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.     

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.     

Thermophilic Campylobacter from fecal samples of animals and their behavior in the ganglioside (GM1)-ELISA, Vero cell test and salt aggregation test

From 397 fecal specimens from apparently healthy and from diarrheic pigs, dogs, cats and cattle 59 strains (= 15%) of thermophilic Campylobacter (C.) spp. were isolated by culture. 39 strains were identified as C. coli and 18 as C. jejuni whereas 2 isolates could not be classified. None of the strains was found to be positive for cytotoxic enterotoxin in the GM1-ELISA. In the Vero-cell test 5 isolates showed a cytotoxic effect. The salt aggregation test (SAT) for indicating cell surface hydrophobicity was positive with 24 strains (5 C. jejuni, 19 C. coli). A correlation of isolation results with clinical manifestation could not be observed.     

Adaption of ELISA for the detection of Campylobacter antibodies and its application in seroepidemiological studies in sheep and cattle herds

ELISA was adapted for the study of antigenic relations among important Campylobacters and for the presence of anti-campylo-bacter antibodies in 394 sheep and 265 cattle. Rabbit anti-C. jejuni, C. coli, G. fetus subsp. fetus and C. laridis heat-stable antigen sera were evaluated against 29 Campylobacter strains and 6 other bacteria. Anti-C. jejuni and G. coli reacted strongly with homologous antigens and weakly with C. fetus subsp. fetus, C. laridis and C. fecalis antigens. C. fetus subsp. fetus serum reacted mainly with its homologous antigen. C. laridis serum showed closer reactivity to C. jejuni than to C. fetus subsp. fetus, C. coli and C. fecalis. Insignificant cross-reactions were observed with Y. enterocolitica, S. dublin and E. aerogenes heat-stable antigens, Ewes vaccinated with C. fetus subsp. fetus bacterin showed higher ELISA titers against C. fetus subsp. fetus antigens than non-vaccinated ewes or rams. Twenty-five percent of the vaccinated animals showed titers as low as 95 % of the non-vaccinated animals. In cattle the lowest antibody titers against C. fetus subsp. fetus, C. jejuni, C. coli and C. laridis antigens were exhibited by the precolostrum sera followed by the postcolostrum and adult sera. These studies demonstrated the applicability of the ELISA test in seroepidemiological investigations concerning the distribution and significance of Campylobacter antibodies in food animal sera.     

Lectin agglutination of thermophilic Campylobacter species

Agglutination tests with lectins indicated differences in the surface composition of strains of the thermophilic (optimum temperature 42 degrees C) Campylobacter species C. coli, C. faecalis, C. hyointestinalis, C. jejuni and C. laridis. All strains examined were agglutinated by the protein-reactive agglutinins of Mangifera indica (mango) and Persea americana (avocado) and a large proportion was also agglutinated by the carbohydrate-reactive lectins of Canavalia ensiformis (Jack bean) and Triticum vulgaris (wheat germ). Reactions with other lectins varied widely between strains, even of the same species and serotype. Lectin agglutination may be useful as a supplementary procedure for characterizing individual Campylobacter isolates for epidemiological purposes.     

Novel employment of lactate dehydrogenase release from porcine aortic endothelial cells (PAEC) as a quantitative marker of cytotoxic activity in thermophilic Campylobacter spp. from human faecal isolates, poultry and environmental sources

The aim of this study was to employ a novel cytotoxicity assay based on primary porcine aortic endothelial cells in combination with a lactate dehydrogenase release assay to quantitatively determine differences in cytotoxin production between Campylobacter jejuni, C. coli, C. lari and urease-positive thermophilic campylobacters (UPTC), isolated from human faeces, animals and environmental sources. Campylobacter isolates totalling 34 and comprising of C. jejuni (n = 24) C. coli (n = 5) and UPTC (n = 4) and C. lari (n = 1) were analysed. The cytotoxic response ranged from 32.15 to 64.47% and 33.08 to 59.41%, for C. jejuni from chicken and human isolates, respectively and there was no statistically significant difference (P > 0.05) in cytotoxic response between C. jejuni isolated from humans and chicken isolates (50.78% versus 50.55% cytotoxicity, respectively). However, there was a difference in response between C. jejuni and C. coli isolated from chickens (50.78% versus 33.22% cytotoxicity, respectively). The greatest cytotoxic response was obtained with the UPTC group of organisms examined (n = 4 isolates) (mean cytotoxic response = 57.11% cytotoxicity. Employment of this cytotoxin assay may help identify virulent strains in poultry that could potentially proceed to cause clinical problems for humans and thus intervention measures targeted at the reduction or elimination of such specific strains, may be sought.     

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.     

Prevalence of thermophilic campylobacters in crows (Corvus levaillantii, Corvus corone) and serogroups of the isolates

A total of 500 fecal droppings of crows collected from a seashore of an ocean bay and from a cemetery on a hill surrounded by a forest were examined for thermophilic campylobacters, and the Skirrow's biovars and Penner's serogroups of the isolates were determined. The organisms were isolated from 169 (62.6%) of 270 seashore crow samples and 106 (46.1%) of 230 cemetery crow samples. During the investigation period from May 1986 to April 1987, the monthly isolation rate of thermophilic campylobacters in the seashore crow varied from 32.0 to 85.0%. C. jejuni, C. coli, and C. laridis were isolated from 150, 21 and 14 samples, respectively. In case of the cemetery crow, the monthly isolation rate varied from 20.0 to 75.0%, and C. jejuni, C. coli, and C. laridis were detected from 80, 12 and 16 samples, respectively. Among 192 strains of C. jejuni selected from 98 seashore and 57 cemetery crow samples, 106 (93.0%) of 114 seashore crow strains and 69 (88.5%) of 78 cemetery crow strains were identified as Skirrow's biovar I. Of 192 strains of C. jejuni serogrouped, 169 strains were classified into 20 serogroups. The Penner's serogroup 2, one of common serogroups among poultry and human isolates in Japan, was the most predominant in crow strains.     

Plasmid profiles of six species of Campylobacter from human beings, swine, and sheep

Twenty-four isolates representing 6 species of Campylobacter were screened for plasmids. A large plasmid with an approximate molecular weight of 38 Mdal was detected in 5 C jejuni isolates originally recovered from diarrheic human beings, in one isolate of C coli recovered from diarrheic pigs, and in 1 isolate of C sputorum ssp mucosalis and 2 isolates of C hyointestinalis recovered from pigs with proliferative enteritis. One isolate of C coli and 1 isolate of C hyointestinalis contained an additional smaller plasmid with an approximate molecular weight of 1.6 Mdal; this plasmid was partially mapped by restriction endonuclease digestion. Fifteen Campylobacter isolates contained no detectable plasmids: 2 C coli, 2 C sputorum ssp mucosalis, 2 C fecalis, 1 C fetus ssp fetus, and 8 C hyointestinalis isolates. In summary, 37.5% of the Campylobacter isolates contained a 38-Mdal plasmid, with 8% having both 38 Mdal and 1.6-Mdal plasmids; 62.5% contained no detectable plasmids.     

Campylobacter jejuni, Campylobacter coli, and cytolethal distending toxin (CDT) genes in common teals (Anas crecca)

To evaluate the presence of Campylobacter spp. and related cdt genes, cloacal swabs were collected from 70 common teals (Anas crecca) and analyzed by culture methods and polymerase chain reaction. In addition, C. jejuni were examined also for the presence of wlaN gene. This is believed to be the first report of Campylobacter spp. in common teal and our results confirm the very common occurrence of C. jejuni (n=40) and C. coli (n=13) in waterfowls. Furthermore, the cdt genes were frequently present in both C. jejuni and C. coli isolated. Moreover, seven C. jejuni isolates carried also the wlaN gene which is presumably involved in the expression of ganglioside mimics in Guillain-Barré syndrome.     

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.     

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.     

The occurrence and characterization of Campylobacter jejuni and C. coli in organic pigs and their outdoor environment

The occurrence and species distribution of thermophilic Campylobacter was investigated in organic outdoor pigs. An increased exposure of outdoor pigs to C. jejuni from the environment may cause a shift from a normal dominance of C. coli to more C. jejuni, which may imply a concern of reduced food safety. Bacteriological methods for determination of Campylobacter excretion level were combined with colony-blot hybridization and real-time PCR for specific detection of C. jejuni in pigs. Campylobacter was isolated from pigs (n=47), paddock environment (n=126) and wildlife (n=44), identified to species by real-time PCR and sub-typed by serotyping (Penner) and pulse-field gel electrophoresis (PFGE) genotyping. All pigs excreted Campylobacter (10(3)-10(7) CFU g(-1) faeces) from the age of 8-13-weeks old. C. jejuni was found in 29% of pigs in three consecutive trials and always in minority to C. coli (0.3-46%). C. jejuni and C. coli were isolated from 10% and 29% of the environmental samples, respectively, while crow-birds and rats harboured C. jejuni. Individual pigs hosted several strains (up to nine serotypes). The paddock environment was contaminated with C. coli serotypes similar to pig isolates, while most of the C. jejuni serotypes differed. C. jejuni isolates of different origin comprised few similar serotypes, just one identical genotype was common between pigs, environment and birds. In conclusion, the occurrence of C. jejuni varied considerably between the three groups of outdoor pigs. Furthermore, transfer of C. jejuni to the outdoor pigs from the nearby environment was not predominant according to the subtype dissimilarities of the obtained isolates.     

Antimicrobial resistance in Campylobacter spp. isolated from Ontario sheep flocks and associations between antimicrobial use and antimicrobial resistance

The objectives of this study were to determine the prevalence of antimicrobial resistance (AMR) in faecal Campylobacter spp. from lambs and adult sheep and associations between antimicrobial use (AMU) and AMR. A total of 275 faecal samples collected during initial and final visits from 51 sheep flocks, including one feedlot, across southern Ontario were tested for the presence of Campylobacter spp. Campylobacter jejuni was detected in 52% (143/275) of the faecal samples, Campylobacter coli in 7% (19/275), Campylobacter lari in 1% (2/275) and 2% (4/275) were non-speciated Campylobacter. Broth microdilution was used to test antimicrobial susceptibility of 162 isolates to nine antimicrobials. Campylobacter jejuni isolates (n = 142) were resistant to tetracycline (39%), ciprofloxacin (4%), nalidixic acid (4%) and telithromycin (1%). C. coli isolates (n = 19) were resistant to tetracycline (74%), and azithromycin, clindamycin, erythromycin, and telithromycin (5%). The C. lari isolate displayed resistance to nalidixic acid. No statistically significant associations were found between AMU and AMR during multivariate modelling in this study.     

Isolation of Campylobacter jejuni and Campylobacter coli from the gall bladder samples of sheep and identification by polymerase chain reaction

In this study, 100 gall bladder samples of sheep slaughtered at an abattoir in Elazi? province were examined for Campylobacter jejuni and Campylobacter coli by culture and polymerase chain reaction (PCR). Preston Campylobacter Agar supplemented with 7% horse blood and Preston Selective Supplement (Oxoid, Hampshire, UK) were used for isolation of the agents. Campylobacter spp. were isolated in 66 samples, and they were identified as 34% C. jejuni and 32% C. coli. A multiplex PCR based upon the use of ceuE gene-specific primers was applied on DNA samples extracted from C. jejuni and C. coli isolates. All C. jejuni and C. coli strains that were positive by culture were also detected to be positive by PCR. This study shows that PCR can be used an alternative, rapid and sensitive test for the identification of C. jejuni and C. coli which threaten human and animal health.     

Phenotypes and genotypes of campylobacter strains isolated after cleaning and disinfection in poultry slaughterhouses

Campylobacter is responsible for human bacterial enteritis and poultry meat is recognised as a primary source of infection. In slaughterhouses, cleaning and disinfection procedures are performed daily, and it has been suggested that disinfectant molecules might select for antibiotic resistant strains if shared targets or combined resistance mechanisms were involved. The aim of the study was to investigate if cleaning and disinfection procedures in poultry slaughterhouses select for antibiotic resistance in Campylobacter jejuni and C. coli and to determine the genotypes of isolates collected after cleaning and disinfection. Nine sampling visits were made to four French slaughterhouses. Samples were collected from transport crates and equipment surfaces, before and after cleaning and disinfection. Minimal inhibitory concentrations of the recovered C. jejuni and C. coli isolates to six antibiotics and two disinfectants were measured. The C. jejuni isolates collected from equipment surfaces after cleaning and disinfection were subjected to PCR-RFLP typing. Twenty-five C. jejuni isolates and 1 C. coli were recovered from equipment surfaces after cleaning and disinfection during five visits to three different slaughterhouses. Those isolates did not show an increased resistance to the tested antibiotics compared to isolates collected before cleaning and disinfection. Only one or two genotypes were recovered after cleaning and disinfection during single visits to each slaughterhouse. This observation suggests that such genotypes may be particularly adapted to survive cleaning and disinfection stress. Understanding the survival mechanisms of Campylobacter should facilitate the implementation of better-targeted strategies and reduce the public health burden associated with Campylobacter infection.     

Prevalence of Campylobacter jejuni and its resistance to antibiotics in poultry in the Czech Republic

Thermotolerant Campylobacter spp., in particular Campylobacter jejuni, are among the most frequently identified pathogens, found to be causing human gastrointestinal infections in Europe, with the Czech Republic being no exception. The presented work aimed at assessing results of the first nationwide monitoring of prevalence and antibiotic resistance of Campylobacter spp. in broiler flocks in the Czech Republic, including a comparison of antibiotic resistance of C. jejuni isolates collected from poultry and the human community. The monitoring was carried out in poultry slaughterhouses in 2006 and 2007. From broilers, cloacal swabs were collected and examined. The human isolates of C. jejuni were acquired from rectal swabs in community patients with diarrhoeal diseases. Suspected isolates of both animal and human origin were confirmed by the PCR methods. Antibiotic resistance to selected anti-microbial agents was tested by the microdilution method. In the monitored period, the prevalence of thermotolerant Campylobacter spp. in broilers in the Czech Republic reached almost 50%. In 2006, C. jejuni was detected in 46% and Campylobacter coli in 3% of the tested samples. In 2007, C. jejuni was found in 43% and C. coli in 2% of the samples. The results of anti-microbial susceptibility testing of C. jejuni showed higher resistance in animals when compared with humans. The only exception was tetracycline with higher resistance in isolates of human origin. The highest resistance detected was to quinolone antibiotics. Resistance to oxolinic acid was 77% in animal and 60% in human isolates, to ciprofloxacin 72% in isolates from poultry and 55% in those from humans. In ampicillin, 26% of poultry isolates and 16% of human isolates were resistant. Moreover, 9% of animal isolates demonstrated resistance to streptomycin, undetected in human isolates. In erythromycin, resistance was found in 6% of poultry and 1% of human isolates.     

Campylobacter in the dog: a clinical and experimental study

Faecal samples from 54 dogs with diarrhoea and 54 control dogs were cultured for Campylobacter, Salmonella and Yersinia species and controlled for enteric viruses. The campylobacter were identified as either C jejuni/coli or C upsaliensis. In the diarrhoeic group 16 dogs (29.6 per cent) were positive for campylobacter, 10 C upsaliensis and six C jejuni/coli. Concomitant infection with parvovirus was evident in six of the dogs with diarrhoea and campylobacter-positive faecal cultures. In the control group 13 dogs (24.1 per cent) were positive for campylobacter; three of the isolates were C upsaliensis and six C jejuni/coli. Four isolates could not be identified. The most prominent clinical findings in naturally occurring cases were an acute onset of vomiting (12 of 16), diarrhoea (16 of 16) which was often haemorrhagic (nine of 16) and a raised rectal temperature. Dogs were infected experimentally with both C jejuni (three dogs) and C upsaliensis (three dogs). The challenge strains could be identified in faecal samples from all the dogs, but clinical signs of diarrhoea were seen in only one dog infected with C jejuni. Soft faeces was passed by one dog infected with C upsaliensis. It is concluded that C jejuni/coli or C upsaliensis are either primary pathogens or, after predisposing factors such as virus infections, act as secondary pathogens. It also seems probable that Campylobacter species are present in the intestinal flora of the normal dog.     

Prevalence of thermophilic campylobacter infections in humans, chickens and crows in Morogoro, Tanzania

Prevalence of thermophilic Campylobacter infections in humans, chickens and crows was determined in a cross-sectional study that was carried out in urban and rural areas of Morogoro region, Tanzania during the period of January 2003 to December 2004. A total of 632 human stool samples, 536 cloacal swabs from local and broiler chickens and 22 intestinal contents from crows were screened for presence of thermophilic campylobacters using Skirrow's protocol. Representative Campylobacter jejuni isolates from human and chicken samples were also analysed by polymerase chain reaction (PCR) as a definitive identification method. The overall prevalence of thermophilic campylobacters was 9.3% (95% CI: 7.2-11.9), 69.8% (95% CI: 65.7-73.6) and 72.7% (95% CI: 49.8-89.3) in humans, chickens and crows respectively. In humans, 59 thermophilic campylobacters were isolated of which 96.6% were C. jejuni and 3.4%Campylobacter coli. There was a significantly (P<0.001) higher prevalence in young individuals (16%) than in adults (7%). Of 341 isolates from chickens, 91.2% were C. jejuni and 8.8% were C. coli. A significantly (P<0.05) higher infection rate was observed in rural local chicken (76%) than in broilers (60%). In crows, of 16 isolates, 93.8% were C. jejuni and 6.2% were C. coli. Definitive identification of C. jejuni by PCR revealed positive results in 74.1% of 243 analysed isolates. Findings in this study indicate high prevalence of thermophilic campylobacters in humans, chickens and crows in Morogoro, and a higher infection rate of C. jejuni than that of C. coli in different animal species. Age of humans and location of chickens were identified as risk factors for thermophilic Campylobacter infections. Positive isolates to biochemical tests that indicated negative results on PCR indicates the additional value of PCR for definitive diagnosis of C. jejuni.     

Dose response and organ invasion of day-of-hatch Leghorn chicks by different isolates of Campylobacter jejuni

Colonization of the ceca and organ invasion by different isolates of Campylobacter jejuni were investigated in day-of-hatch leghorn chicks. This model of Campylobacter colonization of the ceca demonstrates that 1) day-of-hatch birds do not naturally contain cecal Campylobacter, 2) ceca can be colonized with C. jejuni by oral gavage and not by cloacal inoculation; 3) C. jejuni can be recovered from the ceca up until at least 7 days postinoculation, 4) cecal colonization occurs when as little as 10(2) colony-forming units is orally inoculated into chicks, and 5) different C. jejuni isolates vary both in their ability to colonize the ceca and in their ability to invade the liver. These studies demonstrate that we have a working animal model for Campylobacter colonization for day-of-hatch chicks. This animal model is being used to examine intervention strategies such as vaccines by which Campylobacter can be reduced or removed from the food animal.