Increasing Prevalence of Campylobacter jejuni in Feedlot Cattle through the Feeding Period

The prevalence of Campylobacter jejuni in commercial feedlot cattle was monitored throughout the feeding period by repeated bacteriologic culture of feces. Fecal pats (n = 10) in 20 feedlot pens were sampled at 2-weeks interval beginning at entry into the feedlot and continuing until slaughter. The least-squares mean C. jejuni prevalence increased from 1.6% at the first sampling to 61.3% at the final sampling just prior to slaughter. Diverse C. jejuni pulsed-field gel electrophoresis macrorestriction profiles (MRP) were identified among the cattle isolates, but five prevalent MRP and minor variants accounted for >80% of all typed isolates. Chlorination of the water supplied to the water troughs of half of the pens did not affect C. jejuni prevalence in the cattle. Overall, the least-squares mean C. jejuni prevalences were 45.6 and 43.6% in chlorinated and nonchlorinated feedlot pens, respectively. The results of this study demonstrate apparent transmission of C. jejuni among feedlot cattle during the feeding period, unaffected by water chlorination, resulting in a high prevalence of C. jejuni excretion by cattle approaching slaughter.     

Prevalence of Campylobacter spp. in Cattle in Finland and Antimicrobial Susceptibilities of Bovine Campylobacter jejuni Strains

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

Effect of Incubation Temperature on Isolation of Campylobacter jejuni Genotypes from Foodstuffs Enriched in Preston Broth

Preston broth and agar incubated at either 37 or 42°C have been widely used to isolate campylobacters from foodstuffs. The consequences of using either incubation temperature were investigated. Retail packs of raw chicken (n = 24) and raw lamb liver (n = 30) were purchased. Samples were incubated in Preston broth at 37 and 42°C and then streaked onto Preston agar and incubated as before. Two Campylobacter isolates per treatment were characterized. Poultry isolates were genotyped by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and flagellin PCR-restriction fragment length polymorphism, and lamb isolates were genotyped by RAPD only. In total, 96% of the poultry and 73% of the lamb samples yielded campylobacters. The lamb isolates were all Campylobacter jejuni, as were 96% of the poultry isolates, with the remainder being Campylobacter lari. The incubation temperature had no significant effect on the number of positive samples or on the species isolated. However, genotyping of the C. jejuni isolates revealed profound differences in the types obtained. Overall (from poultry and lamb), the use of a single incubation temperature, 37°C, gave 56% of the total number of RAPD C. jejuni genotypes, and hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37°C gave 47% of the PFGE genotypes but 53% were exclusively recovered after incubation at 42°C. Thus, the incubation temperature of Preston media selects for certain genotypes of C. jejuni, and to detect the widest range, samples should be incubated at both 37 and 42°C. Conversely, genotyping results arising from the use of a single incubation temperature should be interpreted with caution.     

Predominant Campylobacter jejuni Sequence Types Persist in Finnish Chicken Production

Consumption and handling of chicken meat are well-known risk factors for acquiring campylobacteriosis. This study aimed to describe the Campylobacter jejuni population in Finnish chickens and to investigate the distribution of C. jejuni genotypes on Finnish chicken farms over a period of several years. We included 89.8% of the total C. jejuni population recovered in Finnish poultry during 2004, 2006, 2007, 2008, and 2012 and used multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) to characterize the 380 isolates. The typing data was combined with isolate information on collection-time and farm of origin. The C. jejuni prevalence in chicken slaughter batches was low (mean 3.0%, CI_95% [1.8%, 4.2%]), and approximately a quarter of Finnish chicken farms delivered at least one positive chicken batch yearly. In general, the C. jejuni population was diverse as represented by a total of 63 sequence types (ST), but certain predominant MLST lineages were identified. ST-45 clonal complex (CC) accounted for 53% of the isolates while ST-21 CC and ST-677 CC covered 11% and 9% of the isolates, respectively. Less than half of the Campylobacter positive farms (40.3%) delivered C. jejuni-contaminated batches in multiple years, but the genotypes (ST and PFGE types) generally varied from year to year. Therefore, no evidence for a persistent C. jejuni source for the colonization of Finnish chickens emerged. Finnish chicken farms are infrequently contaminated with C. jejuni compared to other European Union (EU) countries, making Finland a valuable model for further epidemiological studies of the C. jejuni in poultry flocks.     

Multilocus Sequence Typing for Comparison of Veterinary and Human Isolates of Campylobacter jejuni

Multilocus sequence typing (MLST) has been applied to 266 Campylobacter jejuni isolates, mainly from veterinary sources, including cattle, sheep, poultry, pigs, pets, and the environment, as well as isolates from human cases of campylobacteriosis. The populations of veterinary and human isolates overlap, suggesting that most veterinary sources should be considered reservoirs of pathogenic campylobacters. There were some associations between source and sequence type complex, indicating that host or source adaptation may exist. The pig isolates formed a distinct group by MLST and may well represent a potential pig-adapted clone of C. jejuni. A subset (n = 82) of isolates was reanalyzed with a second MLST scheme which provided a unique set of isolates that had been analyzed at a total of 12 loci. The distribution of isolates among the complexes in each of the two schemes was similar but not identical. In addition to isolates from human outbreaks, one group of isolates that were not epidemiologically linked was also identical at all 12 loci. This group of isolates is believed to represent another stable strain of C. jejuni.     

Multilocus Sequence Typing, Pulsed-Field Gel Electrophoresis, and fla Short Variable Region Typing of Clonal Complexes of Campylobacter jejuni Strains of Human, Bovine, and Poultry Origins in Luxembourg

Campylobacter jejuni is the most common cause of bacterial gastroenteritis in Luxembourg, with a marked seasonal peak during summer. The majority of these infections are thought to be sporadic, and the relative contribution of potential sources and reservoirs is still poorly understood. We monitored human cases from June to September 2006 (n = 124) by molecular characterization of isolates with the aim of rapidly detecting temporally related cases. In addition, isolates from poultry meat (n = 36) and cattle cecal contents (n = 48) were genotyped for comparison and identification of common clusters between veterinary and human C. jejuni populations. A total of 208 isolates were typed by sequencing the fla short variable region, macrorestriction analysis resolved by pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). We observed a high diversity of human strains during a given summer season. Poultry and human isolates had a higher diversity of sequence types than isolates of bovine origin, for which clonal complexes CC21 (41.6%) and CC61 (18.7%) were predominant. CC21 was also the most common complex found among human isolates (21.8%). The substantial concordance between PFGE and MLST results for this last group of strains suggests that they are clonally related. Our study indicates that while poultry remains an important source, cattle could be an underestimated reservoir of human C. jejuni cases. Transmission mechanisms of cattle-specific strains warrant further investigation.     

Amplified Fragment Length Polymorphism Analysis of Campylobacter jejuni Strains Isolated from Chickens and from Patients with Gastroenteritis or Guillain-Barré or Miller Fisher Syndrome

The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships between Campylobacter jejuni strains infecting chickens (n = 54) and those causing gastroenteritis in humans (n = 53). In addition, C. jejuni strains associated with the development of Guillain-Barré syndrome (GBS) (n = 14) and Miller Fisher syndrome (MFS) (n = 4), two related acute paralytic syndromes in human, were included. Strains were isolated between 1989 and 1998 in The Netherlands. The AFLP banding patterns were analyzed with correlation-based and band-based similarity coefficients and UPGMA (unweighted pair group method using average linkages) cluster analysis. All C. jejuni strains showed highly heterogeneous fingerprints, and no fingerprints exclusive for chicken strains or for human strains were obtained. All strains were separated in two distinct genetic groups. In group A the percentage of human strains was significantly higher and may be an indication that genotypes of this group are more frequently associated with human diseases. We conclude that C. jejuni from chickens cannot be distinguished from human strains and that GBS or MFS related strains do not belong to a distinct genetic group.     

Campylobacter jejuni Strains Compete for Colonization in Broiler Chicks

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal^r strain) and one streptomycin-resistant strain (the 02-833L Str^r strain). In vitro binding assays revealed that the C. jejuni F38011 Nal^r and 02-833L Str^r strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str^r strain relative to that of the C. jejuni F38011 Nal^r strain competitively inhibited the binding of the C. jejuni F38011 Nal^r strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str^r strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal^r strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.     

Typing of Campylobacter jejuni and Campylobacter coli isolated from live broilers and retail broiler meat by flaA-RFLP, MLST, PFGE and REP-PCR

We analyzed 100 Campylobacter spp. isolates (C. jejuni and C. coli) from Grenada, Puerto Rico and Alabama, which were collected from live broilers or retail broiler meat. We analyzed these isolates with four molecular typing methods: restriction fragment length polymorphism of the flaA gene (flaA-RFLP), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and automated repetitive extragenic palindromic polymerase chain reaction (REP-PCR) using the DiversiLab system. All methods performed similarly for the typing of C. jejuni and C. coli. The DNA extraction method appears to influence the results obtained with REP-PCR. This method was better for the typing of C. jejuni than C. coli, however both REP-PCR and flaA-RFLP generated types that were indistinguishable between C. jejuni and C. coli and appeared to be random, without any relationship to species, location, or source of isolates. PFGE and MLST generated typing results that had a better correlation with the geographic location of the isolates and showed higher concordance with the Wallace coefficient. The adjusted Rand coefficient did not show higher concordance among the methods, although the PFGE/MLST combination exhibited the highest concordance. PFGE and MLST revealed a better discriminatory power for C. coli isolates than REP-PCR or flaA-RFLP. The use of readily available online tools to calculate the confidence interval of the Simpson's index of diversity and the adjusted Rand and Wallace coefficients helped estimate the discriminatory power of typing methods. Further studies using different C. jejuni and C. coli strains may expand our understanding of the benefits and limitations of each of these typing methods for epidemiological studies of Campylobacter spp.     

Prevalence and Pathogenic Potential of Campylobacter Isolates from Free-Living,Human-Commensal American Crows

Recent studies have suggested a potential role for wild birds in zoonotic transmission of Campylobacter jejuni, the leading cause of gastroenteritis in humans worldwide. In this study, we detected Campylobacter spp. in 66.9% (85/127) of free-ranging American crows (Corvus brachyrhyncos) sampled in the Sacramento Valley of California in 2012 and 2013. Biochemical testing and sequence analysis of 16S rRNA revealed that 93% of isolates (n = 70) were C. jejuni, with cytolethal distending toxin (CDT) and flagellin A genes detected by PCR in 20% and 46% of the C. jejuni isolates (n = 59), respectively. The high prevalence of C. jejuni, coupled with the occurrence of known virulence markers CDT and flagellin A, demonstrates that crows shed Campylobacter spp. in their feces that are potentially pathogenic to humans. Crows are abundant in urban, suburban, and agricultural settings, and thus further study to determine their role in zoonotic transmission of Campylobacter will inform public health.     

Genetic Diversity of Campylobacter jejuni Isolates from Farm Animals and the Farm Environment

The genetic diversity of Campylobacter jejuni isolates from farm animals and their environment was investigated by multilocus sequence typing (MLST). A total of 30 genotypes, defined by allelic profiles (assigned to sequence types [STs]), were found in 112 C. jejuni isolates originating in poultry, cattle, sheep, starlings, and slurry. All but two of these genotypes belonged to one of nine C. jejuni clonal complexes previously identified in isolates from human disease and retail food samples and one clonal complex previously associated with an environmental source. There was some evidence for the association of certain clonal complexes with particular farm animals: isolates belonging to the ST-45 complex predominated among poultry isolates but were absent among sheep isolates, while isolates belonging to the ST-61 and ST-42 complexes were predominant among sheep isolates but were absent from the poultry isolates. In contrast, ST-21 complex isolates were distributed among the different isolation sources. Comparison with MLST data from 91 human disease isolates showed small but significant genetic differentiation between the farm and human isolates; however, representatives of six clonal complexes were found in both samples. These data demonstrate that MLST and the clonal complex model can be used to identify and compare the genotypes of C. jejuni isolates from farm animals and the environment with those from retail food and human disease.     

Production of a Monoclonal Antibody Specific for the Major Outer Membrane Protein of Campylobacter jejuni and Characterization of the Epitope

Campylobacter species are important enteric pathogens causing disease in humans and animals. There is a lack of a good immunological test that can be used routinely to separate Campylobacter jejuni from other Campylobacter species. We produced monoclonal antibodies (MAbs) directed against the major outer membrane protein (MOMP) of C. jejuni using recombinant MOMP as the antigen. One MAb, designated MAb5C4 and of the immunoglobulin G1 isotype, was found to be potentially specific for C. jejuni. Dot blots demonstrated that MAb5C4 reacted with all 29 isolates of C. jejuni tested but did not react with 2 C. jejuni isolates, 26 other Campylobacter spp. isolates, and 19 non-Campylobacter isolates. Western blotting showed that MAb5C4 bound to a single protein band approximately 43 kDa in size, corresponding to the expected size of C. jejuni MOMP. The detection limit of MAb5C4 in a dot blot assay was determined to be about 5 × 10³ bacteria. The epitope on the MOMP was mapped to a region six amino acids in length with the sequence ²¹⁶GGQFNP²²¹, which is 97% conserved among C. jejuni strains but divergent in other Campylobacter spp.; a GenBank search indicated that 95% of C. jejuni isolates will be able to be detected from non-Campylobacter spp. based on the highly specific and conserved region of the GGQFNP polypeptide. The epitope is predicted to be located in a region that is exposed to the periplasm. MAb5C4 is a potentially specific and sensitive MAb that can be used for the specific detection and identification of C. jejuni.     

Antimicrobial Resistance Profiles of Campylobacter jejuni Isolates from Wild Birds in Sweden

In order to determine the occurrence and frequency of resistant strains of the bacterium Campylobacter jejuni and to establish baseline MICs in isolates from an environmental reservoir, the resistance profiles of 10 antimicrobial substances were determined for 137 C. jejuni isolates from wild birds in Sweden. Observed MICs were generally low, with only low to moderate incidence of resistance to the tested compounds. One isolate, however, was resistant to nalidixic acid and ciprofloxacin, indicating that quinolone-resistant genotypes of C. jejuni have the potential to spread to wild bird hosts.     

Correlation between Genotypic Diversity,Lipooligosaccharide Gene Locus Class Variation,and Caco-2 Cell Invasion Potential of Campylobacter jejuni Isolates from Chicken Meat and Humans: Contribution to Virulotyping

Significant interest in studying the lipooligosaccharide (LOS) of Campylobacter jejuni has stemmed from its potential role in postinfection paralytic disorders. In this study we present the results of PCR screening of five LOS locus classes (A, B, C, D, and E) for a collection of 116 C. jejuni isolates from chicken meat (n = 76) and sporadic human cases of diarrhea (n = 40). We correlated LOS classes with clonal complexes (CC) assigned by multilocus sequence typing (MLST). Finally, we evaluated the invasion potential of a panel of 52 of these C. jejuni isolates for Caco-2 cells. PCR screening showed that 87.1% (101/116) of isolates could be assigned to LOS class A, B, C, D, or E. Concordance between LOS classes and certain MLST CC was revealed. The majority (85.7% [24/28]) of C. jejuni isolates grouped in CC-21 were shown to express LOS locus class C. The invasion potential of C. jejuni isolates possessing sialylated LOS (n = 29; classes A, B, and C) for Caco-2 cells was significantly higher (P < 0.0001) than that of C. jejuni isolates with nonsialylated LOS (n = 23; classes D and E). There was no significant difference in invasiveness between chicken meat and human isolates. However, C. jejuni isolates assigned to CC-206 (correlated with LOS class B) or CC-21 (correlated with LOS class C) showed statistically significantly higher levels of invasion than isolates from other CC. Correlation between LOS classes and CC was further confirmed by pulsed-field gel electrophoresis. The present study reveals a correlation between genotypic diversity and LOS locus classes of C. jejuni. We showed that simple PCR screening for C. jejuni LOS classes could reliably predict certain MLST CC and add to the interpretation of molecular-typing results. Our study corroborates that sialylation of LOS is advantageous for C. jejuni fitness and virulence in different hosts. The modulation of cell surface carbohydrate structure could enhance the ability of C. jejuni to adapt to or survive in a host.Campylobacter jejuni is an important human enteric pathogen worldwide (3, 7, 26). Infected humans exhibit a range of clinical spectra, from mild, watery diarrhea to severe inflammatory diarrhea (28). Factors influencing the virulence of C. jejuni include motility, chemotaxis, the ability to adhere to and invade intestinal cells, intracellular survival, and toxin production (28, 30, 52). Besides its role in human enteric illnesses, C. jejuni is a predominant infectious trigger of acute postinfectious neuropathies, such as Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) (1). Significant interest in studying the structure and biosynthesis of the core lipooligosaccharide (LOS) of C. jejuni has resulted from its potential role in these paralytic disorders. Many studies have now provided convincing evidence that molecular mimicry between C. jejuni LOS and gangliosides in human peripheral nerve tissue plays an important causal role in the pathogenesis of GBS/MFS (16, 17, 19, 21).Initial comparative studies of C. jejuni LOS structure and the corresponding DNA sequences of the LOS biosynthesis loci identified eight different LOS locus classes. Three of these classes, A, B, and C, harbor sialyltransferase genes involved in incorporating sialic acid into the LOS (42). Sialylation of the LOS core was found to be associated with ganglioside mimicry and also to affect immunogenicity and serum resistance (21). Recently, Parker et al. (43) identified 11 additional LOS classes on the basis of the sequence at the LOS biosynthesis locus. Their investigation also suggested that the LOS loci of C. jejuni strains are hot spots for genetic exchange, which can lead to mosaicism.Despite evidence on locus variation within C. jejuni LOS classes, PCR-based screening of a collection of 123 clinical and environmental strains showed that almost 60% of C. jejuni strains belong to class A, B, or C (42). Additionally, Godschalk et al. (16) found that 53% (9/17) of GBS-associated C. jejuni strains possessed LOS of class A, while 64% (35/55) of the non-GBS-associated isolates possessed LOS of class A, B, or C, and 62% (13/21) of enteritis-associated Campylobacter strains expressed LOS of class A, B, or C, as well. This relative representation of sialylated LOS classes A, B, and C was hypothesized to be advantageous for C. jejuni in the colonization and infection of various hosts (42, 49). Recently, Louwen et al. (34) demonstrated that C. jejuni strains possessing sialylated LOS (class A, B, or C) invade Caco-2 cells significantly better than nonsialylated strains (with class D or E). Knockout mutagenesis of the LOS sialyltransferase Cst-II in three C. jejuni strains revealed a significant reduction in the invasion potentials of the mutant strains (34). The possible role of LOS in adhesion and invasion was previously highlighted in the work of Perera et al. (44) and Kanipes et al. (29), where a C. jejuni waaF mutant strain showed significant reductions in levels of adherence to and invasion of INT-407 cells.LOS class diversity in C. jejuni strains isolated from chicken meat, an important source of human campylobacteriosis (6, 7, 26), has hardly been studied at all. In addition, the role of LOS class variation in the invasion potential of C. jejuni strains from chicken meat still needs to be explored. The epidemiological relevance of C. jejuni LOS gene screening can be further elaborated by correlating its results with results from other molecular-typing tools (e.g., multilocus sequence typing [MLST] and pulsed-field gel electrophoresis [PFGE]). In the present study, we screened a diverse collection of C. jejuni isolates, from consumer-packaged chicken meats and from sporadic human cases of diarrhea, by PCR for five LOS classes (A, B, C, D, and E). Then we correlated the LOS classes assigned by PCR screening with the genotypes assigned by PFGE and MLST. Finally, we tested the invasion potentials of a representative subset of C. jejuni isolates in relation to their LOS classes and genotypic diversity.     

Antimicrobial Susceptibility Profiles and Molecular Typing of Campylobacter jejuni and Campylobacter coli Isolates from Ducks in South Korea

Campylobacter is a food-borne zoonotic pathogen that causes human gastroenteritis worldwide. Campylobacter bacteria are commensal in the intestines of many food production animals, including ducks and chickens. The objective of the study was to determine the prevalence of Campylobacter species in domestic ducks, and the agar dilution method was used to determine resistance of the isolates to eight antibiotics. In addition, multilocus sequence typing (MLST) was performed to determine the sequence types (STs) of selected Campylobacter isolates. Between May and September 2012, 58 duck farms were analyzed, and 56 (96.6%) were positive for Campylobacter. Among the isolates, 82.1% were Campylobacter jejuni, 16.1% were C. coli, and one was unidentified by PCR. Of the 46 C. jejuni isolates, 87.0%, 10.9%, and 21.7% were resistant to ciprofloxacin, erythromycin, and azithromycin, respectively. Among the C. coli isolates, all 9 strains were resistant to ampicillin, and 77.8% and 33.3% were resistant to ciprofloxacin and azithromycin, respectively. The majority of the Campylobacter isolates were classified as multidrug resistant. Twenty-eight STs were identified, including 20 STs for C. jejuni and 8 STs for C. coli. The most common clonal complexes in C. jejuni were the ST-21 complex and the ST-45 complex, while the ST-828 complex predominated in C. coli. The majority of isolates were of STs noted in ducks and humans from earlier studies, along with seven STs previously associated only with human disease. These STs overlapped between duck and human isolates, indicating that Campylobacter isolates from ducks should be considered potential sources of human infection.     

Molecular Evidence for the Thriving of Campylobacter jejuni ST-4526 in Japan

Campylobacter jejuni is a leading cause of human gastroenteritis worldwide. This study aimed at a better understanding of the genetic diversity of this pathogen disseminated in Japan. We performed multilocus sequence typing (MLST) of Campylobacter jejuni isolated from different sources (100 human, 61 poultry, and 51 cattle isolates) in Japan between 2005 and 2006. This approach identified 62 sequence types (STs) and 19 clonal complexes (CCs), including 11 novel STs. These 62 STs were phylogenetically divided into 6 clusters, partially exhibiting host association. We identified a novel ST (ST-4526) that has never been reported in other countries; a phylogenetic analysis showed that ST-4526 and related STs showed distant lineage from the founder ST, ST-21 within CC-21. Comparative genome analysis was performed to investigate which properties could be responsible for the successful dissemination of ST-4526 in Japan. Results revealed that three representative ST-4526 isolates contained a putative island comprising the region from Cj0737 to Cj0744, which differed between the ST-4526 isolates and the reference strain NCTC11168 (ST-43/CC-21). Amino acid sequence alignment analyses showed that two of three ST-4526 isolates expressed 693aa- filamentous hemagglutination domain protein (FHA), while most of other C. jejuni strains whose genome were sequenced exhibited its truncation. Correspondingly, host cell binding of FHA-positive C. jejuni was greater than that of FHA-truncated strains, and exogenous administration of rFHA protein reduced cell adhesion of FHA-positive bacteria. Biochemical assays showed that this putative protein exhibited a dose-dependent binding affinity to heparan sulfate, indicating its adhesin activity. Moreover, ST-4526 showed increased antibiotic-resistance (nalidixic acid and fluoroquinolones) and a reduced ability for DNA uptake. Taken together, our data suggested that these combined features contributed to the clonal thriving of ST-4526 in Japan.     

Application of the 5′-Nuclease PCR Assay in Evaluation and Development of Methods for Quantitative Detection of Campylobacter jejuni

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional diagnostic testing for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable state. Nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, we present a 5′-nuclease PCR assay for quantitative detection of C. jejuni and describe its evaluation. A probe including positions 381121 to 381206 of the published C. jejuni strain NCTC 11168 genome sequence was identified. When this probe was applied, the assay was positive for all of the isolates of C. jejuni tested (32 isolates, including the type strain) and negative for all other Campylobacter spp. (11 species tested) and several other bacteria (41 species tested). The total assay could be completed in 3 h with a detection limit of approximately 1 CFU. Quantification was linear over at least 6 log units. Quantitative detection methods are important for both research purposes and further development of C. jejuni detection methods. In this study, we used the assay to investigate to what extent the PCR signals generated by heat-killed bacteria interfere with the detection of viable C. jejuni after exposure at elevated temperatures for up to 5 days. An approach to the reduction of the PCR signal generated by dead bacteria was also investigated by employing externally added DNases to selectively inactivate free DNA and exposed DNA in heat-killed bacteria. The results indicated relatively good discrimination between exposed DNA from dead C. jejuni and protected DNA in living bacteria.     

Bacteriophage Therapy To Reduce Campylobacter jejuni Colonization of Broiler Chickens

Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log₁₀ CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens.     

Isolation and characterization of Campylobacter jejuni subsp. jejuni from macaroni penguins (Eudyptes chrysolophus) in the subantarctic region

On Bird Island, South Georgia, albatrosses (n = 140), penguins (n = 100), and fur seals (n = 206) were sampled for Campylobacter jejuni. C. jejuni subsp. jejuni was recovered from three macaroni penguins (Eudyptes chrysolophus). These isolates, the first reported for the subantarctic region, showed low genetic diversity and high similarity to Northern Hemisphere C. jejuni isolates, possibly suggesting recent introduction to the area.     

Detection of Campylobacter jejuni and Salmonella typhimurium in chicken using PCR for virulence factor hipO and invA genes (Saudi Arabia)

Campylobacter jejuni and Salmonella typhimurium are the leading causes of bacterial food contamination in chicken carcasses. Contamination is particularly associated with the slaughtering process. The present study isolated C. jejuni and S. typhimurim from fifty chicken carcass samples, all of which were acquired from different companies in Riyadh, Saudi Arabia. The identification of C. jejuni was performed phenotypically by using a hippurate test and genetically using a polymerase chain reaction with primers for 16S rRNA and hippurate hydrolase (hipO gene). For the dentification of S. typhimurim, a serological Widal test was carried out using serum anti-S. typhimurium antibodies. Strains were genetically detected using invA gene primers. The positive isolates for C. jejuni showed a specific molecular size of 1448 bp for 16S rRNA and 1148 bp for hipO genes. However, the positive isolates of the invA gene exhibited a specific molecular size at 244 bp using polymerase chain reaction (PCR). Comparing sequencing was performed with respect to the invA gene and the BLAST nucleotide isolates that were identified as Salmonella enterica subsp. enterica serovar typhimurium strain ST45, thereby producing a similarity of 100%. The testing identified C. jejuni for hippuricase, GenBank: {"type":"entrez-nucleotide","attrs":{"text":"Z36940.1","term_id":"535805"}}Z36940.1. While many isolates of Salmonella spp. that contained the invA gene were not necessarily identified as S. typhimurim, the limiting factor for the Widal test used antiS. typhimurum antibodies. The multidrug resistance (MDR) of C. jejuni isolates in chickens was compared with the standard C. jejuni strain ATCC 22931. Similarly, S. typhimurium isolates were compared with the standard S. typhimurium strain ATCC 14028.