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(2006). Genetic heterogeneity of the cytolethal distending toxin B (cdtB) gene locus among isolates of Campylobacter lari. British Journal of Biomedical Science: Vol. 63, No. 4, pp. 179-181.

Conventional detection and confirmation methods for Campylobacter jejuni are lengthy and tedious. A rapid hybridization protocol in which a 1,475-bp chromogen-labelled DNA probe (pDT1720) and Campylobacter strains filtered and grown on 0.22-micron-pore-size hydrophobic grid membrane filters (HGMFs) are used was developed. Among the environmental and clinical isolates of C. jejuni, Campylobacter coli, Campylobacter jejuni subsp. doylei, Campylobacter lari, and Arcobacter nitrofigilis and a panel of 310 unrelated bacterial strains tested, only C. jejuni and C. jejuni subsp. doylei isolates hybridized with the probe under stringent conditions. The specificity of the probe was confirmed when the protocol was applied to spiked skim milk and chicken rinse samples. Based on the nucleotide sequence of pDT1720, a pair of oligonucleotide primers was designed for PCR amplification of DNA from Campylobacter spp. and other food pathogens grown overnight in selective Mueller-Hinton broth with cefoperazone and growth supplements. All C. jejuni strains tested, including DNase-producing strains and C. jejuni subsp. doylei, produced a specific 402-bp amplicon, as confirmed by restriction and Southern blot analysis. The detection range of the assay was as low as 3 CFU per PCR to as high as 10(5) CFU per PCR for pure cultures. Overnight enrichment of chicken rinse samples spiked initially with as little as approximately 10 CFU/ml produced amplicons after the PCR. No amplicon was detected with any of the other bacterial strains tested or from the chicken background microflora. Since C. jejuni is responsible for 99% of Campylobacter contamination in poultry, PCR and HGMF hybridization were performed on naturally contaminated chicken rinse samples, and the results were compared with the results of conventional cultural isolation on Preston agar. All samples confirmed to be culture positive for C. jejuni were also identified by DNA hybridization and PCR amplification, thus confirming that these DNA-based technologies are suitable alternatives to time-consuming conventional detection methods. DNA hybridization, besides being sensitive, also has the potential to be used in direct enumeration of C. jejuni organisms in chicken samples.

Shellfish are frequently contaminated by Campylobacter spp, presumably originating from faeces from gulls feeding in the growing or relaying waters. The possible health effects of eating contaminated shellfish were estimated by quantitative risk assessment. A paucity of data was encountered necessitating many assumptions to complete the risk estimate. The level of Campylobacter spp in shellfish meat was calculated on the basis of a five-tube, single dilution MPN and was strongly season-dependent. The contamination level of mussels (

Campylobacter lari is a bacterium associated infrequently with human enteritis. Its differentiation from C. jejuni and C. coli relies on a few biochemical tests whose efficacy is highly questioned; therefore, the incidence and transmission of this pathogen has not been well studied. Two novel oligonucleotide primers for the polymerase chain reaction (PCR) that specifically amplify a 579-bp segment of the 16S rRNA gene of C. lari under optimized conditions were designed. No PCR product was detected when DNA from other strains of Campylobacter, Arcobacter, Helicobacter, Escherichia, Salmonella or Listeria was used as templates. Fast identification of C. lari through the present technique may aid the understanding of its incidence and epidemiology.

In order to gain insight into the epidemiology of Campylobacter lari infection in The Netherlands due to the consumption of raw mussels and oysters, batches of these shellfish were screened for the presence of Campylobacter spp. during a 6 month period in 1993-1994. Apparently, 41 out of 59 batches of mussels and 11 out of 41 batches of oysters were colonized with Campylobacter spp. A subset of the isolates was further characterized by additional phenotypic tests, numerical analysis of electrophoretic protein patterns, and genotyping by random amplification of polymorphic DNA (RAPD). Protein electrophoretic analysis of 39 Campylobacter spp. cultured from 24 batches of mussels and oysters, revealed that all isolates, except two, were C. lari. Two strains with an aberrant protein pattern were identified as C. coli and C. hyointestinalis, respectively. Nalidixic acid susceptible campylobacters (NASC) and urease-positive thermophylic campylobacters (UPTC) did not form separate clusters and should be considered biovars only. Several strains were both urease positive and nalidixic acid susceptible, which represents a new biovar within C. lari. The results of RAPD demonstrated the presence of 3 distinct genetic variants, implying that even within a single batch of shell fish, relatively extensive DNA polymorphisms can be found. It is therefore apparent that this complex group of C. lari is characterized by a high degree of genetic diversity, implying the presence of a heterogenous population of C. lari in crustacean organisms living in marine waters in a restricted area in The Netherlands.

Campylobacter jejuni is responsible for the majority of bacterial foodborne gastroenteritis in the US, usually due to the consumption of undercooked poultry. Research on which avian species transmit the bacterium is limited, especially in the US. We sampled wild birds in three families-Anatidae, Scolopacidae, and Laridae-in eastern North America to determine the prevalence and specific strains of Campylobacter. The overall prevalence of Campylobacter spp. was 9.2% for all wild birds sampled (n = 781). Campylobacter jejuni was the most prevalent species (8.1%), while Campylobacter coli and Campylobacter lari prevalence estimates were low (1.4% and 0.3%, respectively). We used multilocus sequence typing PCR specific to C. jejuni to characterize clonal complexes and sequence types isolated from wild bird samples and detected 13 novel sequence types, along with a clonal complex previously only associated with human disease (ST-658). Wild birds share an increasing amount of habitat with humans as more landscapes become fragmented and developed for human needs. Wild birds are and will remain an important aspect of public health due to their ability to carry and disperse emerging zoonotic pathogens or their arthropod vectors. As basic information such as prevalence is limited or lacking from a majority of wild birds in the US, this study provides further insight into Campylobacter epidemiology, host preference, and strain characterization of C. jejuni.

Campylobacteriosis is an acute infectious diarrheal disease caused by the genus Campylobacter. The infection is mainly due to consumption of raw or undercooked poultry meat, unpasteurized milk, and contaminated water. In recent decades, Campylobacter (especially Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and more recently Campylobacter upsaliensis) have acquired great public health importance worldwide. The incidence of Campylobacter infection varies in both industrialized and developing countries. Campylobacter species are isolated more frequently than combined isolation rates by Salmonella and Shigella species from diarrheic stool samples. Serological and nucleic acid-based assays are used to study the epidemiology of Campylobacter infection. Antibiotic resistance in C. jejuni is considered as an emerging public health problem. Various virulence factors such as flagellin, lipopolysaccharides (LPSs), adhesins, and invasins have been implicated in the pathogenesis of Campylobacter infection. In this review, we present information available through literature search on epidemiology, clinical presentations, and bacteriology of campylobacteriosis.

Campylobacteriosis was the most frequently reported zoonosis in the European Union (EU) in 2010, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari as the most frequently reported species in foodborne outbreaks (FBOs). Relatively sensitive to environmental factors, these species may be present in low numbers. In line with EU policy for food control and FBO detection and in view of the need to reduce response time, we validated an alternative molecular method according to ISO 16140:2003 which establishes the general principle and technical protocol for the validation of alternative methods in the microbiological analysis of food. We used a qualitative real-time PCR commercial kit for the detection of C. jejuni, C. coli, and C. lari in two food categories "fruit and vegetable-based products" and "dairy products". The validation protocol comprises two phases: the first is a method comparison study of the alternative method against the reference method, and the second is an interlaboratory study of each of the two methods. In the first step, ISO 16140:2003 validation examines the following parameters: limit of detection (LOD); relative accuracy, relative specificity and sensitivity; relative detection level (RDL); and inclusivity and exclusivity. Except for LOD, inclusivity and exclusivity, the other steps were performed against the reference method (ISO 10272:2006). The LOD of the real-time PCR method was set at 4CFU/25g or mL for both food categories. Relative accuracy (98.33%), specificity (96.77%), and sensitivity (100%) were recorded for the food category "fruit and vegetable-based products" and 93.3%, 88.24%, 100%, respectively, for "dairy products". The RDL according to Fisher's exact test was p=1 for both food categories, for each level, and each food/strain combination. The interlaboratory study results showed correct identification of all 24 blind samples with both methods by all the participating laboratories. The results show that this commercial kit is suitable for the rapid detection of C. jejuni, C. coli, and C. lari on fruit, vegetables and dairy products and may aid in official controls. In conclusion, the use of alternative methods is recommended for the rapid identification of positive samples and the identification of the possible bacterial source in a FBO within 48 h.

The initial glycan transfer in asparagine-linked protein glycosylation is catalysed by the integral membrane enzyme oligosaccharyltransferase (OST). Here we study the mechanism of the bacterial PglB protein, a single-subunit OST, using chemically synthesized acceptor peptide analogues. We find that PglB can glycosylate not only asparagine but also glutamine, homoserine and the hydroxamate Asp(NHOH), although at much lower rates. In contrast, N-methylated asparagine or 2,4-diaminobutanoic acid (Dab) are not glycosylated. We find that of the various peptide analogues, only asparagine- or Dab-containing peptides bind tightly to PglB. Glycopeptide products are unable to bind, providing the driving force of product release. We find no suitably positioned residues near the active site of PglB that can activate the acceptor asparagine by deprotonation, making a general base mechanism unlikely and leaving carboxamide twisting as the most likely mechanistic proposal for asparagine activation.

Two strains of urease-positive thermophilic Campylobacter (UPTC), CF89–12 and CF89–14, which were identified as UPTC by biochemical characterization, were found for the first time in river water in the Far East, namely, in Japan. The biochemical characteristics were identical to those of strains described previously by Bolton and colleagues. Furthermore, these two strains were positive for arylsulphatase. Consequently, it was demonstrated that UPTC may possibly be differentiated phenotypically from Campylobacter lari by the arylsulphatase test, as well as urease and nalidixic acid tests. Analysis by pulsed-field gel electrophoresis (PFGE) after digestion with Apa I, Sal I and Sma I, which were found to produce distributions of DNA fragments to be suitable for analysis of the genomic DNA from the thermophilic Campylobacter, respectively, demonstrated that these three restriction enzymes produced distributions of a relatively limited number of genomic DNA fragments and also demonstrated that the PFGE profiles obtained with the three restriction enzymes were indistinguishable between the two strains, respectively. The PFGE analysis and conventional fixed-field agarose gel electrophoresis suggested that the both genomes were approximately 1862 kb in length. Even though the two isolates of UPTC were isolated from water in different rivers in Japan, the results suggested that a single strain. as opposed to two distinct strains, was isolated. PFGE profiles after digestion with Sal I and Sma I, respectively, were also demonstrated to be distinctly different among strains isolated in Japan and previously in Europe. This is the first example of the isolation of UPTC from natural sources in countries other than those in Europe.

From the end of April 1991 until the end of 1991, 2209 isolates of Campylobacter spp. have been collected in Plymouth PHL of which 91 (4.1%) were resistant to ciprofloxacin. None of the 91 patients involved had taken a quinolone, but 30/91 (33%) had travelled abroad (16 to the Iberian peninsula) in the three months preceding isolation of the ciprofloxacin-resistant Campylobacter spp. In the case-control study 12/15 (80%) of the cases had recently consumed poultry as had 20/24 (83%) of controls with enteritis due to ciprofloxacin-susceptible Campylobacter spp. A small study of poultry purchased from the supermarket revealed that only 1/37 campylobacters isolated from 64 UK bred chickens was resistant to ciprofloxacin, whereas 7/26 campylobacters isolated from 50 imported chickens were ciprofloxacin-resistant. Of the 75 clinical isolates of ciprofloxacin-resistant Campylobacter spp. subjected to detailed analysis, 68 were Campylobacter jejuni, six were Campylobacter lari, and one was Campylobacter coli. All isolates from man and poultry were resistant to ciprofloxacin, norfloxacin, sparfloxacin and tosufloxacin, and there was an association between fluoroquinolone-resistance and increased MICs of tetracycline. The range of susceptibility to erythromycin and kanamycin were typical of the species. gyrA from C. jejuni P6 (a case with history of travel to Spain) and C. jejuni P16 (isolate from imported chicken) contained point mutations corresponding to an amino acid substitution of isoleucine for threonine at codon 86. It has been suggested that veterinary use of quinolones, notably enrofloxacin, is providing a selective pressure for emergence of resistance to ciprofloxacin amongst human isolates. Now that enrofloxacin has been licensed for use in broiler flocks in the UK, it will be interesting to monitor the prevalence of resistance of campylobacters to quinolones in UK-produced poultry and in UK-acquired human infection.

Lipo-oligosaccharide (LOS) from phenol-water extraction of cells of the Campylobacter lari type strain (ATCC 35221) was separated as a water-insoluble gel of low relative molecular mass (M(r)) from a water-soluble extracellular polysaccharide of high M(r). Structural investigations were performed on the liberated oligosaccharide and the extracellular polysaccharide, variously using 1H, 13C, and 31P NMR spectroscopy, linkage analysis, and fast atom bombardment-mass spectrometry of permethylated derivatives of the glycans and their products of chemical and enzymic degradation. The following structures are proposed for the highly branched region of the LOS: [formula: see text] and for the tetraglycosyl phosphate repeating unit of the extracellular polysaccharide: [-(PO3-)-->3)-beta-D-GlcpNAc-(1-->2)-6-d-alpha-L-gul-Hepp -(1-->2)-3-d-beta-D-threo-Penp-(1-->3)-6-d-alpha-L-gul-He pp-]n

As part of a European research project, the performance of a PCR assay to detect food-borne thermotolerant campylobacters ( Campylobacter jejuni , C. coli , and C. lari ) was evaluated through an international collaborative trial involving 12 participating laboratories. DNA from 10 target and 8 nontarget strains was tested, and the results were reported as the presence of a positive signal after gel electrophoresis. The overall inclusivity (sensitivity) was 93.7%, and the exclusivity (specificity) was 100%. The results indicate that the assay can become an international standard and can be confidently applied in microbiological laboratories.

Although the absence of intervening sequences (IVSs) within the 23S rRNA genes in Campylobacter lari isolates has been described, there are apparently no reports regarding correlations between the nucleotide sequences of 23S rRNA genes and erythromycin (Ery) susceptibility in C. lari isolates. Here, we determined the minimum inhibitory concentrations of 35 C. lari isolates [n = 19 for urease-positive thermophilic Campylobacter (UPTC); n = 16 urease-negative (UN) C. lari] obtained from Asia, Europe, and North America. We found that the 18 isolates were resistant to the Ery (defined as ≧8 μg/mL), and three isolates, UPTC A1, UPTC 92251, and UPTC 504, showed increased resistance (16 μg/mL). No correlations between the IVSs in the helix 45 region within the 23S rRNA gene sequences and Ery resistance were identified in the C. lari isolates examined. In addition, no point mutations occurred at any expected or putative position within the V domain in the isolates. In conclusion, antibiotic resistance against the macrolide erythromycin is mediated through an alternative pathway to that described above.

The present study examines the expression of cytolethal distending toxin (cdt) gene encoding a cytotoxin in Campylobacter lari (n=6 urease-negative [UN] C. lari; n=4 urease-positive thermophilic Campylobacter [UPTC]). When reverse transcription polymerase chain reaction (RT-PCR) was carried out with 10 C. lari isolates using a primer pair to amplify the cdtB gene transcript segment, an approximate 260 bp RT-PCR amplicon was generated with all the isolates. In addition, cdtA, cdtB and cdtC gene operon was identified to be polycistronicly transcribed in the C. lari cells. The cdtB gene translation in the C. lari cells was also confirmed by Western blot analysis. Thus, the cdt gene operon in C. lari organisms, including UN C. lari and UPTC, was expressed at the transcriptional and translational levels in the cells. The present results suggest that all three cdt genes may be functional in the cells.

M. UYTTENDAELE, R. SCHUKKINK, B. VAN GEMEN AND J. DEBEVERE. 1994. NASBAR, an isothermal amplification technique for nucleic acids, was evaluated for the specific identification of Campylobacter jejuni, Camp. coli and Camp. lari. A set of primers and a probe were chosen from the 16S rRNA sequence of Campylobacter. The probe was hybridized in solution with the amplified nucleic acids of 12 Campylobacter species and nine other Gram-negative bacteria. The probe was shown to hybridize specifically to the amplified single-stranded RNA of Camp. jejuni, Camp. coli and Camp. lari in an enzyme-linked gel assay (ELGA). In a Camp. jejuni model system the combination of NASBAR and ELGA was able to detect ca 1000 rRNA molecules. The presence of an excess of Gram-negative bacteria did not influence the sensitivity of detection. A number of 6 cfu of Camp. jejuni, present in a total count of 4 times 106 cfu of Gram-negative bacteria, resulted in a positive hybridization signal.

We report for the first time on the prevalence, antibiotic resistance and RAPD types of Campylobacter species in ducks and duck related environmental samples in Malaysia. Samples were examined by enrichment in Bolton Broth followed by plating onto modified Charcoal Cefoperazone Deoxycholate agar (mCCDA) and/or plating directly onto mCCDA. A total of 643 samples were screened, and the prevalence of Campylobacter spp. in samples from different sources ranged from 0% to 85%. The method of isolation had a significant (P

We evaluated the occurrence of three Campylobacter species--C. jejuni, C. coli, and C. lari--from 333 wild bird fecal samples collected at Tri-State Bird Rescue and Research in Newark, Delaware, in 2008. Using multiplex polymerase chain reaction, we detected C. jejuni from six avian families with an overall prevalence rate of 7.2%. We did not detect any other Campylobacter species. Campylobacter jejuni prevalence ranged widely between different avian families with crows (Corvidae) and gulls (Laridae) having the highest prevalence rates (23% and 25%, respectively).

Molecular cloning, nucleotide sequencing, and characterization of the flaA gene from additional isolates of urease-positive thermophilic Campylobacter (UPTC) were performed. These isolates were obtained from the natural environment in Northern Ireland (n = 9 from mussels) and in England (n = 1 from sea water). All isolates carried the shorter flaA gene, [open reading frames (ORFs), 1,461 to 1,503 base pairs], without any internal termination codons, and did not carry any flaA pseudogenes. The UPTC isolates were well discriminated by the neighbor joining (NJ) phylogenetic tree constructed based on the putative flaA genes ORFs nucleotide sequence information. In addition, the NJ tree constructed based on the flaA-short variable region sequence information discriminated the Campylobacter lari isolates with a similar degree of discrimination power.

Campylobacters other than Campylobacter jejuni or C. coli were isolated in 35% of 343 farms recently analyzed in northern Spain. This study was aimed at identifying at the species level the 120 isolates collected (21 ovine, 52 beef cattle, 44 dairy cattle, and 3 porcine) by species-specific polymerase chain reaction and 16S rRNA gene sequencing analysis. Thus, five species were identified: Campylobacter hyointestinalis (90 isolates), Campylobacter lanienae (12), Campylobacter fetus subsp. fetus (10), Campylobacter lari (1), and Campylobacter sputorum (1). Ambiguous results were obtained for six isolates. Phylogenetic analyses of the 16S rRNA gene sequence placed three of them (cattle isolates) as an intermediate clade between C. hyointestinalis subsp. hyointestinalis and C. fetus, two ovine isolates formed a new clade clustering with Campylobacter concisus despite sharing higher similarity with Campylobacter mucosalis, and one porcine isolate shared similarly high homology with C. lanienae and C. hyointestinalis subsp. lawsonii. C. hyointestinalis was the predominant species, particularly in cattle, but it was also isolated from sheep and swine. C. lanienae was only found in sheep, C. fetus in cattle and sheep, and C. lari in a single dairy cattle farm. Although previously reported, the isolation of C. lari from cattle is not common, and this is the first report of C. lanienae and C. hyointestinalis in sheep. This study demonstrated the wide distribution in livestock of several emerging zoonotic Campylobacter species and provided valuable information on their host animal reservoirs.

Comparison of Cape Town and Skirrow's protocols used in isolation of Campylobacter in humans and broilers was carried out in a cross-sectional study in Morogoro, Tanzania. A total of 176 and 158 human stool and broiler intestinal samples were collected, respectively. While human stool samples were collected from selected health centers, broiler intestinal samples were obtained from selected farms and chicken markets. Samples were inoculated and cultured in duplicate using two protocols and prevalence of Campylobacter were established. In humans, the prevalence of Campylobacter isolates was significantly higher (P

Glycobiology, 21 (1), ISSN:0959-6658

Somatic O (formerly heat-stable) and heat-labile (HL) serotyping methods are commonly used to type Campylobacter jejuni and Campylobacter coli isolates. Although both systems are effective, the labor and time required for each have limited their application. These systems can be simplified by reducing the number of antisera used. To find an appropriate panel of antisera, we determined the distribution of common serotypes in the United States among a representative sample of 298 Campylobacter isolates. The strains, obtained between July 1989 and June 1990 from persons with sporadic cases of diarrhea, were collected from 19 randomly chosen counties in all geographic (census) regions of the United States. All strains were serotyped by the O and HL systems. By phenotypic methods, 288 C. jejuni, 9 hippurate-negative C. jejuni/C. coli, and 1 Campylobacter lari were identified. Of 57 O antisera, 24 typed 252 (84.6%) strains. Of the 55 HL antisera, 23 serotyped 253 (84.9%) strains. All strains were typeable in the unabsorbed O antisera. In the absorbed HL antisera, four strains were nontypeable and 14 were rough and untypeable. In each geographic region, 9 or more O and HL serotypes were found. Serotypes O:1, O:4, and O:13,16,43,50 and HL 1 were identified in all regions. The combination of both schemes gave greater discrimination than either system alone, but the maintenance of both requires a large resource investment. A serotyping scheme incorporating the 24 most prevalent O and 23 most prevalent HL serotypes could be useful for outbreak support and for surveillance. In the near future, we anticipate using a molecular subtyping method in combination with limited serotyping to distinguish Campylobacter strains.

Pulsed-field gel electrophoretic profiles of the respective, undigested and intact chromosomal DNAs, prepared from three species of thermophilic Campylobacter (C. coli, C. jejuni and C. lari) demonstrated that the chromosomal DNAs from C. coli JCM 2529T, four isolated strains of C. coli, C. jejuni JCM 2013 and four isolated strains of C. jejuni migrated to around 1, 900kb, and the chromosomal DNAs from C. lari JCM 2530T migrated to around 1, 640kb. Chromosomal DNAs from 15 isolated strains of C. lari also migrated to almost the same extent as the DNAs from the C. lari type strain to around 1, 640kb. This result clearly demonstrates that pulsed-field gel electrophoresis (PFGE) is useful for the discrimination of C. lari from two other thermophilic species of Campylobacter (C. coli and C. jejuni).When the chromosomal DNAs prepared from the three Campylobacter species were digested with the restriction enzyme ApaI, electrophoretic profiles of the undigested chromosomal DNAs also demonstrated PFGE to be useful for that discrimination.

Four strains of a novel Helicobacter species were isolated from the stomachs of cheetahs (Acinonyx jubilatus) with gastritis. These isolates were phenotypically similar to Helicobacter pylori. The isolates were gram-negative, spiral bacteria which grew under microaerophilic conditions at 37°C, but not at 25 or 42°C, and produced urease, catalase, oxidase, alkaline phosphatase, and gamma-glutamyl transpeptidase. The isolates did not ferment glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, or arabinose; hydrolyze hippurate or indoxyl acetate; or reduce nitrate. They did not produce H2S from triple sugar iron agar, and they did not grow in the presence of 1.0% glycine or 1.5% NaCI. They were resistant to nalidixic acid and sensitive to cephalothin and metronidazole. Cells were typically 0.3 by 2.0 μm and possessed tufts of two to five sheathed, monopolar flagella. The G+C content of strain 90-119 was 30 mol%. Cluster analysis of densitometry scans of polyacrylamide protein gels revealed more than 70% similarity of the cheetah isolates to H. pylori, less than 60% similarity to Helicobacter felis, and less than 50% similarity to Helicobacter mustelae. Complete 16S rRNA sequences were determined for two of the cheetah isolates. Phylogenetic analysis was performed by comparing the cheetah sequences to those of 19 reference strains, including H. pylori, H. felis (two strains), H. mustelae, Helicobacter muridarum, “Flexispira rappini,” Wolinella succinogenes, Campylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter jejuni, Campylobacter lari, Campylobacter rectus, Campylobacter sputorum subsp. bubulus, a Campylobacter sp. (pigisolate), [Bacteroides] gracilis, and [Bacteroides] ureolyticus. The 16S rRNA sequences for 13 of the 19 reference species have not previously been reported. Phylogenetic analysis demonstrated that the cheetah isolates were most closely related to H. pylori (97.4% similarity), H. felis (96.1% similarity), and H. mustelae (93.4% similarity). On the basis of these findings, we propose that these isolates represents a novel species of Helicobacter, which we designate Helicobacter acinonyx. The type strain is 90-119 (CCUG 29263, ATCC 51101).

Helicobacter pylori urease was recovered as a single peak by DEAE-Sepharose column chromatography and Sephacryl S-200 gel filtration. The purified urease was obtained by fast protein liquid chromatography using a Mono Q column. The purified urease preparation gave a single band in polyacrylamide gel disc electrophoresis. Latex particles were sensitized with anti-urease immunoglobulin. The sensitized latex particles were agglutinated with the purified urease and by cell sonicates obtained from 55 strains of H. pylori which were isolated from the gastric mucosa of patients with gastric and duodenal disorders, while they did not react with those obtained from related bacteria known to be urease producers, such as Helicobacter mustelae and urease- positive "Campylobacter lari variants", or by urease of some strains of Enterobacteriae. We have developed a specific and sensitive method for detecting the urease by using the reversed passive latex agglutination technique, in order to identify of the organism.

Since the respective undigested and intact chromosomal DNAs from C. coli JCM2529T, from two strains of C. coli, C. jejuni JCM2013 and from two strains of C. jejuni migrated as fragments of around 1,900 kb and the chromosomal DNAs from C. lari JCM2530T and from eleven strains of C. lari migrated as fragments of around 1,640 kb, pulsed-field gel electrophoresis (PFGE) was clearly demonstrated to be useful for the discrimination of C. lari from two other thermophilic species of Campylobacter (C. coli and C. jejuni). PFGE revealed that SmaI generated nine distinctly different and distinguishable banding profiles from the DNA of 18 strains of C. lari, which included the type strain. Three types of banding profile, namely, A, B and C, exemplified by the nine profiles included those from five, four and three strains of C. lari, respectively. Our present attempt to discriminate and to genome-type C. lari at the chromosomal genomic level using PFGE and SmaI may be a valuable step towards a discriminative and molecular-epidemiologic study of C. lari.

Two sets of PCR primers are constructed to clone the cytochrome P450 structural gene, including putative promoter and terminator structures, and its adjacent genetic loci in Campylobacter lari isolates. The putative open reading frames (ORFs) of the P450 genes from 11 C. lari isolates (n=5 for urease-negative (UN) C. lari; n=6 urease-positive thermophilic campylobacters [UPTC]) examined consisted of 1365 or 1371 bases (455 or 457 amino acid residues), differing from those of the other thermophilic campylobacters (1359 [453] for C. jejuni and C. upsaliensis; 1368 [456] for C. coli). Each of the putative ORFs from the 11 isolates examined was also shown to carry start and stop codons and ribosome binding sites. Two putative promoter structures, consisting of sequences at the -35- and -10-like regions were also identified upstream of the ORFs. A single copy of the P450 gene in the genome was identified with UN C. lari JCM2530(T) and UPTC CF89-12, based on Southern blot hybridisation analysis. In addition, when reverse transcription polymerase chain reaction (RT-PCR) analyses were carried out, the transcription of the P450 structural gene in C. lari organisms in vivo was confirmed. The transcription initiation site for the gene was also determined. High nucleotide sequence similarities (95.2-98.8%) of the full-length P450 structural gene were shown with each of the 12 C. lari isolates. The UN C. lari and UPTC organisms showed similar findings with the neighbour-joining method, based on the sequence information of the P450 structural gene.

The emergence of antimicrobial resistance in Campylobacter spp. has been a growing public health concern globally. The objectives of this study were to determine the prevalence, antimicrobial susceptibility, and genetic relatedness of Campylobacter spp. recovered by the National Antimicrobial Resistance Monitoring System (NARMS) retail meat program. Retail meat samples ( n = 24,566) from 10 U.S. states collected between 2002 and 2007, consisting of 6,138 chicken breast, 6,109 ground turkey, 6,171 ground beef, and 6,148 pork chop samples, were analyzed. A total of 2,258 Campylobacter jejuni , 925 Campylobacter coli , and 7 Campylobacter lari isolates were identified. Chicken breast samples showed the highest contamination rate (49.9%), followed by ground turkey (1.6%), whereas both pork chops and ground beef had C. coli isolates showed higher resistance rates to antimicrobials, with the exception of doxycycline/tetracycline, than those seen for C . jejuni . Pulsed-field gel electrophoresis (PFGE) fingerprinting resulted in 1,226 PFGE profiles among the 2,318 isolates, with many clones being widely dispersed throughout the 6-year sampling period.

We aimed to clarify if the thermophilic Campylobacter lari organisms including urease-negative (UN) C. lari and urease-positive thermophilic Campylobacter (UPTC) can be differentiated at the species and/or subspecies levels by employing the full-length flaA gene and flaA short variable region (SVR) nucleotide sequence information or not. Thermophilic Campylobacter isolates (n = 45) including UN C. lari (n = 17), UPTC (n = 18), and Campylobacter jejuni (n = 10) were well discriminated at the isolate level by the unweighted pair group method using arithmetic means analysis and neighbor joining procedures constructed based on the full-length flaA gene and flaA SVR nucleotide sequence information. Thus, these procedures may possibly be useful for epidemimological studies for C. lari and C. jejuni.

In order to estimate the prevalence of Campylobacter spp. and Salmonella spp. on broiler chicken carcasses and the prevalence of Campylobacter spp. in caeca, 58 French slaughterhouses were investigated in 2008. Enumeration of Campylobacter spp. was also performed in order to study the relation between caeca and carcass contamination. A pool of 10 caeca and one carcass were collected from 425 different batches over a 12-month period in 2008. Salmonella was isolated on 32 carcasses leading to a prevalence of 7.5% ([5.0-10.0](95%CI)). The prevalence of Campylobacter was 77.2% ([73.2-81.2](95%CI)) in caeca and 87.5% ([84.4-90.7](95%CI)) on carcasses. No significant correlation was found between Campylobacter and Salmonella. Positive values of Campylobacter were normally distributed and the average level was 8.05 log(10) cfu/g ([7.94-8.16](95%CI)) in caeca and 2.39 cfu/g ([2.30-2.48](95%CI)) on carcasses. A positive correlation (r = 0.59) was found between the mean of Campylobacter in caeca and on carcasses (p < 0.001). Thus, carcasses from batches with Campylobacter-positive caeca had significantly (p < 0.001) higher numbers of Campylobacter per gram than batches with negative caeca. These results show that Campylobacter can be present in both matrices and reduction in caeca could be a possible way to reduce the amount of bacteria on carcasses. Of the 2504 identifications performed, 3 species of Campylobacter (Campylobacter jejuni, Campylobacter coli and Campylobacter lari) were identified. The main species recovered were C. jejuni and C. coli, which were isolated in 55.3% and 44.5% of positive samples, respectively. These two species were equally represented in caeca but C. jejuni was the most frequently isolated on carcasses with 57.1% and 42.5% of positive carcasses for C. jejuni and C. coli, respectively. This study underlines that target a reduction of Campylobacter on final products requires a decrease of contamination in caeca.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.

P.L. GRIFFITHS. G.S. MORENO AND R.W. A. PARK. 1992. The four species of thermophilic camplyobacters, Campylobacter jejuni, C. coli, C. upsaliensis and C. lari, are difficult to distinguish from each other because of their lack of reactivity in many conventional biochemical and physiological tests. Those tests which do discriminate sometimes give discordant results. Species-specific antibody preparations (APs), capable of discriminating between the thermophilic campylobacter species by dot-ELISA, were raised by inoculation of mice with partially purified membrane protein. The APs produced were absorbed with cells of cross-reactive species and tested by dot-ELISA against reference and natural strains, the identities of which were confirmed by DNA/DNA hybridization. The results showed that such APs could be useful as an alternative to DNA/DNA hybridization for rapid species identification, for example in epidemiological surveys. Western blotting experiments with the APs showed that the specificity of the antibodies was not due to a single antigen.

To overcome some of the deficiencies with current molecular typing schema for Campylobacter spp., we developed a prototype PCR binary typing (P-BIT) approach. We investigated the distribution of 68 gene targets in 58 Campylobacter jejuni strains, one Campylobacter lari strain, and two Campylobacter coli strains for this purpose. Gene targets were selected on the basis of distribution in multiple genomes or plasmids, and known or putative status as an epidemicity factor. Strains were examined with Penner serotyping, pulsed-field gel electrophoresis (PFGE; using SmaI and KpnI enzymes), and multilocus sequence typing (MLST) approaches for comparison. P-BIT provided 100% typeability for strains and gave a diversity index of 98.5%, compared with 97.0% for SmaI PFGE, 99.4% for KpnI PFGE, 96.1% for MLST, and 92.8% for serotyping. Numerical analysis of the P-BIT data clearly distinguished strains of the three Campylobacter species examined and correlated somewhat with MLST clonal complex assignations and with previous classifications of “high” and “low” risk. We identified 18 gene targets that conferred the same level of discrimination as the 68 initially examined. We conclude that P-BIT is a useful approach for subtyping, offering advantages of speed, cost, and potential for strain risk ranking unavailable from current molecular typing schema for Campylobacter spp.

Samples of faeces were taken from 183 healthy pet dogs in a census-based, cross-sectional study in Cheshire; culture methods were used to detect any Campylobacter species and a direct PCR was used to detect Campylobacter upsaliensis . Forty-six of the dogs were positive for C upsaliensis by either culture or direct PCR, giving a prevalence of 25˙1 per cent (95 per cent confidence interval [CI] 19˙0 to 32˙1 per cent). One sample was positive by culture for Campylobacter jejuni (95 per cent CI 0˙0 to 3˙0 per cent) and one for Campylobacter lari . Multivariable logistic regression identified risk factors for the carriage of C upsaliensis by a dog as: living with another dog that also carried C upsaliensis ; being small rather than medium-sized; being less than three years old; living in a household that kept fish; being fed commercial dog treats; and being fed human food titbits, particularly in the dog9s bowl.

Campylobacter spp. are frequently present in the intestinal tract and internal tissues of broiler breeder and broiler chickens. Campylobacter spp. ecology in commercial Leghorn laying hens has not been extensively studied. The objectives of the current study were to determine 1) Campylobacter spp. presence in the reproductive tract, lymphoid organs, liver-gallbladder, and ceca of commercial Leghorn laying hens; 2) species of Campylobacter present; and 3) antimicrobial resistance pattern of Campylobacter isolates. In study 1, three flocks ranging from 94 to 105 wk of age were sampled from a commercial laying complex. In study 2, two flocks, 82 and 84 wk of age, were sampled from a separate complex. Hens were killed, defeathered, aseptically necropsied, and the spleen, liver-gallbladder, ovarian follicles, and upper (infundibulum, magnum, and isthmus) and lower (shell gland and vagina) reproductive tracts were aseptically removed before the ceca. Samples were packed on ice and transported to the laboratory for evaluation. For speciation, a standard BAX real-time PCR method was used while susceptibility testing was performed using US National Antimicrobial Resistance Monitoring System (NARMS) standards and recommended quality control organisms. Isolates were examined for susceptibility using a semi-automated testing system (Sensititer) to the following 9 antimicrobials: azithromycin, clindamycin, ciprofloxacin, erythromycin, florfenicol, gentamicin, nalidixic acid, telithromycin, and tetracycline. In study 1, the isolation rate was 13, 67, 53, 3, 13, and 57% from the ovarian follicles, lower reproductive tract, upper reproductive tract, spleen, liver-gallbladder, and ceca, respectively. In study 2, the isolation rate was 17, 43, 33, 20, 17, and 73% from the ovarian follicles, lower reproductive tract, upper reproductive tract, spleen, liver-gallbladder, and ceca, respectively. Overall, 50% of isolates were Campylobacter jejuni, 49% Campylobacter coli, and 1% Campylobacter lari. In study 1, all of the isolates were pan-susceptible. In study 2, thirty-seven percent of the isolates were resistant to tetracycline. Commercial table egg laying hens housed in colony cages on wire floors had diverse Campylobacter spp. recovered from different tissues and these isolates were not resistant to a broad range of antimicrobials.

A year-long survey of fresh, retail poultry products on sale in Northern Ireland was undertaken to define the prevalence of Campylobacter spp. by using protocols based on ISO (standard) 10272-1:2006. Incubation at 37 and 42 degrees C was undertaken to increase the diversity of isolates obtained. Overall, 652 isolates were identified as Campylobacter spp. by using PCR and amplified fragment length polymorphic typing. Phenotyping wrongly identified 21% of isolates. Prevalences of Campylobacter found were chicken, 91% (n = 336); turkey, 56% (n = 77); and duck, 100% (n = 17). Prevalence rates for chicken produced in Northern Ireland, Scotland, England, and Wales were similar, with a mean value of 91%. The prevalences in product from the latter two countries were much higher than were found in two United Kingdom-wide surveys of chicken. The incubation temperature did not affect the relative proportions of the species isolated (P > 0.05). Campylobacter jejuni composed 64.6% of isolates, Campylobacter coli, 27.4%, and Campylobacter lari, 1%. Most cases of human campylobacteriosis are caused by C. jejuni and C. coli. The overall Campylobacter prevalence results are consistent with Northern Ireland surveys undertaken since 2000, and indicate that United Kingdom strategies to control Campylobacter in chicken have not had a significant effecton the prevalence of this pathogen in retail products on sale in Northern Ireland.

Following TA cloning and sequencing with a novel in silico-designed polymerase chain reaction (PCR) primer pair (f-ClvacJ/r-ClvacJ), approximately 750 base pairs (bp) of promoter and structural gene regions for vacJ and its adjacent genetic loci (approximately 1.14 kbp) were identified in 20 isolates of Campylobacter lari (urease-negative C. lari [n=7]; urease-positive thermophilic Campylobacter [n=13]). The nucleotide sequences of an approximately 70-bp non-coding region, including the typical promoter structure, showed sequence differences at 12 loci among 21 isolates including C. lari RM2100. The putative sigma70 promoter region upstream of the putative open reading frame (ORF), a start codon TTG and a probable ribosome binding site, AGGA, for the vacJ gene were also identified in all 21 C. lari isolates examined. Each ORF for the vacJ terminated with a TAA stop codon. No hypothetical transcriptional terminators were identified within the amplicons. The putative ORFs of the vacJ gene from 21 C. lari isolates consisted of 684 bases, similarly differing from those of the other thermophilic campylobacters (696 bases for C. jejuni RM1221 and NCTC11168 and C. coli RM2228; 690 for C. upsaliensis RM3195). Reverse transcription PCR analysis confirmed the transcription of the vacJ gene in the C. lari cells. A neighbour joining tree suggested a strong molecular discrimination efficacy between UPTC and UN C. lari employing vacJ nucleotide sequence information. The vacJ gene homologue from C. lari organisms appears not to be a lipoprotein signal peptide or a signal peptide in silico.

Cloning, sequencing and characterization of nearly full-length 23S rRNA genes in 12 urease-positive thermophilic Campylobacter (UPTC) isolates were carried out using two novel PCR primer pairs. Nucleotide sequences of the 23S rRNA genes from the 12 isolates were first shown not to carry any intervening sequences (IVSs) in both the 25 and 45 helix regions. Then, two PCR primer sets were designed in silico for amplification of the helix 25 and 45 regions within 23S rRNA gene sequences from Campylobacter lari. No IVSs were identified within the 23S rRNA genes among a total of 53 isolates of C. lari, following PCR amplification, TA cloning and sequencing procedures. Intact 23S rRNA was identified in all 65 C. lari isolates, resulting in no production of the fragmented 23S rRNA. These data suggest that C. lari may not have any opportunity to interact with any other source of IVSs until now, or has been unable to integrate IVSs into their own genomes. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Background The combined sequences encoding a partial and putative rpsI open reading frame (ORF), non-coding (NC) region, a putative ORF for the Campylobacter adhesin to fibronectin-like protein (cadF), a putative Cla_0387 ORF, NC region and a partial and putative Cla_0388 ORF, were identified in 16 Campylobacter lari isolates, using two novel degenerate primer pairs. Probable consensus sequence at the -35 and -10 regions were identified in all C. lari isolates, as a promoter. Results Thus, cadF (-like) gene is highly conserved among C. lari organisms. Transcription of the cadF (-like) gene in C. lari cells in vivo was also confirmed and the transcription initiation site was determined. A peptidoglycan-associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins was completely conserved amongst the putative cadF (-like) ORFs from the C. lari isolates. Conclusion The putative cadF (-like) ORFs from all C. lari isolates were nine amino acid larger than those from C. jejuni, and showed amino acid residues 137 -140 of FALG (50% identity), instead of the FRLS residues of the maximal fibronectin-binding activity site demonstrated within C. jejuni CadF. A neighbor joining tree constructed based on cadF (-like) gene sequence information formed a major cluster consisting of C. lari isolates, separating from the other three thermophilic campylobacters.

In order to elucidate the rate of thermophilic Campylobacter spp. carriage in Danish broiler production and to identify risk factors for occurrence of campylobacter in broiler flocks, a total of 88 randomly selected broiler flocks were tested for campylobacter infection, and a subsequent study of risk factors based on a questionnaire was conducted. The sample material comprised cloacal swabs from live birds before slaughter, and neck skin samples from carcasses at the end of the processing line. A total of 52% of the flocks were found Campylobacter spp.-positive before slaughter. At the end of processing, 24% of the flocks were positive. The species distribution was 87% Campylobacter jejuni, 8% Campylobacter coli and 5% Campylobacter lari. The following parameters were identified as significant risk factors: lack of a hygiene barrier (odds ratio (OR) = 3.1, 1.1 < OR < 9.3), presence of animals in the vicinity of the broiler house on farms with a missing hygiene barrier (OR = 7.0, 1.6 < OR < 33.9), livestock other than chickens on farms with a missing hygiene barrier (OR = 7.6, 1.4 < OR < 44.9), dividing the flock into batches for staggered slaughter (OR = 6.8, 1.2 < OR < 49.3), a down period of less than 14 days (OR = 5.0, 1.2 < OR < 22.6), and feeding purchased wheat rather than home-grown wheat (OR = 3.1, 1.0

A total of 209 well-defined Campylobacter, Helicobacter , and Wolinella strains were tested with a Gen-Probe oligodeoxyribonucleotide probe for Campylobacter jejuni , Campylobacter coli , and Campylobacter lari . Released ribosomal RNA was hybridized with the acridinium-ester-labeled probe. The amount of light proportional to the number of DNA-RNA hybrid molecules was captured by a photo-multiplier and converted to a digital signal. A reading of more than 50,000 relative light units (RLU) was considered positive. After isolates were cultivated, lysis and hybridization procedures were completed in 30 minutes. All 104 strains within the target species were positive, as well as two Campylobacter hyointestinalis strains. The remaining 103 strains, including 15 C hyointestinalis strains, were negative. Our data indicate a sensitivity of 1.0 and specificity of 0.98 (P

Aims: To determine the survival on pasture of Campylobacter spp. naturally present in bovine faeces and compare this with a previously published study using laboratory-cultured Campylobacter spp. Methods and Results: Ten freshly collected cow pats were deposited on pasture during summer, and Campylobacter spp. were enumerated by enrichment broth culture. The counts in three pats were below detection limits. Counts of Campylobacter spp. in the other seven pats fell below detection limits within 14 days. The geometric means of the counts up to 7 days produced a T90 of 2·2 days. Characterization of Campylobacter spp. by PCR and pulsed field gel electrophoresis indicated the presence of at least six genotypes of Campylobacter jejuni, Campylobacter coli and Campylobacter lari. Conclusions: Campylobacter spp. naturally present in cow faeces exhibited a similar survival rate to that previously determined using laboratory-cultured strains. The highly variable counts of naturally occurring Campylobacter spp., and the predominance of lower counts, also support the earlier decision to use laboratory-cultured strains in survival experiments. Significance and Impact of the Study: This study reaffirms the short survival of Campylobacter spp. in cow faeces deposited on pasture. This information will be incorporated into a ‘reservoir model’ for Campylobacter spp. in cow pats on New Zealand pastures.

Campylobacter lari is a member of the epsilon subdivision of the Proteobacteria and is part of the thermotolerant Campylobacter group, a clade that includes the human pathogen C. jejuni. Here we present the complete genome sequence of the human clinical isolate, C. lari RM2100. The genome of strain RM2100 is approximately 1.53 Mb and includes the 46 kb megaplasmid pCL2100. Also present within the strain RM2100 genome is a 36 kb putative prophage, termed CLIE1, which is similar to CJIE4, a putative prophage present within the C. jejuni RM1221 genome. Nearly all (90%) of the gene content in strain RM2100 is similar to genes present in the genomes of other characterized thermotolerant campylobacters. However, several genes involved in amino acid biosynthesis and energy metabolism, identified previously in other Campylobacter genomes, are absent from the C. lari RM2100 genome. Therefore, C. lari RM2100 is predicted to be multiply auxotrophic, unable to synthesize eight different amino acids, acetyl-coA, and pantothenate. Additionally, strain RM2100 does not contain a complete TCA cycle and is missing the CydAB terminal oxidase of the respiratory chain. Defects in the amino acid biosynthetic pathways in this organism could be potentially compensated by the large number of encoded peptidases. Nevertheless, the apparent absence of certain key enzymatic functions in strain RM2100 would be expected to have an impact on C. lari biology. It is also possible that the reduction in the C. lari metabolic machinery is related to its environmental range and host preference.

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

A total of 49 isolates of Campylobacter lari from human, poultry, ducks, pigs, and water were genetically characterized. The species were identified by biotyping and multiplex polymerase chain reaction (PCR). Automatic riboprints were performed with the PstI restriction enzyme and RiboPrinter®. The identification of the isolates was predicted when the corresponding pattern matched one of the patterns of the DuPont identification (DUP-ID) library and was then assigned an identification number. Thirty-five (71.4%) of the isolates were given a DUP-ID number. The isolates from water and animals showed a high degree of similarity to the human strains represented by DUP-PST1-1010, DUP-PST1-1166, DUP-PST1-1178, and DUP-PST1-1081. Some profiles (i.e., DUP-PST1-2021 and DUP-PST1-1184) were found only among the human isolates. Dendrogram analysis using BioNumerics grouped isolates into three main clusters. One of those clusters contained DUP-PST1-2021, DUP-PST1-1184, and DUP-PST1-1081, which was found in both humans and ducks. A second cluster generated DUP-PST1-1010, found in both humans and poultry, and DUP-PST1-1079, found in water. The third cluster consisted of two strains, DUP-PST1-1066 and DUP-PST1-1078, originating in humans, animals, and water. Three human strains and two poultry strains were diverse and formed their own clusters and could not be assigned a DUP-ID number. Because of the similarity of C. lari isolated from humans, poultry, ducks, pigs, and water, as well as the limited knowledge of environmental survival and its virulence factors, special hygienic precautions should be taken to avoid the risk of transmitting Campylobacter.

Campylobacter species are significantly implicated in human gastrointestinal infections. Of 20 species of Campylobacter, C. jejuni, C. coli and C. lari have been considered as the most important causative agents of human infections. In order to better understand the occurrence and epidemiology of these thermophilic Campylobacter species, an improved and rapid detection method is warranted. A novel triplex polymerase chain reaction (PCR) assay was developed based on the variable 16S-23S rDNA internal transcribed spacer (ITS) region to identify and discriminate between these species in water samples.Campylobacter species-specific primers for C. jejuni, C. coli and C. lari derived from highly variable sequences in the ITS region were used. Specificity of the newly designed primers and PCR conditions were verified using other species of Campylobacter as well as 31 different negative control species. The assay was further validated with 97 Campylobacter cultures from water samples.The assay was found to be simple, easy to perform, and had a high sensitivity, specificity and reproducibility. It enabled simultaneous detection and differentiation of multiple Campylobacter species in water samples.Use of the newly developed PCR assay, coupled with a previously developed rapid DNA template preparation step, will enable improved detection capabilities for Campylobacter species in environmental matrices.

A multiplex PCR assay has been developed for the identification of the six commonCampylobactertaxa associated with human gastroenteritis and/or septicaemia, namelyCampylobacter coli,Campylobacter fetus,Campylobacter hyointestinalissubsp.hyointestinalis,Campylobacter jejuni,Campylobacter lariandCampylobacter upsaliensis. The assay was developed using a combination of newly designed and published primers. It provided a specific PCR product for each of the fiveCampylobacterspecies 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 142Campylobacterstrains, the assay correctly identified all strains as 1 of the 6Campylobactertaxa. This multiplex PCR assay is a rapid, simple and practical tool for identification of the sixCampylobactertaxa commonly associated with gastroenteritis and/or septicaemia in humans, and offers an effective alternative to conventional biochemical-based assays.