be a reason why C. jejuni is generally isolated from surface waters more frequently than C. coli. Both species survived better in filtered than in untreated water. This suggests that predation and competition for nutrients affect the survival of both Campylobacter species in the aquatic environment.     

Biochemical and serologic characteristics of Campylobacter jejuni/coli strains causing diarrhea in children

One hundred strains of Campylobacter jejuni/coli isolated from faeces of children with diarrhoea were characterised. Frequency of isolation of these microorganisms from faeces of children with enterocolitis symptoms was evaluated. In this group Campylobacter jejuni/coli constituted 11.4% of all isolates, being the dominant etiologic agent of these infections. Biotype pattern of 100 Campylobacter jejuni/coli strains was determined. Biotype I C.jejuni prevailed and C. coli constituted as much as 35% of all isolated strains. All isolated strains were characterised serologically according to typing scheme of Lior. Seventy four strains were typed and 22 were untypable, out of which four were rough. Two new serotypes were isolated: LIO 71 and LIO 72, LIO 4 and LIO 72 serotypes were the most frequently isolated. Frequency of isolation of Campylobacter jejuni/coli strains were also determined in the period from january 1985 to august 1987.     

Campylobacter jejuni: incidence in processed broilers and biotype distribution in human and broiler isolates.

Campylobacter jejuni was isolated from 18 of 40 processed broiler carcasses and 134 of 327 cloacal swabs obtained at four processing plants in Sydney, Australia. Three of four flocks examined carried C. jejuni. Eighty-two percent of chicken and 98% of human isolates from the area were of identical biotypes.     

The prevalence of campylobacters and arcobacters in broiler chickens

Chicken carcasses from a supermarket and from a poultry abattoir were examined using methods designed to isolate as many strains of campylobacters and related organisms as possible. Strains of arcobacter, but no campylobacters, were isolated from every carcass after enrichment. Campylobacter jejuni subsp. jejuni was isolated from all carcasses examined by direct plating and other Campylobacter -like strains were isolated from nine out of 15 abattoir carcasses by direct plating but not after enrichment. Only the Camp. jejuni subsp. jejuni strains could be identified to species level using a readily available identification scheme and/or a commercial identification kit. Examination of caecal contents from the 15 abattoir poultry yielded Camp. jejuni subsp. jejuni and Campylobacter -like strains from 15 and eight by direct plating, and from six and nine after enrichment, respectively. Four sites in the intestine of the abattoir birds (60 samples) were examined for arcobacters and only one strain was isolated. This indicates that arcobacters are probably not normal inhabitants of the poultry intestine. Poultry is a rich source of other campylobacteria besides the thermophilic Campylobacter spp.     

Lack of evidence for vertical transmission of Campylobacter spp. in chickens

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

Temporal variation and host association in the Campylobacter population in a longitudinal ruminant farm study

Campylobacter jejuni and C. coli were quantified and typed, using multilocus sequence typing (MLST), from fecal samples collected from a mixed cattle and sheep farm during summer. Cattle had a significantly higher prevalence than sheep (21.9% [74/338] and 14.0% [30/214], respectively), but both decreased over time. There were no differences in the average Campylobacter concentrations shed by cattle (600 CFU g(-1)) and sheep (820 CFU g(-1)), although sheep did show a significant temporal reduction in the number of Campylobacter organisms shed in their feces. A total of 21 different sequence types (STs) (97.7% C. jejuni, 2.3% C. coli) were isolated from cattle, and 9 different STs were isolated from sheep (40.6% C. jejuni, 59.4% C. coli). The Campylobacter population in cattle was relatively stable, and the frequencies of genotypes isolated showed little temporal variation. However, the composition of subtypes isolated from sheep did show significant temporal differences. The cattle and sheep consistently showed significant differences in their carriage of Campylobacter species, STs, and CCs despite the fact that both were exposed to the same farming environment. This work has highlighted the patterns of a Campylobacter population on a ruminant farm by identifying the existence of both temporal and between-host variations.     

Prevalence of Campylobacter spp. in cattle in Finland and antimicrobial susceptibilities of bovine Campylobacter jejuni strains

The study investigated the prevalence of Campylobacter spp. in Finnish cattle at slaughter and carcass contamination after slaughter. During the period January to December 2003, bovine rectal fecal samples (n=952) and carcass surface samples (n=948) from 12 out of 15 Finnish slaughterhouses were examined. In total, campylobacters were detected in 31.1% of fecal samples and in 3.5% of carcass surface samples. Campylobacter jejuni was isolated from 19.5%, Campylobacter coli from 2.2%, and presumptive Campylobacter hyointestinalis from 10.8% of fecal samples. Campylobacters were detected in 4.4% and 37.4% of the fecal samples examined both by direct culture and by enrichment (n=730), respectively, suggesting a low level of campylobacters in the intestinal content. A slightly increasing trend was observed in the overall prevalence of campylobacters towards the end of summer and autumn. Seventeen different serotypes were detected among the fecal C. jejuni isolates using a set of 25 commercial antisera for serotyping heat-stable antigens (Penner) of C. jejuni by passive hemagglutination. The predominant serotypes, Pen2 and Pen4-complex, were isolated from 52% of the fecal samples. Subtyping by pulsed-field gel electrophoresis (SmaI) yielded 56 and 20 subtypes out of 330 fecal and 70 carcass C. jejuni isolates, respectively. MICs of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline for 187 C. jejuni isolates were determined using a commercial broth microdilution method. Sixteen (9%) of the isolates were resistant to at least one of the antimicrobials tested. Resistance to nalidixic acid was most commonly detected (6%). No multiresistance was observed.     

Isolation of Campylobacter spp. from pigeon feces by a combined enrichment-filtration technique

A technique combining enrichment in Preston enrichment broth and direct filtration onto chocolate agar was used to isolate Campylobacter species from pigeon feces. Campylobacter jejuni was isolated from 106 of 200 samples tested; 105 strains were isolated by enrichment-filtration, and 84 strains were isolated by direct plating. Most of the strains grew after 48 h at 37 degrees C.     

Isolation of Campylobacter spp. from pigeon feces by a combined enrichment-filtration technique.

A technique combining enrichment in Preston enrichment broth and direct filtration onto chocolate agar was used to isolate Campylobacter species from pigeon feces. Campylobacter jejuni was isolated from 106 of 200 samples tested; 105 strains were isolated by enrichment-filtration, and 84 strains were isolated by direct plating. Most of the strains grew after 48 h at 37 degrees C.     

The isolation and prevalence of campylobacters from dairy cattle using a variety of methods

Faecal samples from 94 dairy cows and 42 calves in three different herds were examined by a variety of techniques for campylobacters. Cefoperazone amphotericin teicoplanin (CAT) agar, modified cefoperazone charcoal deoxycholate agar (mCCDA), Karmali agar, and membrane filtration onto blood agar, were used with and without enrichment in CAT broth. Seventy-nine percent of cattle in herd A carried campylobacters, compared with 40% and 37·5% of cattle in herds B and C, respectively. Most animals carried only one species of Campylobacter . Campylobacter hyointestinalis was isolated most frequently (32% animals positive) with Camp. fetus subsp. fetus and Camp. jejuni subsp. jejuni detected in 11% and 7% of animals, respectively. In addition, a novel biotype of Camp. sputorum was isolated from 60% of 47 cows tested in herd A. Direct plating detected only two of the total of 40 animals positive for campylobacter. Enrichment in CAT broth before membrane filtration onto blood agar or CAT agar were the most successful methods of plating. Campylobacter sputorum was isolated from CAT agar and blood agar but not from mCCDA or Karmali agar. Karmali agar incubated at 30 °C was especially effective for isolating Camp. fetus subsp. fetus .     

Characterization of urease-positive thermophilic Campylobacter subspecies by multilocus enzyme electrophoresis typing

Thirty-one urease-positive thermophilic Campylobacter (UPTC) isolates, including three reference strains (NCTC12892, NCTC12895 and NCTC12896), and three Campylobacter lari isolates, which were isolated from several countries and sources, were compared genotypically by using multilocus enzyme electrophoresis (MLEE). We examined allelic variation around seven enzyme loci, including the adenylate kinase, alkaline phosphatase, catalase, fumarase, malic enzyme, malate dehydrogenase, and L-phenylalanyl-L-leucine peptidase loci. MLEE typing revealed the presence of 23 different electrophoretic types (ETs) among the 31 UPTC isolates, and 14 isolates shared six electrophoretic profiles. Three different ETs were identified for the three C. lari isolates examined, and no ETs were shared by UPTC and C. lari isolates. Quantitative analyses were subsequently performed by using allelic variation data, and the results demonstrated that the mean genetic diversity was 0.655. In conclusion, MLEE demonstrated that the UPTC isolates examined are genetically hypervariable and form a cluster separate from the C. lari cluster.     

Frequency and spatial distribution of environmental Campylobacter spp

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

The occurrence of Campylobacter jejuni in raw cows' milk

Faeces and raw milk from individual cows were examined for the presence of Campylobacter jejuni . After drawing milk, the lactoperoxidase system was inactivated by raising the pH to 7.5. The organism was isolated from 22% of 904 faecal samples and from 4.5% of 904 milk samples. From laboratory experiments it could be concluded that inactivation of the lactoperoxidase system resulted in a better isolation of C. jejuni from raw cows' milk.     

Improved method for the isolation of Campylobacter jejuni and Campylobacter coli bacteriophages

A centrifugation and filtration method of isolating Campylobacter phages has been developed. Forty-nine Campylobacter phages were isolated from 272 effluent samples of which 42 produced lysis with Campylobacter jejuni strains and seven with C. coli strains. Phages were recovered from pig manure, abattoir effluents, human faeces, sewage and poultry manure. Phages were not isolated from water samples, cattle and sheep faeces or farm pasture soil.