Search

Introduction. In May-June 2018, an outbreak of campylobacteriosis involved students and school staff from kindergartens and primary schools in Pescara, southern Italy. Aim. We present details of the epidemiological and microbiological investigation, and the findings of the analytical study, as well as the implemented control measures. Methodology. To identify possible risk factors associated with the observed outbreak, a case control study was conducted using a questionnaire to collect information on the date of symptoms onset, type and duration of symptoms, type of healthcare contact, school attendance, and food items consumed at school lunches during the presumed days of exposure. Attack rates were calculated for each date and school. Logistic regression models were used to estimate the odds ratios of being a case and the odds of illness by food items consumed, respectively. Moreover, we carried out a comparative genomic analysis using whole genome multilocus sequence typing (wgMLST) of Campylobacter jejuni strains isolated during the outbreak investigation to identify the source of the outbreak. Results. Overall, 222 probable cases from 21 schools were identified, and C. jejuni was successfully isolated from 60 patients. The meals in the schools involved were provided by two cooking centres managed by a joint venture between two food companies. Environmental and food sampling, epidemiological and microbiological analyses, as well as a case control study with 176 cases and 62 controls from the same schools were performed to identify the source of the outbreak. The highest attack rate was recorded among those having lunch at school on 29 May (7.8 %), and the most likely exposure was 'caciotta' cheese (odds ratio 2.40, 95 % confidence interval 1.10-5.26, P =0.028). C. jejuni was isolated from the cheese, and wgMLST showed that the human and cheese isolates belonged to the same genomic cluster, confirming that the cheese was the vehicle of the infection. Conclusion. It is plausible that a failure of the pasteurization process contributed to the contamination of the cheese batches. Timely suspension of the catering service and summer closure of the schools prevented further spread.

Purpose: This study was aimed at investigating the occurrence and genetic mechanisms of resistance to ciprofloxacin, tetracycline and erythromycin in clinical isolates of Campylobacter jejuni recovered from human cases of acute gastroenteritis in Turkey., Methodology: MIC values of each antibiotic were determined with the epsilometer test (E-test). Resistance genes/mutations were first screened by PCR and analysed by subsequent DNA sequencing., Results: From a total of 152 C. jejuni isolates tested, 113 (74.3%), 38 (25%) and 9 (5.9%) were found to be resistant to ciprofloxacin, tetracycline and erythromycin, respectively. Sequence analysis of ciprofloxacin-resistant isolates showed that all resistant strains (n=113) carried Thr-86-Ile substition in the gyrA gene, which is the most frequently observed mutation in fluoroquinolone-resistant Campylobacter. All of the tetracycline-resistant isolates (n=38) carried the tetO gene. All of the erythromycin-resistant isolates (n=9) harboured the point mutation A2075G in the 23S rRNA gene, which is the most common mutation conferring macrolide resistance in C. jejuni., Conclusion: The phenotypic susceptibility testing results were found to agree well with those obtained by genetic detection methods for the C. jejuni isolates tested. The findings of this study showed a very high level of resistance to ciprofloxacin and to a lesser extent to tetracycline while resistance to erythromycin remained at a low level. Thus, erythromycin may be considered as the first choice for treatment of Campylobacter infections in this geographical region when indicated.

Purpose and methodology.Campylobacter jejuni is a major zoonotic pathogen that causes food-borne gastroenteritis worldwide. However, there are only a few studies available that have molecularly characterized C. jejuni strains isolated in Brazil. The aim of this study was to genotype 111 C. jejuni strains isolated from sick humans (43), monkey faeces (19), chicken faeces (14), chicken meat (33) and sewage (2) between 1996 and 2016 in Brazil using flaA-SVR (short variable region) sequencing and PFGE. Furthermore, the presence of 16 virulence genes was analysed by PCR., Results: Using PFGE and flaA-SVR sequencing, the 111 C. jejuni strains studied were grouped into three and two clusters, respectively, and some strains of different origin presented a similarity of ≥80 %. In total, 35 flaA-SVR alleles were detected. Alleles gt45, gt49 and gt57 were the most prevalent, in contrast with those frequently described in the PubMLST database. All 111 C. jejuni strains contained the genes flaA, flhA, cadF, docA, cdtA, cdtB, cdtC, iamA, ciaB, sodB, dnaJ, pldA, racR and csrA. The wlaN gene was detected in 11 strains (9.9 %), and the virB11 in just one strain (0.9 %)., Conclusions: In conclusion, the pathogenic potential of the C. jejuni strains studied was highlighted by the high frequency of the majority of the virulence genes searched. The flaA-SVR sequencing and PFGE results showed that some of the strains studied presented a high genotypic similarity, suggesting potential for transmission between animal sources and humans in this country. Altogether, the results characterize further C. jejuni isolates from Brazil, an important producer and exporter of chicken meat.

Given that Campylobacter jejuni is recognized as the most common cause of bacterial gastroenteritis worldwide, recent findings showing comparable levels of Campylobacter concisus in patients with gastroenteritis would suggest that this bacterium is clinically important. The prevalence and abundance of Campylobacter concisus in stool samples collected from patients with acute gastroenteritis was examined using quantitative real-time PCR. The associated virulence determinants exotoxin 9 and zonula occludens toxin DNA were detected for Campylobacter concisus-infected samples using real-time PCR. Campylobacter concisus was detected at high prevalence in patients with gastroenteritis (49.7 %), higher than that observed for Campylobacter jejuni (∼5 %). The levels of Campylobacter concisus were putatively classified into clinically relevant and potentially transient subgroups based on a threshold developed using Campylobacter jejuni levels, as the highly sensitive real-time PCR probably detected transient passage of the bacterium from the oral cavity. A total of 18 % of patients were found to have clinically relevant levels of Campylobacter concisus, a significant number of which also had high levels of one of the virulence determinants. Of these patients, 78 % were found to have no other gastrointestinal pathogen identified in the stool, which strongly suggests a role for Campylobacter concisus in the aetiology of gastroenteritis in these patients. These results emphasize the need for diagnostic laboratories to employ identification protocols for emerging Campylobacter species. Clinical follow-up in patients presenting with high levels of Campylobacter concisus in the intestinal tract is needed, given that it has been associated with more chronic sequelae.

Increasing numbers of Campylobacter hyointestinalis have been isolated from humans and animals with gastroenteritis, although the virulence mechanism of this species remains largely unknown. Here, we show that C. hyointestinalis isolated from a patient with diarrhoea in Thailand produced a novel variant of cytolethal distending toxin (CDT). Sequencing of a 13 965 bp genomic region of C. hyointestinalis carrying the genes coding for Ch-CDT revealed three ORFs of 798, 804 and 537 bp, which code for the Ch-CdtA, Ch-CdtB and Ch-CdtC subunits, respectively. The deduced amino acid sequence of Ch-CdtA showed ∼38.9 % homology with the CdtA of Campylobacter coli, but sequences of Ch-CdtB and Ch-CdtC were homologous to CdtB (65.7 %) and CdtC (33.1 %) of Campylobacter upsaliensis, respectively. Filter-sterilized sonic lysate of C. hyointestinalis demonstrated distension and death of HeLa cells by arresting the cell cycle at the G(2)/M phase and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. Rabbit antiserum raised against recombinant Ch-CdtB was not reactive against the recombinant CdtB protein of Campylobacter jejuni. A reconstituted Ch-CDT holotoxin prepared using each of the recombinant subunit proteins demonstrated distension and death of HeLa cells, suggesting that the C. hyointestinalis isolate indeed produced functionally active Ch-CDT. Furthermore, the immunological distinctiveness of the Ch-CDT produced by C. hyointestinalis and the increasing prevalence of the species in patients and animals with gastroenteritis suggest that this species may be an important emerging zoonotic pathogen.

We report the isolation of a novel helicobacter isolated from the caecum of the Siberian hamster (Phodopus sungorus). Sequence analysis showed 97% sequence similarity to Helicobacter ganmani. In addition, we report the co-infection of these Siberian hamsters with a Campylobacter sp. and a second Helicobacter sp. with 99% sequence similarity to Helicobacter sp. flexispira taxon 8 (Helicobacter bilis), a species isolated previously from patients with bacteraemia. Gross necropsy and histopathology did not reveal any overt pathological lesions of the liver and gastrointestinal tract that could be attributed to the Helicobacter or Campylobacter spp. infections. This is the first helicobacter to be identified in the Siberian hamster and the first report of co-infection of Helicobacter spp. and Campylobacter sp. in asymptomatic Siberian hamsters., (© 2015 The Authors.)

This study aimed to investigate the prevalence of Campylobacter jejuni in potential contamination sources that are not regularly monitored such as free-living urban pigeons and crows, dogs, cats and urban environmental water and to assess the possible impact on the epidemiology of campylobacteriosis in children using multilocus sequence typing (MLST). Campylobacter spp. were detected in 36.2 % of faecal samples of free-living urban birds and in 40.4 % of environmental water samples. A low prevalence of Campylobacter spp. was detected in dogs and cats, with 7.9 and 9.1 %, respectively. Further identification of isolates revealed that environmental water and pet samples were mostly contaminated by other Campylobacter spp. than C. jejuni, whereas C. jejuni was the most prevalent species in faecal samples of free-living birds (35.4 %). This species was the dominant cause of campylobacteriosis in children (91.5 %). In addition, the diversity of C. jejuni MLST types in free-living birds and children was investigated. Clonal complex (CC) 179 was predominant among free-living urban birds; however, only two isolates from children were assigned to this CC. One dog and one child isolate were assigned to the same clonal complex (CC48) and sequence type (ST) 918. The dominant two clonal complexes among the child clinical isolates (CC353 and CC21) were not detected among C. jejuni strains isolated from environmental sources examined in this study. As only two CCs were shared by environmental and child C. jejuni isolates and a high number of novel alleles and STs were found in C. jejuni isolated from free-living urban birds and environmental water, there is probably only a limited link between urban environmental sources and campylobacteriosis in children, particularly in rather cold climatic conditions., (© 2014 The Authors.)

Although Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of human gastrointestinal diseases, other Campylobacter species are also involved in human and animal infections. In this study, we developed a cytolethal distending toxin (cdt) gene-based PCR-RFLP assay for the detection and differentiation of C. jejuni, C. coli, C. fetus, C. hyointestinalis, C. lari, C. helveticus and C. upsaliensis. Previously designed common primers, which can amplify the cdtB gene of C. jejuni, C. coli and C. fetus, were used for detecting seven Campylobacter species and differentiating between them by restriction digestion. The PCR-RFLP assay was validated with 277 strains, including 35 C. jejuni, 19 C. coli, 20 C. fetus, 24 C. hyointestinalis, 13 C. lari, 2 C. helveticus, 22 C. upsaliensis, 3 other Campylobacter spp. and 17 other species associated with human diseases. Sensitivity and specificity of the PCR-RFLP assay were 100 % except for C. hyointestinalis (88 % sensitivity). Furthermore, the PCR-RFLP assay successfully detected and differentiated C. jejuni, C. coli and C. fetus in clinical and animal samples. The results indicate that the PCR-RFLP assay is useful for the detection and differentiation of seven Campylobacter species important for human and animal diseases.

Methyl-accepting chemotaxis proteins (MCPs), also termed transducer-like proteins (Tlps), serve as sensors in bacterial chemotactic signalling, and detect attractants and promote bacterial movement towards suitable sites for colonization. Campylobacter jejuni is a leading cause of human enteritis, but the mechanisms responsible for bacterial chemotaxis and early colonization in the jejunum of hosts are poorly understood. In the present study, we identified several types of bile and sodium deoxycholate (SDC) acting as chemotactic attractants of C. jejuni strain NCTC 11168-O in vitro, in which SDC was the most efficient chemoattractant. In mice with bile duct ligation, the wild-type strain displayed a markedly attenuated ability for colonization. Blockage of Tlp3 or Tlp4 protein with antibody or disruption of the tlp3 or tlp4 gene (Δtlp3 or Δtlp4) caused a significant inhibition of SDC-induced chemotaxis and attenuation for colonization on jejunal mucosa in mice of the bacterium. Disruption of both the genes (Δtlp3/Δtlp4) resulted in the absence of bacterial chemotaxis and colonization, while the tlp-gene-complemented mutants (CΔtlp3 and CΔtlp4) reacquired these abilities. The results indicate that SDC is an effective chemoattractant for C. jejuni, and Tlp3 and Tlp4 are the SDC-specific sensor proteins responsible for the bacterial chemoattraction.

Campylobacter jejuni is the most frequent cause of severe gastroenteritis in the developed world. The major symptom of campylobacteriosis is inflammatory diarrhoea. The molecular mechanisms of this infection are poorly understood compared to those of less frequent disease-causing pathogens. In a previous study, we identified C. jejuni proteins that antibodies in human campylobacteriosis patients reacted with. One of the immunogenic proteins identified (Cj0917) displays homology to carbon starvation protein A (CstA) from Escherichia coli, where this protein is involved in the starvation response and peptide uptake. In contrast to many bacteria, C. jejuni relies on amino acids and organic acids for energy, but in vivo it is highly likely that peptides are also utilized, although their mechanisms of uptake are unknown. In this study, Biolog phenotype microarrays have been used to show that a ΔcstA mutant has a reduced ability to utilize a number of di- and tri-peptides as nitrogen sources. This phenotype was restored through genetic complementation, suggesting CstA is a peptide uptake system in C. jejuni. Furthermore, the ΔcstA mutant also displayed reduced motility and reduced agglutination compared to WT bacteria; these phenotypes were also restored through complementation. Murine dendritic cells exposed to UV-killed bacteria showed a reduced IL-12 production, but the same IL-10 response when encountering C. jejuni ΔcstA compared to the WT strain. The greater Th1 stimulation elicited by the WT as compared to ΔcstA mutant cells indicates an altered antigenic presentation on the surface, and thus an altered recognition of the mutant. Thus, we conclude that C. jejuni CstA is important not only for peptide utilization, but also it may influence host-pathogen interactions.

Active efflux has an important role in the antimicrobial resistance of Campylobacter jejuni and Campylobacter coli. The effects of two putative efflux pump inhibitors (EPIs), phenylalanine-arginine β-naphthylamide and 1-(1-naphthylmethyl)-piperazine, and the effects of inactivation of the cmeB,cmeF and cmeR genes on resistance to a broad range of antimicrobials were studied using the broth microdilution method. The antimicrobials tested in C. jejuni and C. coli were the biocides triclosan, benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and trisodium phosphate, along with the anionic surfactant SDS and the antibiotics erythromycin and ciprofloxacin. Both EPIs partially reversed the resistance to all of these antimicrobials. Differences between these EPIs were seen for substrate preference and reductions in MIC. The MICs of the antimicrobials were reduced in the cmeB and cmeF mutants and increased in the cmeR mutant, with few exceptions. Both of these putative EPIs further decreased the MICs of the antimicrobials in these mutant strains. These data confirm that active efflux is an important mechanism in biocide resistance in C. jejuni and C. coli. At least one non-CmeABC efflux system or reduced uptake is responsible for resistance to biocides.

We describe a case of gastroenteritis caused by Campylobacter concisus. The pathogenic potential of C. concisus has yet to be elucidated. Recent studies indicate an association with enteric disease in immunocompromised patients and inflammatory bowel disease in children. Molecular identification methods may be necessary for identifying certain Campylobacter species because of phenotypic similarity., (© 2012 SGM)

Campylobacter is an important cause of foodborne gastroenteritis. We determined the occurrence of Campylobacter jejuni and Campylobacter coli, using culture-based methods and PCRs targeting virulence-associated genes (VAGs) among children aged ≤14 years who were treated for diarrhoea at emergency rooms in northeastern Brazil. Genomic DNA was extracted directly from stool samples collected from 366 children. A questionnaire was also applied to qualify the clinical conditions presented by each child at the time of admission. C. jejuni and C. coli were detected in 16.4 % (60/366) and 1.4 % (5/366) of the diarrhoeal samples, respectively, by PCR, a much higher proportion than that detected by conventional methods. C. jejuni VAGs were detected in the following proportions of hipO-positive samples: ciaB, 95 % (57/60); dnaJ, 86.7 % (52/60); racR, 98.3 % (59/60); flaA, 80 % (48/60); pldA, 45 % (27/60); cdtABC, 95 % (57/60); and pVir 0 % (0/60). Particular symptoms, such as blood in faeces, vomiting, fever, and/or abdominal pain, were not associated with detection of C. jejuni nor were they associated with any particular VAG or combination of VAGs (P>0.05). C. jejuni and its VAGs were detected in a substantial proportion of the children admitted. Further efforts shall be directed towards elucidating whether these genetic factors or their expressed proteins play a role in Campylobacter pathogenesis.

Prairie dogs (Cynomys ludovicianus) are used to study the aetiology and prevention of gallstones because of the similarities of prairie dog and human bile gallstone composition. Epidemiological and experimental studies have suggested a connection between infection with Helicobacter species and cholesterol cholelithiasis, cholecystis and gallbladder cancer. Ten of the 34 prairie dogs in this study had positive Helicobacter species identified by PCR using Helicobacter genus-specific primers. Ten of 34 prairie dogs had positive Campylobacter species identified in the intestine by PCR with Campylobacter genus-specific primers. Six Helicobacter sp. isolates and three Campylobacter sp. isolates were identified taxonomically by 16S rRNA gene analysis. The prairie dog helicobacters fell into three clusters adjacent to Helicobacter marmotae. On the basis of 16S rRNA gene sequence analysis, three strains in two adjacent clusters were included in the species H. marmotae. Three strains were only 97.1 % similar to the sequence of H. marmotae and can be considered a novel species with the provisional designation Helicobacter sp. Prairie Dog 3. The prairie dog campylobacters formed a single novel cluster and represent a novel Campylobacter sp. with the provisional designation Campylobacter sp. Prairie Dog. They branched with Campylobacter cuniculorum at 96.3 % similarity and had the greatest sequence similarity to Campylobacter helveticus at 97.1 % similarity. Whether H. marmotae or the novel Helicobacter sp. and Campylobacter sp. identified in prairie dogs play a role in cholesterol gallstones or hepatobiliary disease requires further studies.

Campylobacter spp. are common causes of gastrointestinal infections. Campylobacter fetus is a much rarer pathogen in humans, and usually causes bacteraemia and systemic complications in patients with predisposing conditions. We report a case of spondylodiscitis caused by C. fetus subsp. fetus as revealed by vertebral biopsy culture. This identification was confirmed by sequencing the 16S rRNA gene and by phylogenetic analysis. Treatment consisted of 6 weeks antimicrobial therapy combined with a strict initial immobilization, followed by a re-education program. The patient's recovery was uneventful.

To obtain the genotype and antimicrobial susceptibility profiles of Campylobacter jejuni isolates from north China, 93 C. jejuni isolates (56 isolates from patients with diarrhoea, 7 isolates from Guillain-Barré syndrome patients and 30 isolates from chicken stools) were selected for multilocus sequence typing (MLST), PFGE and drug resistance testing. A total of 49 sequence types (STs) were identified from the entire panel of 93 C. jejuni isolates. Fifty-six isolates belonged to 14 clonal complexes, while 37 isolates could not be assigned to any known clonal complex. The most frequently observed clonal complexes were ST-21 (11 isolates), ST-353 (10 isolates) and ST-443 (6 isolates). Fifty-three PFGE SmaI patterns were identified among 93 isolates. No erythromycin-, gentamicin- or streptomycin-resistant isolates were found among the 44 strains isolated in 2008. Resistance to nalidixic acid, levofloxacin and ciprofloxacin was observed in 100 % (44/44) of the tested isolates. This study has shown the genetic characteristics of C. jejuni isolates in north China. In addition, overlapping clonal groups were defined by both MLST and PFGE for C. jejuni human and chicken isolates.

A common problem of both conventional and real-time PCR assays is failure of DNA amplification due to the presence of inhibitory substances in samples. In view of this, our aim was to develop and evaluate internal amplification controls (IACs) for use with an existing duplex real-time PCR assay for Campylobacter coli and Campylobacter jejuni. Both competitive and non-competitive IACs were developed and evaluated. The competitive approach involved a DNA fragment of the coding region of the fish viral haemorrhagic septicaemia virus, flanked by the mapA PCR primers, whilst the non-competitive approach utilized an extra set of universal 16S rDNA primers. Both IAC-PCR assay types were evaluated using cultures of Campylobacter and chicken caecal content samples. Both IACs were sensitive to caecal inhibitors, making them suitable for detecting inhibition which could lead to false-negatives. Results showed that both IACs at optimum concentrations worked well without reducing the overall sensitivity of the PCR assay. Compared to culture, the optimized competitive IAC-PCR assay detected 45/47 positives (sensitivity 93.6 %, specificity 80.1 %); however, it had the advantage over culture in that it could detect mixed infections of C. coli and C. jejuni and was capable of giving a result for a sample within a day.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. The aims of the present blinded study were to measure and compare the in vivo properties of 40 serotyped, biotyped and genotyped C. jejuni isolates from different sources and genetic makeup. An 11-day-old chick embryo lethality assay, which measured embryo deaths and total viable bacteria over 72 h following inoculation of bacteria into the chorioallantoic membrane, revealed a spectrum of activity within the C. jejuni strains. Human and chicken isolates showed similar high virulence values for embryo deaths while the virulence of the bovine isolates was less pronounced. A one-way ANOVA comparison between the capacity of the strains to kill the chick embryos after 24 h with cytotoxicity towards cultured CaCo-2 cells was significant (P=0.025). After inoculation with a Campylobacter strain, mouse ligated ileal loops were examined histologically and revealed degrees of villous atrophy, abnormal mucosa, dilation of the lumen, congestion and blood in lumen, depending on the isolate examined. A 'total pathology score', derived for each C. jejuni strain after grading the pathology features for degree of severity, showed no apparent relationship with the source of isolation. Some relationship was found between amplified fragment length polymorphism groups and total ileal loop pathology scores, and a one-way ANOVA comparison of the mouse pathology scores against total chick embryo deaths after 72 h was significant (P=0.049).

Campylobacter jejuni causes gastroenteritis with a variety of symptoms in humans. In the absence of a suitable animal model, in vitro models have been used to study virulence traits such as invasion and toxin production. In this study, 113 C. jejuni isolates from poultry and poultry-related (n=74) environments as well as isolates from human cases (n=39) of campylobacteriosis and bacteraemia were tested for invasiveness using INT 407 cells. The method was sufficiently reproducible to observe a spectrum of invasiveness amongst strains. As a result, strains were classified as low, high and hyper-invasive. The majority of strains (poultry and human) were low invaders (82 % and 88 %, respectively). High invasion was found for 5 % of human strains and 11 % of poultry-related isolates. However, only 1 % of poultry strains were classified as hyperinvasive compared to 13 % of human isolates (P=0.0182). Of those isolates derived from the blood of bacteraemic patients, 20 % were hyperinvasive, though this correlation was not statistically significant. An attempt was made to correlate invasiveness with the presence of seven genes previously reported to be associated with virulence. Most of these genes did not correlate with invasiveness, but gene cj0486 was weakly over-represented, and a negative correlation was observed for the gene ciaB. This trend was stronger when the two genes were analysed together, thus ciaB(-) cj0486(+) was over-represented in high and hyperinvasive strains, with low invaders more commonly found to lack these genes (P=0.0064).

We developed a loop-mediated isothermal amplification (LAMP) assay for the rapid and simple detection of Campylobacter jejuni and Campylobacter coli. The assay provides a specific LAMP product for each of these two species. The assay correctly identified 65 C. jejuni and 45 C. coli strains, but not 75 non-C. jejuni/coli strains. The sensitivity of the LAMP assay for C. jejuni and C. coli in spiked human stool specimens was 5.6 x 10(3) c.f.u. g(-1) (1.4 c.f.u. per test tube) and 4.8 x 10(3) c.f.u. g(-1) (1.2 c.f.u. per test tube), respectively. When 90 stool specimens from patients with diarrhoea were tested by LAMP and direct plating, the LAMP results showed 81.3 % sensitivity and 96.6 % specificity compared to isolation of C. jejuni and C. coli by direct plating. Further, the LAMP assay required less than 2 h for detection of C. jejuni and C. coli in stool specimens. This LAMP assay is a rapid and simple tool for the detection of C. jejuni and C. coli and will be useful in facilitating the early diagnosis of food poisoning incidents caused by these organisms.

Campylobacter jejuni is a common gastrointestinal bacterial pathogen. Although cytolethal distending toxin (CDT) is proposed to be an important virulence determinant of this pathogen, how CDT(+) and CDT(-) strains differ in their biological properties remains largely unknown. The virulence properties of CDT(+) and CDT(-) strains were studied on HeLa cells and in the suckling mouse model. Presence of the cdtB gene in Campylobacter species was determined by PCR. Five each of CDT(+) and CDT(-) C. jejuni strains were subjected to adherence, invasion and cytotoxicity assay on the HeLa cell line. Bacterial culture supernatants with and without CDT activity were inoculated intragastrically into 2-day-old suckling mice. The mice were sacrificed within 48 h. Histopathological examination of stomach, jejunum, ileum and colon was performed by haematoxylin/eosin staining. cdtB was detected in 88 % and 14 % of C. jejuni and Campylobacter coli strains, respectively. CDT(+) C. jejuni strains adhered to and invaded HeLa cells in significantly higher numbers than CDT(-) strains [CDT(+) vs CDT(-), adherence 2.7 x 10(4)+/-3.5 x 10(4) vs 2.7 x 10(2)+/-1.9 x 10(2); invasion 1.0 x 10(3)+/-1.3 x 10(3) vs 1.4 x 10(1)+/-3.1 x 10(1); P<0.01]. Culture supernatants of all CDT(+) strains demonstrated CDT activity on HeLa cells. Mice inoculated with supernatant containing CDT activity had moderate to severe pathology in different parts of their gastrointestinal tract, with the colon being the major target. Mice inoculated with supernatant lacking CDT activity showed no significant pathology in the gastrointestinal tract. The results demonstrate that CDT(+) C. jejuni strains adhere to and invade epithelial cells more efficiently than CDT(-) strains. CDT is responsible for intestinal pathology and the colon is the major target.

Campylobacter jejuni is a major cause of diarrhoea in humans. However, the pathogenesis of C. jejuni diarrhoea is poorly understood due to the lack of a good animal model of infection. Many animals have been tried with limited success, but a mouse lung model of infection has been found to be satisfactory previously; however, the lung pathology of this model has not been studied. For the purpose of characterizing the histopathological and ultrastructural lesions in the lung of the mouse pulmonary model of C. jejuni infection, C. jejuni strain 81-176 or sterile PBS was intranasally inoculated into BALB/c mice. The infection resulted in a mild illness only, and in an initial predominance of polymorphonuclear cells, followed by the accumulation of macrophages and later the prominence of epithelioid cells. Focal peribronchial pneumonia appeared on day 3, granuloma-like reaction on day 4 and bronchopneumonia on day 5 post-infection. These features developed until day 5 post-infection, but were less consistent afterwards when histopathology was monitored up to 9 days post-infection. Intracellular structures resembling bacteria were observed on days 3 and 5 post-infection, but not on day 7 post-infection. On days 3 and 5 post-infection, degenerative changes were also observed by transmission electron microscopy. The histological changes were not associated with acid-fast bacteria or any fungal elements. The infection was systemic as C. jejuni was isolated from blood and all organ homogenates (lung, spleen, liver, and small and large intestines) at 24 h post-infection. Thereafter, the organism was recovered from the intestine only, thus indicating its predilection for this location. This characterization of pathology should contribute to a better understanding of the animal model and pathogenesis of C. jejuni infection.

Campylobacter jejuni strains develop a high variability of lipooligosaccharide (LOS) structures on the cell surface based on variations in the genetic content of the LOS biosynthesis locus. While the importance of these variations for ganglioside mimicry as a critical factor in the triggering of Guillain-Barré syndrome has already been shown, little work has been done on the investigation of LOS structures and their function in the pathogenesis of gastrointestinal disease. In this study, the presence of several LOS genes in 40 C. jejuni strains with different abilities to colonize the chicken gut and to invade Caco-2 cells was investigated by PCR. Two genes, cgtB and wlaN, encoding putative beta-1,3-galactosyltransferases were detected in most strongly invasive strains and rarely in non-invasive strains. A homopolymeric tract within the wlaN gene resulted in an intact gene product only in strongly invasive strains. The specific function of these genes during LOS biosynthesis is still unknown. cgtB and wlaN gene products are suggested to be involved in development of the colonization and invasion ability of C. jejuni. After a classification of the complete LOS loci, an association between a particular LOS class and colonization and invasion ability of the C. jejuni strain could not be detected. Lack of the pglB gene involved in protein glycosylation in one strain could be responsible for the weak colonization and invasion ability of this strain. There is some evidence that different genetic characteristics were responsible for strong or weak colonization and the invasion ability of C. jejuni strains.

Campylobacter insulaenigrae is a novel species that has been recently only isolated from marine mammals. This is the first report of C. insulaenigrae causing enteritis and septicaemia in a patient with end-stage hepatic and renal disease.

A multiplex PCR assay has been developed for the identification of the six common Campylobacter taxa associated with human gastroenteritis and/or septicaemia, namely Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. The assay was developed using a combination of newly designed and published primers. It provided a specific PCR product for each of the five Campylobacter species and the one subspecies, and each of the PCR products was sufficiently distinguished by a difference in size by agarose gel electrophoresis. On evaluation of efficacy with 142 Campylobacter strains, the assay correctly identified all strains as 1 of the 6 Campylobacter taxa. This multiplex PCR assay is a rapid, simple and practical tool for identification of the six Campylobacter taxa commonly associated with gastroenteritis and/or septicaemia in humans, and offers an effective alternative to conventional biochemical-based assays.

Campylobacter jejuni is one of the leading causes of food-borne gastroenteritis. Because of the high prevalence of C. jejuni in poultry, poultry meat is considered a major source of C. jejuni infections for humans. However, it is not known whether all poultry-associated C. jejuni strains are capable of causing disease in humans. Four different virulence properties of C. jejuni strains were compared between 20 poultry isolates and 24 human isolates. Strains were chosen based on their PFGE pattern to represent a heterogeneous population. The isolates were compared for their ability to invade and induce interleukin-8 (IL-8) production in T84 cells, their production of functional cytolethal distending toxin (CDT) using HEp-2 cells, and their sodium deoxycholate resistance. All four virulence factors were present among strains of human and poultry origin, with strong differences observed among strains. For invasion and IL-8 induction, no difference was observed between the two populations. However, on average, human isolates arrested more HEp-2 cells in their cell cycle than did the poultry isolates (P=0.041), suggesting higher CDT production by the former. The ability to survive 16 000 mug sodium deoxycholate ml(-1) was significantly more pronounced (P=0.006) among human isolates than poultry isolates, although all strains possessed the cmeABC operon. These data suggest that all four virulence properties are widespread among C. jejuni isolates, but that a higher degree of bile-salt resistance and more pronounced CDT production are associated with strains causing enteritis in humans.

Differentiation between Campylobacter jejuni and Campylobacter coli is problematic in clinical specimens due to fastidious growth requirements and limited biochemical tests. This study describes a rapid, multiplex PCR protocol for the direct detection and differentiation of C. jejuni and C. coli in stools. An evaluation was carried out of this multiplex protocol based on the detection of cadF (genus specific), and hipO (C. jejuni) and asp (C. coli) genes, using stool from patients with Campylobacter enteritis and chicken. Protocol sensitivity was assessed and specificity determined using a panel of enteric bacteria, and evaluation of 30 diarrhoeic stool specimens culture negative for Campylobacter. Of the 114 specimens (54 human and 60 chicken) evaluated by the protocol, 70 (61.4 %) were identified as C. jejuni, 35 (30.7 %) as C. coli and 9 (7.9 %) as a mixed infection/colonization with both species. All mixed infections were identified as C. jejuni by culture. Among the stool specimens that were culture negative for Campylobacter, two (6.7 %) were C. jejuni positive by multiplex PCR. The protocol sensitivity limit was 0.015-0.016 ng C. jejuni and C. coli DNA mul(-1) in the specimen. There was no cross-reaction with the reference strains assessed. Comparison of hippurate test and multiplex PCR demonstrated 17 isolates with false-positive hippurate enzymic activity and 7 with false-negative activity. This rapid protocol (turnaround time 6 h) is highly sensitive and specific for direct evaluation of stool for these pathogens. It has significant application for routine clinical diagnostic and epidemiological purposes.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.

The prevalence of tetracycline resistance, tetracycline MICs and tet(O) gene localization were investigated in 83 Campylobacter isolates from patients suffering from acute gastroenteritis in Germany. Combined biochemical and molecular markers identified 74 isolates (89 %) as Campylobacter jejuni, including seven atypical isolates that failed to hydrolyse hippurate, and nine isolates (11 %) as Campylobacter coli. Tetracycline resistance was detected in six out of nine Campylobacter coli isolates (67 %) and 13 out of 74 C. jejuni isolates (18 %). Low-level tetracycline resistance was observed for C. coli (MIC 16 microg ml(-1) for all strains), whereas C. jejuni showed high-level resistance (MIC >256 microg ml(-1) for all strains). Both low- and high-level tetracycline resistance was associated with the presence of the tet(O) gene. In C. jejuni, tet(O) was plasmid-encoded in 54 % of tetracycline-resistant isolates, whereas in C. coli, tet(O) appeared to be located on the chromosome.

The antibiotic sensitivity and the serotype and molecular type (MT) distribution of 41 Campylobacter jejuni strains isolated from individual patients in Tawam Hospital, Al Ain, United Arab Emirates, were investigated. While all strains were sensitive to erythromycin (MIC 0.5-4 mg l(-1)), 35 isolates (85.4 %) exhibited resistance to ciprofloxacin (MIC 8-64 mg l(-1)). All resistant strains carried the Thr-86 to Ile mutation in the gyrase A (gyrA) gene, as shown by mismatch amplification mutation assay (MAMA) and confirmed by sequencing. Based on the partial sequences of gyrA, resistant isolates carried 10 distinct alleles, eight of them representing new variants. Strains were assigned to 30 MTs based on the combined results of PFGE and flaA PCR-RFLP typing. Eight of the 35 ciprofloxacin-resistant strains, isolated over a period of more than 1 year, represented the largest MT, also carrying the same allelic variant of the gyrA gene. These results show that the local incidence of fluoroquinolone resistance among C. jejuni is one of the highest reported worldwide. It was also demonstrated that stable MTs could persist for a relatively long time among the clonally unrelated antibiotic-resistant isolates of C. jejuni. The data also emphasize the need to replace fluoroquinolones as empirical therapy for diarrhoea of undiagnosed aetiology.

Campylobacter fetus causes severe infections in humans and can be isolated from various mammals and reptiles. However, although poultry are considered to be the main reservoir of Campylobacter jejuni, little is known about the presence of C. fetus in poultry. Thus, specific pathogen-free chickens were experimentally inoculated with a mixture of either three non-thermotolerant or four thermotolerant human strains of C. fetus. Faecal samples were regularly sampled after inoculation and caeca and intestines were collected 21 or 40 days after inoculation. All samples were analysed for the presence of Campylobacter using culture techniques. No Campylobacter could be re-isolated. This result strongly suggests that broilers do not play an important part in the C. fetus contamination of humans.

This investigation describes the development of a generally applicable, bioinformatics-driven, single-nucleotide polymorphism (SNP) genotyping assay for the common bacterial gastrointestinal pathogen Campylobacter jejuni. SNPs were identified in silico using the program 'Minimum SNPs', which selects for polymorphisms providing the greatest resolution of bacterial populations based on Simpson's index of diversity (D). The high-D SNPs identified in this study were derived from the combined C. jejuni/Campylobacter coli multilocus sequence typing (MLST) database. Seven SNPs were found that provided a D of 0.98 compared with full MLST characterization, based on 959 sequence types (STs). The seven high-D SNPs were interrogated using allele-specific real-time PCR (AS kinetic PCR), which negates the need for expensive labelled primers or probes and requires minimal assay optimization. The total turnaround time of the SNP typing assay was approximately 2 h. Concurrently, 69 C. jejuni isolates were subjected to MLST and flagellin A short variable region (flaA SVR) sequencing and combined with a population of 84 C. jejuni and C. coli isolates previously characterized by these methods. Within this collection of 153 isolates, 19 flaA SVR types (D=0.857) were identified, compared with 40 different STs (D=0.939). When MLST and flaA SVR sequencing were used in combination, the discriminatory power was increased to 0.959. In comparison, SNP typing of the 153 isolates alone provided a D of 0.920 and was unable to resolve a small number of unrelated isolates. However, addition of the flaA SVR locus to the SNP typing procedure increased the resolving power to 0.952 and clustered isolates similarly to MLST/flaA SVR. This investigation has shown that a seven-member C. jejuni SNP typing assay, used in combination with sequencing of the flaA SVR, efficiently discriminates C. jejuni isolates.

There are no data describing the genetic make-up of Campylobacter strains (an important aetiological agent of diarrhoea) circulating in the Arabian Gulf region. Here, the molecular characterization of two virulence genes in Campylobacter jejuni from Bahrain and the relationship with clinical infection are reported. Molecular screening for cytolethal distending toxin (cdtB) and invasion-associated marker (iam) genes was carried out on C. jejuni stool isolates collected from January 2002 to January 2004 in Bahrain. The molecular characterization was correlated with the patients' socio-demographic and clinical parameters. Of the 96 C. jejuni strains tested, 50 (52 %) were cdtB+/iam+, 30 (31 %) were cdtB+/iam- and 16 (17 %) were cdtB-/iam-. Sixty-nine per cent (66/96) of patients were less than 3 years old, with significantly higher detection of cdtB+/iam+ and cdtB+/iam- strains (P < 0.001 and P < 0.01, respectively) in this age group. Seventy patients (73 %) were symptomatic. In the group that were less than 3 years old, 62 and 85 % of those with cdtB+/iam+ and cdtB+/iam- strains, respectively, were symptomatic compared with 100 % for those over 3 years of age. However, the presence of cdtB-/iam- strains still resulted in clinical infection in the children under 3 years but not in the older patients. This is the first report describing the molecular characterization of virulence genes in Campylobacter isolates from this region. The findings indicate that strains of different virulence genetic make-up are circulating in the population, with children under the age of 3 years being most vulnerable. Further work on the molecular characterization, gene expression and determination of the invasive phenotypes of C. jejuni strains circulating in different regions is needed.

Campylobacter jejuni infection frequently presents as acute enteritis with diarrhoea, malaise, fever and abdominal pain. Vomiting and bloody diarrhoea are reported less frequently. To investigate potential host, micro-organism or environmental factors that might explain the different clinical presentations, the features of laboratory-confirmed Campylobacter jejuni cases presenting with vomiting and/or bloody diarrhoea were compared with cases who did not report either clinical manifestation. Single variable analysis and logistic regression were employed. Explanatory variables included food, water and environmental risks. Cases who reported vomiting and/or bloody diarrhoea tended to suffer a longer illness and were more likely to require hospital admission. Independent risks identified were being a child, female gender, consumption of poultry other than chicken, pre-packed sandwiches and sausages, and reported engineering work or problems with drinking-water supply. A dose-response relationship with vomiting and/or bloody diarrhoea and increasing daily consumption of unboiled tap water was observed also. Vomiting and/or bloody diarrhoea characterized the more severe end of the disease spectrum and might relate to host susceptibility and/or infective dose. The role of unboiled tap water as a potential source of C. jejuni infection in England and Wales requires further investigation.

The genetic heterogeneity among Campylobacter jejuni and Campylobacter coli isolates obtained from apparently healthy cattle and sheep was investigated by random amplified polymorphic DNA (RAPD) analysis. A total of 348 Campylobacter isolates, consisting of C. jejuni (n = 218) and C. coli (n = 130), were analysed. All these isolates were successfully typed by RAPD analysis. The total numbers of band patterns defined by RAPD in cattle and sheep were 42 and 45, respectively. Of the 42 distinct types obtained from cattle, 37 types were observed in C. jejuni isolates (n = 115), and the remaining 5 were in C. coli isolates (n = 30). Of 45 distinct types obtained from sheep, 21 types were observed in C. jejuni isolates (n = 103), and 24 were in C. coli isolates (n = 100). It was concluded that a high degree of heterogeneity existed among the C. jejuni and C. coli isolates of healthy cattle and sheep.

A multiplex-PCR method, specifically designed for application in routine diagnostic laboratories, was developed for the detection of Campylobacter coli and Campylobacter jejuni. Primers were directed towards the following loci: the hippuricase gene (hipO) characteristic of C. jejuni, a sequence partly covering an aspartokinase gene characteristic of C. coli, and a universal 16S rDNA gene sequence serving as an internal positive control for the PCR. The method was tested on 47 C. coli strains and 88 C. jejuni strains, and found to be almost 100% in concordance with biochemical analyses (all except for one C. coli strain), regardless of whether the DNA was prepared from colonies by a simple boiling procedure or by DNeasy Tissue Kit. Pure cultures of C. coli and C. jejuni were identified at 10-100 cells per PCR. When the multiplex-PCR method was used on spiked human stool samples, both strains were identified at 10(5) cells per ml stool. This sensitivity limit was the same whether the DNA was purified by the method of KingFisher mL or QIAamp DNA Stool Kit. When the same spiked stools were grown on modified charcoal cefoperazone deoxycholate agar (mCCDA) plates before PCR, the sensitivity was 100 cells per ml stool, indicating that culturing of campylobacters on mCCDA plates is superior to direct DNA extraction at least when fresh stool samples are analysed by PCR.

The rapid identification of Campylobacter jejuni isolates to strain level would significantly inform the public health investigation of C. jejuni infection. Conceptual advances provided by multilocus sequence typing (MLST) have established the clonal complex as an important epidemiological group at the strain level, enabling accurate and phylogenetically valid strain identification for C. jejuni. The development of real-time PCR assays for allelic discrimination of strain-associated single-nucleotide polymorphisms (SNPs) based upon MLST locus alleles offers one possible approach for rapid strain detection. SNPs defining key alleles diagnostic for the most prevalent clonal complexes were identified following a detailed analysis of the available MLST data. Real-time Taqman allelic discrimination assays designed to detect the SNPs specific for six major clonal complexes, ST-21, ST-45, ST-48, ST-61, ST-206 and ST-257, were developed, allowing the rapid detection of C. jejuni isolates and preliminary strain identification. This will provide an important complementary technique to sequence typing for rapid detection and strain characterization to inform in real-time the public health management and investigation of C. jejuni infections.

The pathogenicity of thermotolerant Campylobacter species, common food-borne pathogens, depends on certain factors unevenly distributed among strains of different origin. The prevalence of such markers has never been examined in a population of Polish Campylobacter strains of human and poultry origin. Therefore, we analysed the presence of the cadF, cdtA, cdtB and cdtC genes and the iam sequence in Campylobacter jejuni (n = 115) and Campylobacter coli (n = 57) isolates from children with diarrhoea and from chicken carcasses. The cadF gene was present in nearly 100% of Campylobacter isolates tested, regardless of their origin or species. In contrast, the iam region was found in 83.3% and 100% of C. coli isolates from children and chickens, respectively, but in only 1.6% and 54.7%, respectively, of C. jejuni isolates. Similarly, the detection rates of cdt genes varied between human and chicken isolates. All three cdt genes were found in nearly all C. jejuni isolates from both children and chickens, but in only 5.6% of human C. coli isolates as compared to 87.2% of chicken C. coli isolates. This different distribution of genetic markers between human and chicken isolates indicates that some Campylobacter infections in children may have additional sources other than contaminated chicken meat.

A membrane-bound, haemolytic phospholipase A(2) (PLA(2)) activity was detected in clinical strains of Campylobacter concisus isolated from children with gastroenteritis. The clinical strains were assigned into two molecular groups (genomospecies) based on PCR amplification of their 23S rDNA. This calcium-dependent, heat-stable, haemolytic PLA(2) activity was detected in strains from both genomospecies. A crude haemolysin extract (CHE) was initially prepared from cellular outer-membrane proteins of these isolates and was further fractionated by ultrafiltration. The haemolytic activity of the extracted fraction (R30) was retained by ultrafiltration using a 30 kDa molecular mass cut-off filter, and was designated haemolysin extract (HE). Both CHE and HE had PLA(2) activity and caused stable vacuolating and cytolytic effects on Chinese hamster ovary cells in tissue culture. Primers for the conserved region of pldA gene (phospholipase A gene) from Campylobacter coli amplified a gene region of 460 bp in all tested isolates, confirming the presence of a homologous PLA gene sequence in C. concisus. The detection of haemolytic PLA(2) activity in C. concisus indicates the presence of a potential virulence factor in this species and supports the hypothesis that C. concisus is a possible opportunistic pathogen.

Although many genes related to the pathogenicity of Campylobacter jejuni have been reported, the relationships between these genes and the sources of strains are not clear. In this study, the presence of 11 pathogenic genes responsible for the expression of adherence, invasion, colonization and cytotoxin production was examined in 111 C. jejuni isolated from human clinical samples, poultry meat, broiler faeces and bovine faeces. For most of the pathogenic genes, no difference in their presence in C. jejuni was found among the sources, but, for racR, wlaN and virB11, there were some variations among sources. The racR gene was present at rates of 98.2 (human clinical samples), 90.5 (poultry meat), 85.7 (broiler faeces) and 76.7 % (bovine faeces). Detection rates for the wlaN gene were 25.0, 23.8, 4.7 and 7.7 % and those for the virB11 gene were 10.7, 9.5, 9.5 and 15.4 % in human clinical samples, poultry meat, broiler faeces and bovine faeces, respectively. One hundred and seven of 111 strains (96.4 %) carried from eight to 10 of the pathogenic genes. These data did not show remarkable differences in the presence of pathogenic genes carried by C. jejuni from various sources.

Eight Campylobacter isolates that were able to grow at 25 degrees C and 42 degrees C and had the same biochemical profile were isolated from the blood of eight immunocompromised patients. Conventional biochemical tests were unable to determine whether they were isolates of thermo-tolerant C. fetus, H2S-negative C hyointestinalis, or a new Campylobacter species. Sequencing of the 16S ribosomal RNA genes showed that all eight isolates had the same nucleotide sequence and this was identical to that of C. fetus (GenBank accession no. AF219233). All eight patients had underlying disease and two died despite antibiotic treatment. Because of the ability of C fetus to grow over a wide range of temperatures and a higher incidence of bacteraemia by this organism than C. jejuni in the past 5 years in Hong Kong, thermo-tolerant C fetus may be an emerging pathogen in immunocompromised patients in the years to come.

Pulsed-field gel electrophoresis (PFGE) was used to analyse 147 isolates collected in two regions of Quebec province (Estrie and Montreal) between March 1998 and Feb. 1999, to determine the utility of molecular strain typing for a population-based collection of Campylobacter jejuni and to compare directly the discriminatory power of SmaI and KpnI restriction digests. With a combination of epidemiological criteria including space and time plus molecular strain typing, 49% of isolates from Estrie and 39% of isolates from Montreal were identified as belonging to a putative cluster. For 41% of the cases, sources were either missing or explicitly unknown; the remaining sources were subject to recall bias. Thus, the evaluation of sporadic cases of campylobacter enteritis by descriptive clinical investigation alone is neither sensitive nor reliable for identifying sources of infection. In the PFGE analysis, KpnI digests provided appreciably greater discriminatory power than SmaI digests. When combining the PFGE analyses with basic epidemiological criteria, 30% of the putative SmaI clusters were inconsistent with the epidemiological criteria compared with 17% of the KpnI clusters. Among the 98 isolates assigned to clusters by SmaI, only 65% gave concordant results with KpnI. In contrast, among the 81 isolates assigned to clusters by KpnI, 92% gave concordant results with SmaI. Finally, clusters that were epidemiologically related to ingestion of raw milk and specific water sources correlated better with the typing results based on KpnI than SmaI. Thus, KpnI is the enzyme of choice for molecular epidemiology studies of C. jejuni. The combination of continuous epidemiological surveillance and molecular strain typing may be useful for identifying new sources and mechanisms of transmission for community-acquired C. jejuni infection andultimately for developing new approaches to prevention.

The pathogenesis of campylobacter infection in man is largely unknown, although cytolethal distending toxin (CDT) has been incriminated as a virulence factor. However, little is known about the cdt genes in Campylobacter spp. isolated from broiler chickens. A total of 350 cloacal swabs was collected and tested by conventional culture and PCR. Of the 114 Campylobacter isolates obtained, 101 (88.6%) were identified as C. jejuni and 13 (11.4%) as C. coli by conventional methods. cdt genes were detected by PCR in all the isolates except one C. jejuni isolate. Cytotoxic effects were produced in a Vero cell line, by 100 of the C. jejuni isolates. In contrast, 10 C. coli isolates produced much lower levels of toxin and 3 produced no detectable toxin. These results confirm the common occurrence of campylobacter infection in chickens and indicate that cdt genes are commonly present in both C. jejuni and C. coli isolates from broilers, but that there are distinct differences in CDT production in these two closely related species.

In order to assess the contribution of cytolethal distending toxin (CDT) to the toxigenicity and pathogenicity of Campylobacter jejuni, the C. jejuni 81-176 and C. jejuni NCTC 11168 CDTs were inactivated by insertional mutation of the cdtB toxin subunit. Cell-free sonicates from isogenic C. jejuni 81-176 cdtB- strains were found to be greatly attenuated in HeLa cytotoxicity assays, whilst still retaining some toxigenicity. Sonicates from a C. jejuni NCTC 11168 cdtB- strain produced no detectable cytotoxicity. When orally administered to adult severe combined immunodeficient (SCID) mice, C. jejuni cdtB mutant strains were unaffected in enteric colonisation abilities but demonstrated impaired invasiveness into blood, spleen and liver tissues. These data suggest that CDT may be the principal toxin produced by this species and that some C. jejuni strains may generate additional toxigenic factor(s) distinct from CDT.

A new commercial serotyping set based on heat-stable Penner's antigens was compared with pulsed-field gel electrophoresis (PFGE) with SmaI and SacII restriction endonucleases. Among 50 isolates of Campylobacter jejuni from Finnish patients, which represented predominant PFGE patterns selected from isolates from sporadic cases and isolates associated with small outbreaks, 11 different serotypes were demonstrated from 43 typable isolates. Several PFGE patterns could be found within one serotype; on the other hand, several serotypes could be demonstrated within one PFGE type. Most isolates originated from sporadic cases; however, some isolates were epidemiologically associated and showed identical serotypes and PFGE patterns. Although the new serotyping set would have been useful in the few epidemic cases studied, several isolates (14%) representing the major PFGE patterns remained untypable or gave weakly positive agglutination reactions only suggesting a plausible serotype (18%). This might restrict the use of the novel serotyping set, at least in Finland.

Two fatal cases of Campylobacter jejuni septicaemia in patients with AIDS were characterised by severe HIV-related immunodeficiency, negative stool cultures and presentation during hospitalisation, developing a clinical picture of fulminant septic shock despite therapy with appropriate antibiotics. Campylobacter spp. are important opportunist pathogens in HIV disease and may cause a septicaemic illness in the absence of enteric disease.

The responses of previously untested immunodeficient mouse strains to campylobacter infection are described. Three strains of adult immunodeficient mice (SCID-Beige, C.B-17-SCID-Beige and RAG-2) were inoculated intragastrically with Campylobacter jejuni NCTC 11168. All mice became heavily colonised, but none developed clinical signs of disease. Immunocompetent BALB/c mice inoculated similarly had much lower colonisation levels. The co-administration of iron dextran had no effect on colonisation levels nor the development of clinical signs of disease. In contrast, C.B-17-SCID-Beige mice, when inoculated with one of a series of 10 clinical isolates of C. jejuni, were more heavily colonised for extended periods (up to 5 months) and approximately 10-20% of the mice became ill with diarrhoea. C. jejuni was detected in mouse faeces throughout at levels of 10(7)-10(9) cfu/g. All mice killed whilst ill with diarrhoea displayed histopathological lesions typical of human campylobacteriosis. Severe pathology was limited to the large intestine and was suggestive of an acute, bacteria-induced inflammation. Although blood was detected in the diarrhoeal stools, no evidence of mucosal epithelial cell invasion was found by immunohistology. No pathology was detected in tissue sections from any of the animals that had not developed signs of disease following C. jejuni inoculation. These immunodeficient mouse strains are readily, and heavily, colonised as adults by C. jejuni. The diarrhoea, although sporadic, was reproducibly produced, and could provide the basis for pathogenicity studies.

High resolution molecular subtyping was applied to Campylobacter jejuni Penner heat-stable (HS) 11 isolates from human infections and other sources. Strains were genotyped by restriction fragment length polymorphism analysis involving PCR-based flagellin gene (flaA) profiling with HinfI and DdeI, and pulsed-field gel electrophoretic (PFGE) profiling with SmaI and KpnI. Fla-genes of the strains were highly conserved as most (95%) had the same fla-profile. PFGE analysis of SmaI digests was more discriminatory with 15 profile subtypes identified, although 36% of isolates had a common profile. The study showed that strains of C. jejuni HS11, unlike those of HS1 and the HS4 complex, were relatively homogeneous at the genomic level and that high resolution molecular techniques were essential for detailed epidemiological subtyping.