Direct quantification of Campylobacter jejuni and Campylobacter lanienae in feces of cattle by real-time quantitative PCR

Campylobacter species are fastidious to culture, and the ability to directly quantify biomass in microbiologically complex substrates using real-time quantitative (RTQ) PCR may enhance our understanding of their biology and facilitate the development of efficacious mitigation strategies. This study reports the use of nested RTQ-PCR to directly quantify Campylobacter jejuni and Campylobacter lanienae in cattle feces. For C. jejuni, the single-copy mapA gene was selected. For C. lanienae, the three-copy 16S rRNA gene was targeted. RTQ-PCR primers were tested alone or they were nested with species-specific primers, and amplification products were detected using the intercalating dye SYBR Green. Nesting did not increase the specificity or sensitivity of C. jejuni quantification, and the limit of quantification was 19 to 25 genome copies ( approximately 3 x 10(3) CFU/g of feces). In contrast, nested RTQ-PCR was necessary to confer specificity on C. lanienae by targeting the 16S rRNA gene. The limit of quantification was 1.8 genome copies ( approximately 250 CFU/g of feces), and there was no discernible difference between the two C. lanienae secondary primer sets evaluated. Detection and quantification of C. jejuni in naturally infested cattle feces by RTQ-PCR were comparable to the results of culture-based methods. In contrast, culturing did not detect C. lanienae in 6 of 10 fecal samples positive for the bacterium and substantially underestimated cell densities relative to nested RTQ-PCR. The results of this study illustrate that RTQ-PCR can be used to directly quantify campylobacters, including very fastidious species, in a microbiologically and chemically complex substrate. Furthermore, targeting of a multicopy universal gene provided highly sensitive quantification of C. lanienae, but nested RTQ-PCR was necessary to confer specificity. This method will facilitate subsequent studies to elucidate the impact of this group of bacteria within the gastrointestinal tracts of livestock and studies of the factors that influence colonization success and shedding.     

Use of PCR for Direct Detection of Campylobacter Species in Bovine Feces

This study reports on the use of PCR to directly detect and distinguish Campylobacter species in bovine feces without enrichment. Inhibitors present in feces are a major obstacle to using PCR to detect microorganisms. The QIAamp DNA stool minikit was found to be an efficacious extraction method, as determined by the positive amplification of internal control DNA added to bovine feces before extraction. With nested or seminested multiplex PCR, Campylobacter coli, C. fetus, C. hyointestinalis, and C. jejuni were detected in all fecal samples inoculated at ≈10⁴ CFU g⁻¹, and 50 to 83% of the samples inoculated at ≈10³ CFU g⁻¹ were positive. At ≈10² CFU g⁻¹, C. fetus, C. hyointestinalis, and C. jejuni (17 to 50% of the samples) but not C. coli were detected by PCR. From uninoculated bovine feces, a total of 198 arbitrarily selected isolates of Campylobacter were recovered on four commonly used isolation media incubated at three temperatures. The most frequently isolated taxa were C. jejuni (152 isolates) and C. lanienae (42 isolates), but isolates of C. fetus subsp. fetus, Arcobacter butzleri, and A. skirrowii also were recovered (≤2 isolates per taxon). Considerable variability was observed in the frequency of isolation of campylobacters among the four media and three incubation temperatures tested. With genus-specific primers, Campylobacter DNA was detected in 75% of the fecal samples, representing an 8% increase in sensitivity relative to that obtained with microbiological isolation across the four media and three incubation temperatures tested. With nested primers, C. jejuni and C. lanienae were detected in 25 and 67% of the samples, respectively. In no instance was DNA from either C. coli, C. fetus, or C. hyointestinalis detected in uninoculated bovine feces. PCR was more sensitive than isolation on microbiological media for detecting C. lanienae (17%) but not C. jejuni. Campylobacters are a diverse and fastidious group of bacteria, and the development of direct PCR not only will increase the understanding of Campylobacter species diversity and their frequency of occurrence in feces but also will enhance the knowledge of their role in the gastrointestinal tract of livestock and of the factors that influence shedding.     

Colonization of Cattle Intestines by Campylobacter jejuni and Campylobacter lanienae

The location and abundance of Campylobacter jejuni and Campylobacter lanienae in the intestines of beef cattle were investigated using real-time quantitative PCR in two studies. In an initial study, digesta and tissue samples were obtained along the digestive tract of two beef steers known to shed C. jejuni and C. lanienae (steers A and B). At the time of slaughter, steer B weighed 540 kg, compared to 600 kg for steer A, yet the intestine of steer B (40.5 m) was 36% longer than the intestine of steer A (26.1 m). In total, 323 digesta samples (20-cm intervals) and 998 tissue samples (3.3- to 6.7-cm intervals) were processed. Campylobacter DNA was detected in the digesta and in association with tissues throughout the small and large intestines of both animals. Although C. jejuni and C. lanienae DNA were detected in both animals, only steer A contained substantial quantities of C. jejuni DNA. In both digesta and tissues of steer A, C. jejuni was present in the duodenum and jejunum. Considerable quantities of C. jejuni DNA also were observed in the digesta obtained from the cecum and ascending colon, but minimal DNA was associated with tissues of these regions. In contrast, steer B contained substantial quantities of C. lanienae DNA, and DNA of this bacterium was limited to the large intestine (i.e., the cecum, proximal ascending colon, descending colon, and rectum); the majority of tissue-associated C. lanienae DNA was present in the cecum, descending colon, and rectum. In a second study, the location and abundance of C. jejuni and C. lanienae DNA were confirmed in the intestines of 20 arbitrarily selected beef cattle. DNA of C. jejuni and C. lanienae were detected in the digesta of 57% and 95% of the animals, respectively. C. jejuni associated with intestinal tissues was most abundant in the duodenum, ileum, and rectum. However, one animal contributed disproportionately to the abundance of C. jejuni DNA in the ileum and rectum. C. lanienae was most abundant in the large intestine, and the highest density of DNA of this bacterium was found in the cecum. Therefore, C. jejuni colonized the proximal small intestine of asymptomatic beef cattle, whereas C. lanienae primarily resided in the cecum, descending colon, and rectum. This information could be instrumental in developing efficacious strategies to manage the release of these bacteria from the gastrointestinal tracts of cattle.     

Frequency and Spatial Distribution of Environmental Campylobacter spp.

Humans are exposed to Campylobacter spp. in a range of sources via both food and environmental pathways. For this study, we explored the frequency and distribution of thermophilic Campylobacter spp. in a 10- by 10-km square rural area of Cheshire, United Kingdom. The area contains approximately 70, mainly dairy, farms and is used extensively for outdoor recreational activities. Campylobacter spp. were isolated from a range of environmental samples by use of a systematic sampling grid. Livestock (mainly cattle) and wildlife feces and environmental water and soil samples were cultured, and isolates were presumptively identified by standard techniques. These isolates were further characterized by PCR. Campylobacter jejuni was the most prevalent species in all animal samples, ranging from 11% in samples from nonavian wildlife to 36% in cattle feces, and was isolated from 15% of water samples. Campylobacter coli was commonly found in water (17%) and sheep (21%) samples, but rarely in other samples. Campylobacter lari was recovered from all sample types, with the exception of sheep feces, and was found in moderate numbers in birds (7%) and water (5%). Campylobacter hyointestinalis was only recovered from cattle (7%) and birds (1%). The spatial distribution and determinants of C. jejuni in cattle feces were examined by the use of model-based spatial statistics. The distribution was consistent with very localized within-farm or within-field transmission and showed little evidence of any larger-scale spatial dependence. We concluded that there is a potentially high risk of human exposure to Campylobacter spp., particularly C. jejuni, in the environment of our study area. The prevalence and likely risk posed by C. jejuni-positive cattle feces in the environment diminished as the fecal material aged. After we took into account the age of the fecal material, the absence or presence of rain, and the presence of bird feces, there was evidence of significant variation in the prevalence of C. jejuni-positive cattle feces between grazing fields but no evidence of spatial clustering beyond this resolution. The spatial pattern of C. jejuni is therefore consistent with that for an organism that is ubiquitous in areas contaminated with cattle feces, with a short-scale variation in infection intensity that cannot be explained solely by variations in the age of the fecal material. The observed pattern is not consistent with large-scale transmission attributable to watercourses, wildlife territories, or other geographical features that transcend field and farm boundaries.     

Prolonged Survival of Campylobacter Species in Bovine Manure Compost

The persistence of naturally occurring campylobacteria in aerobic compost constructed of manure from beef cattle that were administered chlortetracycline and sulfamethazine (AS700) or from cattle not administered antibiotics (control) was examined. Although there were no differences in population sizes of heterotrophic bacteria, the temperature of AS700 compost was more variable and did not become as high as that of control compost. There were significant differences in water content, total carbon (C), total nitrogen (N), and electrical conductivity but not in the C/N ratio or pH between the two compost treatments. Campylobacteria were readily isolated from pen manure, for up to day 15 from control compost, and throughout the active phase of AS700 compost. Campylobacter DNA (including Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis, and Campylobacter jejuni) was detected over the ca. 10-month composting period, and no reductions in quantities of C. jejuni DNA were observed over the duration of the active phase. The utilization of centrifugation in combination with ethidium monoazide (EMA) significantly reduced (>90%) the amplification of C. jejuni DNA that did not originate from cells with intact cell membranes. No differences were observed in the frequency of Campylobacter DNA detection between EMA- and non-EMA-treated samples, suggesting that Campylobacter DNA amplified from compost was extracted from cells with intact cell membranes (i.e., from viable cells). The findings of this study indicate that campylobacteria excreted in cattle feces persist for long periods in compost and call into question the common belief that these bacteria do not persist in manure.Campylobacter jejuni and, to a lesser extent, Campylobacter coli incite serious acute and chronic afflictions. Enteritis caused by C. jejuni (i.e., campylobacteriosis) is the most common cause of bacterial enteritis in Canada (http://dsol-smed.phac-aspc.gc.ca/dsol-smed/ndis/index-eng.php). Although the epidemiology of campylobacteriosis is poorly understood, sporadic outbreaks of campylobacteriosis involving contaminated water have occurred when water treatment has failed. The most serious outbreak in Canada occurred in Walkerton Ontario in 2000; more than 2,300 people became infected with waterborne Escherichia coli O157:H7 and/or C. jejuni originating from cattle feces (3). Alberta, Canada, possesses a very large beef cattle population (≈6 million animals) primarily concentrated in the southern region of the province, and ≈2 million of these animals are in finishing feedlots (1). Large quantities of manure are produced by feedlot cattle. For example, in the Chinook Health Region of Southwestern Alberta in which Lethbridge is situated, there are ≈700,000 cattle in feedlots at any given time, producing ≈12 million kg of manure (fresh weight) per day. Several Campylobacter species, including C. jejuni and C. coli, are frequently shed in beef cattle feces in large numbers (15, 16). Although the impact of cattle-borne campylobacters on human health has not been definitely determined, the southern region of Alberta possesses one of the highest rates of campylobacteriosis in Canada among its human inhabitants, concomitant with the very high density of cattle in this region.Large-scale windrow composting of bovine manure from intensive cattle operations is practiced by some Alberta feedlots. Composting is an aerobic process in which organic matter in manure is stabilized into a humus-like product (30). The process results in water loss and mass reduction, nutrient transformation (22), alteration of physical structure (23), elimination of weed seeds (21), and the inactivation of coliform bacteria (25), protozoan cysts and oocysts (34), and viruses (39). Limited research has investigated the impact of manure management systems, such as aerobic composting, on deactivation of campylobacters. Furthermore, the impact of antimicrobial agents excreted into the manure on the efficacy of the composting process on Campylobacter deactivation has not been investigated. Most studies conducted to date have indicated that campylobacters do not persist well in solid manure once excreted (7, 11, 12, 26, 32, 39). Although it is difficult to isolate or enumerate Campylobacter species within microbiologically complex substrates, molecular detection and/or quantification methods have not been extensively applied to study the persistence of campylobacteria. Furthermore, the persistence of naturally shed campylobacteria has largely been overlooked. Thus, the overall objective of the current study was to measure the ability of campylobacteria naturally shed in bovine feces to persist in manure compost using a combination of culture- and culture-independent methods. Specific objectives were (i) to develop and utilize a centrifugation method to facilitate isolation and detection of DNA from Campylobacter species in bovine manure compost, (ii) to apply qualitative and quantitative PCR methods to evaluate persistence of campylobacteria in compost, (iii) to validate the molecular methods used to amplify DNA from viable cells, and (iv) to contrast the persistence of Campylobacter species in composted manure obtained from beef cattle maintained on a diet supplemented with chlortetracycline and sulfamethazine (AS700) with composted manure from animals not administered antimicrobial agents.     

Correlation of Campylobacter Bacteriophage with Reduced Presence of Hosts in Broiler Chicken Ceca

Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log₁₀ 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log₁₀ 6.9 CFU/g).     

Bacteriophage Receptor Binding Protein Based Assays for the Simultaneous Detection of Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of foodborne gastroenteritis which is occasionally followed by a debilitating neuropathy known as Guillain-Barré syndrome. Rapid and specific detection of these pathogens is very important for effective control and quick treatment of infection. Most of the diagnostics available for these organisms are time consuming and require technical expertise with expensive instruments and reagents to perform. Bacteriophages bind to their host specifically through their receptor binding proteins (RBPs), which can be exploited for pathogen detection. We recently sequenced the genome of C. jejuni phage NCTC12673 and identified its putative host receptor binding protein, Gp047. In the current study, we localized the receptor binding domain to the C-terminal quarter of Gp047. CC-Gp047 could be produced recombinantly and was capable of agglutinating both C. jejuni and C. coli cells unlike the host range of the parent phage which is limited to a subset of C. jejuni isolates. The agglutination procedure could be performed within minutes on a glass slide at room temperature and was not hindered by the presence of buffers or nutrient media. This agglutination assay showed 100% specificity and the sensitivity was 95% for C. jejuni (n = 40) and 90% for C. coli (n = 19). CC-Gp047 was also expressed as a fusion with enhanced green fluorescent protein (EGFP). Chimeric EGFP_CC-Gp047 was able to specifically label C. jejuni and C. coli cells in mixed cultures allowing for the detection of these pathogens by fluorescent microscopy. This study describes a simple and rapid method for the detection of C. jejuni and C. coli using engineered phage RBPs and offers a promising new diagnostics platform for healthcare and surveillance laboratories.     

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 in Bulk Tank Milk and Epidemiology of Campylobacter jejuni in Dairy Herds in Northern Italy

Thermotolerant Campylobacter spp. are frequently the cause of human gastroenteritis and have assumed more importance in Italy following the increased consumption of raw milk. Our objectives were to determine the prevalence and genotypes of Campylobacter spp. in dairy herds and to investigate the possible sources of bulk milk contamination. Bulk milk from dairy herds (n = 282) was cultured for Campylobacter spp. and Enterobacteriaceae. At three Campylobacter jejuni-positive farms, bovine feces, pigeon intestines, milk, and water points were also investigated. Isolates were identified by PCR and genotyped using multilocus sequence typing (MLST). C. jejuni was detected in 34 (12%) bulk milk samples. The strains belonged to 14 sequence types, and the most common clonal complexes were CC-21, CC-48, and CC-403. No association was demonstrated between the presence of C. jejuni and high levels of Enterobacteriaceae in bulk milk. At the three farms examined, C. jejuni was isolated from bovine feces (25/82 [30.5%]), pigeon intestines (13/60 [21.7%]), bulk milk (10/24 [41.7%]), and water points (4/16 [25%]). MLST revealed lineages that were common between milk and bovine feces but distinct between cattle and pigeons. In one herd, C. jejuni with the same genotype was isolated repeatedly from bulk milk and a cow with an udder infection. Our results showed a high prevalence of C. jejuni in bulk milk and suggested that udder excretion, in addition to fecal matter, may be a route of bulk milk contamination. MLST analysis indicated that pigeons are probably not relevant for the transmission of C. jejuni to cattle and for milk contamination.     

Genomic Characterization of Campylobacter jejuni Strain M1

Campylobacter jejuni strain M1 (laboratory designation 99/308) is a rarely documented case of direct transmission of C. jejuni from chicken to a person, resulting in enteritis. We have sequenced the genome of C. jejuni strain M1, and compared this to 12 other C. jejuni sequenced genomes currently publicly available. Compared to these, M1 is closest to strain 81116. Based on the 13 genome sequences, we have identified the C. jejuni pan-genome, as well as the core genome, the auxiliary genes, and genes unique between strains M1 and 81116. The pan-genome contains 2,427 gene families, whilst the core genome comprised 1,295 gene families, or about two-thirds of the gene content of the average of the sequenced C. jejuni genomes. Various comparison and visualization tools were applied to the 13 C. jejuni genome sequences, including a species pan- and core genome plot, a BLAST Matrix and a BLAST Atlas. Trees based on 16S rRNA sequences and on the total gene families in each genome are presented. The findings are discussed in the background of the proven virulence potential of M1.     

Complete Genome Sequence of a Variant of Campylobacter jejuni NCTC 11168

Campylobacter jejuni NCTC 11168 is widely used in research, but at least two variants have been reported. The available genome was sequenced from a variant which later showed a different phenotype and gene expression profile. Here we present the complete genome sequence of a second variant of C. jejuni NCTC 11168.     

Competitive Exclusion of Heterologous Campylobacter spp. in Chicks

Chicken and human isolates of Campylobacter jejuni were used to provide oral challenge of day-old broiler chicks. The isolation ratio of the competing challenge strains was monitored and varied, depending upon the isolates used. A PCR-restriction fragment length polymorphism assay of the flagellin gene (flaA) was used to discriminate between the chick-colonizing isolates. Our observations indicated that the selected C. jejuni colonizers dominated the niche provided by the chicken ceca. Chicken isolates from the flaA type 7 grouping generally had numerical superiority over the human isolates when they were administered in our 1-day-old chick model. Our results suggest that it is possible to use combinations of C. jejuni chicken isolates as a defined bacterial preparation for the competitive exclusion of human-pathogenic C. jejuni in poultry.     

A Real-Time PCR Assay for the Detection of Campylobacter jejuni in Foods after Enrichment Culture

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37°C for 24 h, followed by 42°C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.     

Fingerprinting of Campylobacter jejuni by Using Resolution-Optimized Binary Gene Targets Derived from Comparative Genome Hybridization Studies

The aim of this investigation was to exploit the vast comparative data generated by comparative genome hybridization (CGH) studies of Campylobacter jejuni in developing a genotyping method. We examined genes in C. jejuni that exhibit binary status (present or absent between strains) within known plasticity regions, in order to identify a minimal subset of gene targets that provide high-resolution genetic fingerprints. Using CGH data from three studies as input, binary gene sets were identified with “Minimum SNPs” software. “Minimum SNPs” selects for the minimum number of targets required to obtain a predefined resolution, based on Simpson's index of diversity (D). After implementation of stringent criteria for gene presence/absence, eight binary genes were found that provided 100% resolution (D = 1) of 20 C. jejuni strains. A real-time PCR assay was developed and tested on 181 C. jejuni and Campylobacter coli isolates, a subset of which have previously been characterized by multilocus sequence typing, flaA short variable region sequencing, and pulsed-field gel electrophoresis. In addition to the binary gene real-time PCR assay, we refined the seven-member single nucleotide polymorphism (SNP) real-time PCR assay previously described for C. jejuni and C. coli. By normalizing the SNP assay with the respective C. jejuni and C. coli ubiquitous genes, mapA and ceuE, the polymorphisms at each SNP could be determined without separate reactions for every polymorphism. We have developed and refined a rapid, highly discriminatory genotyping method for C. jejuni and C. coli that uses generic technology and is amenable to high-throughput analyses.     

Detection of Small Numbers of Campylobacter jejuni and Campylobacter coli Cells in Environmental Water, Sewage, and Food Samples by a Seminested PCR Assay

A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as ≤3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection of C. jejuni or C. coli in drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms.     

Reducing Campylobacter jejuni Colonization of Poultry via Vaccination

Campylobacter jejuni is a leading bacterial cause of human gastrointestinal disease worldwide. While C. jejuni is a commensal organism in chickens, case-studies have demonstrated a link between infection with C. jejuni and the consumption of foods that have been cross-contaminated with raw or undercooked poultry. We hypothesized that vaccination of chickens with C. jejuni surface-exposed colonization proteins (SECPs) would reduce the ability of C. jejuni to colonize chickens, thereby reducing the contamination of poultry products at the retail level and potentially providing a safer food product for consumers. To test our hypothesis, we injected chickens with recombinant C. jejuni peptides from CadF, FlaA, FlpA, CmeC, and a CadF-FlaA-FlpA fusion protein. Seven days following challenge, chickens were necropsied and cecal contents were serially diluted and plated to determine the number of C. jejuni per gram of material. The sera from the chickens were also analyzed to determine the concentration and specificity of antibodies reactive against the C. jejuni SECPs. Vaccination of chickens with the CadF, FlaA, and FlpA peptides resulted in a reduction in the number of C. jejuni in the ceca compared to the non-vaccinated C. jejuni-challenged group. The greatest reduction in C. jejuni colonization was observed in chickens injected with the FlaA, FlpA, or CadF-FlaA-FlpA fusion proteins. Vaccination of chickens with different SECPs resulted in the production of C. jejuni-specific IgY antibodies. In summary, we show that the vaccination of poultry with individual C. jejuni SECPs or a combination of SECPs provides protection of chickens from C. jejuni colonization.     

Chromosomal tet(O)-Harboring Regions in Campylobacter coli Isolates from Turkeys and Swine

In turkey-derived Campylobacter coli isolates of a unique lineage (cluster II), the tetracycline resistance determinant tet(O) was chromosomal and was part of a gene cassette (transposon) interrupting a Campylobacter jejuni-associated putative citrate transporter gene. In contrast, the swine-derived C. coli strain 6461 harbored a chromosomal tet(O) in a different genomic location.     

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.