Search

Background Campylobacter is one of the leading causes of human bacterial gastroenteritis worldwide. Campylobacter infections are most often associated with the consumption of raw milk, undercooked poultry, and contaminated water. Objective The RapidChek®Campylobacter test system (PTM number 052201) was validated for the detection of Campylobacter jejuni, C. coli, and C. lari in raw ground chicken, chicken carcass rinse, and turkey carcass sponges. Methods The method uses a proprietary enrichment medium. Following aerobic enrichment, an immunochromatographic test strip is inserted into the tube containing the enrichment, developed for 20 min, and interpreted. Campylobacter-inoculated food samples were tested by the method, as well as the USDA/FSIS cultural reference method; Isolation and Identification of Campylobacter jejuni/coli/lari from Poultry Rinse, Sponge and Raw Product Samples MLG 41.04. The candidate method was also confirmed by an alternative cultural method. The RapidChek method was tested with 50 Campylobacter strains comprised of C. jejuni, C. coli, and C. lari, and 30 non-target strains. Results A total of 80 low-level spiked samples were tested by both methods in the study. The candidate method yielded 49 presumptive positives: all presumptive results were confirmed culturally. The reference method produced a total of 41 confirmed positive results. No difference between the alternate confirmation method and reference confirmation method was observed. Probability of detection analysis demonstrated no significant differences in the number of positive samples detected by the candidate method and cultural reference method. The RapidChek method detected all 50 Campylobacter strains and none of the 30 non-target strains, including Campylobacter spp. other than C. jejuni, C. coli, and C. lari. Conclusion The candidate method performed as well as the reference method in the detection of C. jejuni, C. coli, and C. lari in raw ground chicken, chicken carcass rinse, and turkey carcass sponges. Highlights Aerobic enrichment of selected matrixes for 48 h yielded reliable presumptive results for Campylobacter.

Members of the Campylobacter lari group are causative agents of human gastroenteritis and are frequently found in shellfish, marine waters, shorebirds, and marine mammals. Within a One Health context, we used comparative genomics to characterize isolates from a diverse range of sources and geographical locations within Europe and Australia and assess possible transmission of food, animal, and environmental isolates to the human host. A total of 158 C. lari isolates from Australia, Denmark, France, and Germany, which included 82 isolates from human stool and blood, 12 from food, 14 from domestic animal, 19 from waterbirds, and 31 from the environment were analyzed. Genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance (AMR) traits was carried-out. Most of the isolates belonged to C. lari subsp. lari (Cll; 98, 62.0%), while C. lari subsp. concheus and C. lari urease-positive thermotolerant Campylobacter (UPTC) were represented by 12 (7.6%) and 15 (9.5%) isolates, respectively. Furthermore, 33 (20.9%) isolates were not assigned a subspecies and were thus attributed to distant Campylobacter spp. clades. Whole-genome sequence-derived multilocus sequence typing (MLST) and core-genome MLST (cgMLST) analyses revealed a high genetic diversity with 97 sequence types (STs), including 60 novel STs and 14 cgMLST clusters (≤10 allele differences), respectively. The most prevalent STs were ST-21, ST-70, ST-24, and ST-58 (accounting for 13.3%, 4.4%, 3.8%, and 3.2% of isolates, respectively). A high prevalence of the 125 examined virulence-related loci (from 76.8 to 98.4% per isolate) was observed, especially in Cll isolates, suggesting a probable human pathogenicity of these strains. IMPORTANCE Currently, relatedness between bacterial isolates impacting human health is easily monitored by molecular typing methods. These approaches rely on discrete loci or whole-genome sequence (WGS) analyses. Campylobacter lari is an emergent human pathogen isolated from diverse ecological niches, including fecal material from humans and animals, aquatic environments, and seafood. The presence of C. lari in such diverse sources underlines the importance of adopting an integrated One Health approach in studying C. lari population structure for conducting epidemiological risk assessment. This retrospective study presents a comparative genomics analysis of C. lari isolates retrieved from two different continents (Europe and Australia) and from different sources (human, domestic animals, waterbirds, food, and environment). It was designed to improve knowledge regarding C. lari ecology and pathogenicity, important for developing effective surveillance and disease prevention strategies.

We investigate the role of black-headed gulls (Chroicocephalus ridibundus), an omnivorous species that is among the most likely wild bird candidates for transmission of zoonotic agents, as a potential reservoir ofCampylobacterspp. Colonies with different anthropogenic pressures were studied to examine differences in exposure to sources ofCampylobacterbetween rural and urban birds. We recordedCampylobacterspp. in 4.87% of adult black-headed gulls and 2.22% of their chicks after analysing 1036 cloacal swabs collected over two breeding seasons in three colonies in northern Poland.Campylobacter jejuniwas found most frequently (85.72%), andCampylobacter lariandCampylobacter coliwere much scarcer. Prevalence ofCampylobacterdid not differ significantly between black-headed gulls breeding in urban (4.27%) and rural (3.80%) habitats. Almost all isolates from chicks and adults were susceptible to azithromycin (97.62%) and erythromycin (95.24%), but fewer to tetracycline (50.00%) and ciprofloxacin (47.62%).Campylobacterprevalence was unrelated to the date of sampling. Our study indicates that black-headed gulls are carriers of resistant to antibioticsCampylobacterand they can contaminate natural waterbodies with their faeces, which poses a threat to human and farm animal health.

Consumption of

Zebra finches (Taeniopygia guttata) are laboratory animal species commonly used for modeling neurobiology and learning. Historically, using bacterial culture, biochemical analysis, and 16S ribosomal RNA gene sequencing, bacterial isolates from feces of finches housed at Massachusetts Institute of Technology had been presumptively diagnosed as Campylobacter jejuni, which is commonly isolated from both domestic and wild birds. Although the zebra finches were not clinically affected, C. jejuni is a known zoonotic pathogen that causes gastroenteritis in humans worldwide. Human transmission is predominantly foodborne and associated with the consumption of contaminated poultry; however, humans can also become infected from contact with C. jejuni-infected reservoir hosts. Because C. jejuni-infected finches pose a risk to research personnel, a study was undertaken to investigate the prevalence and taxonomic identification of Campylobacter spp. present in the finch colony. Campylobacter spp. were isolated from a total of 26 finch fecal samples collected in 2003, 2010, and 2017. 16S ribosomal RNA sequencing of all isolates determined that they shared 99% identity with either C. jejuni or Campylobacter lari. Sixteen of the isolates were subjected to further biochemical characterization and atpA and rpoB gene sequence analysis. Based on these analyses, three clusters of Campylobacter species were identified. The draft whole-genome sequences were determined for one representative isolate from each cluster. A pan-genomic phylogenetic tree, average nucleotide identity, digital DNA-DNA hybridization, and orthologous gene analyses indicated that each isolate was its own novel species, distinct from C. jejuni and other avian Campylobacter species. We have named these novel species Campylobacter taeniopygiae, Campylobacter aviculae, and Campylobacter estrildidarum, and in each novel species, we identified virulence genes suggesting their pathogenic and zoonotic potential.

Consumption of Campylobacter contaminated food or water is a leading cause of human acute gastroenteritis. Campylobacter jejuni, Campylobacter coli, and Campylobacter lari account for over 95% of total Campylobacter infections. A multiplex quantitative polymerase chain reaction (qPCR) for simultaneous identification of C. jejuni, C. coli, and C. lari was developed for use with the SmartCycler II system.We evaluated and combined previously described primers and probes for Campylobacter detection, designed a new internal amplification control, and optimized the multiplex qPCR for the detection of C. jejuni, C. coli, and C. lari.This method was 100% specific when tested against a panel of 32 target Campylobacter strains and 31 non-Campylobacter reference strains. Furthermore, there was no cross-reactivity with seven strains from four nontarget Campylobacter species. The amplification efficiency of each target in this multiplex qPCR was over 90%, and each coefficient of linearity was greater than 0.99. With artificially mixed genomic DNA, this method detected as few as two, three, and two genome copies of C. jejuni, C. coli, and C. lari, respectively. This method was also able to detect these three Campylobacter species in artificially contaminated milk with a sensitivity of five spiked cells of each target per reaction.The three Campylobacter targets were simultaneously identified using artificially mixed genomic DNA and spiked raw milk. This SmartCycler-based multiplex qPCR is a rapid, specific, and sensitive method to identify C. jejuni, C. coli, and C. lari.

The aim of this study was to identify and characterize thermophilic Campylobacter species in faecal samples from goats in Khartoum State, Sudan, by application of multiplex polymerase chain reaction. Campylobacteriosis is a zoonotic disease of global concern, and the organisms can be transmitted to human via food, water and through contact with farm animals and pets. There are five clinically related Campylobacter species: Campylobacter jejuni (C. jejuni). Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter fetus. Conventional cultural methods to diagnose campylobacteriosis are tedious and time consuming. Wide ranges of genes have been reported to be used for PCR-based identification of Campylobacter spp. We used a multiplex PCR assay to simultaneously detect genes from the major five clinically significant Campylobacter spp. The genes selected were hipO (hippuricase) and 23S rRNA from glyA (serine hydroxymethyl transferase) from each of C. jejuni. C. coli, C. lari, and C. upsaliensis; and sapB2 (surface layer protein) from C. fetus subsp. fetus. The assay was used to identify Campylobacter isolates recovered from 336 cultured faecal samples from goats in three localities in Khartoum State. C. coli was the most predominant isolate (234; 69.6%), followed by C. jejuni (19; 5.7%), C. upsaliensis (13; 3.9%), C. fetus subsp. fetus (7; 2.1%) and C. lari (6; 1.8%). Twenty-nine goats showed mixed infection with Campylobacter spp., 21 of which harbored two Campylobacter spp., while eight animals were infected with three species. Ten out of twelve goats that displayed diarrhea harbored C. coli only. C. coli, C. jejuni and C. upsaliensis showed significant variation with localities. The prevalence of C. coli was significantly higher (87; 25.9%) in goats from Omdurman, whereas C. jejuni and C. upsaliensis were significantly higher (11; 3.3%, 9; 2.7%) in goats from Khartoum. The multiplex PCR assay was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing the isolates, even in mixed cultures. The study demonstrated the significance of goats as reservoirs in the dissemination of Campylobacter spp. which could be considered as potential agent of caprine enteritis and abortion as well as contamination of the wider environment posing serious public health concern in Khartoum State.

A multiplex PCR assay for simultaneous detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari was developed and validated to assess the occurrence of these bacteria in fresh chicken meat and by-products in Bangkok, Thailand, by using a new combination of four previously published PCR primers for C. jejuni, C. coli, C. lari, and a universal 16S rDNA gene as an internal control. The specificity was determined by using 13 strains of other bacteria. With pure culture DNA, the detection limit was 0.017 ng/PCR for C. jejuni and C. coli and was 0.016 ng/PCR for C. lari. It can detect 10 CFU of C. jejuni, C. coli, and C. lari in 2 g of chicken meat within a 16-h enrichment time. Our multiplex PCR assay was applied for identification of Campylobacter spp. in 122 supermarket samples and 108 fresh market samples. Of the 230 samples evaluated by multiplex PCR, 54.0, 3.3, and 10.7% of supermarket samples were positive for C. jejuni, C. coli, and mixed C. jejuni and C. coli, respectively, and 56.5 and 33.3% of fresh market samples were positive for C. jejuni and mixed C. jejuni and C. coli, respectively. No sample was positive for C. lari. Fresh market samples had significantly higher C. jejuni and C. coli contamination than those from supermarkets (relative risk: 1.3; P = 0.0001). Compared with the culture method (a gold standard), the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of multiplex PCR were 97.7, 86.8, 96.1, 92.0, and 95.2%, respectively. No significant difference was observed between results from two methods (P = 0.55). Therefore, the established multiplex PCR was not only rapid and easy to perform but had a high sensitivity and specificity to distinguish between C. jejuni, C. coli, and C. lari, even in samples containing mixed contamination. Our study indicated that fresh chicken meat and by-products from fresh markets were significantly less hygienic than those from supermarkets.

Full-length tlyA gene and its adjacent genetic loci from the urease-positive thermophilic Campylobacter (UPTC) CF89-12 [approximately 15,000 base pairs (bp) in length], as well as a reference strain Campylobacter lari RM2100 (approximately 9,000 bp), were analyzed. The possible open-reading frame of tlyA from UPTC CF89-12 was shown to have 720 bp with a calculated molecular mass of approximately 26.7 kDa. Using a primer pair designed in silico, a total of approximately 1.1 kbp consisting of putative promoter region, structural gene for tlyA, and its adjacent genetic loci were identified in all 17 C. lari isolates [n = 13 for UPTC; n = 4 for urease-negative (UN) C. lari]. Although sequence differences were demonstrated at approximately 20 loci within the 90 bp non-coding (NC) region, including the putative promoter structure candidates immediately upstream of the tlyA gene among the 18 isolates including C. lari RM2100, no sequence differences were identified within the NC region among the five UN C. lari isolates examined. A start codon ATG and a probable ribosome-binding site, AGGC(T)GG(A), for the tlyA gene were identified in all 18 isolates, including C. lari RM2100. The putative intrinsic ρ-independent transcriptional terminator structure candidate was also identified for the tlyA gene in both UPTC CF89-12 and C. lari RM2100. Additionally, the hemolysis assay was performed with some of the C. lari isolates. The tlyA gene nucleotide sequence data may possibly be useful for discrimination between UN C. lari and UPTC organisms, as well as for the differentiation among the four thermophilic Campylobacter species.

1. The aim of the present study was to evaluate the antimicrobial resistance of Campylobacter strains (C. jejuni, C. coli and C. lari) isolated from broiler carcasses processed in the State of Paraná, Brazil. 2. Rates of microbial resistance and susceptibility were assessed by both Disk Diffusion (DD) and Etest (Minimum Inhibitory Concentration) techniques. Antibiotics were tested using DD (12 antibiotics) and/or MIC (7 antibiotics) methods. 3. A total of 95.8% of the strains were resistant to at least two agents. In terms of multidrug resistance, 75% of strains were resistant to three or more groups of antibiotics. The highest rates of resistance were detected for cefalotin, ciprofloxacin, tetracycline and nalidixic acid. A high rate of susceptibility of the strains to erythromycin (95.8%) was found confirming that this is considered the agent of choice for treating campylobacteriosis. Comparison of the microbial resistance and susceptibility, as determined simultaneously by the two methods, found the techniques to be statistically equivalent for 5 out of the 6 antibiotics tested. 4. The results of this study suggest the need for adopting measures to control the use of antibiotics in broiler production to prevent multidrug resistance of Campylobacter strains and reduce the risk of serious human diseases caused by the consumption of contaminated chicken meat.

The thermotolerant species Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis are the causative agents of the human illness called campylobacteriosis. This infection represents a threat for the health of consumers in Europe. It is well known that poultry meat is an important food vehicle of Campylobacter infection. As emerged from the reported scientific literature published between 2006 and 2016, poultry meat sold at retail level in Europe represents an important source of the pathogen. The contamination level of poultry meat sold at retail can vary depending on pre- and post-harvest factors. Among the pre-harvest measures, strict biosecurity practices must be guaranteed; moreover, among post-harvest control measures scalding, chilling and removal of faecal residues can reduce the contamination level of Campylobacter. An additional issue is represented by increasing proportion of Campylobacter isolates resistant to tetracyclines, ciprofloxacin, and nalidixic acid, thus feeding a serious concern on the effectiveness of antibiotic treatment for human campylobacteriosis in a near future.

Summary Campylobacter causes acute gastroenteritis in people worldwide and is frequently isolated from food, animals and the environment. The disease is predominately food-borne but many routes of transmission and sources of infection have been described, including contact with pets. The prevalence of Campylobacter spp. in dogs and cats varies widely, and data on New Zealand pets are limited. This study aimed to investigate the prevalence of Campylobacter spp. in dogs, cats and retail raw meat pet food products in New Zealand and to characterize Campylobacter jejuni isolates using multilocus sequence typing (MLST). Ninety dogs and 110 cats examined at the Massey University Veterinary Teaching Hospital for elective procedures, and fifty locally purchased retail raw meat pet diets were sampled. Two culture protocols combining Bolton broth enrichment and mCCDA and CAT agars in a microaerobic atmosphere at 42°C and 37°C with species identification using PCR were performed. The prevalence of Campylobacter spp., C. jejuni, Campylobacter upsaliensis and Campylobacter helveticus was 36%, 13%, 23% and 1% in dogs and 16%, 5%, 5% and 7% in cats, respectively. One dog had Campylobacter lari confirmed, and three dogs and one cat had multiple Campylobacter spp. detected. Significantly more animals tested positive using CAT than mCCDA agar (P

The presence of Campylobacter species was studied in three Antarctic penguin species, Adélie (Pygoscelis adeliae), chinstrap (Pygoscelis antarctica) and gentoo (Pygoscelis papua). A total of 390 penguins were captured in 12 different rookeries along the Antarctic Peninsula with differences in the amount of human visitation: six colonies were highly visited [Stranger Point, King George Island (P. papua and P. adeliae); Hannah Point, Livingston Island (P. papua and P. antarctica); Deception Island (P. antarctica); and Paradise Bay, Antarctic Peninsula (P. papua)], and six colonies were rarely visited [Devil’s Point, Byers Peninsula, Livingston Island (P. papua); Cierva Cove, Antarctic Peninsula (P. papua); Rongé Island (P. papua and P. antarctica); Yalour Island (P. adeliae); and Avian Island (P. adeliae)]. A total of 23 strains were isolated from penguins from nine different rookeries. Campylobacter lari subsp. lari was isolated from eight samples (seven from P. papua and one from P. adeliae); C. lari subsp. concheus from 13 (ten from P. adeliae and three from P. antarctica) and C. volucris from two samples (both from P. papua). We did not find any significant differences in the prevalence of Campylobacter spp. between the populations in highly and rarely visited areas. This is the first report of C. lari subsp. concheus and C. volucris isolation from penguins in the Antarctic region., This study has been funded by Spanish Ministry of Science and Innovation and European Regional Development Fund under the Projects CGL2004-01348, POL2006-05175, CGL2007-60369 and CTM2011-24427 during writing. This is a contribution to the PINGUCLIM project.

This large-scale study compared incubation temperatures (37°C versus 42°C) to study the detection of thermophilic Campylobacter species, including Campylobacter jejuni , C. coli , and C. lari , in various surface water samples and bird fecal droppings around Hamilton Harbor, Lake Ontario. The putative culture isolates obtained from incubation temperatures of 37 and 42°C were confirmed by Campylobacter genus- and species-specific triplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer (ITS) region. A total of 759 water, wastewater, and bird fecal dropping samples were tested. Positive amplification reactions for the genus Campylobacter were found for 454 (60%) samples incubated at 37°C, compared to 258 (34%) samples incubated at 42°C. C. jejuni (16%) and C. lari (12%) were detected significantly more frequently at the 42°C incubation temperature than at 37°C (8% and 5%, respectively). In contrast, significantly higher rates of C. coli (14%) and other Campylobacter spp. (36%) were detected at the 37°C incubation temperature than at 42°C (8% and 7%, respectively). These results were consistent across surface water, wastewater, and bird fecal dropping samples. At times, Campylobacter spp. were recovered and detected at 37°C (3% for C. jejuni , 10% for C. coli , and 3% for C. lari ) when the same samples incubated at 42°C were negative. A significantly higher rate of other Campylobacter spp. was detected only at 37°C (32%) than only at 42°C (3%). These results indicate that incubation temperature can significantly influence the culturability and detection of thermophilic and other fastidious Campylobacter spp. and that a comprehensive characterization of the Campylobacter spp. in surface water, wastewaters, or bird fecal droppings will require incubation at both 37 and 42°C.

A real time quantitative PCR combined with propidium monoazide (PMA) treatment of samples was implemented to quantify live C. jejuni, C. coli and C. lari on broiler chicken carcasses at selected processing steps in the slaughterhouse. The samples were enumerated by culture for comparison. The Campylobacter counts determined with the PMA-qPCR and the culture method were not concordant. We conclude that the qPCR combined with PMA treatment of the samples did not fully reduce the signal from dead cells.

Presence or absence of Campylobacter spp. in water of five rivers upstream from an intake point for drinking water production was investigated, and isolates genetically compared with human, pig and poultry isolates in order to determine their source. River water and drinking water obtained from these rivers were sampled one time per month, over a period of one year, and tested for Campylobacter. Isolates were typed by PFGE. Campylobacter was not detected in treated drinking water, but 50% of the river samples were contaminated. Contamination was observed on the four seasons. In total, 297 Campylobacter isolates were collected and generated 46 PFGE profiles. Campylobacter jejuni was the most frequently detected species in samples (74.1% of the isolates), followed by Campylobacter coli (17.8%) and Campylobacter lari (8.1%). Forty-two of the 46 PFGE profiles were unique. Only one genotype was detected three times in a river during the year and four genotypes in two different rivers. When compared to animal and human Campylobacter PFGE profiles, 14, 11 and one Campylobacter genotypes from water were genetically closed to human, poultry, and pig Campylobacter genotypes, respectively. The Campylobacter population displayed a high level of genetic diversity, suggesting that contamination originated from various origins. Human, poultry and pig were sources of contamination of the river by Campylobacter. Finally, no Campylobacter were detected in drinking water, indicating that the risk of outbreaks due to consumption of drinking water is low.

The antibacterial activity of ethanolic extracts of selected Thai medicinal plants (Rhodomyrtus tomentosa (Aiton) Hassk., Quercus infectoria G. Olivier, and Eleutherine americana Merr.) against Campylobacter spp. was investigated. Sixty-five Campylobacter, including 39 isolates from humans and 26 isolates from chicken samples, were tested. Reference Campylobacter spp. that are commonly encountered in gastroenteritis were included. The ethanolic extract of E. americana demonstrated good antibacterial activity against all the tested isolates. Inhibition zones ranged from 10 to 37 mm. Minimum inhibitory concentration (MIC) of the extract against Campylobacter isolates from humans and chicken samples ranged from 31.25 to 500 μg/mL and 62.50 to 1,000 μg/mL, respectively. The minimum bactericidal concentration ranged from 31.25 to 1,000 μg/mL for isolates from humans and 125 to 1,000 μg/mL from chicken isolates. The bactericidal activity of the ethanolic extracts of E. americana against important Campylobacter spp., including Campylobacter coli MUMT 18630, Campylobacter fetus ATCC 27374, Campylobacter jejuni ATCC 81176, Campylobacter lari ATCC 43675, and Campylobacter upsaliensis DMST 19055, were assessed at MIC, 2 MIC, and 4 MIC by counting viable cells after various time intervals. At 4 MIC, the level of the tested isolates decreased by 2 to 5 log-fold within 8 h. The ethanolic extract of E. americana demonstrated antibacterial activity against all Campylobacter spp. from both human and chicken isolates. Further investigation of this plant species may provide an alternative medicine for Campylobacter infection and an effective food additive to prevent the infection.

Salmonella and Campylobacter are common bacterial pathogens associated with human gastro-enteritis; and raw poultry is considered to be an important source of these bacteria. To evaluate whether the Salmonella serovars and Campylobacter spp. bacteria could be monitored for the purpose of microbial presence, enumeration and antimicrobial resistance in raw poultry, 152 poultry carcasses were randomly selected from 10 markets in retail outlets of Phnom Penh during March 2006 to February 2007. The majority of poultry samples was contaminated by Salmonella serovars (88.2%) and Campylobacter spp. (80.9%). A very high contamination of Salmonella was found at 3-4 log₁₀ CFU/g for 22.4% of samples and of Campylobacter at 7-8 log₁₀ CFU/g for 1.3% of samples. Fifty nine different Salmonella serovars contaminated 134 poultry carcasses; five most prevalent serovars covered 29.1% of serovars isolates (Anatum, Typhimurium, Corvallis, Stanley and Enteritidis). Three Campylobacter species contaminating 123 raw poultry were Campylobacter jejuni (50.0%), Campylobacter coli (29.0%) and Campylobacter lari (21.0%). High antibiotic resistance percentages were found among Salmonella serovars and Campylobacter spp. isolates. This study revealed that raw poultry at the retail outlets in Phnom Penh markets are contaminated with high prevalences of food-borne pathogens, and communicating the importance of minimizing this risk in reducing human infections.

The presence of Campylobacter spp. was investigated in 41 Antarctic fur seals ( Arctocephalus gazella ) and 9 Weddell seals ( Leptonychotes weddellii ) at Deception Island, Antarctica. Infections were encountered in six Antarctic fur seals. The isolates, the first reported from marine mammals in the Antarctic region, were identified as Campylobacter insulaenigrae and Campylobacter lari .

Campylobacter infections have been reported at prevalences ranging from 2 to 50% in a range of wild bird species, although there have been few studies that have investigated the molecular epidemiology of Campylobacter spp. Consequently, whether wild birds are a source of infection in humans or domestic livestock or are mainly recipients of domestic animal strains and whether separate cycles of infection occur remain unknown. To address these questions, serial cross-sectional surveys of wild bird populations in northern England were carried out over a 2-year period. Fecal samples were collected from 2,084 wild bird individuals and screened for the presence of Campylobacter spp. A total of 56 isolates were recovered from 29 birds sampled at 15 of 167 diverse locales. Campylobacter jejuni , Campylobacter lari , and Campylobacter coli were detected by PCR, and the prevalences of different Campylobacter spp. in different avian families ranged from 0% to 33%. Characterization of 36 C. jejuni isolates by multilocus sequence typing revealed that wild birds carry both livestock-associated and unique strains of C. jejuni . However, the apparent absence of unique wild bird strains of C. jejuni in livestock suggests that the direction of infection is predominantly from livestock to wild birds. C. lari was detected mainly in wild birds sampled in an estuarine or coastal habitat. Fifteen C. lari isolates were analyzed by macrorestriction pulsed-field gel electrophoresis, which revealed genetically diverse populations of C. lari in Eurasian oystercatchers ( Haematopus ostralegus ) and clonal populations in magpies ( Pica pica ).

A polyphasic study was undertaken to clarify the taxonomic position of Campylobacter lari-like strains isolated from shellfish and humans. The diversity within the strain collection was initially screened by means of fluorescent amplified fragment length polymorphism analysis and whole-cell protein electrophoresis, revealing the existence of two clusters distinct from C. lari and other Campylobacter species. The divergence of these clusters was confirmed by phenotypic analysis and by 16S rRNA and hsp60 gene sequence analysis. Phylogenetic analysis identified C. lari, Campylobacter jejuni, Campylobacter coli and Campylobacter insulaenigrae as the closest phylogenetic neighbours of both taxa. DNA-DNA hybridizations revealed that one cluster, comprising 10 strains, represented a novel Campylobacter species, for which the name Campylobacter peloridis sp. nov. is proposed, with 2314BVA(T) (=LMG 23910(T) =CCUG 55787(T)) as the type strain. The second cluster, comprising six strains, represents a novel subspecies within the species C. lari, for which the name Campylobacter lari subsp. concheus subsp. nov. is proposed, with 2897R(T) (=LMG 21009(T) =CCUG 55786(T)) as the type strain. The description of C. lari subsp. concheus has the effect of automatically creating the subspecies Campylobacter lari subsp. lari subsp. nov. (type strain LMG 8846(T)=NCTC 11352(T)).

Conventional procedures for isolation of thermophilic Campylobacter spp. from chicken are complex, labor intensive, and time-consuming. The objective of this study was to create a novel Campylobacter culturing apparatus. A main concept of the device was based on the ability of Campylobacter to pass through a 0.45 microm pore size filter in viscous media. Preliminary study demonstrated that only viable Campylobacter moved through the membrane filter and could multiply in the enrichment culture. C. jejuni, C. coli, C. lari, and C. upsaliensis in the chicken samples were detected at cell concentrations as low as 10 cfu/g, after 24 h incubation at 42 degrees C. In total, 84 retail chicken samples were comparatively studied using both conventional method and apparatus. Sixteen samples (19.05%) were positive by the apparatus method; 14 (16.66%) of these positive samples contained C. coli and 2 (2.38%) contained C. jejuni. With the conventional method, 7 (8.33%) samples were positive 7 (8.33%) with C. coli. In conclusion, the apparatus detected more positive samples than did the conventional culture method.

This study aims to clarify the molecular characteristics of the urease gene operon from urease-positive thermophilic campylobacters (UPTC) obtained from different sources and in various countries. Sequence heterogeneity was observed for the promoter structures at the -35-like region among the 12 isolates examined. The most probable TTG start codon was suggested for the ureB and ureH genes, and for the ureA, E, F and G genes, ATG was suggested among all the isolates examined. Overlap was detected between ureA and ureB and between ureB and ureE among all the isolates examined. UPTC is the first example of an overlap between the two structural genes ureA and ureB. When the completely sequenced open reading frames (ORFs) for ureE, ureF, ureG and ureH were identified, non-coding regions between ureE and ureF, ureF and ureG, and ureG and ureH were also demonstrated. All six start codons of the six urease genes were demonstrated to be preceded by Shine-Dalgarno sequences among all the isolates examined. The Cys-His sequence corresponding to urease active sites were aligned perfectly and fully conserved among the three UPTC isolates examined. A putative and intrinsic p-independent transcriptional terminator was identified to be identical among all the isolates examined. A partial and putative ORF of about 200 bp in length showing high sequence similarity to GTP cyclohydrolase I was observed downstream of ureH.

Polymerase chain reaction (PCR) amplicons (approximately 2.5 kbp) encoding a cdt gene operon and two partial and putative open reading frames (ORFs) were identified in six urease-negative (UN) Campylobacter lari isolates using a new PCR primer pair constructed in silico. Three closely spaced and putative ORFs for cdtA, cdtB and cdtC, two putative promoters and a hypothetically intrinsic p-independent transcription terminator were found in the operon. Each ORF commenced with an ATG start codon and terminated with a TGA stop codon for cdtA and cdtB and a TAA for cdtC. Interestingly, an overlap of four nucleotides was detected between cdtA and cdtB and the non-coding region of six base pairs occurring between cdtB and cdtC. The start codons for the three cdt genes were preceded by Shine-Dalgarno sequences. Although nucleotide sequence differences were identified at seven loci in the cdtA gene, six in cdtB and two in cdtC among the seven isolates (including C. lari RM2100), no polymorphic sites occurred in the putative promoters, hypothetically intrinsic transcription terminator and the three ribosome binding sites among the seven isolates. All nine amino acid residues specific for both Escherichia coli cdtB and mammalian DNase I were completely conserved in the cdtB gene locus in the 26 C. lari isolates, as well as in C. jejuni and C. coli. No PCR amplicons were generated with urease-positive thermophilic campylobacters (UPTC; n=10) using the primer pair.

Aim: To examine the occurrence, diversity and transmission of Campylobacter in a poultry slaughterhouse. Methods and Results: During a 4-week period, a slaughterhouse was sampled alternately during slaughtering and the following mornings post-disinfection. Samples were taken from poultry at six stages in the slaughter process and from 25 environmental sites. For positive broiler flocks slaughtered on one occasion, 92% and 48% of the environmental sites were positive during slaughter and post-disinfection, respectively. For positive laying hen flocks slaughtered on three occasions, 8–56% and 12–20% of the environmental sites were positive during slaughter and post-disinfection, respectively. Genetic fingerprinting by amplified fragment length polymorphism (AFLP) of the 109 isolates obtained resulted in 28 different AFLP clones. Five AFLP clones were present for more than 1 week. Conclusions: Slaughtering of Campylobacter-positive broilers resulted in extensive contamination of the slaughterhouse, including the air. A high proportion of the laying hen flocks were Campylobacter positive, but these caused less environmental contamination than the broilers. This, together with the freezing of all layer carcasses, results in a lower public health risk from laying hens, when compared with broilers. Significance and Impact of the Study: When slaughtering Campylobacter-positive broilers, the implementation of preventive measures is important to reduce contamination of negative carcasses and to protect the workers against infection.

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals ( Phoca vitulina ) and a harbor porpoise ( Phocoena phocoena ) in Scotland, and the other describes the isolation of Campylobacter jejuni , Campylobacter lari , and an unknown Campylobacter species from northern elephant seals ( Mirounga angustirostris ) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42°C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae , with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus . Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.

Cloning, sequencing and molecular characterisation of a cryptic plasmid, pUPTC237, from a urease-positive thermophilic Campylobacter (UPTC) isolate obtained from the natural environment in Northern Ireland is reported in this study. Based on the determined DNA sequence, the pUPTC237 DNA was identified as a circular molecule of 3828 bp with a G+C content of 29.5%. As with other plasmid DNAs from Gram-negative bacteria, pUPTC237 contained an A+T-rich region (A+T content: 95%), followed by multiple direct tandem repeat units of 22 bp, characteristic of a replication origin and iteron sequence. A possible open reading frame (ORF)-1 was located upstream of the A+T-rich region and the iteron sequence that encoded a 460 amino acid protein similar to the mobilisation (mob) protein and two putative promoter structure sequences at the -35 and -10 regions and a possible ribosome binding site occurred upstream of the start codon for the ORF-1. Moreover, three possible ORFs (a short ORF-2 encoding 26 amino acids, similar to repA; an ORF-3 encoding 341 amino acids, similar to repB; and an ORF-4 encoding 96 amino acids with unknown function) were also identified. There are also two putative promoter structures for these three ORFs at the -35 and -10 regions upstream of the possible ORF-2. A possible transcription termination region was identified downstream of ORF-4. Northern blot hybridisation analysis suggested that these four ORFs constitute an operon and generate a messenger RNA (mRNA) transcript.

Campylobacter species, particularly thermophilic campylobacters, have emerged as a leading cause of human foodborne gastroenteritis worldwide, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari responsible for the majority of human infections. Although most cases of campylobacteriosis are self-limiting, campylobacteriosis represents a significant public health burden. Human illness caused by infection with campylobacters has been reported across Canada since the early 1970s. Many studies have shown that dietary sources, including food, particularly raw poultry and other meat products, raw milk, and contaminated water, have contributed to outbreaks of campylobacteriosis in Canada. Campylobacter spp. have also been detected in a wide range of animal and environmental sources, including water, in Canada. The purpose of this article is to review (i) the prevalence of Campylobacter spp. in animals, food, and the environment, and (ii) the relevant testing programs in Canada with a focus on the potential links between campylobacters and human health in Canada.

Traditionally fluorescence in situ hybridization (FISH) has been performed with labeled DNA oligonucleotide probes. Here we present for the first time a high affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting thermotolerant Campylobacter spp. using FISH. Thermotolerant Campylobacter spp, including the species Campylobacter coli, Campylobacter jejuni and Campylobacter lari, are important food and water borne pathogens. The designed PNA probe (CJE195) bound with higher affinity to a previously reported low affinity site on the 16S rRNA than the corresponding DNA probe. PNA also overcame the problem of the lack of affinity due to the location of the binding site and the variation of the target sequence within species. The PNA probe specificity was tested with several bacterial species, including other Campylobacter spp. and their close relatives. All tested C. coli, C. jejuni and C. lari strains were hybridized successfully. Aging of the Campylobacter cultures caused the formation of coccoid forms, which did not hybridize as well as bacteria in the active growth phase, indicating that the probe could be used to assess the physiological status of targeted cells. The PNA FISH methodology detected C. coli by membrane filtration method from C. coli spiked drinking water samples.

We studied the prevalence of the omp50 gene and the Omp50 protein in Campylobacter strains. Immunodetection assays and DNA-DNA hybridizations showed that most C. coli strains tested were negative and most C. jejuni and C. lari strains tested were positive. A PCR assay was developed, using the omp50 gene as a species-specific target. We propose a combination of a hippurate test and an omp50 assay to perform identification of Campylobacter species.

A total of 139 surface water samples from seven lakes and 15 rivers in southwestern Finland were analyzed during five consecutive seasons from autumn 2000 to autumn 2001 for the presence of various enteropathogens ( Campylobacter spp., Giardia spp ., Cryptosporidium spp., and noroviruses) and fecal indicators (thermotolerant coliforms, Escherichia coli , Clostridium perfringens , and F-RNA bacteriophages) and for physicochemical parameters (turbidity and temperature); this was the first such systematic study. Altogether, 41.0% (57 of 139) of the samples were positive for at least one of the pathogens; 17.3% were positive for Campylobacter spp. (45.8% of the positive samples contained Campylobacter jejuni , 25.0% contained Campylobacter lari , 4.2% contained Campylobacter coli , and 25.0% contained Campylobacter isolates that were not identified), 13.7% were positive for Giardia spp., 10.1% were positive for Cryptosporidium spp., and 9.4% were positive for noroviruses (23.0% of the positive samples contained genogroup I and 77.0% contained genogroup II). The samples were positive for enteropathogens significantly ( P < 0.05) less frequently during the winter season than during the other sampling seasons. No significant differences in the prevalence of enteropathogens were found when rivers and lakes were compared. The presence of thermotolerant coliforms, E. coli , and C. perfringens had significant bivariate nonparametric Spearman's rank order correlation coefficients ( P < 0.001) with samples that were positive for one or more of the pathogens analyzed. The absence of these indicators in a logistic regression model was found to have significant predictive value (odds ratios, 1.15 × 10 8 , 7.57, and 2.74, respectively; P < 0.05) for a sample that was negative for the pathogens analyzed. There were no significant correlations between counts or count levels for thermotolerant coliforms or E. coli or the presence of F-RNA phages and pathogens in the samples analyzed.

Four hundred pork livers from bacon pigs (37 herds) obtained at six pig-processing plants were studied to assess the Campylobacter contamination rate. Deep tissue areas were sampled immediately after evisceration. Approximately 6% of livers were infected with Campylobacter spp., including Campylobacter coli (67%), Campylobacter jejuni (30%), and Campylobacter lari (3%). The 60 resulting isolates (39 C. coli isolates, 19 C. jejuni isolates, and 2 C. lari isolates) employed in this study were characterized at the subspecies level in a comparison of eight phenotyping schemes, including four biotyping, two serotyping, and two phage-typing schemes. The Skirrow-Benjamin biotyping scheme produced two biotypes for C. jejuni, i.e., biotype 2 (95%) and biotype 1 (5%). The Lior biotyping scheme subdivided C. coli into biotype 1 (41%) and biotype 2 (59%), while biotype 4 was the dominant type (95%) for C. jejuni. The Roop scheme allowed further differentiation of C. coli into three biovars, i.e., biovar 1 (57%), biovar 2 (40%), and biovar 3 (3%), and it subdivided C. jejuni into two biotypes, i.e., biovar 1 (95%) and biovar 2 (5%). Preston biotyping produced the largest degree of subspecies differentiation, with 18 C. coli biotypes and 7 C. jejuni biotypes being identified. The most common were biotypes 2650 and 6030, representing 18 and 42% of all C. coli and C. jejuni isolates, respectively. The Penner-Hennessy serotyping scheme successfully serotyped 89% of the isolates, with 10 serotypes being identified; 30% of the serotypeable isolates were accounted for by Penner 23, followed by Penner 20 (16%) and Penner 39 (14%). The Lior serotyping scheme successfully serotyped only 45% of the strains, and eight serogroups were identified, with Lior 36 (31%), Lior 20 (23%), and Lior 5 being the most frequent. The Preston scheme and the Khakhria-Lior phage-typing scheme were able to type 16 and 25% of the isolates, respectively. The Preston scheme produced three phage groups, i.e., 69 (56%), 90 (22%), and 116 (22%), and the Khakhria-Lior scheme also produced three phage types, i.e., 44 (40%), 27 (33%), and 37 (20%), as well as atypical lysis patterns (7%). The results of this study demonstrate the role of Preston biotyping in the phenotyping of isolates, particularly in diagnostic laboratories that have no access or limited access to molecular typing equipment.

Consecutive isolates of quinolone-resistant campylobacter isolated over a 5 year period (1990-1995) from the faeces of patients with enteritis in Plymouth, UK, were examined for the epidemiology of mutations in gyrA (n = 127). In addition, clinical isolates and poultry isolates from Germany, The Netherlands and other regions of the UK collected before 1995 were examined for mutations in the quinolone resistance-determining region of gyrA by single- stranded conformational polymorphism analysis and direct sequencing of a 270 bp fragment of PCR-generated DNA. The majority of isolates (173/208) carried a mutation at codon 86 in gyrA resulting in substitution of Ile for Thr; all of these were resistant to ciprofloxacin (MIC ≥ 2 mg/L). One isolate of Campylobacter jejuni had a mutation at Asp-90, and another had a double muta- tion at Thr-86 and Pro-104. Only two resistant isolates showed no mutation in gyrA. A novel gyrA sequence was amplified from two Campylobacter lari and one C. jejuni, which exhibited a valine at codon 86. Only 8/192 isolates had changes in gyrB; all were shown to relate to silent mutations in gyrB and presumably reflect natural polymorphisms in the gene.

A total of 1,794 migrating birds trapped at a coastal site in southern Sweden were sampled for detection of Campylobacter spp. All isolates phenotypically identified as Campylobacter jejuni and a subset of those identified as non- C. jejuni were identified to the species level by PCR-based techniques. C. jejuni was found in 5.0% of the birds, Campylobacter lari was found in 5.6%, and Campylobacter coli was found in 0.9%. An additional 10.7% of the tested birds were infected with hippurate hydrolysis-negative Campylobacter spp. that were not identified to the species level. The prevalence of Campylobacter spp. differed significantly between ecological guilds of birds. Shoreline-foraging birds feeding on invertebrates and opportunistic feeders were most commonly infected (76.8 and 50.0%, respectively). High prevalence was also shown in other ground-foraging guilds, i.e., ground-foraging invertebrate feeders (11.0%), ground-foraging insectivores (20.3%), and plant-eating species (18.8%). Almost no Campylobacter spp. were found in ground-foraging granivores (2.3%), arboreal insectivores (0.6%), aerial insectivores (0%), or reed- and herbaceous plant-foraging insectivores (3.5%). During the autumn migration, a high proportion of samples from juveniles were positive (7.1% in passerines, 55.0% in shorebirds), indicating transmission on the breeding grounds or during the early part of migration. Prevalence of Campylobacter spp. was associated with increasing body mass among passerine bird species. Furthermore, prevalence was higher in short-distance migrants wintering in Europe than in long-distance migrants wintering in Africa, the Middle East, or Asia. Among ground-foraging birds of the Muscicapidae, those of the subfamily Turdinae (i.e., Turdus spp.) showed a high prevalence of Campylobacter spp., while the organism was not isolated in any member of the subfamily Muscicapinae (i.e., Erithacus and Luscinia ). The prevalence of Campylobacter infection in wild birds thus seems to be linked to various ecological and phylogenetic factors, with great variations in carriership between different taxa and guilds.

A study was conducted to investigate the prevalence of Salmonella and Campylobacter from farm to slaughter. The efficiency of trimming and water spray (490 to 588 kPa pressure) on the removal of visible fecal contamination from broiler carcasses before chilling was also investigated. Drag swabs were used to sample litter from the farm houses. Samples of ceca and carcasses without and with visible fecal contamination before and after trimming or spray washing of fecal contamination were taken during slaughter of the flocks previously visited at the farms. There was a low prevalence of Salmonella on the litter from the farms (5%) and cecum and carcasses (0%). However, Campylobacter jejuni and Campylobacter coli were present in farms' litter (100 and 58.8%, respectively), cecum samples (100 and 70.6%, respectively), and carcasses with (58.8 and 11.6%, respectively) and without (17.6 and 9.8%, respectively) visible fecal contamination. There was high prevalence of C. jejuni but at low counts and low prevalence and high counts of C. coli. Campylobacter lari was not detected in any sample. Trimming the visible fecal contamination decreased the prevalence of C. jejuni but increased occurrence of C. coli. Trimming did not reduce the counts of Campylobacter and of hygiene indicator microorganisms on the carcasses. Water shower reduced the counts of hygiene indicator microorganisms by 20%. Therefore, control measures for preventing introduction of Campylobacter and the use of good hygienic conditions are needed to warrant the microbiological quality and safety of broiler carcasses.

Southern hybridisation shows that urease-negative (UN) Campylobacter lari JCM2530(T) carries two putative major outer membrane protein (MOMP) genes. Sequences of approximately 2.1 kbp, encoding non-coding (NC) regions, with possible open reading frames (ORFs) for MOMP (porA1 or porA2) of approximately 1.2 kbp, NC regions and partial and putative Cla_0435 or Cla_1109 ORFs were identified in all five UN C. lari isolates examined, following polymerase chain reaction (PCR) cloning and sequencing. Each putative MOMP structural gene carried start and stop codons and ribosome binding sites of 1236-1278 bp in length. The putative sigma70 transcriptional promoter and the hypothetical rho-independent transcription terminator structures were also seen. Using Northern hybridisation, there was in vivo monocistronic MOMP gene transcription. In addition, in a Japanese urease-positive thermophilic Campylobacter (UPTC) CF89-12 strain, the porA1 gene locus, including an extra gene (approximately 2000 bp in length) was identified. The extra gene may occur within the porA1 gene locus in the eight UPTC isolates of the 23 C. lari isolates examined. Thus, a genetic heterogeneity occurred within the porA1 gene locus from some of the C. lari organisms including the UPTC CF89-12.

Aims: The differences between phenotyping and genotyping (polymerase chain reaction– restriction fragment length polymorphism) of Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis were assessed. Methods and Results: A total of 51, 63 and 88 strains from dogs, pigs and humans, respectively, were examined. The strains were first typed by biochemical methods, then by PCR–RFLP using AluI and Tsp509I. None of the strains were typed as Camp. lari by the PCR–RFLP. The biggest differences were found in the identification of Camp. jejuni and Camp. coli. The main discrepancies were caused with the hippurate hydrolysis test and sensitivity to cephalothin and nalidixic acid. Strains which were identified biochemically as Camp. coli and by digestion with AluI as Camp. jejuni (eight strains) were tested for the presence of the hippuricase gene. Conclusion: The PCR typing results showed the presence of the hippuricase gene as unique to Camp. jejuni. Significance and Impact of the Study: A reliable identification of Campylobacter spp. should be supplemented with a molecular method.

Aim: To estimate the prevalence of thermotolerant Campylobacter spp. in commercially reared partridges (Perdix perdix) in southern Italy. Methods and Results: Cloacal swabs of partridges (n = 240), equally distributed between male and female birds, from a game bird farm located in the Southern Italy were examined for the prevalence of thermotolerant Campylobacter spp. The samples were processed in order to detect thermotolerant Campylobacter spp. by culture methods. The positive samples were then confirmed by multiplex polymerase chain reaction. Thermotolerant Campylobacter spp. were isolated from 118 (49·2%) of the 240 cloacal swabs examined. As proved by PCR, 100% of the strains were identified as Campylobacter coli (118/118), and 15 (12·7%) out of the 118 positive samples were also positive for Campylobacter jejuni. In contrast, Campylobacter lari was not identified. Adult partridges showed a significantly higher prevalence (P

Summary: The internal spacer region (ISR) between the 16S and 23S rRNS genes of Campylobacter was investigated by PCR fragment length typing and DNA sequencing of clinical and chicken wild-type isolates. PCR fragment length typing showed one fragment of 859 nt in length for the 12 strains of Campylobacter coli investigated. Thirty-six of the Campylobacter jejuni subsp. jejuni strains possessed one fragment, which varied in size between 727 and 802 nt. Three strains showed two fragments between 501 and 923 nt. Strains of C. jejuni subsp. doylei, Campylobacter lari and Campylobacter upsaliensis possessed one or two fragments with lengths different from those of C. coli and C. jejuni subsp. jejuni. DNA sequences were obtained from 54 nt downstream of rrs up to rrl of four strains of C. coli, eight strains of C. jejuni subsp. jejuni, and one strain each of C. jejuni subsp. doylei and C. lari, selected to represent the different biotypes of Campylobacter. ISR lengths determined by PCR fragment length typing and DNA sequencing corresponded for 12 strains. For two strains of C. coli, PCR fragment length typing underestimated ISR lengths by 159 and 193 nt, probably related to incomplete resolution of the distal helical structures, which were not fully denatured during PAGE. For the 14 strains and the published C. jejuni subsp. jejuni sequence, the first 206-211 nt were conserved and included the two tRNA genes in the characteristic tRNAAla to tRNAlle order separated by a short 8-9 nt spacer region. Within the region downstream of tRNAlle, conserved regions were identified which allowed a separation of C. lari from C. coli and C. jejuni but not separation of C. coli from C. jejuni. The 69-282 nt longer variable regions in C. coli strains allowed separation of this species from C. jejuni, confirming results obtained by PCR typing. Certain nucleic acid positions in variable regions were related to the Lior biotypes. Sequence information from ISRs of more strains is needed to ascertain if separation of species and biotypes will be possible for diagnostic purposes.

A survey of the isolation rate and population size of thermophilic campylobacters in lambs at slaughter was carried out to determine the seasonal variation of thermophilic campylobacters in ovine hosts. Isolation rates determined by enrichment methods were always higher than those using direct plating onto selective agar and showed that Campylobacter could be isolated from 91·7% (n = 360) of samples from the small intestine of the lambs. Enumerations (MPN), done monthly over a 2-year period, averaged 4·00 log 10 (n = 1080, S.D. 0·16) campylobacters g−1 fresh weight (fw) intestinal contents with some samples giving values higher than 7 log MPN gfw−1. These results show that the prevalence of thermophilic campylobacters in sheep intended for slaughter is much higher than previously reported. Statistical analyses showed that there was a significant seasonal periodicity in the Campylobacter populations in the small intestines of lambs at slaughter (P = 0·004) but that there was no statistical relationship with environmental parameters such as minimum and maximum temperature, rainfall or hours of sunshine. In an analysis along the length of the intestinal tract of lambs, campylobacters were isolated from 80% of small intestine and 30% of rumen samples, but not from the true stomach, large intestine or caecal samples. A survey of sheep grazing on salt marsh, fell (upland) and farm pasture showed that the isolation rate of thermophilic campylobacters in fresh faeces was substantially lower (29·3% (n = 420)) than that from the small intestine of lambs at slaughter. No significant difference (>0·05) was found between the isolation rate of campylobacters from faeces sampled in late spring/early summer and autumn, nor between the different types of grazing.

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.

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.

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.

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.

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.